SMART MODE:

NORMAL GENOMIC

• Conner JM, Darracq MA, Roberts J, Tuszynski MH
• Nontropic actions of neurotrophins: subcortical nerve growth factor gene delivery reverses age-related degeneration of primate cortical cholinergic innervation.
• Proc Natl Acad Sci U S A. 2001; 98: 1941-6
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• Normal aging is associated with a significant reduction in cognitive function across primate species. However, the structural and molecular basis for this age-related decline in neural function has yet to be defined clearly. Extensive cell loss does not occur as a consequence of normal aging in human and nonhuman primate species. More recent studies have demonstrated significant reductions in functional neuronal markers in subcortical brain regions in primates as a consequence of aging, including dopaminergic and cholinergic systems, although corresponding losses in cortical innervation from these neurons have not been investigated. In the present study, we report that aging is associated with a significant 25% reduction in cortical innervation by cholinergic systems in rhesus monkeys (P < 0.001). Further, these age-related reductions are ameliorated by cellular delivery of human nerve growth factor to cholinergic somata in the basal forebrain, restoring levels of cholinergic innervation in the cortex to those of young monkeys (P = 0.89). Thus, (i) aging is associated with a significant reduction in cortical cholinergic innervation; (ii) this reduction is reversible by growth-factor delivery; and (iii) growth factors can remodel axonal terminal fields at a distance, representing a nontropic action of growth factors in modulating adult neuronal structure and function (i.e., administration of growth factors to cholinergic somata significantly increases axon density in terminal fields). These findings are relevant to potential clinical uses of growth factors to treat neurological disorders.

• Shooter EM
• Early days of the nerve growth factor proteins.
• Annu Rev Neurosci. 2001; 24: 601-29
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• Adult male mouse submaxillary glands served as the preferred starting material for the isolation of the nerve growth factor (NGF) proteins in most of the isolation studies done. Two types of NGF proteins were isolated from extracts of the gland, a high-molecular-weight 7S NGF complex and a low-molecular-weight protein variously called NGF, betaNGF, or 2.5S NGF. The latter, which mediated all known biological functions of NGF, were closely related forms of a basic NGF dimer in which the N and C termini of two monomers (chains) were modified by proteolytic enzymes to different extents with no effect on biological activity. The betaNGF dimer showed a novel protein structure in which the two chains interacted non-covalently over a wide surface. Correspondingly, the betaNGF dimer was found to be unusually stable and the form through which NGFs actions were mediated at physiological concentrations. The betaNGF dimer was one of three subunits in 7S NGF; the other two were the gamma subunit, an arginine esteropeptidase or kallikrein, and the alpha subunit, an inactive kallikrein. Two zinc ions were also present in the complex and contributed greatly to its stability. There was much debate about whether 7S NGF was a specific protein complex of interacting subunits and, if so, what functions it might play in the biology of NGF. Observations of the inhibition of the enzyme activity of the gamma subunit and of the biological activity of betaNGF in 7S NGF were important in determining that 7S NGF was a naturally occurring complex and the sole source of NGF in the gland extract or in saliva. Specific interactions between the active site of the gamma subunit and the C-terminal arginine residues of the NGF chains, confirmed in the three-dimensional structure of 7S NGF, suggested a role for the gamma subunit in pro-NGF processing during the assembly of 7S NGF. In spite of the detailed knowledge of 7S NGF structure, no information on the role of this complex in the neurobiology of NGF has emerged. With the exception of the submaxillary gland of an African rodent, no other source of NGF has been convincingly shown to synthesize the alpha and gamma subunits, and they may well be irrelevant to NGFs actions.

• Stanisz AM, Stanisz JA
• Nerve growth factor and neuroimmune interactions in inflammatory diseases.
• Ann N Y Acad Sci. 2000; 917: 268-72
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• Discovered almost 50 years ago, nerve growth factor (NGF) has been extensively studied in various biological systems. NGF has recently been suggested to play an important role in mediating and/or regulating immune response, in addition to its trophic and tropic effects on nerve growth and regeneration It is clear that in complex interactions between immune cells and nervous system NGF plays a central role. We have only just begun to identify and understand the direct mechanisms by which NGF activates target cells, the precise identity of the target cells, and the particular factors released from target cells. Nerve growth factor together with possibly other neurotrophins such as BDNF (brain-derived nerve growth factor), GDNF (glial-derived nerve growth factor) or NT3 are important modulators of immunity. More detailed studies are needed at the receptor, mediator and cellular levels to better understand the neuroimmunomodulatory properties of neurothrophins and NGF. The nature of the involvement of NGF in inflammation and inflammatory diseases remains a particularly interesting question. By blocking NGF or mediators released upon NGF activation, we are able to control the progress of inflammation, thereby opening many therapeutic opportunities for the future.

• Apfel SC
• Neurotrophic factors and pain.
• Clin J Pain. 2000; 16: 711-711
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• Neurotrophic factors have been shown to play significant roles in the transmission of physiologic and pathologic pain. Nerve growth factor appears to be particularly important. It is crucial for the development of sympathetic and small fiber sensory neurons that serve as nociceptors. It stimulates the expression and release of neuropeptides involved in pain transmission, and interacts with cellular and molecular mediators of inflammation. Blockade with nerve growth factor antiserum demonstrates the critical role of the growth factor in mediating inflammatory hyperalgesia. Administration of nerve growth factor to rodents results in the rapid onset of hyperalgesia. Although the exact mechanism is unknown, several possibilities have been proposed. In clinical trials for the treatment of Alzheimer disease and peripheral neuropathy, induction of pain has been the major adverse event. When administered intracebrebroventricularly, a dull constant back pain resulted. Subcutaneous injection of nerve growth factor induces injection site hyperalgesia, as well as generalized myalgias and arthralgias. Whether the mechanisms underlying these adverse events are identical to those associated with the hyperalgesia in rodents is unknown. In addition to nerve growth factor, other growth factors, such as brain-derived neurotrophic factor and glial cell-derived neurotrophic factor, may be involved in pain pathways. Their precise roles are still being defined, but evidence suggests that they may have particular relevance to neuropathic pain. Understanding the role all these factors play may change the way we approach the treatment of pain in general, and neuropathic pain in particular.

• Otten U, Marz P, Heese K, Hock C, Kunz D, Rose-John S
• Cytokines and neurotrophins interact in normal and diseased states.
• Ann N Y Acad Sci. 2000; 917: 322-30
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• Neurotrophins (NTs) such as nerve growth factor (NGF) as well as cytokines, for example, interleukin-6 (IL-6), are communicators between the nervous and immune systems. There is evidence for mutual interactions between NTs and cytokines. Strategies are being developed to elucidate the molecular mechanism/s of interactions and to understand how cytokines are involved in health and disease. Analysis of underlying signaling pathways in glial cells indicates that different transcription factors, such as NF-kappa B, cAMP-responsive-element binding protein (CREB), and activator protein 1 (AP-1), are involved in NT induction. IL-6 and NTs of the NGF family are coexpressed at sites of nerve injury. Interactions of these factors could modulate both neuronal de- and regeneration: IL-6 in conjunction with its soluble IL-6 receptor induces a specific pattern of NTs in astrocytes in defined brain regions. This indicates that the IL-6 system mediates a local supply of NTs that participate in diverse CNS functions, such as protection of neurons from insults, neuronal survival, and neuroimmune responses.

• Bersani G, Iannitelli A, Fiore M, Angelucci F, Aloe L
• Data and hypotheses on the role of nerve growth factor and other neurotrophins in psychiatric disorders.
• Med Hypotheses. 2000; 55: 199-207
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• Nerve growth factor (NGF) was discovered and characterized for its role on the growth, differentiation and maintenance of specific neurons of the peripheral nervous system. Subsequent studies revealed that NGF is synthesized and released within the central nervous system and exerts a trophic and functional role on basal forebrain cholinergic neurons; it is involved in a protective role following brain insults induced by an epileptic status, seizure, as well as surgical and chemical lesions.More recently our collaborative studies provided evidence that NGF is implicated in neurobehavioral response including cerebral alterations associated with psychiatric disorders. In this brief review, ongoing and emerging data are presented and discussed.

• Heinrich G, Lum T
• Fish neurotrophins and Trk receptors.
• Int J Dev Neurosci. 2000; 18: 1-27

• Saragovi HU, Gehring K
• Development of pharmacological agents for targeting neurotrophins and their receptors.
• Trends Pharmacol Sci. 2000; 21: 93-8
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• Neurotrophins comprise a family of protein growth factors that control the survival, growth, and/or differentiation of neurons and several other cell populations derived from the neuroectoderm. Neurotrophins and their receptors are important targets for the therapy of human disease, with potential applications ranging from the treatment of chronic or acute neurodegeneration to pain and cancer. Neurotrophins have been used clinically but are poor pharmacological agents. Consequently, approaches to develop pharmacological agents that target neurotrophins, their receptors or neurotrophin signaling pathways have been attempted.

• Aloe L, Simone MD, Properzi F
• Nerve growth factor: a neurotrophin with activity on cells of the immune system.
• Microsc Res Tech. 1999; 45: 285-91
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• Numerous studies published in the last two decades provide evidence that nerve growth factor (NGF), a polypeptide originally discovered because of its neurotrophic activity, acts on a variety of cells of the immune system, including mast cells, eosinophils, and B and T lymphocytes. NGF has been shown to increase during inflammatory responses, autoimmune disorders, parasitic infections, and allergic diseases. Moreover, stress, which is characterized also by activation of a variety of immune cells, causes a significant increase in basal plasma NGF levels. Recently published studies reveal that hematopoietic progenitor cells seem to be able to produce and/or respond to NGF. We report these data and discuss the hypothesis of the possible implication of NGF on the functional activities of immune cells.

• Varela-Nieto I, Silos-Santiago I
• Introduction to structural and functional studies on nerve growth factor.
• Microsc Res Tech. 1999; 45: 206-206

• Aloe L
• Rita Levi-Montalcini: a brief biographic view of past and present studies on nerve growth factor.
• Microsc Res Tech. 1999; 45: 207-9

• Niewiadomska G, Malecki M
• [Signal transduction of nerve growth factor (NGF) and its TrkA and p75NTR receptors]
• Postepy Biochem. 1999; 45: 21-31

• Shamovsky IL, Ross GM, Riopelle RJ, Weaver DF
• The interaction of neurotrophins with the p75NTR common neurotrophin receptor: a comprehensive molecular modeling study.
• Protein Sci. 1999; 8: 2223-33
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• Neurotrophins are a family of proteins with pleiotropic effects mediated by two distinct receptor types, namely the Trk family, and the common neurotrophin receptor p75NTR. Binding of four mammalian neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), to p75NTR is studied by molecular modeling based on X-ray structures of the neurotrophins and the extracellular domain of p55TNFR, a homologue of p75NTR. The model of neurotrophin/receptor interactions suggests that the receptor binding domains of neurotrophins (loops I and IV) are geometrically and electrostatically complementary to a putative binding site of p75NTR, formed by the second and part of the third cysteine-rich domains. Geometric match of neurotrophin/receptor binding domains in the complexes, as characterized by shape complementarity statistic Sc, is comparable to known protein/protein complexes. All charged residues within the loops I and IV of the neurotrophins, previously determined as being critical for p75NTR binding, directly participate in receptor binding in the framework of the model. Principal residues of the binding site of p75NTR include Asp47, Lys56, Asp75, Asp76, Asp88, and Glu89. The additional involvement of Arg80 and Glu53 is specific for NGF and BDNF, respectively, and Glu73 participates in binding with NT-3 and NT-4/5. Neurotrophins are likely to induce similar, but not identical, conformational changes within the p75NTR binding site.

• Lamballe F
• [Neurotrophins and the central nervous system]
• Rev Neurol (Paris). 1999; 155: 1084-7

• Kishi T et al.
• Crystal structure of neuropsin, a hippocampal protease involved in kindling epileptogenesis.
• J Biol Chem. 1999; 274: 4220-4
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• Neuropsin is a novel serine protease, the expression of which is highly localized in the limbic areas of the mouse brain and which is suggested to be involved in kindling epileptogenesis and hippocampal plasticity. The 2.1-A resolution crystal structure of neuropsin provides the first three-dimensional view of one of the serine proteases highly expressed in the nervous system, and reveals a serine protease fold that exhibits chimeric features between trypsin and nerve growth factor-gamma (NGFgamma), a member of the kallikrein family. Neuropsin possesses an N-glycosylated "kallikrein loop" but forms six disulfide bonds corresponding to those of trypsin. The ordered kallikrein loop projects proline toward the active site to restrict smaller residues or proline at the P2 position of substrates. Loop F, which participates in forming the S3/S4 sites, is similar to trypsin rather than NGFgamma. The unique conformations of loops G and H form an S1 pocket specific for both arginine and lysine. These characteristic loop structures forming the substrate-binding site suggest the novel substrate specificity of neuropsin and give a clue to the design of its specific inhibitors.

• Wiesmann C, Ultsch MH, Bass SH, de Vos AM
• Crystal structure of nerve growth factor in complex with the ligand-binding domain of the TrkA receptor.
• Nature. 1999; 401: 184-8
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• Nerve growth factor (NGF) is involved in a variety of processes involving signalling, such as cell differentiation and survival, growth cessation and apoptosis of neurons. These events are mediated by NGF as a result of binding to its two cell-surface receptors, TrkA and p75. TrkA is a receptor with tyrosine kinase activity that forms a high-affinity binding site for NGF. Of the five domains comprising its extracellular portion, the immunoglobulin-like domain proximal to the membrane (TrkA-d5 domain) is necessary and sufficient for NGF binding. Here we present the crystal structure of human NGF in complex with human TrkA-d5 at 2.2 A resolution. The ligand-receptor interface consists of two patches of similar size. One patch involves the central beta-sheet that forms the core of the homodimeric NGF molecule and the loops at the carboxy-terminal pole of TrkA-d5. The second patch comprises the amino-terminal residues of NGF, which adopt a helical conformation upon complex formation, packing against the 'ABED' sheet of TrkA-d5. The structure is consistent with results from mutagenesis experiments for all neurotrophins, and indicates that the first patch may constitute a conserved binding motif for all family members, whereas the second patch is specific for the interaction between NGF and TrkA.

• Fawcett JR, Chen X, Rahman YE, Frey WH
• Previously reported nerve growth factor levels are underestimated due to an incomplete release from receptors and interaction with standard curve media.
• Brain Res. 1999; 842: 206-10
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• The 1996 research report by Hoener et al. [M.C. Hoener, E. Hewitt, J. M. Conner, J.W. Costello, S. Varon, Nerve growth factor (NGF) content in adult rat brain tissue is several-fold higher than generally reported and is largely associated with sedimentable fractions, Brain Res. 728 (1996) 47-56.] compares levels of nerve growth factor (NGF) found in rat brain by assaying both supernatant and pellet to previously reported data. However, Hoener et al. miscalculated when converting values previously reported in the literature to units of picogram per milliliter. Regardless of this mistake, the method of tissue extraction does affect the extent of release of NGF, which must be maximized in order to accurately determine NGF levels in the central nervous system. We now report that accurate measurement of NGF levels is not only affected by the incomplete release of NGF from receptors, but also the medium in which the standard curve is run. It is the combination of these two variables that has led to the underestimation of NGF levels in previous research.

• Lederhaas G
• Neurotrophins.
• Reg Anesth Pain Med. 1999; 24: 250-1

• Frade JM, Barde YA
• Nerve growth factor: two receptors, multiple functions.
• Bioessays. 1998; 20: 137-45
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• Nerve growth factor (NGF) was characterized over 4 decades ago, and like the other neurotrophins subsequently discovered, it is best known for its trophic role, including the prevention of programmed cell death in specific populations of neurones in the peripheral nervous system. This property can be accounted for by the activation of a tyrosine kinase receptor. NGF also regulates neuronal function, as illustrated by its role in pain and inflammation, and in synaptic plasticity. Finally, NGF recently was shown to activate the neurotrophin receptor p75 (p75NTR), a receptor with no intrinsic catalytic activity and with similarities to members of the tumor necrosis factor receptor family. During normal development, the activation of p75NTR by NGF actually kills cells in the central nervous system. One remarkable property of NGF is then that it controls cell numbers in opposite ways in the developing nervous system, a result of its unique ability to activate two different receptor types.

• Culotti JG, Merz DC
• DCC and netrins.
• Curr Opin Cell Biol. 1998; 10: 609-13
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• Recent advances highlight the versatility and complexity of this highly conserved axon and cell migration guidance system. Characterization of netrin mutant phenotypes in worm, fly and mouse all suggest that netrins play local as well as long-range roles in guidance. Evidence from multiple sources also indicates that the netrin receptor DCC can mediate both attractive and repulsive responses to netrins.

• Chao M, Casaccia-Bonnefil P, Carter B, Chittka A, Kong H, Yoon SO
• Neurotrophin receptors: mediators of life and death.
• Brain Res Brain Res Rev. 1998; 26: 295-301
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• The mechanism of action of NGF has continued to provide a challenging and formidable problem in signal transduction. NGF can bind independently to two different receptors, the trkA tyrosine kinase receptor and the p75 neurotrophin receptor, which are involved in many different signaling events. In addition to promoting cell differentiation survival, NGF can paradoxically be an inducer of cell death. Several receptor mediated mechanisms are proposed to explain how NGF might act as a trophic factor and as a cell killer. The survival and cell death properties of the receptors are dependent upon the relative ratio of receptors and the persistent nature of the signaling events.

• Tessarollo L
• Pleiotropic functions of neurotrophins in development.
• Cytokine Growth Factor Rev. 1998; 9: 125-37
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• Neurotrophins are soluble growth factors known mainly for their roles in regulating the development of the mammalian nervous system. Two types of receptors mediate the actions of these polypeptides: the Trk family of tyrosine kinase receptors and the so-called p75 low-affinity NGF receptor. Neurotrophins and their receptors are highly expressed in the nervous system. Gene targeting approaches in the mouse have uncovered some of their functions in promoting survival and developmental maturation of certain types of neurons of the peripheral and central nervous system, confirming their critical role in neural development. Furthermore, the phenotypes observed in these mutants have demonstrated the specificity of the interactions between neurotrophins and their receptors. These families of genes are also widely expressed in a variety of non-neuronal systems throughout development, including the cardiovascular, endocrine, reproductive and immune systems. Our knowledge of neurotrophin functions in non-neuronal tissues is still fragmented and mostly indirect. Nevertheless, there is increasing evidence that neurotrophins may have broader physiological effects besides regulating neuronal survival and differentiation. Analysis of mice lacking neurotrophins or neurotrophin receptors promises to provide avenues for elucidating these functions.

• Wigger G, Masabanda J, Stranzinger G, Fries R
• A molecular marker for the bovine ciliary neurotrophic factor receptor gene (CNTFR) maps to 8q21.
• Anim Genet. 1998; 29: 64-64

• Smith RE, Sadun AA
• Clearing the cornea with nerve growth factor.
• N Engl J Med. 1998; 338: 1222-3

• Butte MJ, Hwang PK, Mobley WC, Fletterick RJ
• Crystal structure of neurotrophin-3 homodimer shows distinct regions are used to bind its receptors.
• Biochemistry. 1998; 37: 16846-52
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• Neurotrophin-3 (NT-3) is a cystine knot growth factor that promotes the survival, proliferation, and differentiation of developing neurons and is a potential therapeutic for neurodegenerative diseases. To clarify the structural basis of receptor specificity and the role of neurotrophin dimerization in receptor activation, the structure of the NT-3 homodimer was determined using X-ray crystallography. The orthorhombic crystals diffract to 2.4 A, with dimer symmetry occurring about a crystallographic 2-fold axis. The overall structure of NT-3 resembles that of the other neurotrophins, NGF and BDNF; each protomer forms a twisted four-stranded beta sheet, with three intertwined disulfide bonds. There are notable differences, however, between NT-3 and NGF in the surface loops and in three functionally important regions, shown in previous mutagenesis studies to be critical for binding. One such difference implies that NT-3's binding affinity and specificity depend on a novel hydrogen bond between Gln 83, a residue important for binding specificity with TrkC, and Arg 103, a residue crucial for binding affinity with TrkC. NT-3's extensive dimer interface buries much of the otherwise solvent-accessible hydrophobic surface area and suggests that the dimeric state is stabilized through the formation of this hydrophobic core. A comparison of the dimer interface between the NT-3 homodimer and the BDNF/NT-3 heterodimer reveals similar patterns of hydrogen bonds and nonpolar contacts, which reinforces the notion that the evolutionarily conserved neurotrophin interface resulted from the need for receptor dimerization in signal initiation.

• Casaccia-Bonnefil P, Kong H, Chao MV
• Neurotrophins: the biological paradox of survival factors eliciting apoptosis.
• Cell Death Differ. 1998; 5: 357-64
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• Neurotrophins are target-derived soluble polypeptides required for neuronal survival. Binding of neurotrophins to Trk receptor tyrosine kinases initiate signaling cascades that promote cell survival and differentiation. All family members bind to another receptor (p75NTR), which belongs to the tumor necrosis factor superfamily. Hence, nerve growth factor (NGF) and related trophic factors are unique in that two separate receptor types are utilized. Although the biological function of p75NTR has been elusive, it has been suggested to mediate apoptosis of developing neurons in the absence of Trk receptors. This presents a tantalizing paradigm, in which life-death decisions of cells are dependent upon the expression and action of two different receptors with distinctive signaling mechanisms. In the presence of TrkA receptors, p75 can participate in the formation of high affinity binding sites and enhanced NGF responsiveness leading to a survival signal. In the absence of TrkA receptors, p75 can generate, in only specific cell populations, a death signal. Here we discuss the unique features and implications of this unusual signal transduction system.

• Reinshagen M et al.
• Commercial mouse and human nerve growth factors contain nerve growth factor prohormone isoforms.
• J Neurosci Methods. 1997; 76: 75-81
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• Highly sensitive chemiluminescence immunoblot analysis was utilized to examine the purity of mouse 2.5S-, beta- and 7S nerve growth factors as well as that of recombinant human beta-nerve growth factor obtained from commercial vendors. Three polyclonal antisera and two monoclonal antibodies to 13 kDa nerve growth factor (2.5S NGF and beta-NGF) were employed for assessing the purity of each preparation. In addition, polyclonal antisera against two prepro-NGF specific domains were used for immunoblotting analysis to ascertain the identity of high molecular weight nerve growth factor immunoreactive proteins as prohormones. Both the mouse and human NGF preparations contained 53 and 60 kDa immunoreactive proteins. Of these, the mouse 60 kDa and the human 53 kDa proteins strongly immunoreacted with both prepro-nerve growth factor specific domain antibodies suggesting that they are two NGF prohormone isoforms. In addition, both the mouse and human nerve growth factor preparations contained proteins that were immunoreactive to polyclonal antisera and monoclonal antibodies to mouse 2.5S and/or beta-NGF. High molecular weight aggregates of prohormones were also observed in mouse and human nerve growth factor samples. In summary, none of the ten NGF samples examined were pure as stated. Our study cautions investigators in the field to be aware of the presence of nerve growth factor prohormones and other proteins in various mouse and human nerve growth factors sold commercially.

• Kullander K, Kaplan D, Ebendal T
• Two restricted sites on the surface of the nerve growth factor molecule independently determine specific TrkA receptor binding and activation.
• J Biol Chem. 1997; 272: 9300-7
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• Nerve growth factor (NGF) and neurotrophin-3 (NT-3) mediate activities such as survival, differentiation, and proliferation in various subsets of neurons. In this report, we define precisely the residues in human NGF responsible for NGF biological activity and binding specificity to the neurotrophin receptor TrkA. In earlier studies we defined five amino acid residues of NGF which confer NGF-like activity to NT-3 when replacing corresponding residues in the 120-amino acid long NT-3 molecule. Using this gain-of-function strategy we report the further dissection of this functional epitope. We also define another motif separated topographically in the NGF dimer and determined to be independently responsible for NGF specificity. The first of the two motifs determined to elicit NGF specificity is defined by the residues Val-48, Pro-49, and Gln-96, which are situated in the two top beta-loops of NGF. The second motif is represented by residues Pro-5 and Phe-7 situated in the proximal part of the NH2 terminus. Both motifs contain structurally important residues revealing a novel principle, where specificity for neurotrophin ligand-receptor interactions could be determined by variable residues modifying the conformation of the neurotrophin backbone. These findings will enhance further the possibility of mimicking NGF with low molecular weight compounds.

• Ross GM et al.
• Zinc alters conformation and inhibits biological activities of nerve growth factor and related neurotrophins.
• Nat Med. 1997; 3: 872-8
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• A role for Zn2+ in a variety of neurological conditions such as stroke, epilepsy and Alzheimer's disease has been postulated. In many instances, susceptible neurons are located in regions rich in Zn2+ where nerve growth factor (NGF) levels rise as a result of insult. Although the interaction of Zn2+ with this neurotrophin has previously been suggested, the direct actions of the ion on NGF function have not been explored. Molecular modeling studies predict that Zn2+ binding to NGF will induce structural changes within domains of this neurotrophin that participate in the recognition of TrkA and p75NTR. We demonstrate here that Zn2+ alters the conformation of NGF, rendering it unable to bind to p75NTR or TrkA receptors or to activate signal transduction pathways and biological outcomes normally induced by this protein. Similar actions of Zn2+ are also observed with other members of the NGF family, suggesting a modulatory role for this metal ion in neurotrophin function.

• Cunningham ME, Stephens RM, Kaplan DR, Greene LA
• Autophosphorylation of activation loop tyrosines regulates signaling by the TRK nerve growth factor receptor.
• J Biol Chem. 1997; 272: 10957-67
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• Many receptor tyrosine kinases possess an "activation loop" containing three similarly placed tyrosine autophosphorylation sites. To examine their roles in the TRK NGF receptor, these residues (Tyr-670, Tyr-674, and Tyr-675) were mutated singly and in all combinations to phenylalanine and stably expressed in Trk-deficient PC12nnr5 cells. All mutant receptors showed significantly diminished nerve growth factor (NGF)-stimulated autophosphorylation, indicating impaired catalytic activity. NGF-induced neurite outgrowth exhibited dose-responsive behavior when transfectants were compared by relative receptor expression and exhibited a functional hierarchy: wild type > Y670F >/= Y674F >> Y675F >/= YY670/674FF = YY670/675FF >> YY674/675FF > YYY670/674/675FFF. NGF-induced tyrosine phosphorylation of Shc, ERKs, and SNT and immediate early gene inductions generally paralleled neurogenic potential. However, activation of phosphatidylinositol 3'-kinase and tyrosine phosphorylation of phospholipase Cgamma-1 was essentially abolished. The latter effect appears due to selective inability of the mutated TRKs to autophosphorylate the tyrosine residue (Tyr-785) required for binding phospholipase Cgamma-1 and indicates that the "activation loop" tyrosines participate in NGF-dependent changes in receptor conformation. Our findings stress the importance that expression levels play in assessing the consequences of receptor mutations and that all three activation loop tyrosines have roles regulating both overall and specific NGF-mediated signaling through TRK.

• Aloe L, Micera A, Bracci-Laudiero L, Vigneti E, Turrini P
• Presence of nerve growth factor in the thymus of prenatal, postnatal and pregnant rats.
• Thymus. 1997; 24: 221-31
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• The presence of Nerve Growth Factor (NGF) and the expression of low- and high-affinity NGF receptor were investigated in prenatal, postnatal and pregnant rats. Using ELISA and immunohistochemistry it was found that both NGF and its receptors are present in the medulla of the thymus and are more strongly expressed in pre- and early postnatal life and nearly absent in adult and ageing rats. It was also found that during pregnancy, which is characterised by an involution of the cortex and hypertrophy of the medulla, the level of NGF in the thymus increases. The present study showed not only that NGF is produced in a specific compartment of the thymus, the medulla, but that its synthesis declines with age, following thymus involution. These results suggest that NGF may be critically involved in the proliferation and differentiation of thymic cells.

• Pincelli C, Yaar M
• Nerve growth factor: its significance in cutaneous biology.
• J Investig Dermatol Symp Proc. 1997; 2: 31-6
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• Work in the past 10 y has greatly expanded our knowledge regarding the role of neurotrophins and, in particular, nerve growth factor, in skin. During fetal development, neurotrophins are expressed both in the mesenchyme and the epithelium of developing skin, and they play a trophic role supporting the survival of innervating neurons. Nerve growth factor appears to have an important role also in the adult organism. It serves as a regulatory molecule during skin inflammation and repair, affecting both the neuronal and the immune systems. Recent studies also suggest that nerve growth factor is an important autrocrine growth factor and survival factor for keratinocytes, cells that express both high- and low-affinity receptors for nerve growth factor. Neural crest-derived melanocytes also express receptors for nerve growth factor. Nerve growth factor appears to have a major role during skin injury, e.g., preventing melanocyte apoptotic cell death following UV irradiation. Thus, epidermal events interact to maintain the number and function of both melanocytes and keratinocytes in skin. These recent insights into the mechanisms underlying nerve growth factor effects in skin may eventually result in the development of novel therapeutic modalities.

• Yang QF, Fan M, Wan XC
• [Research progresses on signal transduction of neurotrophins]
• Sheng Li Ke Xue Jin Zhan. 1997; 28: 209-13
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• Binding of neurotrophins and their receptors lead to dimerization and autophosphorylation of trks. Activated trkA initiates the Ras pathway and finally opens the transcriptions of immediate early genes and delayed response genes or participates directly in physiological responses. Target-derived neurotrophins bind to and induce phosphorylation of trk receptors at the axonal terminal. Active trk or NT-trk or other signal molecules can be retrogradely transported along the axon to transduct messages to neuronal nucleus. There are local autocrine and paracrine mechanisms besides target-derived NTs. Following nervous system injury, increased gene expressions of NTs and their receptors and increased retrograde axonal transport are helpful to survive and regenerate for injured neurons. Lacking NTs and their receptors will result in serious abnormal development of nervous system of mice.

• Wei F, Zhang J
• [Biological actions of nerve growth factor in pain sensation]
• Sheng Li Ke Xue Jin Zhan. 1997; 28: 172-4

• Davies AM
• Neurotrophins: the yin and yang of nerve growth factor.
• Curr Biol. 1997; 7: 3840-3840
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• In the fifty years since its discovery, a substantial body of work has established that nerve growth factor plays a key role in promoting the survival of neurons during development; the recent demonstration that it can also promote cell death therefore comes as a surprise.

• Furukawa Y, Furukawa S
• [Neurotrophins and their receptors]
• Tanpakushitsu Kakusan Koso. 1997; 42: 470-80

• Ryden M, Ibanez CF
• A second determinant of binding to the p75 neurotrophin receptor revealed by alanine-scanning mutagenesis of a conserved loop in nerve growth factor.
• J Biol Chem. 1997; 272: 33085-91
• Display abstract

• In the neurotrophin family, variable regions contain solvent-accessible residues important for receptor binding specificity, whereas many of the conserved residues are buried in hydrophobic cores or in the dimer interface. A stretch of six amino acids (from Asp-72 to Asn-77) in nerve growth factor (NGF) represents an exception to this general rule. These residues are highly conserved and yet form an exposed hydrophilic loop region away from other known determinants of receptor binding. We have investigated the functional importance of this region in NGF using alanine-scanning mutagenesis. Individual mutation of Asp-72, Lys-74, or His-75 to alanine (mutants D72A, K74A, and H75A, respectively) reduced the binding affinity for the p75 neurotrophin receptor by 4-10-fold. Only the D72A mutant showed an additional impairment in binding to the TrkA receptor, which was accompanied by reduced biological activity in PC12 cells, indicating a structural and/or conformational effect of this mutation. Replacement of Ser-73 or Asn-77 with alanine (mutants S73A and N77A, respectively) had no measurable effects on receptor binding. The triple mutant K74A/H75A/N77A exhibited properties that were consistent with the combined effects of the individual mutations, namely impaired binding to p75 without deficits in its interaction with TrkA. In contrast, in the triple mutant D72A/S73A/K74A, the simultaneous replacement of Asp-72 and Lys-74 with alanine had a compensatory effect such that binding to both p75 and TrkA was comparable to that of wild-type NGF, despite the deficits seen in the individual replacements. This molecule, however, was produced at low levels, and its biological activity in sympathetic ganglion explants was reduced, which, together with results from TrkA phosphorylation assays, indicated a reduced stability during prolonged culture conditions. Taken together, these data reveal a second region of interaction with the p75 receptor in NGF with the positively charged residues Lys-74 and His-75 as candidate points of contact. In addition, Asp-72 appears to be a structurally important side chain for stabilizing the conformation of the loop through interactions with neighboring residues.

• Kullander K, Kylberg A, Ebendal T
• Specificity of neurotrophin-3 determined by loss-of-function mutagenesis.
• J Neurosci Res. 1997; 50: 496-503
• Display abstract

• Neurotrophin-3 (NT-3) is a member of the family of neurotrophic factors, which also includes nerve growth factor (NGF) and which have specific activities on different subsets of vertebrate neurons. The aim of this study was to determine which residues in NT-3 direct its specificity to the cognate TrkC receptor. It was possible to replace 80% of the residues in NT-3 with NGF residues without loss of specific activity. Residues D72, Y85, R87, W101, S107, and A111, together with either the residues F12, V18, V20, M37, V42, F54, and K57 or the variable regions IV and V, accounted for the specificity of NT-3. It is concluded that NGF and NT-3 use overlapping as well as separated regions for determination of specificities for their cognate receptors TrkA and TrkC, respectively.

• Sun J, Liu T
• [Nerve growth factor family and peripheral auditory nerve system]
• Zhonghua Er Bi Yan Hou Ke Za Zhi. 1997; 32: 311-4

• Hempstead BL, Chao MV
• Entering the domain of neurotrophin binding.
• Nat Biotechnol. 1997; 15: 623-4

• Gargano N, Levi A, Alema S
• Modulation of nerve growth factor internalization by direct interaction between p75 and TrkA receptors.
• J Neurosci Res. 1997; 50: 1-12
• Display abstract

• While the central role played by TrkA in nerve growth factor (NGF) signalling has been established by dissecting its signal transduction pathways, insight into the mechanism of action of p75LNR, the low-affinity neurotrophin receptor, has only recently been achieved. The relative contribution of p75LNR and TrkA to the constitution of high-affinity receptors for NGF and, similarly, with TrkB an TrkC to the formation of those for other neurotrophins, is presently under debate. Some form of collaboration in mediating neurotrophin activities has been observed between the Trk and p75LNR receptors, but only recent indirect evidence indicates a molecular interaction. In the present work, we have ectopically coexpressed p75LNR and TrkA in Sf9 insect cells by using baculovirus vectors, and show a direct association between the two NGF receptors. In addition, we show that the intracellular and extracellular domains of both receptors contribute to this interaction. Finally, we demonstrate that NGF becomes endocytosed in TrkA-expressing cells but not in p75LNR-expressing cells, and that such function can be modulated by the presence of the intracellular domain of p75LNR receptor.

• Holden PH et al.
• Immunoglobulin-like domains define the nerve growth factor binding site of the TrkA receptor.
• Nat Biotechnol. 1997; 15: 668-72
• Display abstract

• Nerve growth factor (NGF) is involved in the development and maintenance of the nervous system. NGF binds with high affinity to the extracellular region of the tyrosine kinase receptor TrkA. This domain comprises leucine and cysteine rich motifs, followed by two immunoglobulin like (Ig-like) domains. We describe the expression and purification of recombinant Ig-like domains. Fluorescence and circular dichroism spectroscopy show that the protein is folded into a compact globular structure and contains mainly beta-sheet secondary structure. Recombinant protein binds to NGF and can inhibit NGF bioactivity both in vitro and in vivo.

• Eng M et al.
• Formulation development and primary degradation pathways for recombinant human nerve growth factor.
• Anal Chem. 1997; 69: 4184-90
• Display abstract

• The chemical and physical stabilities of recombinant human nerve growth factor (NGF) in aqueous solution were investigated between 5 and 37 degrees C and at pH 4.2-5.8. NGF chemical stability decreased with a decrease in pH due to Asp60-Pro61 cleavage, with the stability being greater in acetate buffer than in succinate buffer at each pH investigated. Aggregation was a significant degradation pathway at 37 degrees C, with the aggregation rate being greatest in succinate buffer at pH 5.8. Quantitation of NGF degradation by cation-exchange chromatography was complicated by the rearrangement of the NGF monomer variants into various mixed dimers over time. Treatment with dilute acid brought the dimer distribution rapidly to equilibrium, allowing NGF degradation to be accurately quantitated. An acetate-buffered formulation at pH 5.5 was investigated in more detail. To assist in degradation product identification, NGF degradation was accelerated with base, hydrogen peroxide, and temperature. These degradation products were shown to coelute on RP-HPLC with the variants found when the protein was stored at -70, 5, and 25 degrees C. By electrospray mass spectrometry, peptide maps, and LC/MS, these degradation products were shown to be monooxidized (Met37) and dioxidized (Met37 and Met92) NGF, with Met37 being more labile, deamidated NGF (Asn45), and NGF with Asp93 isomerized to beta-Asp93. NGF can be stored in pH 5.5 acetate buffer at 5 degrees C for 1.5 years with less than 10% conversion to these degradation products, with Asp93 isomerization being the primary degradation pathway.

• Catala M
• [Neurotrophins and the development of the peripheral nervous system]
• Rev Neurol (Paris). 1997; 155: 73-5

• Tong Y, Wang H, Chen W
• [Cloning and sequencing of the gene for premature beta nerve growth factor]
• Zhongguo Ying Yong Sheng Li Xue Za Zhi. 1997; 13: 316-8
• Display abstract

• The entire coding sequence of premature nerve growth factor (NGF) beta subunit was amplified from Genomic DNA extracted from peripheral white blood cells of Chinese by polymerase chain reaction (PCR). The PCR products were cloned into a premade T-vector (linearized plasmid pBluescript II SK (+) with a T base at 3' terminal), followed by automatic sequencing with double strand plasmids. The nucleotide sequence coding for the mature NGF is the same as that reported by Europeans, but there is a base difference within the proper region, resulting in an amino acid replacement in premature NGF.

• Kruttgen A, Heymach JV Jr, Kahle PJ, Shooter EM
• The role of the nerve growth factor carboxyl terminus in receptor binding and conformational stability.
• J Biol Chem. 1997; 272: 29222-8
• Display abstract

• The role of the nerve growth factor (NGF) carboxyl terminus in the function of NGF is not well understood. Previous work showed that deletion of residues 112-120 abolished NGF bioactivity. Several mutagenesis studies, however, have localized the binding sites of the two NGF receptors, p75 and TrkA, to other regions of the NGF molecule. To investigate the role of the NGF COOH terminus, we performed a detailed structure-function analysis of this region by deleting stepwise each of the nine COOH-terminal residues as well as constructing six point mutants. We found that point mutations within the 111-115 region, but not deletion of residues 116-120, significantly decreased NGF bioactivity, as determined by TrkA tyrosine phosphorylation and neurite outgrowth from PC12 cells. Mutation of the absolutely conserved Leu112 led to severely disrupted p75 binding on A875 cells but had only a modest effect on TrkA binding to MG87-TrkA fibroblasts. This suggests that the p75 binding surface is more extended than previously believed and includes not only charged residues within loops 1 and 5 but also spatially discontinuous, uncharged residues in a region where the NH2 and COOH termini are in close proximity. Unexpectedly, deletion of COOH-terminal residues beyond Ala116 led to significantly decreased stability. These results demonstrate that residues 111-115, but not residues 116-120, are important for both the structural stability and biological activity of NGF.

• Frade JM, Rodriguez-Tebar A, Barde YA
• Induction of cell death by endogenous nerve growth factor through its p75 receptor.
• Nature. 1996; 383: 166-8
• Display abstract

• During development, neuronal survival is regulated by the limited availability of neurotrophins, which are proteins of the nerve growth factor (NGF) family. Activation of specific trk tyrosine kinase receptors by the neurotrophins blocks programmed cell death. The trkA-specific ligand NGF has also been shown to activate the non-tyrosine kinase receptor p75, a member of the tumour necrosis factor (TNF) receptor and Fas (APO-1/CD95) family. Here we report that, early in development, endogenous NGF causes the death of retinal neurons that express p75 but not trkA. These results indicate that, as with cells of the immune system, the death of neurons in the central nervous system can also be induced by ligands, and that the effect of NGF on cell fate depends on the type of receptor expressed by developing neurons.

• Dyck PJ
• Nerve growth factor and diabetic neuropathy.
• Lancet. 1996; 348: 1044-5

• Kostiza T, Meier J
• Nerve growth factors from snake venoms: chemical properties, mode of action and biological significance.
• Toxicon. 1996; 34: 787-806
• Display abstract

• An overview of nerve growth factors (NGF) and their receptor system is presented. This general introduction deals with mouse and human NGF, since most of the fundamental knowledge is based on these mammalian proteins. Great emphasis is laid on the nonneuronal NGF effects which are well discussed for their possible toxinological function. The snake venom NGFs were reviewed with respect to their purification, classification and properties. From the date of their discovery about 40 years ago, researchers have been interested in the possible function of mouse salivary and snake venom NGFs. The physiological relevance of snake venom NGF is therefore widely discussed on the base of experimental facts and hypothetical considerations.

• Carter BD, Dechant G, Frade JM, Kaltschmidt C, Barde YA
• Neurotrophins and their p75 receptor.
• Cold Spring Harb Symp Quant Biol. 1996; 61: 407-15

• Osambaev CI
• [Nerve growth factor activity in the rat regenerating liver tissue]
• Biull Eksp Biol Med. 1996; 121: 345-7

• Guo M, Meyer SL, Kaur H, Gao JJ, Neet KE
• Mutational studies of conserved residues in the dimer interface of nerve growth factor.
• Protein Sci. 1996; 5: 447-55
• Display abstract

• An understanding of the structure-function relationship of nerve growth factor (NGF) requires precise knowledge of all the residues and regions that participate in NGF receptor binding, receptor activation, and biological activity. Seven recombinant human NGF mutants having alanine substituted for residues located either in the NGF dimer interface or beta-strand region were studied to determine the role of each amino acid residue in NGF biological activity. F86A, T91A, R100A, and R103A remained nearly full active with 61, 120, 91, and 73% of wild-type activity, respectively, in the PC12 cell bioassay. Hydrophobic core and dimer interface residues Y52, F53, and F54 were studied in more detail. Y52A and F54A were expressed in very low levels, suggesting that these two residues may be important for protein stability. Y52A retained full biological activity (91%). F53A had a 20- and 70-fold reduction in biological activity and TrkA phosphorylation, respectively, with only a 5- to 10-fold effect on TrkA binding and no effect on low-affinity receptor binding. F54A had significantly decreased TrkA phosphorylation and biological activity (40-fold). The results suggest that F53 and F54 may play a structural role in TrkA receptor activation subsequent to binding.

• Yuen EC, Howe CL, Li Y, Holtzman DM, Mobley WC
• Nerve growth factor and the neurotrophic factor hypothesis.
• Brain Dev. 1996; 18: 362-8
• Display abstract

• The discovery of nerve growth factor (NGF) over 40 years ago led to the formulation of the "Neurotrophic Factor Hypothesis". This hypothesis states that developing neurons compete with each other for a limited supply of a neurotrophic factor (NTF) provided by the target tissue. Successful competitors survive; unsuccessful ones die. Subsequent research on NTFs has shown that NTF expression and actions are considerably more complex and diverse than initially predicted. Even for NGF, different regulatory patterns are seen for different neuronal populations. As would be predicted by the "Neurotrophic Factor Hypothesis", NGF levels critically regulate basal forebrain cholinergic neuron size and neurochemical differentiation. In contrast, the level of trkA, the NGF receptor, regulates these properties in caudate-putamen cholinergic neurons. Understanding NTF regulation and actions on neurons has led to their use in clinical trials of human neurological diseases. NTFs may emerge as important therapies to prevent neuronal dysfunction and death.

• Fainzilber M et al.
• CRNF, a molluscan neurotrophic factor that interacts with the p75 neurotrophin receptor.
• Science. 1996; 274: 1540-3
• Display abstract

• A 13.1-kilodalton protein, cysteine-rich neurotrophic factor (CRNF), was purified from the mollusk Lymnaea stagnalis by use of a binding assay on the p75 neurotrophin receptor. CRNF bound to p75 with nanomolar affinity but was not similar in sequence to neurotrophins or any other known gene product. CRNF messenger RNA expression was highest in adult foot subepithelial cells; in the central nervous system, expression was regulated by lesion. The factor evoked neurite outgrowth and modulated calcium currents in pedal motor neurons. Thus, CRNF may be involved in target-derived trophic support for motor neurons and could represent the prototype of another family of p75 ligands.

• Bergner A et al.
• Crystal structure of a coagulogen, the clotting protein from horseshoe crab: a structural homologue of nerve growth factor.
• EMBO J. 1996; 15: 6789-97
• Display abstract

• The clotting cascade system of the horseshoe crab (Limulus) is involved in both haemostasis and host defence. The cascade results in the conversion of coagulogen, a soluble protein, into an insoluble coagulin gel. The clotting enzyme excises the fragment peptide C from coagulogen, giving rise to aggregation of the monomers. The crystal structure of coagulogen reveals an elongated molecule that embraces the helical peptide C fragment. Cleavage and removal of the peptide C would expose an extended hydrophobic cove, which could interact with the hydrophobic edge of a second molecule, leading to a polymeric fibre. The C-terminal half of the coagulogen molecule exhibits a striking topological similarity to the neurotrophin nerve growth factor (NGF), providing the first evidence for a neurotrophin fold in invertebrates. Similarities between coagulogen and Spatzle, the Drosophila ligand of the receptor Toll, suggest that the neurotrophin fold might be considered more ancient and widespread than previously realized.

• Furukawa Y, Furukawa S, Hayashi K
• [Biochemistry of neurotrophins]
• Seikagaku. 1996; 68: 14-30

• Hayashi K, Inoue S, Ikeda K
• Purification and characterization of nerve growth factors (NGFs) from the snake venoms.
• Adv Exp Med Biol. 1996; 391: 403-16

• Longo FM, Mobley WC
• Minimized hormones grow in stature.
• Nat Biotechnol. 1996; 14: 1092-1092

• Woo SB, Timm DE, Neet KE
• Alteration of NH2-terminal residues of nerve growth factor affects activity and Trk binding without affecting stability or conformation.
• J Biol Chem. 1995; 270: 6278-85
• Display abstract

• The role of the NH2-terminal region of nerve growth factor (NGF) was studied with an NGF delta 9/13 deletion mutant, overexpressed in a baculovirus system, and mouse NGF truncated at Met-9 by cleavage with CNBr (des-(1-9)-NGF). Structural studies have been performed on the purified proteins, in addition to biological activity assessment, in order to determine effects of such modifications on global conformation and stability. The activity of NGF delta 9/13 was reduced below detectable levels, and the activity of the des-(1-9)-NGF form was decreased by at least a 50-fold in a PC12 bioassay. Competitive binding of NGF delta 9/13 to low affinity receptors on PC12 cells was not impaired; however, the mutant was not capable of competing for the cold chase-stable, high affinity binding of NGF to the cells. The binding of NGF delta 9/13 to Sf21 cells ectopically expressing the TrkA NGF receptor was also abolished. Thus, deletion of residues 9-13 significantly altered the binding affinity for the high affinity receptors on PC12 cells and for the TrkA receptor, but not for the low affinity receptor. Neither the secondary structure, determined by circular dichroism, nor the conformational stability determined by equilibrium denaturation of NGF delta 9/13 was altered as compared with wild type NGF. Slight conformational and stability perturbations of des-(1-9)-NGF were revealed by the same analysis; however, these changes were found to reflect the influence of the formic acid treatment, not the truncation of 9 residues. Our results support the conclusion that the NH2-terminal domain encompassing residues 1-9 and 9-13 is essential for maintaining the binding capability of NGF for high affinity TrkA receptors. Moreover, conformational and stability data show that the functional results of these modifications of the NH2-terminal region are directly due to receptor binding and not to secondary effects of improper folding or other indirect structural changes.

• Scully JL, Otten U
• NGF: not just for neurons.
• Cell Biol Int. 1995; 19: 459-69
• Display abstract

• Nerve growth factor (NFF) is the prototypic member of a family of related neurotrophins (Nts). Although originally defined by its actions in the peripheral and central nervous systems, recent data indicate the presence of extensive interactions between NGF and the endocrine and immune systems steroid hormones are able to modulate the neurosomal expression NGF, while functional NGF receptors have been detected on cells of the immune system, and increased levels of NGF protein are found during the acute phase of diseases with a significant inflammatory component. These wider functions are likely to be of concern in any attempted therapeutic use of NGF.

• Anand P, Foley P, Navsaria HA, Sinicropi D, Williams-Chestnut RE, Leigh IM
• Nerve growth factor levels in cultured human skin cells: effect of gestation and viral transformation.
• Neurosci Lett. 1995; 184: 157-60
• Display abstract

• Extracts of cultured human keratinocytes and fibroblasts were assayed for nerve growth factor-like immunoreactivity (NGF) by a specific enzyme-linked immunoabsorbant assay. NGF levels were higher in primary cultured keratinocytes than in freshly isolated keratinocytes or culture through multiple passages. Viral transformation of keratinocytes with the human papilloma virus (HPV16) significantly increased NGF levels, whilst transformation with the simian virus (SV40), which induces simple epithelial differentiation, reduced the concentration of NGF. Passaged epidermal keratinocytes contained more than twice as much NGF as did passaged fibroblasts. Oral keratinocytes and fibroblasts, and psoriatic fibroblasts, all from high turnover tissues, did not contain significantly different levels of NGF in culture than dermal keratinocytes or fibroblasts. Foetal fibroblasts contained five times as much NGF as did adult fibroblasts. These results suggest that basal keratinocytes are a major but not sole source of NGF in human skin, and that NGF may play a role in human skin development.

• LeSauteur L, Wei L, Gibbs BF, Saragovi HU
• Small peptide mimics of nerve growth factor bind TrkA receptors and affect biological responses.
• J Biol Chem. 1995; 270: 6564-9
• Display abstract

• Small monomeric cyclic analogs that mimic the beta-turn regions of nerve growth factor (NGF) were designed and synthesized. Potent competitive antagonists were derived from the NGF beta-turn C-D, which inhibited [125I] NGF binding to TrkA receptors and specifically inhibited optimal NGF-mediated neurite outgrowth in PC12 cells. The cyclic beta-turn A'-A" analog also inhibited NGF binding to TrkA receptors but with lower potency. These data indicate that beta-turns C-D and A'-A" are critical for TrkA binding and may confer neurotrophin receptor specificity. Furthermore, structural requirements for binding are absolute, because unconstrained analogs derived from the same regions had no effect. Compounds that mimic NGF will be useful in deciphering the interactions of NGF and its receptors and in rational drug design.

• McDonald NQ, Panayotatos N, Hendrickson WA
• Crystal structure of dimeric human ciliary neurotrophic factor determined by MAD phasing.
• EMBO J. 1995; 14: 2689-99
• Display abstract

• Ciliary neurotrophic factor (CNTF) promotes the survival and differentiation of developing motor neurons and is a potential therapeutic for treating neurodegeneration and nerve injury. The crystal structure of human CNTF has been determined at 2.4 A resolution using multi-wavelength anomalous diffraction (MAD) phasing from a single Yb3+ ions. The structure reveals that CNTF is dimeric, with a novel anti-parallel arrangement of the subunits, not previously observed for other cytokines. Each subunit adopts a double crossover four-helix bundle fold, in which two helices contribute to the dimer interface, whilst two different helices show pronounced kinks. Analysis of the electrostatic surface of CNTF identified residues within these kinked helices that may contact the CNTF receptor-alpha. Solution experiments show that CNTF dimerizes at concentrations > 40 microM. Such dimers are likely to be relevant to the storage of CNTF in the peripheral nerve given the high concentrations present in this tissue. However, it is unlikely that they play a role in engaging the three distinct receptor subunits that comprise the CNTF receptor, given the low concentration of extracellular CNTF and its high potency.

• Huang BR, Cai LW
• [Progress in the study of nerve growth factor family and its receptors]
• Sheng Li Ke Xue Jin Zhan. 1995; 26: 115-20
• Display abstract

• There were several noticeable progress in the study of the neurotrophic factors, their receptors and the neuronal apoptosis. Firstly, several members of the nerve growth factor family have been identified: NGF, BDNF, NT-3 and NT-4/5. Secondly, the relationship between the three-dimensional structure of the NGF and its function and evolution has been revealed. Thirdly, two kinds of the neurotrophin family receptors were characterized, they are low-affinity NGFR (p75 NGFR) and the trk proto-oncogene product such as p140trkA, p145trkB and p145trkC. Induction of apoptosis by the low-affinity NGFR was also found. Finaly it is possible to use the neurotrophin for treating the degenerative neurological illnesses.

• Kourounakis A, Bodor N
• Quantitative structure activity relationships of catechol derivatives on nerve growth factor secretion in L-M cells.
• Pharm Res. 1995; 12: 1199-204
• Display abstract

• PURPOSE. Although many catechol derivatives are potent stimulators of Nerve Growth Factor synthesis in L-M cells, not much is known about their mechanism of action. In order to obtain a Quantitative Structure Activity Relationship (QSAR), AM1 quantum mechanical calculations were performed on a group of 23 catechol derivatives with different levels of activity. METHODS. A set of 18 parameters/descriptors were obtained by AM1 quantum mechanical calculations for each catechol derivative. Linear combinations of the calculated descriptors were fitted to the activity (as extracted from literature data) of the compounds by using simple or multiple regression analysis. RESULTS. Good correlation with activity was obtained for specific parameters such as the adiabatic ionization potential and other 'oxidation'-related descriptors of the molecules while poor correlation was observed for most of the other parameters as, for example, for log P. CONCLUSIONS. Our results show that activity is associated with parameters related to the oxidation of the catechol derivatives, strongly supporting recent literature suggesting that an oxidative process is involved in their action.

• Isanbaev CI
• [The hyperinnervation phenomenon and the gradient distribution of nerve-growth-factor activity in the tissue of the regenerating rat liver]
• Arkh Patol. 1995; 57: 64-7
• Display abstract

• An increase in the number and fluorescence intensity of liver nerve structures was recorded using fluorescent-histochemical method following 50% resection of the left lobe of the liver. Simultaneous isolation of liver NGF showed its activity in the operated lobe to have had gradient distribution, which motivated successful growth of nerve fibers to the zone of regeneration and development of hyperinnervation in it.

• Soderstrom S, Ebendal T
• In vitro toxicity of methyl mercury: effects on nerve growth factor (NGF)-responsive neurons and on NGF synthesis in fibroblasts.
• Toxicol Lett. 1995; 75: 133-44
• Display abstract

• The effect of methyl mercury chloride (MeHgCl) on the chick sympathetic and sensory dorsal root ganglia was studied in a biological in vitro assay. These cultures were not affected by the addition of MeHg up to a concentration of 2 microM. However, after an addition of 4-5 microM MeHg the capability of the neurons to respond to added nerve growth factor (NGF) was completely inhibited. The effect of MeHg was also examined in a fibroblast cell line, mouse 3T3 cells. After the addition of mercury to the culture medium at concentrations as low as 0.1 microM, an elevated production of the NGF protein was observed. However, NGF mRNA measured in the individual fibroblast cells by in situ hybridization was found to be reduced to about 80% of the control in the low level at day 2 of exposure. These results suggest that the release of NGF is actively enhanced from the 3T3 cells by addition of low levels of mercury. The results thus show that MeHg at low to moderate concentrations has adverse effects on NGF responses in cultured neurons and moreover alter levels of NGF production in cells, suggestive of mechanisms for mercury toxicity in the developing nervous system.

• McDonald NQ, Chao MV
• Structural determinants of neurotrophin action.
• J Biol Chem. 1995; 270: 19669-72
• Display abstract

• Five decades of research on NGF have led to the discovery of a small family of evolutionarily conserved proteins, which have vital functions in the survival and neuronal development of specific neuronal populations. The generation of mice lacking neurotrophin expression has recapitulated classic experiments using anti-NGF antibodies to dissect the physiological effects of trophic factor deprivation (73). Very similar outcomes resulted from both the NGF immunodepletion experiments and the transgenic mouse experiments. The genetic results also verify the structural predictions made from binding results in heterologous cells. The findings in cell culture and animal experiments clearly indicate the efficacy of neurotrophic factors for promoting the survival of prominent neuronal populations such as sensory and motor neurons. The high degree of conservation of neurotrophin structure is accompanied by a surprising variation in the amino acid contacts used by each neurotrophin with p75 and the trk receptor family members. It is this variation that may provide specificity for each ligand-receptor complex. The future challenge will be to make use of this knowledge to design effective therapeutic strategies to treat neurodegeneration and nerve injury.

• May AC, Johnson MS
• Improved genetic algorithm-based protein structure comparisons: pairwise and multiple superpositions.
• Protein Eng. 1995; 8: 873-82
• Display abstract

• Three major improvements to a previously described method for automatic protein structure comparison are described. First, a limit to translations for the rigid-body superposition is now assigned according to the dimensions of the structures being compared. Second, examination of the effect of the gap penalty on the derivation of a sequence alignment corresponding to a given structure superposition has led to a method to evaluate alternative structure-based sequence alignments. Third, the pairwise procedure has been generalized to multiple structure alignment. This implementation of rigid-body superposition can recognize well documented distant relationships which hitherto have required consideration of additional features and properties as well as those relationships between proteins of different sizes. A much larger common scaffold or framework between six globins can be extracted than that obtained using a standard algorithm for multiple structure superposition.

• Rush RA, Mayo R, Zettler C
• The regulation of nerve growth factor synthesis and delivery to peripheral neurons.
• Pharmacol Ther. 1995; 65: 93-123
• Display abstract

• Recent experiments have provided new insights into the way in which the synthesis of nerve growth factor (NGF) is regulated. In particular, a variety of agents have been found to influence the cellular concentration of mRNANGF and NGF both in vitro and in vivo. However, no clear mechanism has been found to indicate the existence of a feedback regulation of NGF synthesis by effector tissue innervation. We have argued in this review that some form of feedback control is likely to exist between the innervation and the cells producing the factor. One such possibility, the regulation of the availability of NGF by control of its secretion, is discussed.

• Fantini F, Magnoni C, Bracci-Laudiero L, Pincelli C TE
• Nerve growth factor is increased in psoriatic skin.
• J Invest Dermatol. 1995; 105: 854-5

• Sagoh M et al.
• Accumulation of nerve growth factor in cerebrospinal fluid and biological activity following neurosurgery.
• Neurol Med Chir (Tokyo). 1995; 35: 431-7
• Display abstract

• Perioperative nerve growth factor (NGF) levels in cerebrospinal fluid (CSF) of patients with acoustic neurinoma (14 cases), tentorial meningioma (1 case), or subarachnoid hemorrhage (1 case) were examined. Preoperative NGF levels in CSF were below the level of detection in all patients. However, NGF was found to accumulate transiently in CSF following neurosurgery. Pre- and postoperative CSF obtained from a patient with acoustic neurinoma enhanced the proliferation of astrocytes in neuronal cell cultures derived from embryonic rat cortex grown in serum-free defined medium, and increased choline acetyltransferase activity of cholinergic neurons derived from embryonic rat septal area and brainstem. The effect of postoperative CSF on septal and brainstem neurons was more potent than that of preoperative CSF. These results indicate that NGF and non-NGF-type neurotrophic activities accumulate in the CSF following neurosurgery. These neurotrophic activities are probably important in the regeneration of damaged neural networks in the central nervous system.

• Binkley J, Allen P, Brown DM, Green L, Tuerk C, Gold L
• RNA ligands to human nerve growth factor.
• Nucleic Acids Res. 1995; 23: 3198-205
• Display abstract

• High affinity RNA ligands to human nerve growth factor (NGF) were selected from pools of random RNA using SELEX [Tuerk, C. and Gold, L. (1990) Science, 249, 505-510]. Nerve growth factor, which is a protein required for the development of neurons, is not known to bind nucleic acids as part of its natural function. We describe two of the selected RNA molecules in detail. One of them is highly structured, folding into a pseudoknot with an additional hairpin-loop; this structure provides salt-resistant binding to NGF. The other is unstructured and elevated salt concentrations inhibit its binding. These molecules compete with each other for NGF binding. Our RNAs may furnish useful diagnostic tools for the study of an important neurotrophic protein; additionally, they illustrate another example of the potential for nucleic acids to take part in novel binding interactions.

• Gilad GM, Gilad VH
• Chemotaxis and accumulation of nerve growth factor by microglia and macrophages.
• J Neurosci Res. 1995; 41: 594-602
• Display abstract

• Astrocytes and microglia play a critical role in the reaction of the central nervous system (CNS) to trauma. Although both astrocytes and microglia can produce it, accumulation of immunoreactive nerve growth factor (the prototype neurotrophin important for the survival of several classes of neurons) was observed selectively in cultured microglia and macrophages, rather than in astrocytes. Furthermore, microglia were found to display chemotaxis toward a localized source of nerve growth factor and, as demonstrated by autoradiography, take up extracellular nerve growth factor. These findings suggest that microglia, the brain's own macrophages, participate in the regulation of nerve growth factor availability in a site-specific manner. This novel function may assume a general importance both in the CNS and the peripheral nervous system at critical times after trauma when this neurotrophin is needed for nerve cell survival.

• Carlson SL, Albers KM, Beiting DJ, Parish M, Conner JM, Davis BM
• NGF modulates sympathetic innervation of lymphoid tissues.
• J Neurosci. 1995; 15: 5892-9
• Display abstract

• Immune tissues are known to be innervated by the sympathetic nervous system, but little is known of what directs the innervation to specific tissue compartments. This report examines the sympathetic innervation of immune tissues in transgenic mice that overexpress nerve growth factor (NGF) in skin and other epithelial structures. NGF transgenic mice exhibited dramatic hyperinnervation in the splenic marginal zone, and the medulla and capsule of peripheral lymph nodes. In contrast, the transgenic mesenteric lymph nodes showed no hyperinnervation. This difference correlated with the location of these nodes; peripheral lymph nodes drain skin where the transgene was expressed while mesenteric lymph nodes drain non-transgene-expressing structures. In addition, the level of innervation correlated with the level of NGF peptide content as assayed by ELISA (3- and 13-fold increase in transgenic spleen and axillary lymph nodes, respectively; no increase in mesenteric nodes) and immunocytochemistry. RT-PCR showed that the NGF transgene was not being expressed in the immune tissues, suggesting that immune tissues can concentrate transgene-produced NGF. It was also demonstrated that the change in innervation had functional consequences. The mitogen response to concanavalin A (ConA) by spleen cells was decreased in the transgenics suggesting that elevated catecholamines or NGF can modulate the proliferative response of these cells. These mice demonstrate that NGF can modulate the sympathetic innervation and function of the immune system.

• McNeeley PA et al.
• Interleukin-1 beta induces nerve growth factor release and gene expression in human glioblastoma cells.
• Proc West Pharmacol Soc. 1995; 38: 63-6

• Ibanez CF, Ryden M
• [Nerve growth factors in future therapies. Protein mutation broadens the range of therapeutic possibilities]
• Lakartidningen. 1995; 92: 2191-4
• Display abstract

• The development and maintenance of the vertebrate nervous system requires a continuous supply of a number of polypeptide hormones known as neurotrophic factors. The ability of neurotrophic factors to promote the survival of peripheral and central neurons during development and after neuronal damage has aroused interest in these molecules as potential therapeutic agents for the treatment of nervous lesions and neurodegenerative diseases. Understanding of the molecular basis of the biological specificity of neurotrophic polypeptides has provided insight into their mechanisms of action and allowed strategies to be developed for the rational design of derivatives and analogues with specific pharmacological properties.

• Inagaki S, Furuyama T, Iwahashi Y
• Identification of a member of mouse semaphorin family.
• FEBS Lett. 1995; 370: 269-72
• Display abstract

• Grasshopper semaphorin I (Sema I) and its related proteins, chick collapsin and mouse Sema III contribute to the axon guidance by their repellent actions [5,9,12]. We have identified a member of semaphorin gene family from the mouse brain and named it M-Sema F. The N-terminal encodes a semaphorin domain that is similar between Sema I-III [6] followed by a single putative immunoglobulin-like domain, a transmembrane domain, and a proline-rich intracellular domain. M-Sema F mRNA is expressed widely in the nervous tissues during development. These suggest that M-Sema F is a transmembrane member of the semaphorin family of the vertebrate which may function in the developing neuronal network.

• Chapman BS, Kuntz ID
• Modeled structure of the 75-kDa neurotrophin receptor.
• Protein Sci. 1995; 4: 1696-707
• Display abstract

• Motifs in ligand-binding domains of the neurotrophin (NTR) and lymphotoxin (TNFR-I) receptors define a family of receptors that mediates programmed cell death. We have explored relationships of architecture and function in this family through a molecular model of NTR, also called p75NGFR or LANR. Modeling by homology took advantage of four modular subdomains in the crystal structure of TNFR-I that also occur in NTR. Hypothetical complexes between the model and a ligand structure (for nerve growth factor, NGF) were then examined using docking software. NTR appears to bind in the dimer interface of NGF, making two sets of contacts. NTR subdomains III and IV provide the ligand-contact surfaces, in contrast to TNFR, in which subdomains II and III contact TNF-beta. NTR subdomain II appears to have been evolutionarily modified, potentially contributing to an interface between receptor subunits. These and other specific predictions of the model will require experimental confirmation.

• Paus R, Luftl M, Czarnetzki BM
• Nerve growth factor modulates keratinocyte proliferation in murine skin organ culture.
• Br J Dermatol. 1994; 130: 174-80
• Display abstract

• Despite the fact that several cell types residing in or travelling through the skin are targets and/or sources of nerve growth factor (NGF), little is known about the role of NGF in skin development, physiology and disease. Employing a previously defined skin organ culture assay for studying the proliferation of murine keratinocytes in their natural tissue environment, we have assessed the effect of murine NGF (7S) on keratinocyte proliferation in intact skin derived from two defined stages of the murine hair cycle. We found that 10-200 ng/ml NGF stimulated epidermal keratinocyte proliferation in organ-cultured C57 BL-6 mouse skin in the telogen phase of the hair cycle. Follicle keratinocyte proliferation was stimulated by 100 ng/ml NGF in telogen skin organ culture, but this concentration of NGF inhibited both epidermal and follicle keratinocyte proliferation in organ culture of anagen skin. The latter inhibitory effect of NGF was abrogated by co-incubation with neutralizing anti-NGF antibodies or with the protein kinase C inhibitor staurosporine. The proliferation-modulatory effects of NGF were associated with the induction of significant mast cell degranulation, and were inhibited by cromoglycate co-administration. This is the first report of a modulatory, hair cycle-dependent effect of NGF on keratinocyte proliferation in situ, which may require the presence of mast cells. Our study supports the notion of auto- and paracrine functions of NGF in murine skin physiology, which can be further assessed in the physiologically relevant mouse model delineated here.

• Baldwin AN, Shooter EM
• Disulfide mutants of the binding domain of the rat low affinity nerve growth factor receptor (p75NGFR).
• J Biol Chem. 1994; 269: 11456-61
• Display abstract

• The binding domain of the low affinity nerve growth factor receptor (p75NGFR) is built from four "cysteine repeats," with almost identical patterns of half-cystine residues. To study the pattern of disulfide bridging within each cysteine repeat, we have mutated pairs of cysteine residues, primarily in the fourth repeat, and have tested the relative ability of each mutant to be cross-linked to the nerve growth factor after transient expression in COS 7 cells. The results give an indication of the relative importance of different disulfide bonds for the stability of the active conformation. Immunofluorescent staining shows that all of these proteins, even those that may be partially misfolded, are expressed to varying degrees on the surface of COS cells. They can also be detected by the monoclonal antibody MC192 on a Western blot following electrophoresis on a nondenaturing gel. Some cannot, however, be detected by MC192 on a Western blot after heat denaturation in SDS. The ability to refold under these conditions correlates in general with the ability of the mutant protein to bind nerve growth factor on the surface of COS cells.

• Lewin GR, Mendell LM
• Regulation of cutaneous C-fiber heat nociceptors by nerve growth factor in the developing rat.
• J Neurophysiol. 1994; 71: 941-9
• Display abstract

• 1. Previous work has indicated that nerve growth factor (NGF) is an important factor for the development of nociceptive A-delta fibers. Here we asked if the availability of NGF during the first 2 wk of development can influence the phenotypic development of unmyelinated afferent C-fibers. 2. To do this, we treated newborn rats with antibodies to NGF from postnatal day (PND) 2-14, a treatment known not to lead to cell death, or with exogenous NGF (PND 0-14). Untreated litter mates served as controls. When the animals were mature (5-20 wk later), they were anesthetized (urethan, 1.25 g/kg ip) and single-unit recordings were made from sural nerve C-fibers in dorsal root filaments. To obtain an unbiased estimate of the physiological types present, an electrocutaneous search technique was employed to isolate the location of the putative receptive field of the C-fiber being studied within the sural nerve territory. This technique enabled us to acquire a sample of C-fibers that was unbiased by natural search stimuli which are often damaging to skin. 3. We found that the proportion of C-fibers that could be driven by noxious heat and mechanical stimuli (C-mechanoheat fibers, C-MH) was reduced from 28% (11/40 fibers) of the sample in controls to 10% (3/31 fibers) in anti-NGF treated animals. The C-MH fibers appeared to have been replaced by a novel type of pressure receptor with an unusually low mechanical threshold (mean 0.86 +/- 0.58 compared with 13.0 +/- 8.3 g for control mechanonociceptors).(ABSTRACT TRUNCATED AT 250 WORDS)

• Kullander K, Ebendal T
• Neurotrophin-3 acquires NGF-like activity after exchange to five NGF amino acid residues: molecular analysis of the sites in NGF mediating the specific interaction with the NGF high affinity receptor.
• J Neurosci Res. 1994; 39: 195-210
• Display abstract

• Despite the large sequence similarity around 55-60% among the known NGF-related neurotrophins, the members display different activities on different subset of neurons. Recent studies have shown that the various neurotrophins are ligands with high affinity to different receptors of the Trk family of tyrosine kinase receptors. We wanted to elucidate what specific parts of NGF replaced in neurotrophin-3 (NT-3) would result in NGF-like receptor binding and biological activity. By studying evolutionarily conserved amino acid sequences not shared by NT-3 and NGF and excluding parts which have been examined in earlier work with NGF and BDNF chimeras as well as taking advantage of the crystallographic data available for NGF, we decided to exchange three specific blocks of two or three amino acids in the human NT-3 backbone for the corresponding residues in NGF. The NGF residues Asn-Ile-Asn (43-45), Val-Phe (48,49) and Gln-Ala-Ala (96-98) were combined in pairs and are all shown to contribute NGF-like activity in the context of NT-3. The most efficient NGF-like transformation was obtained by the exchange of Pro-Val and Leu-Val-Gly in NT-3 to the NGF residues Val-Phe and Gln-Ala-Ala. This mutant reached 90% NGF activity, based on survival of sympathetic neurons, stimulation of fibre outgrowth from sympathetic ganglia, the ability to block high affinity NGF binding to PC12 cells and phosphorylation of gp140trk. Thus, the three mutants with paired combinations of the NGF residues as well as the NT-3 housing all three blocks of NGF residues were able to mimic NGF activity. This activity is gained, although the mutated neurotrophin proteins do not lose the original NT-3 activity as ascertained by the stimulation of neurite outgrowth from the Remak ganglion. The three mutated sites are situated in two beta-loops at one end of the NGF molecule, forming a cleft that could specifically interact with high affinity to the signalling NGF receptor gp140trk.

• Nechad M, Ruka E, Thibault J
• Production of nerve growth factor by brown fat in culture: relation with the in vivo developmental stage of the tissue.
• Comp Biochem Physiol Comp Physiol. 1994; 107: 381-8
• Display abstract

• The regulation of nerve growth factor (NGF) production is still poorly understood. We attempt here to determine whether brown adipose tissue (BAT), which is densely innervated by sympathetic nerve fibres and can be induced to grow in adult rats by simple cold exposure, has the ability to produce NGF and thus to stimulate the growth of its innervation and, if so, whether this NGF activity is developmentally regulated. BAT at various stages of development was cocultured with NGF-sensitive sympathetic ganglia in the presence or absence of antiserum to NGF. Both undifferentiated BAT from newborn hamster and differentiated BAT from newborn rat induced neurite outgrowth by producing a neurotrophic factor which was biologically and immunologically indistinguishable from mouse submandibular gland beta (2.5S) NGF. Newborn hamster BAT and BAT from adult rats exposed to cold for 1 or 2 days showed the same high level of NGF activity, whereas the activities of newborn rat BAT, BAT from adult rats at normal room temperature or cold-exposed for 3 days or more were not significantly different from each other and were only about half as high. Newborn hamster BAT and BAT from adult rats cold-exposed for 1 or 2 days are both characterized in vivo by a rapidly increasing mitotic activity of the stromal-vascular cells, which distinguishes them from the other developing tissues tested. Our observations, therefore, suggest a relationship between NGF synthesis and proliferative activity.

• Loy R, Taglialatela G, Angelucci L, Heyer D, Perez-Polo R
• Regional CNS uptake of blood-borne nerve growth factor.
• J Neurosci Res. 1994; 39: 339-46
• Display abstract

• Nerve growth factor (NGF), in addition to being a neurotrophic substance, has effects on the endocrine and immune systems. For example, intravenous injection of NGF results in a cascade of events leading to an increase in glucocorticoid secretion. While this response appears to be mediated centrally, there has been no evidence that circulating NGF has access to the CNS. Using intravenous injections of 125I-NGF, we find specific uptake at 1 hr but none at 6 hr, into homogenates of the basal forebrain, cerebellum, frontal cortex, hippocampus, and olfactory bulb. By autoradiography, uptake is localized to circumventricular organs, deep layers of the cerebellum, and all layers of the hippocampal region CA1, but not the dentate gyrus. Thus, uptake of blood-borne NGF could affect the hypothalamic-pituitary-adrenal axis via binding to NGF receptors present in the hippocampus. However, the sources of endogenous NGF, the mechanism of access through the blood-brain barrier, the eventual fate of NGF entering from the blood, and the physiological significance of this uptake remain to be elucidated.

• Aloe L, Skaper SD, Leon A, Levi-Montalcini R
• Nerve growth factor and autoimmune diseases.
• Autoimmunity. 1994; 19: 141-50
• Display abstract

• The initiation of a humoral immune response to a foreign antigen is a complex biologic process involving the interaction of many cell types and their secreted products. Autoimmune diseases, which are characterized by an abnormal activation of the immune system, probably result from the failure of normal self-tolerance mechanisms. The etiology of such illnesses, however, is far from being understood. While there have been extensive studies on the participation of the immune and endocrine systems in autoimmune diseases, few have dealt with nervous system-mediated immunoregulation in such situations. Evidence continues to grow suggesting that nerve growth factor (NGF), first identified for its activity in promoting the growth and differentiation of sensory and sympathetic neurons, may exert a modulatory role on neuroimmunoendocrine functions of vital importance in the regulation of homeostatic processes. Newly detected NGF-responsive cells belong to the hemopoietic-immune system and to populations in the brain involved in neuroendocrine functions. NGF levels are elevated in a number of autoimmune states, along with increased accumulation of mast cells. NGF and mast cells both appear to be involved in neuroimmune interactions and tissue inflammation. Moreover, mast cells themselves synthesize, store, and release NGF, proposing that alterations in normal mast cell behaviors may provoke maladaptive neuroimmune tissue responses whose consequences could have profound implications in inflammatory disease states, including those of an autoimmune nature. This review focuses on these cellular events and presents a working model which attempts to explain the close interrelationships of the neuroendocrinoimmune triade via a modulatory action of NGF.

• Pincelli C, Fantini F, Giannetti A
• Nerve growth factor and the skin.
• Int J Dermatol. 1994; 33: 308-312

• Li Y, Huang B, Cai L
• [Amplification, cloning and sequencing of beta nerve growth factor gene in the Chinese population]
• Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 1994; 16: 334-8
• Display abstract

• Genomic DNA was extracted from white blood cells. Using the genomic DNA as template, the gene encoding nerve growth factor (beta-NGF) in the Chinese population was amplified using polymerase chain reaction (PCR). The beta-NGF gene obtained by PCR was then cloned into plasmid pUC 12 which was digested with SmaI. After transformation, screening and identification, 8 clones containing the beta-NGF gene were analyzed. Nucleotide sequence of the NGF was determined by dideoxy-nucleotide chain-termination method. The sequencing result showed that there are 6 differences between the beta-NGF sequence of the Chinese population and previous reported beta-NGF sequence.

• Kuroda T, Ueda M, Nakano M, Saeki M
• Altered production of nerve growth factor in aganglionic intestines.
• J Pediatr Surg. 1994; 29: 288-92
• Display abstract

• Nerve growth factor (NGF), a target-derived neurotrophic molecule, is required specifically by sympathetic and dorsal root ganglion cells for their survival and maturation during embryonic and early postnatal development. In the present study, the NGF expression was studied both at the protein and mRNA level in normal and aganglionic intestines of Piebald-strain mice and also in 10 human specimens using immunohistochemical and reverse transcriptase polymerase chain reaction (RT-PCR) techniques. In the aganglionic intestines of the mice, immunoreactive NGF was found on the giant nerve fibers in the submucosal layer, but not found in the mucosal layer. In the mRNA study, the signal for NGFmRNA was less intense in the aganglionic rectum of the congenitally megacolonic mice than in the rectum of the normal mice. In contrast, the distal dilated colon of the congenitally megacolonic mice had a more intense signal for NGFmRNA than did the colon of normal mice. The results obtained from human specimens were compatible with the findings in the Piebald mice; the distal colons harvested from the patients with Hirschsprung's disease (or its allied disease) had a uniformly more intense signal for NGFmRNA than did the normal colons. The results of this study may indicate that NGF production is altered in the aganglionic intestines and also in the "transitional zone" in Hirschsprung's disease. The altered production of NGF may be useful in increasing the accuracy of diagnosis of Hirschsprung's disease.

• Jippo T et al.
• Poor response of cultured mast cells derived from mi/mi mutant mice to nerve growth factor.
• Blood. 1994; 84: 2977-83
• Display abstract

• Decreased numbers of mast cells and abnormalities in the phenotype of mast cells are observed in the skin of mi/mi mutant mice. Recently, the mi locus was identified to encode a novel member of the basic-helix-loop-helix-leucine zipper protein family of transcription factors. Since nerve growth factor (NGF) has been reported to influence the proliferation and the phenotype of cultured mast cells (CMCs), we compared the effect of NGF between mi/mi and control normal (+/+) CMCs. Addition of NGF to the suboptimal dose of recombinant murine interleukin-3 (rmIL-3) increased the plating efficiency of +/+ CMCs, but not of mi/mi CMCs. Although +/+ CMCs were berberine sulfate-negative when cultured with rmIL-3 alone, +/+ CMCs became berberine sulfate-positive when cultured in the presence of both rmIL-3 and NGF, which suggests increased heparin content. In contrast, NGF did not influence the phenotype of mi/mi CMCs. +/+ CMCs significantly bound 125I-NGF, but mi/mi CMCs did not, which suggests a defect of NGF receptors in mi/mi CMCs. Both p75 and p140 molecules are known to be involved in the formation of NGF receptors. Although the expression of p140 messenger (m)RNA was comparable between +/+ and mi/mi CMCs, the expression of p75 mRNA was significantly lower in mi/mi CMCs than in +/+ CMCs. Taken together, the poor response of mi/mi CMCs to NGF appeared to be attributable to the impaired transcription of the p75 gene.

• Tria MA, Fusco M, Vantini G, Mariot R
• Pharmacokinetics of nerve growth factor (NGF) following different routes of administration to adult rats.
• Exp Neurol. 1994; 127: 178-83
• Display abstract

• The suggested potential for therapeutic use of nerve growth factor (NGF) in the treatment of toxic and degenerative disorders of the nervous system indicates a need to determine its pharmacokinetics. To this end, murine NGF was administered to adult rats and multiple blood samples were withdrawn at intervals. NGF levels, determined in plasma samples by a two-site enzyme immunoassay, were used to determine the pharmacokinetics of NGF. These studies demonstrate that murine NGF has a distribution half-life of about 5.4 min and an elimination half-life of 2.3 h following intravenous injection. When administered by subcutaneous (sc) injection, the elimination half-life is prolonged to 4.5 h. Administration of NGF by sc continuous infusion, using mini-osmotic pumps, provides stable, dose-related levels of circulating NGF within few days from pump implantation. Upon removal of the pump, NGF levels show a rapid decay (t1/2 about 1.5 h) followed by a slow elimination phase (t1/2 about 150 h). These pharmacokinetic parameters might serve for selection of an appropriate administration route and dose regimen that would optimize schedule-dependent expression of NGF therapeutic activity.

• Scott SA, Crutcher KA
• Nerve growth factor and Alzheimer's disease.
• Rev Neurosci. 1994; 5: 179-211
• Display abstract

• Nerve growth factor (NGF) is a well-characterized protein that exerts pharmacological effects on a group of cholinergic neurons known to atrophy in Alzheimer's disease (AD). Considerable evidence from animal studies suggests that NGF may be useful in reversing, halting, or at least slowing the progression of AD-related cholinergic basal forebrain atrophy, perhaps even attenuating the cognitive deficit associated with the disorder. However, many questions remain concerning the role of NGF in AD. Levels of the low-affinity receptor for NGF appear to be at least stable in AD basal forebrain, and the recent finding of AD-related increases in cortical NGF brings into question whether endogenous NGF levels are related to the observed cholinergic atrophy and whether additional NGF will be useful in treating this disorder. Evidence regarding the localization of NGF within the central nervous system and its presumed role in maintaining basal forebrain cholinergic neurons is summarized, followed by a synopsis of the relevant aspects of AD neuropathology. The available data regarding levels of NGF and its receptor in the AD brain, as well as potential roles for NGF in the pathogenesis and treatment of AD, are also reviewed. NGF and its low affinity receptor are abundantly present within the AD brain, although this does not rule out an NGF-related mechanism in the degeneration of basal forebrain neurons, nor does it eliminate the possibility that exogenous NGF may be successfully used to treat AD. Further studies of the degree and distribution of NGF within the human brain in normal aging and in AD, and of the possible relationship between target NGF levels and the status of basal forebrain neurons in vivo, are necessary before engaging in clinical trials.

• Maness LM, Kastin AJ, Weber JT, Banks WA, Beckman BS, Zadina JE
• The neurotrophins and their receptors: structure, function, and neuropathology.
• Neurosci Biobehav Rev. 1994; 18: 143-59
• Display abstract

• The neurotrophins are a family of polypeptides that promote differentiation and survival of select peripheral and central neurons. Nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, and neurotrophin-5 are included in this group. In recent years, tremendous advances have been made in the study of these factors. This has stimulated our review of the field, characterizing the neurotrophins from initial isolation to molecular analysis. The review also discusses their synthesis, localization, and responsive tissues, in both the periphery and CNS. The complex receptor interactions of the neurotrophins are also analyzed, as are putative signal transduction mechanisms. Discussion of the observed and postulated involvement in neuropathological disorders leads to the conclusion that the neurotrophins are involved in the function and dysfunction of the nervous system.

• Katoh-Semba R, Semba R, Kato H, Ueno M, Arakawa Y, Kato K
• Regulation by androgen of levels of the beta subunit of nerve growth factor and its mRNA in selected regions of the mouse brain.
• J Neurochem. 1994; 62: 2141-7
• Display abstract

• Our previous studies showed that the concentration of the beta subunit of nerve growth factor (beta-NGF) in nervous tissues is higher in male than in female mice. To identify the brain regions that are affected by androgens, the amounts of beta-NGF protein and its mRNAs were measured in male, female, and castrated male CD-1 mice and testicular feminization mice at 3-4 months of age. Among tissues examined, the hypophysis of males contained the highest average concentration of beta-NGF protein. In most regions of the brain, individual levels were more variable in males than in females. However, after the castration, such variations in beta-NGF levels disappeared. Average levels of beta-NGF protein in males were higher in the cerebellum (eightfold higher), olfactory bulb (12-fold higher), hypothalamus (sixfold higher), and hypophysis (72-fold higher) than those in corresponding regions of females. No significant differences were observed in levels of beta-NGF protein in the hippocampus, cerebral cortex, striatum, septum, and brainstem. The castration of male mice caused a reduction in levels of beta-NGF protein in the hypothalamus and hypophysis, but not in the cerebellum and olfactory bulb, to the female levels. The concentrations of beta-NGF protein in testicular feminization mice were similar to those in female CD-1 mice in all regions.(ABSTRACT TRUNCATED AT 250 WORDS)

• Salikhov RS, Parilis II, Khafizova MG, Kazanov EI, Khamidov DK
• [Determination of unique peptide fragments in nerve growth factors]
• Mol Biol (Mosk). 1994; 28: 201-3
• Display abstract

• A new classificational method based on the availability of peptide fragments unique for a certain protein family is presented. Seven common peptide fragments unique for the nerve growth factor amino acid sequence are determined, and two of them are found in the neurotrophic factor from pig brain.

• Wang MH, Gonias SL, Skeel A, Wolf BB, Yoshimura T, Leonard EJ
• Proteolytic activation of single-chain precursor macrophage-stimulating protein by nerve growth factor-gamma and epidermal growth factor-binding protein, members of the kallikrein family.
• J Biol Chem. 1994; 269: 13806-10
• Display abstract

• Promacrophage-stimulating protein (MSP) is an 80-kDa protein that acquires biological activity after cleavage at an Arg-Val bond to a disulfide-linked alpha beta heterodimer by serine proteases of the intrinsic coagulation cascade. These proteases, which include serum kallikrein, factor XIIa and factor XIa, are members of the trypsin family of serine proteases. We now report that two other members of the family, nerve growth factor-gamma (NGF-gamma) and epidermal growth factor-binding protein (EGF-BP), cleave and activate pro-MSP to the disulfide-linked alpha beta heterodimer. Cleavage of 1.5 nM pro-MSP by 1 nM NGF-gamma or EGF-BP at 37 degrees C was almost complete within 30 min. These concentrations of enzyme are about 2 orders of magnitude less than is required for cleavage by serum kallikrein or factor XIIa. Cleavage of pro-MSP to MSP was associated with a conformational change in the protein, because the cleaved product, but not pro-MSP, was detected by a sandwich enzyme-linked immunoassay. Cleavage caused the appearance of biological activity, as measured by chemotactic activity of MSP for resident peritoneal macrophages, by MSP-induced macrophage shape change, and by stimulation of macrophage ingestion of C3bi-coated erythrocytes. These findings suggest the possibility of cooperative interactions between NGF-gamma or EGF-BP and pro-MSP in inflammation and wound healing.

• Zimmermann A, Sutter A, Stephani U
• Evidence for an NGF-induced autocrine neurotrophic potential of glial cells in nervous system development.
• Glia. 1994; 12: 81-5
• Display abstract

• NGF receptor bearing peripheral glial cells, purified from chicken embryo dorsal root ganglia, were found to secrete neurite growth promoting activity (NGPA) but no NGF (detection limit 1 pM). If, however, minute concentrations of NGF (2 pM) were added to the glial cultures, an autocrine response was observed. Levels of NGF-like activity in the medium rose up to 50-fold. Induction of this autocrine response occured within a narrow range of NGF concentrations. NGPA production was not affected by addition of NGF. Coculture with neurons influenced the neurotrophic factor production. The data suggest that glial cells can "sense" traces of NGF and regulate its availability during neural development.

• Holland DR, Cousens LS, Meng W, Matthews BW
• Nerve growth factor in different crystal forms displays structural flexibility and reveals zinc binding sites.
• J Mol Biol. 1994; 239: 385-400
• Display abstract

• Murine beta-nerve growth factor (beta NGF) is a 118 amino acid residue polypeptide which, as a functional dimer, plays an important role in the survival and development of certain neuronal populations. The structure of the bis-desocta1-8 form of murine beta NGF has been determined in two different crystal modifications using X-ray methods. The two crystal forms, with space groups P2(1)2(1)2(1) and C2, were grown from 18 to 20% polyethylene glycol 8000 and 100 mM Pipes (pH 6.1) with zinc acetate concentrations of 1 mM and 100 mM, respectively. The C2 structure was solved by multiple isomorphous replacement using four heavy-atom derivatives and was refined to a crystallographic residual of 17.9% and 2.5 A resolution. The crystals contain three beta NGF monomers per asymmetric unit. Two monomers form a dimer related by a non-crystallographic 2-fold axis of symmetry. The third monomer also forms a dimer that is very similar, but with a crystallography related monomer as a partner. The electron density clearly defines residues 12 through 115 for all three monomers but the extreme N and C-terminal residues (9 to 11, 116 to 118) are ill defined in some cases. The P2(1)2(1)2(1) structure was solved by molecular replacement using the C2 structure as a search model and was refined to a crystallographic residual of 19.7% at 2.8 A resolution. This crystal form contains two monomers per asymmetric unit, again arranged as a non-crystallographic 2-fold-related dimer. The N and c termini are also variably defined. The core of each of the five monomers, which forms a cysteine knot motif, is very similar in all structures. Also, the dimer structures are very similar to one another, whether the monomers are related by crystallographic or non-crystallographic symmetry. However, three of the four loop regions that extend from the core of each monomer display substantial variability in conformation, even between monomers of the same dimer. This structural variability in the putative receptor binding regions suggests that structural malleability might be important in allowing the ligands to bind to different receptors with different affinities.(ABSTRACT TRUNCATED AT 400 WORDS)

• Davies AM
• Neurobiology. Tracking neurotrophin function.
• Nature. 1994; 368: 193-4

• Brewster WJ, Fernyhough P, Diemel LT, Mohiuddin L, Tomlinson DR
• Diabetic neuropathy, nerve growth factor and other neurotrophic factors.
• Trends Neurosci. 1994; 17: 321-5
• Display abstract

• Diabetic neuropathy typically presents as an insidious symmetrical distal degenerative disease of peripheral nerves. A failure of neurotrophic factors to regulate neuronal phenotype might be expected to result in such a clinical picture. Experimentally, diabetic rats show reduced expression of target-derived nerve growth factor as well as reduced expression of neuronal genes that are responsive to nerve growth factor. The latter is corrected by administration of exogenous nerve growth factor. Thus, deficient neurotrophic support might contribute to the pathogenesis of diabetic neuropathy, and any successful treatment might include exogenous neurotrophins or other strategies to correct their deficiency of action.

• Leon A et al.
• Mast cells synthesize, store, and release nerve growth factor.
• Proc Natl Acad Sci U S A. 1994; 91: 3739-43
• Display abstract

• Mast cells and nerve growth factor (NGF) have both been reported to be involved in neuroimmune interactions and tissue inflammation. In many peripheral tissues, mast cells interact with the innervating fibers. Changes in the behaviors of both of these elements occur after tissue injury/inflammation. As such conditions are typically associated with rapid mast cell activation and NGF accumulation in inflammatory exudates, we hypothesized that mast cells may be capable of producing NGF. Here we report that (i) NGF mRNA is expressed in adult rat peritoneal mast cells; (ii) anti-NGF antibodies clearly stain vesicular compartments of purified mast cells and mast cells in histological sections of adult rodent mesenchymal tissues; and (iii) medium conditioned by peritoneal mast cells contains biologically active NGF. Mast cells thus represent a newly recognized source of NGF. The known actions of NGF on peripheral nerve fibers and immune cells suggest that mast cell-derived NGF may control adaptive/reactive responses of the nervous and immune systems toward noxious tissue perturbations. Conversely, alterations in normal mast cell behaviors may provoke maladaptive neuroimmune tissue responses whose consequences could have profound implications in inflammatory disease states, including those of an autoimmune nature.

• Ricceri L, Alleva E, Calamandrei G
• Impairment of passive avoidance learning following repeated administrations of antibodies against nerve growth factor in neonatal mice.
• Neuroreport. 1994; 5: 1401-4
• Display abstract

• CD-1 Swiss mice were used to study the effects of repeated intracerebroventricular (i.c.v.) injections, on postnatal days (PND) 3, 6, 9, of rabbit antiserum (50 micrograms) raised against murine nerve growth factor (NGF) both on locomotor activity in an open field test (PND 10) and on a passive avoidance learning test (PND 11 and 12). While no difference was evident in locomotor activity levels between anti-NGF and control pups, an impairment of anti-NGF pups was detectable in the passive avoidance performance. Removal of endogenous NGF seems to produce a detrimental effect on a behavioural performance which is known to be under central cholinergic control, confirming previous reports on the role of NGF in early neurobehavioural responding in altricial rodents.

• Hirota M et al.
• Changes in nerve growth factor content of the submaxillary gland in the genetically dystrophic (mdx) mouse.
• J Neurol Sci. 1994; 121: 176-82
• Display abstract

• We evaluated the nerve growth factor (NGF) contents in the submaxillary gland of the mdx mouse, a model for Duchenne muscular dystrophy (DMD), and found that the NGF and NGF mRNA contents in this organ, where extraordinarily high amounts of NGF are synthesized and stored independently of development or maintenance of the nervous system, were markedly elevated in the male mdx mouse at 8 and 11 weeks of age. However, the NGF content of this organ in 4-week-old male mdx mice was lower than that of control mice although statistical significance of difference was not observed. In the mdx female mouse, the submaxillary NGF content was significantly lower than that of the normal mouse at 4 weeks of age, but was similar to that of the normal at 8 and 11 weeks of age. The amounts of epidermal growth factor (EGF), another protein that is known to be sexually and developmentally regulated in the mouse submaxillary gland like NGF, was found to be also significantly increased in this organ of the male mdx mouse at 8 and 11 weeks of age, and to be significantly decreased in that of the female mdx mouse at 4 weeks of age. The parallel changes in NGF and EGF contents during development of the submaxillary gland suggest that the mdx mouse suffers from some abnormality in the development of this organ.(ABSTRACT TRUNCATED AT 250 WORDS)

• Barde YA
• Neurotrophic factors: an evolutionary perspective.
• J Neurobiol. 1994; 25: 1329-33

• Ibanez CF
• Structure-function relationships in the neurotrophin family.
• J Neurobiol. 1994; 25: 1349-61
• Display abstract

• The study of structure-function relationships in the neurotrophin family has in recent years increased our understanding of several important aspects of neurotrophin function. Site-directed mutagenesis studies have localized amino acid residues important for binding to the low-affinity (p75LNGFR), as well as to the members of the Trk family of tyrosine kinase receptors. A cluster of positively charged residues has been shown to form a surface for binding to p75LNGFR in all four neurotrophins. Differences in the spatial distribution of these charges among the different neurotrophins may explain some of their distinct binding properties. Elimination of these positive charges drastically reduces binding to p75LNGFR but not to the Trk family members, and it does not impair the biological properties of the neurotrophins in vitro, arguing that binding to and activation of Trk receptors is sufficient to mediate the biological responses of neurotrophins. In contrast, the binding sites to Trk receptors appear to be formed by discontinuous stretches of amino acid residues distributed throughout the primary sequence of the molecule. These include the N-terminus, some of the variable loop regions and a beta-strand. Despite their apparent distribution, when viewed in the three-dimensional structure of NGF, these residues appear grouped on one side of the neurotrophin dimer, delineating a continuous surface extending approximately parallel to the twofold symmetry axis of the molecule. Two symmetrical surfaces are formed along the axis of the neurotrophin dimer providing a model for ligand-mediated receptor dimerization. In the neurotrophin family, co-evolution of cognate ligands and Trk receptors has developed specific contacts through different residues in the same variable regions of the neurotrophins. Thus, binding specificity is determined by the cooperation of distinct active and inhibitory binding determinants that restrict ligand-receptor interactions. Binding determinants to the Trk receptors can be manipulated independently in a rational fashion to create neurotrophin analogues with novel ligand-binding properties. In this way, second-generation chimeric neurotrophins with multiple specificities (pan-neurotrophins) have been engineered which may have valuable applications in the treatment of neurodegeneration and nerve damage.

• Aloe L, Tirassa P, Alleva E
• Cold water swimming stress alters NGF and low-affinity NGF receptor distribution in developing rat brain.
• Brain Res Bull. 1994; 33: 173-8
• Display abstract

• We have previously shown that the nerve growth factor (NGF) is released into the bloodstream following intraspecific fighting behaviour and that the level released correlates with the number of fighting episodes. We subsequently reported that NGF and its messenger RNA are present in identified hypothalamic nuclei and increase following intermale fighting behaviour. This report provides data showing that in 16-day-old rats cold water swimming stress (CWSS) alters the distribution of low-affinity NGF-Receptors (p75NGFR) and NGF levels in the central nervous system. A significant increase of NGF level was observed in the cortex, while the p75NGFR immunoreactivity decreased in neurons of the septum, nucleus basalis and striatum. Choline acetyltransferase activity in forebrain tissues remained at baseline levels. Our result suggests that NGF and p75NGFR, involved in the development and differentiation of the nervous system, are affected by stress.

• Pincelli C et al.
• Expression and function of nerve growth factor and nerve growth factor receptor on cultured keratinocytes.
• J Invest Dermatol. 1994; 103: 13-8
• Display abstract

• Keratinocytes, a key cellular component both for homeostasis and pathophysiologic processes of the skin, secrete a number of cytokines and are stimulated by several growth factors. Nerve growth factor (NGF) is synthesized in the skin and basal keratinocytes express the low-affinity nerve growth factor receptor (NGF-R). We present evidence that normal human keratinocytes in culture express the low- and the high-affinity NGF-R both at the mRNA level, as determined by reverse-transcription polymerase chain reaction and at the protein level, as shown by cytofluorimetric analysis. NGF significantly stimulates the proliferation of normal human keratinocytes in culture in a dose-dependent manner. This effect can be prevented by the addition of both an anti-NGF neutralizing antibody and a high-affinity NGF-R (trk) specific inhibitor, the natural alkaloid K252a. By contrast, keratinocyte proliferation is not inhibited by an anti-low-affinity NGF-R monoclonal antibody, thus suggesting that NGF effect on human keratinocytes is mediated by the high-affinity NGF-R. Moreover, NGF mRNA is expressed in normal human keratinocytes and NGF is secreted by keratinocytes in increasing amounts during growth, as detected by enzyme-linked immunosorbent assay. These results suggest that NGF could act as a cytokine in human skin and take part in disorders of keratinocyte proliferation.

• Kasaian MT, Jacobberger JW, Neet KE
• Flow cytometric analysis of fluorescein-labeled nerve growth factor binding to A875 human melanoma cells.
• Exp Cell Res. 1994; 210: 77-85
• Display abstract

• The interaction of fluorescein-labeled nerve growth factor (NGF) with human melanoma cells (A875) has been studied in order to assess better methodology for rigorous NGF binding studies. The NGF was modified at a single carboxyl group with iodoacetamidofluorescein after reaction with carbodiimide and cystamine. The modified NGF showed full binding competence in competition with radiolabeled NGF and full biological activity in neurite outgrowth assays compared to native NGF. Binding to unfixed, viable cells was assayed using flow cytometry. This method offers the advantage that unbound ligand need not be separated from that which is cell-associated, thus avoiding perturbation of the binding equilibrium, and accurate, extensive statistical analysis is possible. Binding of fluorescein-NGF was mainly specific and saturable, with analysis by three methods of data treatment indicating a Kd of 0.8 to 3 nM at 4 degrees C. Time-based data acquisition allowed a continuous time course for binding to be generated. Binding reached a steady-state level within 5 min of exposure of the cells to the ligand. Kinetic and steady-state results obtained using fluorescein-NGF agree well with previous data produced by 125I-NGF binding studies. The main limitation of the flow cytometric method in the NGF system is the relative lack of sensitivity compared to the binding of radiolabeled NGF, partially due to unusual quenching of the fluorophore bound to NGF.

• Lindsay RM
• Neurotrophins and receptors.
• Prog Brain Res. 1994; 103: 3-14

• Shih A, Laramee GR, Schmelzer CH, Burton LE, Winslow JW
• Mutagenesis identifies amino-terminal residues of nerve growth factor necessary for Trk receptor binding and biological activity.
• J Biol Chem. 1994; 269: 27679-86
• Display abstract

• Limited proteolysis and site-directed mutagenesis of human nerve growth factor (hNGF) was utilized to determine the role of the NH2 terminus in p140TrkA (TrkA) receptor function. Purified (6-118)hNGF, representing deletion of the first 5 NH2-terminal residues, is 9-fold less potent than (1-118)hNGF in displacing 125I-hNGF from TrkA. The deletion of a further 4 residues to form (10-118)hNGF is nearly 300-fold less potent. (6-118)hNGF is only 2-4-fold less potent than (1-118)hNGF in eliciting TrkA autophosphorylation and PC12 cell neurite outgrowth, suggesting that the first 5 residues of the NH2 terminus are necessary for full TrkA binding activity but may be less critical for potent receptor activation and intracellular signaling. In contrast, purified mutant H4D results in a nearly 1000-fold loss in TrkA binding at 4 degrees C relative to (1-118)hNGF, at least 10-fold less potency of TrkA autophosphorylation, and 30-fold loss of potency in PC12 cell differentiation. NH2-terminal hBDNF/hNGF and hNT3/hNGF chimeric mutants further affected these activities suggesting that the NH2-terminal sequence specificity of hNGF contributes to structural interactions necessary for TrkA receptor binding and ligand-induced signaling. The potency of binding of all hNGF variants to the low affinity NGF receptor p75 was largely unaffected indicating distinct structural contributions of the NH2-terminal region of hNGF to the binding to TrkA versus p75.

• Hantzopoulos PA, Suri C, Glass DJ, Goldfarb MP, Yancopoulos GD
• The low affinity NGF receptor, p75, can collaborate with each of the Trks to potentiate functional responses to the neurotrophins.
• Neuron. 1994; 13: 187-201
• Display abstract

• NGF and the other neurotrophins all bind to the low affinity NGF receptor (LNGFR). Although early studies suggested that the LNGFR was absolutely required for the formation of a functional neurotrophin receptor, current evidence indicates that the Trk family of receptor tyrosine kinases, in the absence of the LNGFR, can directly bind to and mediate responses to the neurotrophins. Here we describe a functional approach, in fibroblasts, designed to assay for the ability of the LNGFR to potentiate Trk-mediated responses to the neurotrophins. We report that although collaboration between the LNGFR and the Trks could be detected in this system, a truncated form of the LNGFR displayed a much more dramatic ability to interact functionally with each of the Trks, potentiating masked autocrine loops as well as responses to limiting amounts of exogenously provided neurotrophins.

• Ikeda T, Puro DG
• Nerve growth factor: a mitogenic signal for retinal Muller glial cells.
• Brain Res. 1994; 649: 260-4
• Display abstract

• Knowledge of the effects of nerve growth factor (NGF) on glia is limited. A CNS site where NGF-glial interactions may occur is the retina. NGF is endogenous to the retina, and the retinal Muller glial cells have NGF receptors. Here, we examined the possibility that NGF may be a mitogen for Muller glial cells, which often proliferate in response to pathophysiological conditions. Experiments were performed on cultured glial cells from the adult human retina. Exposure of cultured Muller glial cells to 2.5 S NGF under serum-free conditions resulted in a concentration-dependent increase in cell number and bromodeoxyuridine incorporation into nuclei. The half-maximally effective concentration was 0.04 ng/ml (1.5 pM), consistent with activation of high affinity NGF receptors. K252a, a blocker of the neurotrophin family of tyrosine kinase-linked receptors, potently inhibited the proliferative effect of NGF. Transforming growth factor beta-2, another growth factor endogenous to the retina, inhibited the mitogenic response to NGF. These findings indicate that human Muller glial cells in culture express functional NGF receptors and that the response of Muller cells to NGF can be modulated by other growth factors.

• Fahnestock M
• The role of kallikreins in growth factor processing: the kallikrein gene family from the African rodent Mastomys.
• Braz J Med Biol Res. 1994; 27: 1885-90
• Display abstract

• 1. Kallikreins are trypsin-like serine proteases which have been implicated in the biosynthesis of a number of growth factors and hormones. Mastomys, an African rodent, is the only mammal other than mouse known to contain high levels of kallikreins and growth factors in its submandibular gland. Mastomys has a large kallikrein family similar to mouse and rat. Cloning and sequencing of several of these cDNAs demonstrates a high degree of homology with mouse and rat kallikreins, but careful analysis suggests that it will not be possible to determine the function of these kallikreins by sequence information alone. 2. It is not known whether a Mastomys kallikrein processes and binds to nerve growth factor (NGF) as in mouse submandibular gland. Mastomys NGF is bound to a protein similar in size, charge, and lack of esterase activity to alpha-NGF, a mouse kallikrein. However, the Mastomys NGF complex does not contain a proteolytically active kallikrein as does the mouse NGF complex. Thus, the identification of kallikreins as growth factor processing enzymes and the presence of a kallikrein family member in high-molecular-weight complexes in species other than mouse is still in question.

• Oderfeld-Nowak B, Bacia A
• Expression of astroglial nerve growth factor in damaged brain.
• Acta Neurobiol Exp (Warsz). 1994; 54: 73-80
• Display abstract

• This article provides an overview of different brain insults leading to induction of astroglial NGF expression. It discusses mechanisms of induction of astroglial NGF in a damaged brain and a possible role of its expression in pathophysiology of Alzheimer's disease. It emphasizes that the pharmacological manipulation of astroglial production of NGF may serve as a possible therapeutic strategy to ameliorate the pathological changes occurring after brain damage.

• Santambrogio L et al.
• Nerve growth factor production by lymphocytes.
• J Immunol. 1994; 153: 4488-95
• Display abstract

• Nerve growth factor (NGF) is a neurotrophic protein essential for the maintenance and growth of peripheral sympathetic neurons and basal forebrain cholinergic neurons. Recently, NGF has also been shown to have effects on cells of the immune system. In a search for extra neural sources of NGF, we detected NGF-specific mRNA in mouse T lymphocytes of both the CD4+ and CD8+ phenotypes with the use of an RNase protection assay, PCR, and DNA sequence analysis. In CD4+ cells, NGF was present in both Th1 and Th2 Ag-specific clones, but an increase of NGF-specific message was detected after antigenic stimulation only in Th2 clones. NGF mRNA was also detected in splenic B lymphocytes and in a cell line derived from a murine follicular center cell lymphoma. Translation into protein and secretion of NGF were demonstrated by Western blot analysis. The secreted NGF is in an active form capable of inducing differentiation of PC12 cells into sympathetic-like neurons. Furthermore, conditioned medium from clones or lines positive for NGF mRNA was capable of inducing p140 tyrosine kinase autophosphorylation in 3T3 fibroblasts transfected with cDNA encoding for the tyrosine kinase family NGF receptor. We conclude that lymphocytes synthesize and secrete NGF either as a para-autocrine factor acting on the immune system itself, or as a factor for the maintenance of peripheral neurons.

• Ilag LL, Lonnerberg P, Persson H, Ibanez CF
• Role of variable beta-hairpin loop in determining biological specificities in neurotrophin family.
• J Biol Chem. 1994; 269: 19941-6
• Display abstract

• The neurotrophins are members of a family of structurally and functionally related neurotrophic factors that control the development and maintenance of vertebrate neurons. The crystal structure of nerve growth factor (NGF), the prototypic member of this family, contains three pairs of anti-parallel beta-strands connected by beta-hairpin loops, which contain most of the variable residues among the four neurotrophin proteins. Recently, amino acid residues in these variable loop regions have been implicated in the interaction between NGF and its signal-transducing receptor TrkA. In NGF, residues 40-49 (variable region II) span a very flexible and solvent-accessible beta-hairpin loop that is highly variable between different neurotrophins. To investigate the role of this domain in determining biological specificities in the neurotrophin family, we constructed a series of chimeric molecules by exchanging this variable region among three neurotrophins (i.e. NGF, neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4)) that bind and activate three different Trk receptors (i.e. TrkA, -C, and -B, respectively). The chimeric molecules were analyzed for their ability to activate different Trk receptor subtypes and to promote the survival of subpopulations of peripheral neurons expressing specific types of Trk mRNAs. Exchange of the 40-49 variable loop region between NGF and NT-3 resulted in molecules capable of activating both TrkA and TrkC receptors and of rescuing neurons containing TrkA and TrkC mRNAs, indicating that this loop plays an important role in determining biological specificities in these two neurotrophins. Furthermore, variable region II from NT-4 conferred the ability to differentiate nnr5 PC12-TrkB cells to a chimeric NT-3 molecule that was originally incapable of eliciting a response in these cells. In contrast, exchanges between NGF and NT-4 did not suffice to generate molecules with broader biological specificities, suggesting that other regions in these molecules are also required. Our results support the evolutionary relationships between the three polypeptides deduced from structural comparisons.

• Blaber M, Isackson PJ, Holden HM, Bradshaw RA
• Synthetic chimeras of mouse growth factor-associated glandular kallikreins. II. Growth factor binding properties.
• Protein Sci. 1993; 2: 1220-8
• Display abstract

• Six chimeric constructs of the sequentially similar growth factor-associated kallikreins-epidermal growth factor binding protein (EGF-BP) and the gamma-subunit of nerve growth factor (gamma-NGF)--have been expressed, and their ability to generate complexes with epidermal growth factor (EGF) and beta-NGF, analogous to the high molecular weight forms (7S NGF and HMW-EGF) found in the mouse submaxillary gland, evaluated. The chimeras are distinguished by the interchange of three regions composing the amino, middle, and carboxyl terminal regions that encompass four surface loops possibly involved in specific growth factor interactions. Native beta-NGF (along with native alpha-NGF) formed complexes indistinguishable from naturally occurring 7S NGF, characterized by an alpha 2 beta gamma 2 structure (where beta-NGF is itself a dimer), with recombinant (r) gamma-NGF and with a chimera in which the amino terminal region from EGF-BP was substituted. Two other chimeras containing either the middle or carboxyl terminal regions of gamma-NGF showed weaker ability to form 7S complexes. Thus, all chimeras containing two segments from gamma-NGF retained at least some ability to form the 7S complex. rEGF-BP reacted weakly with EGF, but the chimera composed of the amino and middle segments of EGF-BP and the carboxyl terminal segment of gamma-NGF formed a nativelike HMW-EGF complex. None of the other chimeras appeared to bind EGF. These results identify amino acid positions within each kallikrein that participate in strong growth factor interactions and demonstrate that, outside of active site contacts, different regions of the kallikreins are involved in the binding of EGF and beta-NGF, respectively.

• Aloe L et al.
• Level of nerve growth factor and distribution of mast cells in the synovium of tumour necrosis factor transgenic arthritic mice.
• Int J Tissue React. 1993; 15: 139-43
• Display abstract

• We have recently reported that nerve growth factor (NGF) increases in the synovium of patients affected by arthritis, as well as in animal models. We report here that the synovium of transgenic arthritic mice expressing human tumour necrosis factor (TNF) contains numerous mast cells (MC) and that their appearance is a phenomenon which was correlated to the local increase in NGF level. These findings provide further evidence that NGF plays a role in inflammation and suggest a functional link between NGF and MC.

• Li C, Chen J, Wang R, Cheng H, Lin J, Yang L
• [PCR amplification of nerve growth factor (beta-NGF) gene in human brain]
• Hua Xi Yi Ke Da Xue Xue Bao. 1993; 24: 353-5
• Display abstract

• Nerve growth factor, an important nutrition factor for neuronal survival, growth, development and maintenance of neuronal functions, consists of three kinds of subunit (alpha 2, beta and gamma 2), but only the subunit beta-NGF has the biological activity. beta-NGF coding sequence is located in a single exon of NGF gene. In our study, a pair of specific primers (29 mers) has been designed and synthesized with ABI 318 A DNA synthesizer. The upstream primer includes an initiation codon and the 1st to 20th base of the exon. The downstream primer includes the complementary sequence of 334 to 354 base of the exon and a stop codon. The full-length DNA fragment of beta-NGF gene has been successfully obtained from human brain cDNA library in lambda ZAPII/EcoRI and human brain genomic DNA respectively by using polymerase chain reaction. The reaction program consists of denaturing at 93 degrees C for 1 min, annealing at 55 degrees C for 1 min, and extending at 72 degrees C for 2 min. Both amplified products of 350 bp in length coincide with the exon coding sequence and are enough for the analysis of restriction mapping, DNA sequencing and construction of expression vector.

• Ruberti F, Bradbury A, Cattaneo A
• Cloning and expression of an anti-nerve growth factor (NGF) antibody for studies using the neuroantibody approach.
• Cell Mol Neurobiol. 1993; 13: 559-68
• Display abstract

• 1. The neuroantibody approach, based on the expression of selected monoclonal antibodies by cells of the nervous system, has recently been described (Cattaneo and Neuberger, 1987; Piccioli et al., 1991). In order to apply this experimental strategy to study the role of nerve growth factor (NGF) in the central nervous system (CNS), we exploited the monoclonal antibody (mAb), alpha D11, which neutralizes very efficiently the biological activity of NGF, both in vitro and in vivo (Cattaneo et al., 1988). 2. The alpha D11 antibody chains were cloned and expressed in COS cells as rat/human chimaeric proteins. The cloned antibody was shown to display all the properties of the parental alpha D11 antibody, including its ability to neutralize NGF biological activity. 3. This will allow us to engineer the expression of recombinant alpha D11 antibodies in the CNS, to study the role of NGF in the developing and adult nervous system. This approach can be extended to other neurotrophic factors for which neutralizing monoclonal antibodies are available.

• Ritter AM, Lewin GR, Mendell LM
• Regulation of myelinated nociceptor function by nerve growth factor in neonatal and adult rats.
• Brain Res Bull. 1993; 30: 245-9
• Display abstract

• The role of nerve growth factor (NGF) as a survival factor for sensory neurons during embryonic life has been well documented. Here we examine the actions of NGF or antisera against NGF (anti-NGF) on physiologically identified sensory neurons with myelinated axons later in life, after the dependence on NGF for survival ends. We find that the effects of NGF and anti-NGF are specific for sensory neurons which are nociceptors. Treatments were found to affect the biophysical properties, the development, or the physiological function of myelinated nociceptors. They also affect the animal's behavioral response to noxious stimulation, depending upon when the treatments were given: neonatally, from 2-5 weeks of age, or chronically, beginning at birth. Thus, we find that the actions of NGF are specific for nociceptors but that the function of this neurotrophic factor changes according to the developmental age of the animal.

• Hamburger V
• The history of the discovery of the nerve growth factor.
• J Neurobiol. 1993; 24: 893-7
• Display abstract

• The Nerve Growth Factor (NGF) is the progenitor of a family of growth factors which is still expanding. The history of its discovery is very colorful; it is a rare combination of scientific reasoning, intuition, fortuities, and good luck. In addition, I believe that the collaboration of three scientists with very different backgrounds contributed to the success: I had grown up in a laboratory of experimental embryology, Dr. Levi-Montalcini came from neurology, and Dr. Stanley Cohen was from biochemistry. The decision where to begin the history of a discovery is always arbitrary. I shall give my reasons why I begin this story with my wing bud extirpations on chick embryos and the analysis of the effects of the operation on the development of spinal nerve centers, published in 1934. Of course, I am aware of the fact that the analysis of neurogenesis had been pioneered by Dr. R. G. Harrison and his students at Yale University since the beginning of this century. It should be mentioned that their experiments had been done on amphibian embryos. My own interest in problems of neurogenesis dates back to my Ph.D. thesis in the Zoology Department of Professor H. Spemann at the University of Freiburg in (the Federal Republic of) Germany; it dealt with the influence of the nervous system on the development of limbs in frog embryos. After I had obtained some inconclusive results I did the crucial experiment of producing nerveless legs. I removed the lumbar part of the spinal cord and the spinal ganglia before the outgrowth of nerve fibers. The nerveless legs developed normally in every respect, but the muscles atrophied eventually.

• Lewin GR, Mendell LM
• Nerve growth factor and nociception.
• Trends Neurosci. 1993; 16: 353-9
• Display abstract

• Nerve growth factor (NGF) is thought of as a target-derived factor responsible for the survival and maintaining the phenotype of specific sets of peripheral and central neurons during development and maturation. Recently, using physiological techniques, we have shown that specific functional types of nociceptive sensory neurons require NGF, first for survival during development in utero and then for their normal phenotypic development (but not survival) in the early postnatal period. In adulthood, the physiological role of NGF changes dramatically and here it may serve as a link between inflammation and hyperalgesia. Despite apparent changes in NGF's mode of action as the animal matures, it always interacts specifically with nociceptive sensory neurons.

• Tuttle JB, Creedon DJ
• Pharmacology of nerve growth factor output by target cells.
• Ann N Y Acad Sci. 1993; 692: 273-6

• Guroff G
• Nerve growth factor as a neurotrophic agent.
• Ann N Y Acad Sci. 1993; 692: 51-9

• Di Marco E et al.
• Nerve growth factor binds to normal human keratinocytes through high and low affinity receptors and stimulates their growth by a novel autocrine loop.
• J Biol Chem. 1993; 268: 22838-46
• Display abstract

• Normal human keratinocytes synthesize and secrete biologically active nerve growth factor (NGF) in a growth regulated fashion (Di Marco, E., Marchisio, P. C., Bondanza, S., Franzi, A. T., Cancedda, R., and De Luca, M. (1991) J. Biol. Chem. 266, 21718-21722). Here we show that the same human keratinocytes bind NGF via low and high affinity receptors. In parallel with the course of NGF synthesis, the expression of low affinity NGF receptor (p75NGFr) decreases when a confluent, differentiated, and fully stratified epithelium is obtained. In skin sections, p75NGFr is present in basal keratinocytes and absent from suprabasal, terminally differentiated cells. The trkA protooncogene product (p140trkA), a component of the NGF receptor, is not expressed by keratinocytes. Instead, keratinocytes express a new member of the trk family (that we termed trkE), which generates 3.9-kilobase transcripts. Keratinocyte-derived NGF plays a key role in the autocrine epidermal cell proliferation. This has been proven by (i) direct effect of NGF on [3H]thymidine incorporation, (ii) inhibition of autocrine keratinocyte growth by monoclonal antibodies (alpha D11) inhibiting human NGF biological activity, and (iii) inhibition of autocrine keratinocyte proliferation by a trk-specific inhibitor, the natural alkaloid K252a. These data provide evidence that NGF, in addition to its effect as a survival and differentiation factor, is a potent regulator of cell proliferation, at least in human epithelial cells.

• Tuveri MA, Passiu G, Mathieu A, Aloe L
• Nerve growth factor and mast cell distribution in the skin of patients with systemic sclerosis.
• Clin Exp Rheumatol. 1993; 11: 319-22
• Display abstract

• Nerve growth factor (NGF), a well-characterized neurotrophic factor, induces an increase in the number of mast cells (MCs) in the peripheral tissues of developing rats, as well as histamine release in fully differentiated MCs. Since MCs increase in the dermis of patients with early systemic sclerosis (SSc), we examined the distribution of NGF in the skin of patients affected by SSc. Immunohistochemical studies showed that NGF distribution was more intense in the dermis of patients with SSc than in the skin of controls. A possible correlation between MCs and NGF in disease activity is hypothesized.

• Figueiredo BC, Otten U, Strauss S, Volk B, Maysinger D
• Effects of perinatal hypo- and hyperthyroidism on the levels of nerve growth factor and its low-affinity receptor in cerebellum.
• Brain Res Dev Brain Res. 1993; 72: 237-44
• Display abstract

• Deficits or excesses of thyroid hormones during critical periods of mammalian cerebellar development can lead to profound biochemical and morphological abnormalities in this system. The goal of this study was to investigate the effects of perinatal hypo- and hyperthyroidism on the ontogeny of nerve growth factor (NGF) and its low-affinity receptor (p75NGFR) in the rat cerebellum. The concentration of NGF and of p75NGFR immunoreactivity (IR) were measured, several days after birth, in cerebella of rats which had received propylthiouracil (PTU) or thyroxine. NGF concentration was markedly enhanced only on postnatal day 2 (P2) in hyperthyroid rats, whereas in hypothyroid (PTU-treated) rats NGF values were similar to age-matched controls. These observations suggest that thyroid hormone affects NGF synthesis during early periods of cerebellar development. In Purkinje cells of control animals, p75NGFR IR peaked at P10. In hypothyroid rats, the expression of p75NGFR was retarded, peaking at P15, whereas in hyperthyroid rats it was advanced, peaking at P8. The increased p75NGFR IR found in Purkinje cell bodies and the delayed disappearance of p75NGFR IR from the external granular layer of hypothyroid rats suggest different roles for thyroid hormone in the developing cerebellum. We conclude that p75NGFR and NGF are independently regulated by thyroid hormone during critical periods of cerebellar development. The effect of thyroid hormone deficiency on p75NGFR content in Purkinje cells may involve complex mechanisms such as impaired efficiency of axonal transport.

• Pousset F
• Cytokines and the brain. Bischenberg, October 11-15 1992.
• Eur Cytokine Netw. 1993; 4: 57-61
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• The congress on cytokines, held in Bischenberg (October 11-15, 1992), was marked by the quality of the presentations and a limited number of participants which helped to foster communication. Recent data on biological activities of cytokine and cytokine receptor interactions of the oldest members of this family were reported as well as the mechanisms of inhibition of their activities. Other reports highlighted the growing number of cytokines identified in the brain, their central distribution, and the relationship between peripheral and central compartments of cytokines. But this meeting was also marked by a number of reports, based on different experimental models, that the biological activities of cytokines were of physiological relevance in fever, neuropathologies and behaviour. Interesting discussions raised fundamental questions about whether cytokines could be considered as neurotrophic factors, the role of the blood brain barrier and the signals regulating transmission between the immune and nervous systems. The implication of cytokines in central functions reinforces the concept of a central immune response encompassing soluble and cellular interactions between cerebral cells themselves and between cerebral and immune cells.

• Raffioni S, Bradshaw RA, Buxser SE
• The receptors for nerve growth factor and other neurotrophins.
• Annu Rev Biochem. 1993; 62: 823-50

• Drinkwater CC, Barker PA, Suter U, Shooter EM
• The carboxyl terminus of nerve growth factor is required for biological activity.
• J Biol Chem. 1993; 268: 23202-7
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• A series of mouse nerve growth factor (NGF) deletion mutants have been constructed using in vitro mutagenesis to define domains of the protein essential for its activity. Deletions of the amino or carboxyl termini of mature NGF or of an internal domain, which normally produces a surface-exposed reverse turn, have been analyzed. Mutants with deletions in the amino terminus or in the reverse turn retain significant biological activity, whereas, in contrast, a mutant NGF lacking the seven most carboxyl-terminal amino acids is appropriately synthesized but shows no measurable biological activity. These results suggests that the flexible carboxyl tail of NGF, and perhaps other neurotrophins, plays a crucial role in mediating receptor recognition and/or ligand binding.

• Fahnestock M, Lu Q
• Nerve growth factor synthesis by mouse submandibular gland cells in culture.
• Brain Res. 1993; 621: 339-42
• Display abstract

• Mouse submandibular gland (SMG) cells in culture rarely retain functional properties of SMG cells in vivo. We demonstrate that both primary SMG cells and the mouse SMG cell line SCA-9 secrete biologically active nerve growth factor (NGF). However, primary cells secrete 40-fold more NGF than SCA-9 cells, demonstrating that SCA-9 cells cannot substitute for primary SMG cells for the study of SMG NGF in vitro.

• Fabian RH, Hulsebosch CE
• Plasma nerve growth factor access to the postnatal central nervous system.
• Brain Res. 1993; 611: 46-52
• Display abstract

• The accessibility of macromolecules to the postnatal mammalian central nervous system is a subject of controversy and importance. One of the biologically important macromolecules in neural development, nerve growth factor (NGF), is thought to be unable to cross the blood-brain barrier in postnatal mammals. In this study, I125 labeled 7S NGF was systemically administered to postnatal rats and was found to accumulate in the brain in significant levels. In addition, brains from saline perfused rats contained intact 7S NGF as determined by gel autoradiography. Radiolabeled NGF was found to be localized to specific areas corresponding to p75 NGF receptor distribution in the hippocampus and cortex by autoradiography. Circulating plasma NGF has access to the developing postnatal neuraxis where it may exert a humoral trophic function.

• Luppi P et al.
• NGF is released into plasma during human pregnancy: an oxytocin-mediated response?
• Neuroreport. 1993; 4: 1063-5
• Display abstract

• The presence of biologically active nerve growth factor (NGF) in the peripheral circulation of women during pregnancy, labour and lactation was investigated. Using a sensitive immunoenzymatic assay (ELISA), we found an approximately five-fold increase in plasma NGF levels during labour and lactation compared with the concentrations found at the term of gestation or in control healthy women. Since labour and lactation are characterized by activation of the hypothalamo-pituitary-adrenal axis and by high plasma levels of the neurohypophyseal hormone oxytocin, and since the intravenous injection of oxytocin in female rats causes a 176% increase in the hypothalamic levels of NGF, it is possible that the increased amount of circulating NGF is correlated with one or both of these events.

• Yamaguchi K, Sasano A, Urakami T, Tsuji T, Kondo K
• Stimulation of nerve growth factor production by pyrroloquinoline quinone and its derivatives in vitro and in vivo.
• Biosci Biotechnol Biochem. 1993; 57: 1231-3
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• Pyrroloquinoline quinone (PQQ), which is a cofactor of microbial quinoprotein enzymes, was found to be a potent enhancer of nerve growth factor (NGF) production in vitro. One of PQQ derivatives, oxazopyrroloquinoline trimethylester, had little activity in vitro, but increased the NGF content in rat brain in vivo.

• Yamaguchi K et al.
• Stimulation of nerve growth factor synthesis and secretion by fellutamide A in vitro.
• Biosci Biotechnol Biochem. 1993; 57: 195-9
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• Fellutamide A, a tripeptide derivative from Penicillium fellutanum was found to be a potent enhancer of nerve growth factor (NGF) synthesis and secretion in vitro. This compound enhanced production of NGF in L-M cells, rat brain cells, and rat glial cells. The mode of action of this compound was suggested to be different from that of a known NGF inducer, epinephrine, as inducing activities of fellutamide A and epinephrine were additive when they were admixed at the concentration which gave saturation in its respective activity. Furthermore, fellutamide A but not epinephrine induced production of NGF in the rat brain cells. NGF-inducing activity of several tripeptides and other compounds related to fellutamide A was examined.

• Conner JM, Varon S
• Nerve growth factor immunoreactivity in the anterior pituitary of the rat.
• Neuroreport. 1993; 4: 395-8
• Display abstract

• Nerve growth factor (NGF) has long been recognized for its neurotrophic actions upon sensory and sympathetic neurons of the PNS and upon cholinergic neurons of the CNS basal forebrain. Much evidence, however, suggests that potential reciprocal interactions between NGF and various endocrine systems may also occur. In the present investigation we used immunohistochemical techniques to investigate whether NGF is found within the adult rat pituitary, thereby suggesting a possible interaction within this critical neuroendocrine gland. Our results indicate that NGF immunoreactivity is present only within the anterior lobe of the male and female rat pituitary and is exclusively localized within cells immunoreactive for thyroid stimulating hormone (TSH).

• Murase K, Hattori A, Kohno M, Hayashi K
• Stimulation of nerve growth factor synthesis/secretion in mouse astroglial cells by coenzymes.
• Biochem Mol Biol Int. 1993; 30: 615-21
• Display abstract

• We examined the effect of coenzymes such as PQQ, pyrroloquinoline quinone; TOPA, 3-(2,4,5-trihydroxyphenyl)-DL-alanine, and lipoic acid on nerve growth factor (NGF) synthesis in mouse astroglial cells, BALB c/3T3 cells, and WS-1 cells in culture. These coenzymes had a stimulating effect on NGF synthesis without causing cytotoxicity. Especially PQQ showed the strongest activity of promoting NGF synthesis in astroglial cells, whereas lipoic acid had the strongest effect on BALB c/3T3 cells. The activity may not due to the catechol ring or 1,4-benzoquinone ring, but due to the oxidative or reductive activity. These results suggest that these coenzymes may play a role in NGF synthesis and neuronal survival through the stimulating effect of the NGF synthesis in brain and such compounds are good candidates as NGF inducers.

• Gorbunova NB, Kaliunov VN
• [Measurement of the level of nerve growth factor in murine blood serum]
• Vopr Med Khim. 1993; 39: 47-50
• Display abstract

• Different values of nerve growth factor (NGF) concentrations, estimated in mice blood, were obtained as a result of the following reasons: 1) use of various procedures for estimation, 2) interaction of beta-NGF with high molecular blood serum proteins and 3) presence of a variety of the nerve growth proteins in mice blood: from complex to subunit forms of the protein including intermediate associations.

• Blaber M, Isackson PJ, Burnier JP, Marsters JC Jr, Bradshaw RA
• Synthetic chimeras of mouse growth factor-associated glandular kallikreins. I. Kinetic properties.
• Protein Sci. 1993; 2: 1210-9
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• A series of six chimeric proteins, composed of fragments corresponding to either one or the other of the growth factor-associated mouse glandular kallikreins-epidermal growth factor binding protein (EGF-BP) and the gamma-subunit of nerve growth factor (gamma-NGF)--were expressed in Escherichia coli and isolated, and their kinetic properties were characterized. The assembly of these synthetic proteases involved the substitution of regions of the proteins containing four specific surface loops that have been postulated to influence both kinetic specificity and the formation of growth factor complexes. The substrates utilized in the kinetic characterization of these chimeric kallikreins were tripeptide nitroanilides representing carboxyl termini of both the EGF and beta-NGF mature hormones, putative processing sites for these kallikreins in the precursors. Characterization of these hybrid enzymes demonstrates that Km and kcat kinetic constants may be independently affected by the regions utilized in construction of these chimeric kallikreins. Specifically, loop 1, located in the amino terminal region (Bode, W., et al., J. Mol. Biol. 164, 237-282, 1983), in gamma-NGF enhanced the kcat for substrates containing threonine in the P2 position, as is the case during the processing of the carboxy terminus of the beta-NGF precursor. Also, the central regions of the kallikreins containing loop 2 and the kallikrein loop dictated the generally inverted Km and kcat kinetic constants observed between EGF-BP and gamma-NGF. Finally, in gamma-NGF the autolysis loop, found in the carboxyl terminal region, functions to lower the Km kinetic constant for a variety of substrates. The results allow previously characterized kinetic differences between EGF-BP and gamma-NGF to be interpreted in terms of specific regions of the proteins and identify a subset of amino acid positions responsible for these functional characteristics.

• Bax B, Blaber M, Ferguson G, Sternberg MJ, Walls PH
• Prediction of the three-dimensional structures of the nerve growth factor and epidermal growth factor binding proteins (kallikreins) and an hypothetical structure of the high molecular weight complex of epidermal growth factor with its binding protein.
• Protein Sci. 1993; 2: 1229-41
• Display abstract

• We have predicted the three-dimensional structures of the serine protease subunits (gamma-NGF, alpha-NGF, and EGF-BP) of the high molecular weight complexes of nerve growth factor (7S NGF) and epidermal growth factor (HMW-EGF) from the mouse submandibular gland (from the X-ray crystal structures of two related glandular kallikreins). The conformations of three of the six loops surrounding the active site are relatively well defined in the models of gamma-NGF and EGF-BP, but three other loops are likely to have flexible conformations. Although the amino acid sequence of alpha-NGF is closely related to those of gamma-NGF and EGF-BP, it is catalytically inactive. Model-building studies on alpha-NGF suggested that mutations (in alpha-NGF) just prior to the active site serine (195) and an unusual N-terminal sequence are consistent with alpha-NGF having a zymogen-like conformation (similar to that in chymotrypsinogen). An hypothetical model of the quaternary structure of HMW-EGF has been constructed using this model of EGF-BP and the NMR structure of murine EGF. The C-terminal arm of EGF was modeled into the active site of EGF-BP based on data indicating that the C-terminal arginine of EGF occupies the S1 subsite of EGF-BP. Data suggesting one of the surface loops of EGF-BP is buried in the HMW-EGF complex and symmetry constraints were important in deriving a schematic model. A molecular docking program was used to fit EGF to EGF-BP.

• Hefti F, Lapchak PA
• Pharmacology of nerve growth factor in the brain.
• Adv Pharmacol. 1993; 24: 239-73

• Katoh-Semba R, Semba R, Kashiwamata S, Kato K
• An acceleration of age-related increases in levels of the beta-subunit of nerve growth factor in selected tissues from senescence-accelerated mice (SAM-P/8).
• J Mol Neurosci. 1993; 4: 107-15
• Display abstract

• An investigation was made of age-related changes in levels of the beta-subunit of nerve growth factor (beta-NGF) in selected tissues and of testosterone in serum in senescence-accelerated mice (SAM-P/8) and in the control mice (senesence-resistant mice; SAM-R/1). The concentrations of testosterone in serum were higher in SAM-P/8 than in SAM-R/1 at ages 2 and 4 mo. The level of beta-NGF in the thymus from SAM-R/1 increased with age, resulting in a statistically significant difference in its level between mice at ages 2 and 12 mo. By contrast, there was a transient increase in SAM-P/8 at around age 4 mo with a subsequent decrease. Consequently, significant differences were apparent in levels of beta-NGF between the two types of mouse at ages 2 and 4 mo. Similar results were obtained in the adrenal gland and testis. Compared to SAM-R/1 at age 2 mo, the average concentrations of beta-NGF in the hypophysis were higher in SAM-R/1 at ages 4 and 8 mo and in SAM-P/8 at all ages. In other tissues tested, no remarkable differences were detected. Our present results indicate that, in SAM-P/8, the elevation in levels of beta-NGF in the thymus, adrenal gland, testis, and hypophysis occurs in the early period of life compared to the control mice. Possible dysfunction of the disorder of hypophysis is discussed.

• Maysinger D, Piccardo P, Filipovic-Grcic J, Cuello AC
• Microencapsulation of genetically engineered fibroblasts secreting nerve growth factor.
• Neurochem Int. 1993; 23: 123-9
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• We demonstrated that genetically modified fibroblasts can be encapsulated into biocompatible, biodegradable spheres retaining their viability and capacity to continuously secrete nerve growth factor (NGF) for at least two months. Genetically engineered rat fibroblasts producing NGF were encapsulated in an alginate-polylysine gel with the ultimate objective of improving transplantation methodologies. Cultures were suspended in a sodium alginate solution and the suspension was extruded drop-wise into a solution of calcium chloride. Morphological properties of the spheres were assessed by light and electron microscopy. The spheres had a homogenous external membrane, without fibroblasts, protruding from the surface of the capsular membrane. The NGF determinations in culture media showed that encapsulated fibroblasts continued to synthesize NGF for at least 60 days. We also confirmed that secreted NGF was biologically active, by assessing the induction of choline acetyltransferase (ChAT) activity in dissociated embryonic rat septal cultures. These results encourage further studies using in vivo models to determine the value of applying microencapsulated genetically modified cells secreting trophic factors as a therapeutic strategy for central nervous system (CNS) injuries.

• Neveu I, Jehan F, Jandrot-Perrus M, Wion D, Brachet P
• Enhancement of the synthesis and secretion of nerve growth factor in primary cultures of glial cells by proteases: a possible involvement of thrombin.
• J Neurochem. 1993; 60: 858-67
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• Newborn rat brain astrocytes cultured in vitro in a chemically defined medium are shown to secrete enhanced levels of nerve growth factor (NGF) when they are exposed to various types of proteases. Proteolytic enzymes such as alpha-thrombin or collagenase induce a continuous, dose-dependent enhancement of the levels of cell-secreted NGF. Incubation of astrocytes for a 24-h period with 300 ng/ml of alpha-thrombin (approximately 9 nM, or 1 U/ml) results in an increase of the levels of cell-secreted NGF by a factor of three- to fourfold, and at doses 10 times higher, stimulation by a factor of up to four- to fivefold was observed. This phenomenon reflects an enhancement of the cellular pool of NGF mRNA, already noticeable after 3 h of treatment, which is preceded by a temporary activation of protooncogenes encoding transcription factors of the AP-1 family, such as c-fos, c-jun or junB. Trypsin, plasmin, alpha-chymotrypsin, or elastase also enhanced, to different extents, the levels of cell-secreted NGF. However, unlike alpha-thrombin or collagenase, these enzymes cause, above a critical concentration, an extensive cell detachment from the solid support, and this is accompanied by a decrease of their activity on the production of NGF, so that their dose-response curves are bell shaped. Stimulation was maximal at those concentrations that cause a limited loosening of the cell-substratum interactions, as evidenced by a retraction of some cell processes after 24 h of treatment. Studies of the effect of alpha-thrombin indicate that the proteolytic activity itself is required to enhance the production of NGF by astrocytes. Inactivation of alpha-thrombin with D-phenyl-alanyl-L-propyl-L-arginine chloromethyl ketone, phenylmethylsulfonyl fluoride, antithrombin III, or hirudin results in a marked decrease of the stimulatory effect. Furthermore, the prolonged presence of alpha-thrombin is required to elicit a maximal effect on the levels of extracellular NGF, which was observed after 48 h of treatment. It is known that some effects of alpha-thrombin require binding to the cell surface. We found that gamma-thrombin, which still has some proteolytic activity but has lost its ability to bind to the cell surface, is almost as potent as alpha-thrombin in promoting the release of NGF. It is concluded that the effect of thrombin on NGF synthesis is essentially mediated by its proteolytic activity.(ABSTRACT TRUNCATED AT 400 WORDS)

• Alleva E, Aloe L, Bigi S
• An updated role for nerve growth factor in neurobehavioural regulation of adult vertebrates.
• Rev Neurosci. 1993; 4: 41-62
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• Increasing attention has been focused on the role(s) of nerve growth factor (NGF) in neurobehavioural regulations of adult vertebrates. This interest springs from the emerging evidence that NGF is a "regulator" of physiological processes belonging to the three main homeostatic systems: the nervous, immune and endocrine systems. In fact, the spectrum of action of the NGF molecule is not restricted to neuronal cell types (central basal forebrain; peripheral sensory and sympathetic neurons) but extends also to nonneuronal cells. In mice intermale aggressive behaviour enhances serum NGF levels and promotes its synthesis in some hypothalamic areas. Other types of social events are able to cause NGF release, particularly under stress conditions. The achievement of a social role (dominant vs subordinate) is due to a functional loop involving salivary NGF release-->enhanced production of adrenal hormones-->submissive behaviour-->NGF release. In humans, plasma platelet-derived growth factor (PDGF) increases following mental stress. The aim of this review is to give an updated survey on NGF roles in neurobehavioural regulations of adult animals.

• Mitsiadis TA, Couble P, Dicou E, Rudkin BB, Magloire H
• Patterns of nerve growth factor (NGF), proNGF, and p75 NGF receptor expression in the rat incisor: comparison with expression in the molar.
• Differentiation. 1993; 54: 161-75
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• Nerve growth factor (NGF), a target-derived neurotrophic substance, may have broader biological functions in various types of non-neuronal differentiating cells. The effects of NGF are dependent on initial binding of NGF to specific cell-surface receptors (p75NGFR and p140prototrk) on responsive cells. The continuously growing rat incisor offers an excellent model demonstrating defined territories of differentiation of specific cell populations. We used immunohistochemistry to determine sites of NGF, proNGF and p75NGFR accumulation in the rat incisor, whereas NGF mRNA expression was visualized by in situ hybridization in the developing rat molar and incisor. Strictly similar patterns of NGF mRNA, proNGF and NGF expression were observed in differentiating cells responsible for the production of the main structural matrices of the tooth. Thus, proNGF-like and NGF-like immunoreactivity, as well as the NGF mRNA signal were observed in preameloblasts and young ameloblasts of the dental epithelium and in polarizing odontoblasts of the dental mesenchyme. In contrast, the distribution of p75NGFR was correlated with differentiation event only in dental mesenchyme: polarizing odontoblasts expressed p75NGFR whereas the molecule was absent in functional odontoblasts. In dental epithelium, the restricted expression of p75NGFR in ameloblast precursor cells was correlated with proliferative phenomena. The patterns of proNGF, NGF and p75NGFR expression in epithelium and mesenchyme implicate both an autocrine and paracrine mode of action of the NGF molecule in dental tissues. The findings reported here are important for understanding NGF action in specific dental cell populations and suggest that this molecule is involved in the cascade of events that directs tooth development.

• Eto Y
• [Signal cascade of NGF and its application in the treatment of brain injury and neural tumor]
• Tanpakushitsu Kakusan Koso. 1993; 38: 1603-7

• Murakami A, Tatsuno T, Kimura T, Noguchi H, Nakamura M
• Regulation of nerve growth factor secretion in L-M cells by catechol derivatives.
• Neurosci Res. 1993; 17: 71-5
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• We investigated the mechanism responsible for the stimulation of nerve growth factor (NGF) secretion by catechol derivatives in L-M cells, using L-threo-3,4-dihydroxyphenylserine (L-DOPS). Treatment of the cells with L-DOPS increased the NGF content in the L-M cell medium by approximately 3-fold. This stimulatory effect was not blocked by a decarboxylase inhibitor, or by alpha- or beta-adrenergic blockers. Intracellular cAMP levels were not changed by exposure to L-DOPS. The antioxidants, ascorbic acid and sodium pyrosulfite, completely prevented the stimulatory effect of L-DOPS, and radical scavengers (superoxide dismutase plus catalase) caused a significant partial inhibition of the response to L-DOPS. Quinone derivatives (adrenochrome, 4-n-propyl-1,2-benzoquinone), which are the oxidative products of the catechol derivatives, increased the NGF content in the medium, and their potency was greater than that of the catechol derivatives themselves. These findings suggest that L-DOPS and other catechol derivatives might be oxidized in the medium to form quinone derivatives, and that it is these which predominantly express a stimulatory effect on NGF secretion by a novel cAMP-independent mechanism in L-M cells.

• Marchetti D, Menter D, Jin L, Nakajima M, Nicolson GL
• Nerve growth factor effects on human and mouse melanoma cell invasion and heparanase production.
• Int J Cancer. 1993; 55: 692-9
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• The role of growth factor networks in regulating the progression of human melanocytes towards tumorigenicity and ultimately the malignant phenotype is poorly understood. In particular, the autocrine and paracrine influences that modulate cellular invasion and extracellular matrix degradative enzymes of melanoma cells remain undefined at the molecular level. We report here that nerve growth factor (NGF) can modify some metastasis-associated cellular properties of human and mouse melanoma cells. Treatment of early-passage human metastatic melanoma cells (MeWo) or their variants (3S5, 70W) with biologically active 2.5S NGF resulted in (a) delayed density-dependent inhibition of melanoma cell growth; (b) increased in vitro invasion through a reconstituted basement membrane; and (c) time- and dose-dependent induction of heparanase, a heparan-sulfate-specific endo-beta-D-glucuronidase associated with human melanoma metastasis. These effects of NGF were most marked in the 70W brain-colonizing cells (70W > MeWo > 3S5). The NGF enhancement of heparanase secretion was not species-specific, since it was also observed in murine B16 melanoma cells; the highest NGF stimulation of heparanase was found in brain-colonizing murine B16-B15b variant (B16-B15b > B16-BL6, B16-F10, B16-F1). NGF also increased the invasive capacity of the human 70W and murine B16-B15b sublines in a chemoinvasion assay performed with filters coated with purified heparan sulfate proteoglycan (HSPG). The enhancement of chemotactic response and heparanase production was detected at NGF concentrations sufficient to fully saturate both low- and high-affinity NGF receptors (NGFR), the neurotrophin receptor (p75) and the trkA gene product, respectively. The results suggest that, in addition to the effects of NGF on cellular development and differentiation within the peripheral and central nervous systems, NGF can exert changes in the invasive properties of neuroectoderm-derived melanoma cells.

• Aloe L, Tuveri MA, Levi-Montalcini R
• Nerve growth factor and distribution of mast cells in the synovium of adult rats.
• Clin Exp Rheumatol. 1992; 10: 203-4

• Domenici L, Parisi V, Maffei L
• Exogenous supply of nerve growth factor prevents the effects of strabismus in the rat.
• Neuroscience. 1992; 51: 19-24
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• It has recently been reported that exogenous supply of nerve growth factor prevents the effects of monocular deprivation both in rats and in cats. Here we have extended these experiments to the case of strabismus. Repeated intraventricular injections of nerve growth factor were performed in rats made surgically strabismic early in the critical period. At the end of the critical period the ocular dominance distribution of visual cortical neurons was assessed in strabismic untreated, strabismic nerve growth factor-treated and strabismic Cytochrome C-treated (control) rats by means of extracellular recordings. We found that in rats surgical strabismus causes a consistent loss of binocular neurons. By contrast the treatment with nerve growth factor maintains the normal ocular dominance distribution of neurons in the primary visual cortex. We conclude that nerve growth factor exogenously supplied prevents the effects induced by surgical strabismus in rats and suggest that nerve growth factor has a role in visual cortical plasticity.

• Timm DE, Vissavajjhala P, Ross AH, Neet KE
• Spectroscopic and chemical studies of the interaction between nerve growth factor (NGF) and the extracellular domain of the low affinity NGF receptor.
• Protein Sci. 1992; 1: 1023-31
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• Nerve growth factor (NGF) interacts with a cell surface receptor on responsive neurons to initiate a series of cellular events leading to neuronal survival and/or differentiation. The first step in this process is the binding of NGF to a low affinity and/or a high affinity receptor. In the present report, we have studied the conformation and stability of recombinant receptor extracellular domain (RED) from the human low affinity receptor and the structural basis of its interaction with NGF. Circular dichroism (CD) studies indicate that the RED is primarily random coil in nature with little regular secondary structure. Thermal stability studies have shown that this irregular conformation is a specific structure that can undergo a reversible two-state thermal denaturation with a concomitant fluorescent and CD change. During heating at 100 degrees C for 15 min, the structure of RED is sufficiently unfolded for a reducing agent, dithiothreitol, to inactivate the receptor toward NGF binding and cross-linking. The complex formation between the RED and NGF has been examined by differential CD measurements, and we have shown that a small, reproducible change in conformation occurs in RED or NGF upon interaction. These results are interpreted in terms of the initiation of NGF cell surface binding and possible modes of signal transduction.

• Traverso S, Morchio R, Tamone G
• Neuronal growth and the Steiner problem.
• Riv Biol. 1992; 85: 405-18
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• In this paper we conjecture that neuronal networks develop following an optimality principle. We point out that a neuronal outgrowth in culture may be seen as the solution of a classical optimization problem: the "Steiner Problem". A neuron might grow minimizing a "cost", which may be determined by the viscoelastic properties of the neuron cytoplasm. We then discuss the role of chemiotactic factors such as the Nerve Growth Factor (NGF) in an optimized neuronal development in vivo. Finally we suggest, with some mathematical arguments, that the optimization of the elastic forces in the growing neuron may give rise to a "fractal" structure.

• Koyama J, Inoue S, Ikeda K, Hayashi K
• Purification and amino-acid sequence of a nerve growth factor from the venom of Vipera russelli russelli.
• Biochim Biophys Acta. 1992; 1160: 287-92
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• Nerve growth factor (NGF) was purified from the venom of Vipera russelli russelli by Sephadex G-50 gel filtration, S-Sepharose column chromatography and Blue-Sepharose CL-6B column chromatography. The purified NGF was found to be a glycoprotein, whose apparent molecular mass was estimated to be about 17.5 kDa by SDS-PAGE. The amino-acid sequence was determined by a combination of conventional methods. The V. r. russelli NGF was composed of 117 amino-acid residues with one residue, Asn-21, being N-linked glycosylated and the molecular mass of its protein portion was calculated to be 13,280 Da.

• Lewin-Kowalik J, Sieron AL, Barski JJ
• [Recent views on the role of nerve growth factor and other molecules with well documented neuronotrophic action]
• Postepy Hig Med Dosw. 1992; 46: 497-519
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• In the present paper recent data of the structure, regulation of production and mechanisms of action of the nerve growth factor and other proteins of well documented neurotrophic influence were reviewed. The purpose of this article was also to discuss the possible role of these molecules in the pathogenesis of some neurological diseases and their potential therapeutic importance.

• Kilmon J
• Snake venom: "gentler" purification provides attractive nerve growth factor source.
• Am Biotechnol Lab. 1992; 10: 1820-1820

• Schmelzer CH et al.
• Biochemical characterization of recombinant human nerve growth factor.
• J Neurochem. 1992; 59: 1675-83
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• Recombinant human nerve growth factor (rhNGF) was expressed and secreted by Chinese hamster ovary cells and purified to homogeneity using ion-exchange and reversed-phase (RP) chromatography. The isolated product was shown to be consistent with a 120-amino-acid residue polypeptide chain by amino acid composition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), RP-HPLC, and mass spectrometry and with an N-terminal sequence consistent with that expected from the cDNA for human nerve growth factor. By size-exclusion chromatography, rhNGF behaves like a noncovalent dimer. Limited enzymatic digests of the 120-residue monomer produced additional species of 118 (trypsin, removal of the C-terminal Arg119-Ala120 sequence) and 117 (trypsin plus carboxypeptidase B, removal of the C-terminal Arg118-Arg119-Ala120 sequence) residues. Each of these species was isolated by high-performance ion-exchange chromatography and characterized by amino acid and N-terminal sequence analyses, SDS-PAGE, RP-HPLC, and mass spectrometry. All three species were present in the digests as both homodimeric and heterodimeric combinations and found to be equipotent in both the chick dorsal root ganglion cell survival and rat pheochromocytoma neurite extension assays.

• Saltiel AR, Decker SJ
• Diversity in cellular signaling for nerve growth factor and insulin: variations on a common theme.
• J Invest Dermatol. 1992; 98: 1720-1720
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• Numerous similarities exist in the cellular signaling events observed for insulin and nerve growth factor. Because the two hormones share many functional properties, and exhibit similar effects on neurons, the possibility of common early signaling events has been explored. Many studies have focused on the important role of protein phosphorylation. Two distinct but related mechanisms are discussed that may mediate, in part, the ability of these two hormones to regulate the activities of protein kinases and phosphatases.

• Levi A, Alema S
• The mechanism of action of nerve growth factor.
• Annu Rev Pharmacol Toxicol. 1991; 31: 205-28

• McDonald NQ, Lapatto R, Murray-Rust J, Gunning J, Wlodawer A, Blundell TL
• New protein fold revealed by a 2.3-A resolution crystal structure of nerve growth factor.
• Nature. 1991; 354: 411-4
• Display abstract

• Nerve growth factor (NGF) is a member of an expanding family of neurotrophic factors (including brain-derived neurotrophic factor and the neurotrophins) that control the development and survival of certain neuronal populations both in the peripheral and in the central nervous systems. Its biological effects are mediated by a high-affinity ligand-receptor interaction and a tyrosine kinase signalling pathway. A potential use for NGF and its relatives in the treatment of neurological disorders such as Alzheimer's disease and Parkinson's disease requires an understanding of the structure-function relationships of NGF. NGF is a dimeric molecule, with 118 amino acids per protomer. We report the crystal structure of the murine NGF dimer at 2.3-A resolution, which reveals a novel protomer structure consisting of three antiparallel pairs of beta strands, together forming a flat surface. Two subunits associate through this surface, thus burying a total of 2,332 A. Four loop regions, which contain many of the variable residues observed between different NGF-related molecules, may determine the different receptor specificities. A clustering of positively charged side chains may provide a complementary interaction with the acidic low-affinity NGF receptor. The structure provides a model for rational design of analogues of NGF and its relatives and for testing the NGF-receptor recognition determinants critical for signal transduction.

• McDonald NQ, Blundell TL
• Crystallization and characterization of the high molecular weight form of nerve growth factor (7 S NGF).
• J Mol Biol. 1991; 219: 595-601
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• A high molecular weight form of nerve growth factor (7 S NGF) has been crystallized in two crystal forms from polyethylene glycol 4000 by the vapour diffusion technique. The orthorhombic form A belongs to the space group P2(1)2(1)2(1) and has cell dimensions of a = 95.6, b = 96.5 and c = 147.0 A. With synchrotron X-ray radiation, these crystals diffract to 2.8 A resolution. They contain an intact 7 S NGF complex in the asymmetric unit. The tetragonal form B, which grows at similar conditions to the A form, belongs to the space group P4(1)2(1)2 (or P4(3)2(1)2) with unit cell dimensions of a = 97.4, b = 97.4 and c = 308.3 A. These crystals diffract to 3.6 A resolution and contain one 7 S complex per asymmetric unit. Native X-ray data have been collected to 3.3 A for the A form and to 5.0 A for the B form, both using synchrotron radiation.

• Baraldi M, Zanoli P, Truzzi C, Paro M, Italiano G, Prosdocimi M
• Urine retention due to intra-spinal cord injection of colchicine in rats: improved recovery of bladder function by monosialoganglioside GM1 and nerve growth factor administration.
• Funct Neurol. 1991; 6: 235-8
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• Intra-spinal cord injection of a low dose of colchicine (2 micrograms/rat) at the lumbar level affects the micturition reflex leading to voiding suppression, bladder hypertrophy and overflow incontinence which lasts about four weeks. The administration of nerve growth factor and monosialoganglioside GM1 normalizes urine output within 3 days and improves recovery of the bladder contraction tested by a cystometric analysis.

• Carriero F, Campioni N, Cardinali B, Pierandrei-Amaldi P
• Structure and expression of the nerve growth factor gene in Xenopus oocytes and embryos.
• Mol Reprod Dev. 1991; 29: 313-22
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• A large part of the coding portion of the Xenopus nerve growth factor (NGF) gene has been identified and cloned by the use of a chicken cDNA probe and its sequence has been determined. Comparison of the derived amino acid sequence of mature Xenopus NGF with that of other species showed a high conservation, whereas comparison of the prepropeptide showed large divergent regions alternated with short conserved regions. Expression of the NGF gene was examined during development of oocytes and embryos. Surprisingly, NGF mRNA was found in the oocyte; it is present in small previtellogenic as well as in fully grown oocytes. NGF mRNA, passed to the embryo at fertilization, is degraded before the gastrula stage and starts accumulating again around the stage of the neurula. The association of NGF mRNA with polysomes is indicative of NGF synthesis during oogenesis. In fact, by using antibodies against mouse NGF it was possible to reveal NGF molecules present as precursors. These molecules accumulate during oogenesis and are maintained in the embryos up to the blastula stage; a very faint band corresponding to a smaller size peptide is sometimes detected. A maternal role for the NGF can be proposed, although a possible activity of NGF in the oocyte cannot be ruled out.

• Yan Q, Settle SL, Wilkins MR
• Hypotension induced by intravascular administration of nerve growth factor in the rat.
• Clin Sci (Colch). 1991; 80: 565-9
• Display abstract

• 1. The observation that the area postrema expresses a high level of nerve-growth-factor-receptor immunoreactivity prompted an investigation of the effects of nerve growth factor on autonomic function in the rat. 2. Bolus injection of pharmacological doses of the peptide via the femoral vein led to reproducible, dose-dependent falls in blood pressure. 3. Administration via the vertebral artery, a more direct route to the brainstem, did not appear to lower the threshold dose required to induce hypotension. Furthermore, pretreatment with hexamethonium did not inhibit the hypotensive response to nerve growth factor. 4. Administration of a second dose of nerve growth factor after recovery from the first injection produced little or no fall in blood pressure. Similarly, pretreatment with the mast cell degranulating agent, compound 48/80, rendered the animal refractory to nerve growth factor. 5. The fall in blood pressure induced by nerve growth factor was markedly attenuated by pretreatment with chlorpheniramine. 6. It is concluded that the fall in blood pressure induced by intravenous administration of pharmacological doses of nerve growth factor is mediated by vasoactive substances, particularly histamine, released from mast cells.

• Taiwo YO, Levine JD, Burch RM, Woo JE, Mobley WC
• Hyperalgesia induced in the rat by the amino-terminal octapeptide of nerve growth factor.
• Proc Natl Acad Sci U S A. 1991; 88: 5144-8
• Display abstract

• Nerve growth factor (NGF) in the mouse submandibular gland undergoes cleavage of its amino-terminal octapeptide when salivation is induced by epinephrine. The significance of this event is uncertain; cleaved NGF demonstrates bioactivity and no function has been attributed to the octapeptide produced (NGF-OP; Ser-Ser-Thr-His-Pro-Val-Phe-His). Enzyme inhibition studies indicating structural relatedness of NGF-OP and bradykinin (BK) prompted us to determine whether NGF-OP would elicit BK-like actions. We found that like BK, NGF-OP induced a decrease in mechanical nociceptive threshold (i.e., produced hyperalgesia) in the hairy skin of the rat. This effect was dose-dependent and sequence-specific; like BK it was attenuated by sympathectomy and indomethacin pretreatment. However, NGF-OP actions appeared to be distinct from those for BK in that tissue injury was required for NGF-OP to induce hyperalgesia. Furthermore, we found no evidence that NGF-OP bound to or activated BK receptors. Our data indicate that NGF-OP is a distinct mediator of hyperalgesia. We suggest that NGF-OP alters pain threshold in the injured target regions of NGF-responsive neurons.

• Fahnestock M
• Structure and biosynthesis of nerve growth factor.
• Curr Top Microbiol Immunol. 1991; 165: 1-26
• Display abstract

• Most of our knowledge about NGF comes from extensive study of the mouse submaxillary gland protein. NGF from this source is isolated as a high molecular weight complex consisting of beta-NGF and two subunits, alpha and gamma, belonging to the kallikrein family of serine proteases. There are few other tissues where NGF is found in sufficient quantities for protein purification and study, although new molecular biological techniques have accelerated the study of NGFs from a variety of species and tissues. Mouse submaxillary gland NGF is synthesized as a large precursor that is cleaved at both N- and C-terminals to produce mature NGF. This biologically active molecule can be further cleaved by submaxillary gland proteases. The roles of the alpha and gamma subunits in the processing of the beta-NGF precursor, the modulation of the biological activity of beta-NGF, and the protection of mature beta-NGF from degradation have been well studied in the mouse. However, the apparent lack of alpha and gamma subunits in most other tissues and species and the existence of a large family of murine kallikreins, many of which are expressed in the submaxillary gland, challenge the relevance of murine high molecular weight NGF as a proper model for NGF biosynthesis and regulation. It is important therefore to identify and characterize other NGF complexes and to study their subunit interactions, biosynthesis, processing, and regulation. This review points out a number of other species and tissues in which the study of NGF has just begun. At this time, there exist many more questions than answers regarding the presence and the functions of NGF processing and regulatory proteins. By studying NGF in other species and tissues and comparing the processing and regulation of NGF from several sources, we will discover the unifying concepts governing the expression of NGF biological activity.

• Ridgway RL, Syed NI, Lukowiak K, Bulloch AG
• Nerve growth factor (NGF) induces sprouting of specific neurons of the snail, Lymnaea stagnalis.
• J Neurobiol. 1991; 22: 377-90
• Display abstract

• Nerve growth factor (NGF) was examined for its ability to elicit sprouting by adult molluscan neurons. Motoneurons and interneurons (but not neurosecretory cells) from Lymnaea exhibited a sprouting response to murine 2.5S NGF in defined medium with a half-maximal response at about 150 ng/mL. Furthermore, an NGF antiserum blocked sprouting by all normally responsive neurons. We tested whether an NGF-like molecule is a component of conditioned medium (CM) by attempting to preabsorb its sprout-inducing activity with NGF antiserum. Treatment of CM with immune (but not nonimmune) serum largely blocked the response of motoneurons, but not that of neurosecretory cells, to CM. We conclude that NGF exerts neurotrophic activity on specific adult Lymnaea neurons, and suggest the possibility that an NGF-like molecule may exist in the molluscan nervous system.

• Barnett J et al.
• Physicochemical characterization of recombinant human nerve growth factor produced in insect cells with a baculovirus vector.
• J Neurochem. 1991; 57: 1052-61
• Display abstract

• Recombinant human nerve growth factor (rhNGF) secreted by insect cells was purified by ion-exchange and reversed-phase chromatography to near homogeneity. The N-terminus of the secreted molecule was analogous to that of mouse salivary gland NGF. In its native conformation, the insect cell produced rhNGF molecules were homodimers consisting of 120 amino acid polypeptide chains. Mature rhNGF was found not to be significantly glycosylated (less than 0.08 mol of N-acetylglucosamine/mol of protein). The rhNGF was homogeneous with regard to molecular weight and amino acid sequence. Isoelectric focusing resolved the rhNGF into one major and one minor component. Because rhNGF from insect cells can be obtained in large quantities, purified to near homogeneity, and is similar to natural NGF with regard to physicochemical properties and biological activity, it is suitable for further evaluation in animal models as a therapeutic molecule for neurodegenerative diseases such as Alzheimer's disease.

• Fahnestock M et al.
• beta-NGF-endopeptidase: structure and activity of a kallikrein encoded by the gene mGK-22.
• Biochemistry. 1991; 30: 3443-50
• Display abstract

• Mouse nerve growth factor (NGF) is cleaved at a histidine-methionine bond to release an NH2-terminal octapeptide (NGF1-8). The enzyme responsible, beta-NGF-endopeptidase, is structurally and functionally similar to gamma-NGF and epidermal growth factor-binding protein (EGF-BP) and cleaves mouse low molecular weight kininogen to produce bradykinin-like activity. These data have suggested that, like gamma-NGF and EGF-BP, beta-NGF-endopeptidase is a mouse glandular kallikrein. Evidence for a physiological role for NGF1-8 encouraged studies to further characterize the structure and function of this enzyme. Purified beta-NGF-endopeptidase migrated as a single band on isoelectric focusing and reducing SDS-polyacrylamide gels. As was expected, it removed NGF1-8 from NGF. Interestingly, enzymatic activity on an artificial substrate, and on NGF, was inhibited by NGF1-8 and by bradykinin. These studies further supported the view that beta-NGF-endopeptidase acts on both NGF and kininogen. The first 30 NH2-terminal amino acids of beta-NGF-endopeptidase were sequenced. This analysis demonstrated that the enzyme is encoded by the gene designated mGK-22 (Evans et al., 1987). The sequence of this gene corresponds to that of EGF-BP type A (Anundi et al., 1982; Drinkwater et al., 1987), and so studies were performed to determine whether or not beta-NGF-endopeptidase participates in EGF complex formation. Chromatographic and kinetic data gave no evidence that beta-NGF-endopeptidase is an EGF-binding protein. Our studies suggest that contamination of high molecular weight (HMW) EGF preparations with beta-NGF-endopeptidase erroneously led to earlier designation of the product of mGK-22 as an EGF-BP.(ABSTRACT TRUNCATED AT 250 WORDS)

• Bothwell M
• Tissue localization of nerve growth factor and nerve growth factor receptors.
• Curr Top Microbiol Immunol. 1991; 165: 55-70

• Ebendal T et al.
• Human nerve growth factor: biological and immunological activities, and clinical possibilities in neurodegenerative disease.
• Adv Exp Med Biol. 1991; 296: 207-25

• Perez-Polo JR et al.
• Nerve growth factor and neuronal cell death.
• Mol Neurobiol. 1990; 4: 57-91
• Display abstract

• The regulation of neuronal cell death by the neuronotrophic factor, nerve growth factor (NGF), has been described during neural development and following injury to the nervous system. Also, reduced NGF activity has been reported for the aged NGF-responsive neurons of the sympathetic nervous system and cholinergic regions of the central nervous system (CNS) in aged rodents and man. Although there is some knowledge of the molecular structure of the NGF and its receptor, less is known as to the mechanism of action of NGF. Here, a possible role for NGF in the regulation of oxidant--antioxidant balance is discussed as part of a molecular explanation for the known effects of NGF on neuronal survival during development, after injury, and in the aged CNS.

• Blaber M, Isackson PJ, Bradshaw RA
• The characterization of recombinant mouse glandular kallikreins from E. coli.
• Proteins. 1990; 7: 280-90
• Display abstract

• A system has been developed for the expression in E. coli of 12 of the 14 expressed mouse submandibular gland kallikreins as cassettes subcloned directly from cDNA. Using the epidermal growth factor binding protein (mGK-9) and the gamma-subunit of nerve growth factor (mGK-3), as test cases, mature processed forms, obtained as functionally active proteins, as well as various precursor forms, were isolated. The expression system described allows rapid isolation of kallikrein protein from corresponding cDNA with yields of approximately 1.0 mg of purified protein from 10 g of initial cell paste. This expression system will facilitate structure/function studies of the mouse glandular kallikrein gene family and help elucidate the regions of the mature proteins responsible for the diverse catalytic behavior and growth factor interactions observed in this family of proteins.

• Black IB, DiCicco-Bloom E, Dreyfus CF
• Nerve growth factor and the issue of mitosis in the nervous system.
• Curr Top Dev Biol. 1990; 24: 161-92

• Ebendal T, Olson L, Persson H
• [Nerve growth factor as a therapeutic agent: transplantation of brain tissue as a possible treatment of neurodegenerative disorders]
• Lakartidningen. 1990; 87: 1773-4
• Display abstract

• An account of experiments focused on the treatment of neurodegenerative disease by means of cell grafts, in which genetically modified cells that release abundant nerve growth factor are described, and their transplantation to the rat brain reported. The cells can survive in the rat brain and exert potent effects on cholinergic neurons both in the host cortex, basal forebrain and striatum, and in tissue grafts.

• Kaisho Y, Yoshimura K, Nakahama K
• Cloning and expression of a cDNA encoding a novel human neurotrophic factor.
• FEBS Lett. 1990; 266: 187-91
• Display abstract

• A cDNA encoding a novel human neurotrophic factor (designated nerve growth factor-2; NGF-2) was cloned from a human glioma cDNA library using a synthetic DNA corresponding to human nerve growth factor (NGF). The cloned cDNA encodes a polypeptide composed of 257 amino acid residues including a prepro-sequence of 138 residues and a mature region of 119 residues. The amino acid sequence of human NGF-2 exhibits 58% similarity with that of human NGF. Conditioned medium of COS-7 cells transfected with an expression plasmid for human NGF-2 cDNA supported the survival of sensory neurons isolated from dorsal root ganglia of embryonic chicks. A 1.5 kb of NGF-2 mRNA can be detected from an early development stage in rat brain, by Northern blotting analysis.

• Senut MC, Lamour Y, Brachet P, Dicou E
• Neuronal localization of the nerve growth factor precursor-like immunoreactivity in the cortex and hippocampus of the rat brain.
• Adv Exp Med Biol. 1990; 265: 133-9

• Alleva E, Calamandrei G
• Polypeptide growth factors in mammalian development: some issues for neurotoxicology and behavioral teratology.
• Neurotoxicology. 1990; 11: 293-303
• Display abstract

• Protein molecules known as Growth Factors (GFs) appear to play an important regulatory role in a number of CNS functions. In particular Nerve Growth Factor (NGF), the best characterized among about 30 GF molecules, is endowed with specific activities on cholinergic and peptidergic CNS neurons. Indexes of neurobehavioral maturation are accelerated in the mouse by neonatal NGF exposure, while a similar treatment with EGF exerts both growth-promoting and growth-inhibiting effects on somatic and behavioral development. Scopolamine hyperactivity is enhanced around weaning by NGF pretreatment. Data are discussed along with (a) NGF prevention of cholinergic system damage and (b) the molecular mechanism of endogenously-triggered CNS repair processes.

• Ibanez CF, Hallbook F, Ebendal T, Persson H
• Structure-function studies of nerve growth factor: functional importance of highly conserved amino acid residues.
• EMBO J. 1990; 9: 1477-83
• Display abstract

• Selected amino acid residues in chicken nerve growth factor (NGF) were replaced by site-directed mutagenesis. Mutated NGF sequences were transiently expressed in COS cells and the yield of NGF protein in conditioned medium was quantified by Western blotting. Binding of each mutant to NGF receptors on PC12 cells was evaluated in a competition assay. The biological activity was determined by measuring stimulation of neurite outgrowth from chick sympathetic ganglia. The residues homologous to the proposed receptor binding site of insulin (Ser18, Met19, Val21, Asp23) were substituted by Ala. Replacement of Ser18, Met19 and Asp23 did not affect NGF activity. Modification of Val21 notably reduced both receptor binding and biological activity, suggesting that this residue is important to retain a fully active NGF. The highly conserved Tyr51 and Arg99 were converted into Phe and Lys respectively, without changing the biological properties of the molecule. However, binding and biological activity were greatly impaired after the simultaneous replacement of both Arg99 and Arg102 by Gly. The three conserved Trp residues at positions 20, 75 and 98 were substituted by Phe. The Trp mutated proteins retained 15-60% of receptor binding and 40-80% of biological activity, indicating that the Trp residues are not essential for NGF activity. However, replacement of Trp20 significantly reduced the amount of NGF in the medium, suggesting that this residue may be important for protein stability.

• Iwane M et al.
• Production, purification and characterization of biologically active recombinant human nerve growth factor.
• Biochem Biophys Res Commun. 1990; 171: 116-22
• Display abstract

• The human NGF gene was isolated and inserted downstream from murine leukemia virus LTR in a plasmid having dihydrofolate reductase cDNA. The expression plasmid was introduced into CHO cells. Selection of the transformants for the resistance to methotrexate gave a CHO cell line which produced human NGF at a level of 4 mg/L in the culture medium. The recombinant human NGF was purified to near homogeneity from the culture supernatant. The NH2-terminal amino acid sequence, the COOH-terminal amino acid (Ala), and the amino acid composition of the human NGF were identical to those deduced from the nucleotide sequence of the human NGF gene. The recombinant human NGF was composed of 120 amino acid residues. Three disulfide linkages were determined to be Cys15-Cys80, Cys-58-Cys108, and Cys68-Cys110; the locations were identical to those in the mouse 2.5S NGF molecule. The specific biological activity of the recombinant human NGF was comparable with that of authentic mouse 2.5S NGF as determined by stimulation of neurite outgrowth from PC12 cells.

• McDonald NQ, Lapatto R, Murray-Rust J, Blundell TL
• X-ray crystallographic studies on murine nerve growth factor.
• J Cell Sci Suppl. 1990; 13: 19-30
• Display abstract

• The largest and best characterised family of neurotrophic growth factors is that of nerve growth factor (NGF) and its relatives. In order to understand the relation of structure and function, we have undertaken X-ray analyses of murine NGF. The active component beta-NGF crystallises as hexagonal bipyramids that give good X-ray diffraction data using a synchrotron to 2.3A resolution. We have prepared several heavy atom derivatives that are being used in the method of multiple isomorphous replacement to solve the phase problem and determine the three-dimensional structure. We have also prepared crystals of the precursor, 7S NGF, which is a complex of three different subunits of composition alpha 2 beta 2 gamma 2. We have collected X-ray data to 3A resolution on two crystal forms with related cell dimensions and orthorhombic spacegroups. Detailed analyses of the structures of NGF in these crystal forms, taken together with data on sequence and biological activity, should give clues concerning the role of the precursor complex in storage and assist the identification of the surface region involved in receptor binding.

• Kaliunov VN, Petrusenko GP, Fomichenko KV
• [The effect of nerve growth factor, guanethidine and their combined use on nuclease activity in animal tissues]
• Vopr Med Khim. 1989; 35: 117-20
• Display abstract

• Activity of ribo- and deoxyribonucleases RNAases I and II, DNAases I and II was studied in brain hemispheres, liver, kidney and heart tissues of one-month-old rats, which were administered daily, beginning from birth either with nerve growth factor 15 micrograms/kg, guanethidine 30 micrograms/kg or with these compounds simultaneously at the doses mentioned above. Activity of the nucleases studied was altered in nervous tissue and in vegeto-dependent tissues after separate treatment with both nerve growth factor and guanethidine, while their simultaneous administration caused slight normalization but not to complete recovery of the patterns studied.

• Ebendal T, Persson H, Larhammar D, Lundstromer K, Olson L
• Characterization of antibodies to synthetic nerve growth factor (NGF) and proNGF peptides.
• J Neurosci Res. 1989; 22: 223-40
• Display abstract

• Sequence data for the mature nerve growth factor (NGF) protein and its precursor are available from molecular cloning of the NGF gene in several species, including mice, humans, rats, and chickens. Hydrophilicity analysis of the predicted rat and chicken prepro-NGF was carried out to locate putative antigenic determinants. Eight peptides were selected and synthesized based on hydrophilicity profiles. Two peptides represent sequences in the rat (and mouse) pro-NGF, one peptide (our peptide P3) represents a highly conserved region of the mature NGF protein (identical in humans, mice, rats, and chickens), two peptides are specific for the mature chicken NGF, and the remaining three peptides are specific for the mature rat NGF (each with only one amino acid substitution compared with corresponding segments of the mouse NGF). For immunization, the peptides were conjugated to keyhold limpet hemocyanin and used to produce antisera in rabbits. After bleeding, peptide-specific antibodies were purified on affinity columns prepared by coupling each of the synthetic peptides. The different peptide antisera and affinity-purified antibodies then were characterized by enzyme-linked immunoassay (ELISA) and immunohistochemistry of the male mouse submandibular gland, a rich exocrine source of NGF. ELISA analysis showed that all peptide antisera bound two to four orders of magnitude better than normal rabbit serum to a coat of their proper peptide. The higher binding was retained by the purified peptide antibodies compared with normal rabbit immunoglobulin. Specific tests, in which one peptide antiserum was checked against different peptide coats in the ELISA, also showed two to four orders of magnitude higher binding of antibodies to the proper synthetic peptide. The peptide antibodies also were tested for their ability to bind to native mouse beta NGF coated to the immunoplates. Only antibodies raised to the conserved P3 peptide recognized native NGF to an extent similar to that obtained with polyclonal anti-NGF antibodies. Conversely, P3 was well recognized by several different NGF antisera. Immunohistochemically, both peptide antisera against the pro-NGF stained the perinuclear cytoplasm in the basal part of the cells of the granulated convoluted tubules in the mouse submandibular gland.(ABSTRACT TRUNCATED AT 400 WORDS)

• Mutoh T, Guroff G
• The role of phosphorylation in the action of nerve growth factor.
• Biofactors. 1989; 2: 71-6
• Display abstract

• Although there is a great deal of information available about the biological effects of nerve growth factor, the detailed mechanism(s) by which it works is still not known. Some general outlines, however, are becoming clear. It is known that a number of second messenger systems are recruited and there is evidence that this recruitment has functional consequences. There is also increasingly strong evidence that nerve growth factor causes the activation of parallel pathways of phosphorylative cascades leading to changes in the phosphorylation and, consequently, in the function of key proteins throughout the cell. It can be suggested that changes in the phosphorylation of nuclear proteins initiate the alterations in gene expression known to occur after nerve growth factor treatment, which underlie the dramatic changes in the overall biology of nerve growth factor-responsive cells.

• Schwarz MA, Fisher D, Bradshaw RA, Isackson PJ
• Isolation and sequence of a cDNA clone of beta-nerve growth factor from the guinea pig prostate gland.
• J Neurochem. 1989; 52: 1203-9
• Display abstract

• The guinea pig prostate gland contains high levels of nerve growth factor similarly to the mouse submandibular gland. Nerve growth factor from the guinea pig prostate gland cross-reacts weakly with antisera directed against mouse nerve growth factor, is associated with different proteins, and may be processed by a different mechanism. We have isolated a full-length cDNA clone for nerve growth factor from a library prepared from RNA of the guinea pig prostate gland. The guinea pig cDNA contains 1,075 nucleotides and is very similar to the shorter of two predominant nerve growth factor transcripts present in the mouse submandibular gland. The cDNA sequence predicts a precursor protein of 241 amino acids that is 86% identical to the mouse amino acid sequence. Only 10 amino acid changes are present in the C-terminal region corresponding to the mature 118 amino acid beta-nerve growth factor of the mouse. Dibasic amino acid processing sites that are present at the N- and C-termini of the mature protein sequence and two other dibasic amino acid sites, representing potential processing sites within the propeptide, are all conserved, suggesting a similar mechanism of processing.

• Snider WD, Johnson EM Jr
• Neurotrophic molecules.
• Ann Neurol. 1989; 26: 489-506
• Display abstract

• Neurotrophic molecules have a profound influence on developmental events such as naturally occurring cell death, differentiation, and process outgrowth. Despite their striking effects on developing neurons, a role for these molecules in the pathogenesis or therapy of neurological disease has not yet been defined. However, a variety of recent advances promise to provide the techniques necessary to assess the potential relevance of neurotrophic molecules to clinical neurology. In this article we review recent investigations into the biological effects, regulation of production, and mechanisms of action of the best characterized trophic molecule, nerve growth factor. In addition we review studies characterizing brain-derived neurotrophic factor and other putative neurotrophic molecules. Finally, we discuss how pharmacological effects of these molecules may be relevant to the therapy of disease states as well as neural regeneration.

• Dalseg AM, Bock E
• [Nerve growth factor (NGF)--the current state of knowledge]
• Nord Med. 1989; 104: 45-7
• Display abstract

• As Nerve Growth Factor (NGF) is probably the growth factor most extensively studied so far, it serve as a model of other growth factors. NGF regulates the survival of specific populations of neurons during critical periods of their development. NGF is also essential to the development and maintenance of specific functional properties of these neurons and to the ability of certain neurons to regenerate.

• Dreyfus CF
• Effects of nerve growth factor on cholinergic brain neurons.
• Trends Pharmacol Sci. 1989; 10: 145-9
• Display abstract

• Nerve growth factor (NGF) is a fully characterized molecule, well known for its actions in the differentiation and maintenance of peripheral neurons. However, recent studies suggest that its actions are not limited to the periphery, but may extend to the CNS. In particular, this trophic agent appears to affect development and survival of a variety of brain cell populations. Noteworthy are its actions on cholinergic neurons that degenerate in Alzheimer's disease and Huntington's chorea. However, studies of NGF receptor sites suggest that effects of NGF may also extend to non-cholinergic cell groups. Cheryl Dreyfus summarizes these data and points to future work necessary to define further the underlying mechanisms of action and to examine the function of NGF on diverse brain populations.

• Hefti F, Hartikka J, Knusel B
• Function of neurotrophic factors in the adult and aging brain and their possible use in the treatment of neurodegenerative diseases.
• Neurobiol Aging. 1989; 10: 515-33
• Display abstract

• This review summarizes the current knowledge of characterized neurotrophic factors, including nerve growth factor (NGF) which serves as paradigmatic example when studying novel molecules. Special consideration is given to the function of neurotrophic factors in the adult and aging brain. Strategies are discussed for the eventual development of pharmacological applications of these molecules in the treatment of neurodegenerative diseases.

• Ebendal T
• [Regulation of the supply of nerve growth factor results in good development of neural graft]
• Lakartidningen. 1989; 86: 1074-1074

• Oda T, Ohta M, Inoue S, Ikeda K, Furukawa S, Hayashi K
• Amino acid sequence of nerve growth factor purified from the venom of the Formosan cobra Naja naja atra.
• Biochem Int. 1989; 19: 909-17
• Display abstract

• Nerve growth factor (NGF) was isolated from the venom of the Formosan cobra (Naja naja atra). The amino acid sequence was determined by a combination of conventional methods. The total number of amino acid residues was 116, giving a molecular mass of 13,057 Da. The sequence was identical with that deduced from the nucleotide sequence of an NGF cDNA from the venom gland of Naja naja siamensis, reported by Selby et al. [J. Neurosci. Res., 18, 293-298 (1987)].

• Lahtinen T, Soinila S, Lakshmanan J
• Biological demonstration of nerve growth factor in the rat pituitary gland.
• Neuroscience. 1989; 30: 165-70
• Display abstract

• Explants of anterior, intermediate and posterior pituitary lobes of rats at various ages evoked an intense nerve fibre outgrowth when co-cultured with neonatal sympathetic superior cervical ganglia in a collagen gel medium. Freezing and thawing of pituitary lobes, prior to culture, did not abolish their growth-promoting effect. Since the addition of antiserum to nerve growth factor in the culture medium inhibited totally the stimulative action of both fresh and frozen pituitary explants it is suggested that the stimulation is mediated by nerve growth factor or an immunologically related molecule. Based on the present results it is concluded that nerve growth factor is synthesized by both the anterior, intermediate and posterior lobes throughout the postnatal period, as well as in adult rats. The function of nerve growth factor in the pituitary gland is discussed.

• Large TH et al.
• Structure and developmental expression of the nerve growth factor receptor in the chicken central nervous system.
• Neuron. 1989; 2: 1123-34
• Display abstract

• Chicken nerve growth factor (NGF) receptor cDNAs have been isolated and sequenced in an effort to identify functionally important receptor domains and as an initial step in determining the functions of the NGF receptor in early embryogenesis. Comparisons of the primary amino acid sequences of the avian and mammalian NGF receptors have identified several discrete domains that differ in their degree of conservation. The highly conserved regions include an extracellular domain, likely to be involved in ligand binding, in which the positions of 24 cysteine residues and virtually all negatively charged residues are conserved; a transmembrane region, including flanking stretches of extracellular and cytoplasmic amino acids, which has properties suggesting it interacts with other proteins; and a cytoplasmic PEST sequence, which may regulate receptor turnover. Transient expression of NGF receptor mRNA has been seen in many regions of the developing CNS. Experiments suggest that both NGF and its receptor help regulate development of the retina.

• Kumar S
• Nerve growth factor and neuroblastoma.
• J Histochem Cytochem. 1988; 36: 1359-60

• Yu ZY
• [Advances in the research on nerve growth factor]
• Sheng Li Ke Xue Jin Zhan. 1988; 19: 226-30

• Edwards RH, Selby MJ, Garcia PD, Rutter WJ
• Processing of the native nerve growth factor precursor to form biologically active nerve growth factor.
• J Biol Chem. 1988; 263: 6810-5
• Display abstract

• In the mouse submaxillary gland beta nerve growth factor (beta-NGF) forms a complex with two members of the kallikrein family of serine proteases, termed the alpha- and gamma-subunits of NGF. We demonstrate that the beta-NGF precursor produced in mammalian cells via a recombinant vaccinia virus can be cleaved by stoichiometric quantities of the gamma-subunit to produce beta-NGF. Trypsin in catalytic quantities also produces native beta-NGF. Proper cleavage depends critically on the conformation of the precursor. beta-NGF has at least 10-fold more biological activity than its precursor.

• Fung LC, De Boni U
• Modulation of nuclear rotation in neuronal interphase nuclei by nerve growth factor, by gamma-aminobutyric acid, and by changes in intracellular calcium.
• Cell Motil Cytoskeleton. 1988; 10: 363-73
• Display abstract

• Nuclear rotation (NR) is typically measured as motion of nucleoli within nuclei of cells in vitro. This occurs in cycling cells. However, its observation in neurons arrested in interphase indicates that mechanisms related to mitosis are not a prerequisite. We have recently shown that NR occurs in three dimensions within the nuclear space, that it occurs within the space delineated by the outer nuclear membrane and that it includes chromatin domains in addition to nucleoli and have postulated that this motion of chromatin domains is related to changes in gene expression. We now show that exposure of dorsal root, sensory neurons in vitro to nerve growth factor (NGF) or to gamma-aminobutyric acid (GABA), agents which alter gene expression, and to agents causing redistribution of calcium, such as EGTA and the calcium ionophore A23187, significantly alters NR. The NGF increased the mean rate of NR and did so at a time after exposure when activity of RNA polymerases have been shown to rise. Exposure to GABA resulted, within minutes, in shifts of the nucleolus within the three-dimensional space of the nucleus, associated in some neurons with significant, sigmoidal increases in the rate of NR. The calcium ionophore A23187 as well as chelation of extracellular calcium with EGTA similarly increased rates. Importantly, excess calcium, with EGTA remaining present, returned NR of all nucleoli to rates not different from controls. This indicates that the increase in NR seen with EGTA is specific to the chelation of calcium and not an nonspecific response to EGTA. It is difficult to link the action of agents which alter gene expression or transmembrane ion balance with changes in NR. Nevertheless, in support of our hypothesis, the results presented here show that agents known to alter gene expression, alter NR in a temporally coincident manner and that they do so, possibly, by calcium-dependent mechanisms.

• Drinkwater CC, Evans BA, Richards RI
• Kallikreins, kinins and growth factor biosynthesis.
• Trends Biochem Sci. 1988; 13: 169-72

• Hefti F, Knusel B
• Chronic administration of nerve growth factor and other neurotrophic factors to the brain.
• Neurobiol Aging. 1988; 9: 689-90
• Display abstract

• Intracerebral chronic administration of nerve growth factor (NGF) is able to counteract age-related cholinergic dysfunction. This finding suggests that intracerebral administration of neurotrophic factors may become a generally valuable approach when attempting to ameliorate age-related neuronal deficits in experimental animals and humans. This commentary briefly discusses possible strategies for the development of such "neurotrophic factor treatments."

• Whittemore SR, Friedman PL, Larhammar D, Persson H, Gonzalez-Carvajal M, Holets VR
• Rat beta-nerve growth factor sequence and site of synthesis in the adult hippocampus.
• J Neurosci Res. 1988; 20: 403-10
• Display abstract

• A rat beta-nerve growth factor (NGF) genomic sequence encoding the entire 3' exon of preproNGF was cloned, and its nucleotide sequence was determined. Rat NGF shows very high homology with other known NGFs in both the prepropeptide and the 3' untranslated regions. The presumptive signal sequence, the cysteine residues important for tertiary structure, possible glycosylation sites, and dibasic amino acids required for proteolytic cleavage to mature NGF are conserved across species. Comparison of the hydrophobicity plots and amino acid sequences revealed an evolutionary divergent domain on the external surface of NGF, which may account for the poor immunologic crossreactivities of the various NGFs. In situ hybridization with a rat-specific oligodeoxynucleotide indicated high levels of NGF mRNA synthesis in both hippocampal granule and pyramidal cell layers. These results are consistent with one role for NGF in the CNS as a neuronally released, retrogradely transported neurotrophic factor for basal forebrain cholinergic neurons.

• Pezzati P, Mueller A, Bienenstock J, Stanisz AM
• Neuropeptides, nerve growth factor, and mucosal immunity.
• Brain Behav Immun. 1988; 2: 311-7

• Fahnestock M, Bell RA
• Molecular cloning of a cDNA encoding the nerve growth factor precursor from Mastomys natalensis.
• Gene. 1988; 69: 257-64
• Display abstract

• Mastomys natalensis is an African rat that has high levels of nerve growth factor (NGF) in its submaxillary glands. Like in the mouse, Mastomys NGF is found as a high-molecular-weight complex. However, the Mastomys complex differs from the mouse complex, in that the gamma-subunit is either missing or is less tightly bound in the Mastomys NGF complex. In the mouse, the gamma-subunit has been implicated in the processing of the beta-NGF precursor. The possible lack of gamma-subunits in the Mastomys NGF high-molecular-weight complex suggested that the Mastomys beta-NGF precursor might differ from the mouse beta-NGF precursor in some of its processing sites. In particular, Mastomys beta-NGF might lack the C-terminal dipeptide cleavage site implicated in beta-gamma subunit interactions in mouse NGF. In order to test this hypothesis, we isolated and sequenced a cDNA clone for Mastomys beta-NGF. We report here the cloning and sequencing of a cDNA coding for beta-NGF from Mastomys natalensis. The cDNA library was prepared from Mastomys submaxillary gland mRNA and the beta-NGF clone was isolated using a mouse cDNA as a probe. The nucleotide sequence of Mastomys beta-NGF is 95% homologous to that of mouse beta-NGF. In particular, the Mastomys beta-NGF precursor contains the same three C-terminal residues as the mouse, suggesting that the Mastomys beta-NGF precursor could interact with a gamma-like subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

• Lakshmanan J, Burns C, Smith RA
• Molecular forms of nerve growth factor in mouse submaxillary glands.
• Biochem Biophys Res Commun. 1988; 152: 1008-14
• Display abstract

• By use of immunoblot analysis, we demonstrate the presence of a nerve growth factor with a relative molecular mass (Mr) of 53,000 in submaxillary glands of adult male and female mice in addition to the 13,000 form previously discovered in this tissue. Both the Mr 53,000 and 13,000 forms of nerve growth factor are present at equal concentrations in salivary glands of male mice while the Mr 53,000 form is present at a greater concentration than the Mr 13,000 form in the glands of female mice. Testosterone pretreatment, however, specifically augments the Mr 13,000 form of nerve growth factor in the salivary glands of female mice. The existence of nerve growth factor with molecular weight of 53,000, which is higher than the molecular size predicted for the two precursor forms of nerve growth factor, suggest the possibility that one of the nerve growth factor precursors or a product derived from it may undergo post-translational modification in submaxillary glands of mice.

• Davies AM
• The emerging generality of the neurotrophic hypothesis.
• Trends Neurosci. 1988; 11: 243-4

• Orts Llorca F
• [Nerve growth factor and the history of its discovery]
• An R Acad Nac Med (Madr). 1988; 105: 483-8

• Paalme VA, Arumiae UV, Siigur IR, Neuman TE, Shepilov SA
• [Salivary glands of venomous and nonvenomous snakes synthesize and secrete the nerve growth factor]
• Dokl Akad Nauk SSSR. 1988; 301: 754-6

• Kaliunov VN, Ermilov LG, Shmalei SV
• [The function of the cholinoreactive systems of the postsynaptic neuron under the action of nerve growth factor]
• Nerv Sist. 1988; 27: 41-8

• Sieron A, Lewin-Kowalik J
• [Nerve growth factor, its chemical structure and biological function]
• Postepy Biochem. 1988; 34: 173-82

• Despres G, Giry N, Romand R
• Immunohistochemical localisation of nerve growth factor-like protein in the organ of Corti of the developing rat.
• Neurosci Lett. 1988; 85: 5-8
• Display abstract

• The presence of nerve growth factor (NGF)-like protein was investigated in the cochlea of the developing rat between birth and postnatal day 30, by the indirect immunofluorescence technique. Nerve growth factor-like protein could be detected from birth up to day 8. The immunostaining was localised within the hair mainly above their nuclei. No NGF-like immunoreactivity was observed in spiral ganglion cells. The data suggest that NGF acts as a neurotrophic factor, especially for efferent endings in the developing cochlea.

• Young M, Blanchard MH, Sessions F, Boyle MD
• Subunit structure of high molecular weight mouse nerve growth factor.
• Biochemistry. 1988; 27: 6675-81
• Display abstract

• Studies from several laboratories have shown that mouse submandibular glands and mouse saliva contain nerve growth factor (NGF) as part of a high molecular weight oligomeric macromolecule composed of three different subunits, termed alpha, beta, and gamma. The beta-subunit is the nerve growth-promoting protein. The gamma-subunit is a serine protease class enzyme of highly restricted substrate specificity. The alpha-subunit has no known function. This high molecular weight form of nerve growth factor is also a Zn(II)-containing metalloprotein. In the present study, measurements of multiple physicochemical parameters have been used to deduce the subunit structure of high molecular weight NGF. Results demonstrate that it contains two alpha-, one beta- and one gamma-subunit together with one tightly bound Zn(II) ion per molecule.

• Matsuda Y
• [Nerve growth factor and S6 kinase]
• Seikagaku. 1987; 59: 347-52

• Finn PJ, Ferguson IA, Wilson PA, Vahaviolos J, Rush RA
• Immunohistochemical evidence for the distribution of nerve growth factor in the embryonic mouse.
• J Neurocytol. 1987; 16: 639-47
• Display abstract

• The distribution of nerve growth factor-like immunoreactivity has been examined in the embryonic mouse with special reference to the CNS. The intensity of the immunoreactive stain was found to be greatest on embryonic days 15 and 16. The antigen is widespread and present in high concentrations in both the PNS and CNS. Most intense staining was detected in cranial nerve tracts, hippocampus, developing white matter of the spinal cord and tegmentum. Lower intensities were found within diencephalic regions, spinal cord grey matter, medullary fibre tracts and cerebellum. These results support the increasing evidence suggesting that the trophic molecule nerve growth factor has an important role to play in the development of central as well as peripheral neurons.

• Romani S, Moroder L, Wunsch E
• Synthesis of the trypsin fragment 10-25/75-88 of mouse nerve growth factor. I. The protected tetradecapeptide 75-88.
• Int J Pept Protein Res. 1987; 29: 99-106
• Display abstract

• Cyanogen bromide cleavage followed by trypsin digestion of mouse nerve growth factor (NGF) allowed the isolation of a double chain unsymmetrical cystine peptide of high neurotrophic activity. The presence of tryptophan residues severely limits the synthetic approaches for selective disulfide bridging. The strategy applied for such a purpose as well as the preparation of the suitably protected tetradecapeptide corresponding to sequence 75-88 as key intermediate for the synthesis of the NGF fragment 10-25/75-88 are described.

• Romani S et al.
• Synthesis of the trypsin fragment 10-25/75-88 of mouse nerve growth factor. II. The unsymmetrical double chain cystine peptide.
• Int J Pept Protein Res. 1987; 29: 107-17
• Display abstract

• The synthesis of the unsymmetrical double chain cystine peptide corresponding to sequence 10-25/75-88 of mouse nerve growth factor is described. The synthetic product was inactive in all bioassays examined; consequently, this portion of the NGF molecule did not represent or contain a lower molecular weight form of the neurotrophic factor.

• Selby MJ, Edwards R, Sharp F, Rutter WJ
• Mouse nerve growth factor gene: structure and expression.
• Mol Cell Biol. 1987; 7: 3057-64
• Display abstract

• The organization and biologically significant sequences of the entire mouse nerve growth factor (NGF) gene have been determined. The gene spans 45 kilobases and contains several small 5' exons. Transcription of the gene results in four different mRNA species, which can be accounted for by alternative splicing and independent initiation from two promoters. These transcripts encode proteins which have divergent N termini and the NGF moiety at their C termini. The levels of the various NGF transcripts have been determined in different tissues and throughout postnatal development. We have also examined the expression of these transcripts in the brain in response to specific early sensory deprivation. The results suggest that the expression of NGF mRNA during postnatal development is regulated independently of the formation of complex neural networks.

• Heumann R, Bandtlow C, Davies A, Korsching S, Thoenen H
• The structure-function relationship of the nerve growth factor molecule and the regulation of its synthesis.
• Biochem Soc Trans. 1987; 15: 131-2

• Levi-Montalcini R
• The nerve growth factor: thirty-five years later.
• EMBO J. 1987; 6: 1145-54

• Neet KE, Fanger MW, Baribault TJ
• Derivation of monoclonal antibody to nerve growth factor.
• Methods Enzymol. 1987; 147: 186-94

• Heumann R
• Regulation of the synthesis of nerve growth factor.
• J Exp Biol. 1987; 132: 133-50
• Display abstract

• Through nearly 40 years of research nerve growth factor (NGF) has become a paradigm for neurotrophic factors. NGF is synthesized and released from innervated target tissues in limiting amounts, thereby regulating the cell number and the differentiated properties of responsive neurones. Three distinct cell types are responsive to NGF: peripheral sensory and sympathetic neurones and certain types of central cholinergic ones. The effects of NGF are mediated through interaction with a specific receptor which activates a transmembrane second-messenger system. NGF synthesis is regulated during development and in the adult animal. In the developing whisker pad, NGF synthesis commences with its sensory innervation, but sensory neurones lack NGF receptors at the stage when their fibres are growing to their target. These findings indicate that NGF does not attract sensory nerve fibres chemotactically to their target fields during development, but is involved in target-controlled neuronal cell death and in regulation of the density of innervation of target tissues. In non-neuronal cells of the sciatic nerve, NGF synthesis is up-regulated during development and after nerve lesion. Thus the changes in NGF levels after lesion in the adult animal are consistent with the hypothesis that the non-neuronal cells relapse into an earlier developmental stage. Regenerating fibres penetrating into the distal nerve stump restore the low adult levels of NGF. Recent evidence indicates that macrophages invading the nerve after transection produce signals which increase NGF synthesis.

• Korsching S, Thoenen H
• Two-site enzyme immunoassay for nerve growth factor.
• Methods Enzymol. 1987; 147: 167-85

• Glinsky GV, Ivanova AB, Vinnitsky VB, Arumae U, Siigur J, Neuman T
• Nerve growth factor may be the metastasis growth factor.
• Ann N Y Acad Sci. 1987; 496: 656-9

• Selby MJ, Edwards RH, Rutter WJ
• Cobra nerve growth factor: structure and evolutionary comparison.
• J Neurosci Res. 1987; 18: 293-8
• Display abstract

• We have cloned and sequenced an NGF cDNA from the cobra venom gland. The structure of the predicted cobra precursor resembles that of the mouse prohormone, with a highly conserved mature NGF protein at the C-terminus. In the non-NGF moiety, a putative signal peptide and two sets of basic residues presumably reflecting cleavage sites that are more conserved, while large interstitial regions are poorly conserved. This moiety may be involved in defining the folding of the precursor for subsequent proteolytic maturation and/or encode a bioactive polypeptide(s). Primer extension analysis indicates a single NGF transcript in the cobra venom gland.

• Black IB
• Trophic molecules and evolution of the nervous system.
• Proc Natl Acad Sci U S A. 1986; 83: 8249-52
• Display abstract

• Although recent work has reemphasized the general importance of ontogeny in evolution, underlying developmental molecular mechanisms are largely undefined. What heritable ontogenetic mechanisms result in the evolution of new morphologies and functions? Such questions are particularly difficult in the nervous system, in which each of 10(11) neurons forms approximately equal to 10(4) specific interconnections. I propose that specific heritable, trophic interactions during development, which determine cell survival and pathway size, form a substrate for neural evolution. This model is based on the observation that neurons are vastly overproduced during ontogeny; neurons, their pathways and connections are dependent on target-derived trophic factors for developmental survival; and co-innervating, functionally and anatomically distinct neural populations compete for common trophic factors for survival. Focusing on sympathetic and sensory neurons, which require the target-derived, trophic protein nerve growth factor at different times for developmental survival, and which innervate common targets, different classes of ontogenetic evolutionary mechanisms may be characterized. Evolution may occur from heritable changes in the structure of trophic gene products or altered timing of expression. Molecular mechanisms underlying heterochrony are thereby described. The model is directly applicable to evolution of the brain and is testable in a variety of situations.

• Johnson D et al.
• Expression and structure of the human NGF receptor.
• Cell. 1986; 47: 545-54
• Display abstract

• The nucleotide sequence for the human nerve growth factor (NGF) receptor has been determined. The 3.8 kb receptor mRNA encodes a 427 amino acid protein containing a 28 amino acid signal peptide, an extracellular domain containing four 40 amino acid repeats with six cysteine residues at conserved positions followed by a serine/threonine-rich region, a single transmembrane domain, and a 155 amino acid cytoplasmic domain. The sequence of the extracellular domain of the NGF receptor predicts a highly ordered structure containing a negatively charged region that may serve as the ligand-binding site. This domain is conserved through evolution. Transfection of a full-length cDNA in mouse fibroblasts results in stable expression of NGF receptors that are recognized by monoclonal antibodies to the human NGF receptor and that bind [125I]NGF.

• Fahnestock M, Brundage S, Shooter EM
• The sequence of a cDNA clone coding for a novel kallikrein from mouse submaxillary gland.
• Nucleic Acids Res. 1986; 14: 4823-35
• Display abstract

• Mouse submaxillary gland contains many proteolytic enzymes, the most widely studied of which are the kallikreins. This gland also contains high levels of nerve growth factor (NGF), which is isolated as a complex of three subunits, alpha, beta, and gamma. We report here the cloning and sequence analysis of a novel kallikrein from mouse submaxillary gland. Antibodies directed against the alpha subunit precipitate the product of this clone, but do not precipitate the homologous gamma subunit. This new kallikrein is therefore closely related to alpha NGF, yet in contrast to the alpha subunit, its sequence suggests it has proteolytic activity.

• Marx JL
• Nerve growth factor acts in brain.
• Science. 1986; 232: 1341-2

• Vinores SA
• Nerve growth factor modification of the ethylnitrosourea model for multiple schwannomas.
• Ann N Y Acad Sci. 1986; 486: 124-31
• Display abstract

• The administration of ethylnitrosourea (ENU) to pregnant rats late in gestation or to neonatal rats results in the induction of Schwann cell tumors in a high percentage of perinatally exposed animals. Exogenous administration of nerve growth factor (NGF) significantly reduces the number of Schwann cell tumors and other neurogenic tumors developing in ENU-treated rats. Administration of antibodies directed against NGF prior to neonatal ENU exposure results in a substantial increase in the incidence of Schwann cell tumors, particularly in the trigeminal nerves of both rats and mice. Transplacental ENU treatment causes early neoplastic proliferation (ENP) at 90 days of age in the Schwann cell population of trigeminal nerves in nearly all exposed rats. A variety of NGF treatment protocols (single or multiple inoculations or microinfusion prior to or following ENU exposure) resulted in a significant reduction in ENU-induced ENP in trigeminal nerves. These results indicate that NGF may convey protection either directly or indirectly, by an unknown mechanism, to Schwann cells and other supportive neural cells by reducing their sensitivity to ENU-induced neoplastic transformation.

• Pintar JE, Sonnenfeld KH, Fisher J, Klein RS, Kreider B
• Molecular and immunocytochemical studies of neurofibromas and related cell types.
• Ann N Y Acad Sci. 1986; 486: 96-106

• Lucas CA, Kreutzberg GW
• Regulation of acetylcholinesterase secretion from neuronal cell cultures.--1. Actions of nerve growth factor, cytoskeletal inhibitors and tunicamycin.
• Neuroscience. 1985; 14: 349-60
• Display abstract

• The release of enzymatically active acetylcholinesterase was measured from two types of neuronal cell cultures. Evidence is presented that primary cultures of chick optic lobe and clonal rat PC12 cells released acetylcholinesterase by similar mechanisms. The secretion was selective for the tetrameric or larger molecule forms of acetylcholinesterase. Thus the chick primary cultures released the so-called G4 isoenzyme of acetylcholinesterase. The rat PC12 cells, after pretreatment with nerve growth factor, secreted mainly G4 acetylcholinesterase and a small amount of an isoenzyme sedimenting at 16S. The rate of secretion of acetylcholinesterase was regulated by the three types of agents used. (1) Acetylcholinesterase secretion was markedly increased by nerve growth factor. (2) Microtubule inhibitors (colchicine, taxol and nocodazole) suppressed the rate of release of acetylcholinesterase. (3) Acetylcholinesterase release was also decreased by an inhibitor of protein glycosylation, tunicamycin. We provide evidence that these three types of agents regulate acetylcholinesterase secretion not by a direct effect on secretion, but by modulatory effects on the synthesis, intracellular transport and turnover of the enzyme. (1) Nerve growth factor acts on acetylcholinesterase secretion via increased synthesis of messenger RNA. (2) Microtubule inhibitors act by preventing intracellular transport. (3) Tunicamycin acts by decreasing the intracellular activity of acetylcholinesterase, possibly via enhanced proteolytic breakdown.

• Stach RW, Wagner BJ, Ennulat DJ, Perez-Polo JR
• Characteristics of the nerve growth factor receptors and a nerve growth factor receptor--nerve growth factor covalent complex.
• Int J Neurosci. 1985; 26: 87-94
• Display abstract

• Nerve growth factor is a polypeptide hormone that is required for the normal growth and development of the embryonic sensory and sympathetic nervous systems. On these cells, there are two different receptors for the nerve growth factor. Recently, these receptors have been isolated from three cell types and shown to have essentially the same binding characteristics. Molecular weights for receptors from two of these cell types, embryonic sensory and rat pheochromocytoma cells, have been determined. In addition, the formation of a covalent nerve growth factor, nerve growth factor receptor complex, has been investigated on embryonic sensory and sympathetic neurons. The formation of this covalent complex containing 125I-beta nerve growth factor is prevented by the addition of excess unlabeled nerve growth factor or by the addition of sodium fluoride and dinitrophenol. This complex forms at 4 degrees, 22 degrees, and 37 degrees, indicating that it is occurring on the cell surface. A disulfide bond is involved in the formation of this covalent complex.

• Evans BA, Richards RI
• Genes for the alpha and gamma subunits of mouse nerve growth factor are contiguous.
• EMBO J. 1985; 4: 133-8
• Display abstract

• Here we describe the structure and linkage of genes encoding the alpha and gamma subunits of mouse nerve growth factor (NGF). These genes are members of the highly homologous glandular kallikrein multigene family. Together with the beta subunit, the alpha and gamma proteins constitute the high mol. wt. (7S) form of NGF isolated from mouse submandibular gland. The gamma subunit is an active serine protease and is thought to cleave pro-beta-NGF to generate the mature growth factor. The alpha subunit has no detectable proteolytic activity, but is essential for the stable formation of 7S NGF. Lack of enzyme activity of the alpha subunit can be attributed, at least in part, to the deletion of 15 nucleotides in a highly conserved coding region which is normally involved in the activation of serine proteases from their inactive zymogen form.

• Dunbar JC, Bradshaw RA
• Nerve growth factor biosynthesis: isolation and characterization of a guinea pig prostate kallikrein.
• J Cell Biochem. 1985; 29: 309-19
• Display abstract

• Guinea pig prostate contains one major soluble esteropeptidase activity. The protein has been purified and characterized and found to be a glycoprotein comprised of a single polypeptide chain. The molecular has a similar Km for lysine and arginine synthetic substrates, although the Vmax for arginine is much greater than that for lysine. Amino-terminal sequence analysis has also revealed a marked degree of homology to mouse gamma-nerve growth factor (NGF) and the kallikrein family of serine proteases. In contrast to gamma-NGF, however, the guinea pig enzyme does not appear to form stable complexes with beta-NGF.

• Banks BE, Pearce FL, Springer CJ, Vernon CA
• On the immunology of nerve growth factor.
• Neurosci Lett. 1985; 61: 127-30
• Display abstract

• We report some experiments on the immunological properties of nerve growth factor from the venom of Heloderma horridum and from bull seminal vesicles. On the basis of these results, taken together with results already in the literature, we propose an operational definition of the term nerve growth factor.

• Hatanaka H
• [Nerve growth factor]
• Tanpakushitsu Kakusan Koso. 1984; 29: 1937-54

• Bradshaw RA, Dunbar JC, Isackson PJ, Kouchalakos RN, Morgan CJ
• Nerve growth factor: mechanism of action.
• Symp Fundam Cancer Res. 1984; 37: 87-101
• Display abstract

• Although the detailed mechanism of action of NGF as it occurs in vivo still requires considerable clarification, a model in which NGF is generated by end organs of responsive neurons and interacts with these neurons to provide trophic stimulation and maintenance of viability seems highly plausible. The demonstration of NGF synthesis by these cells, including the details of the process, is required, and information regarding the events following the formation of the hormone-receptor complex at the plasma membrane must be obtained. However, the contribution of internalization and the possible role of the internalized receptor as a second messenger for generating the long-term effects of the hormone are particularly appealing. Any findings with NGF will almost certainly provide additional important insights into other polypeptide growth factors, particularly those substances found in the neurotrophic subset.

• Hazen-Martin DJ, Simson JA
• Immunocytochemical localization of nerve growth factor: effects of fixation.
• J Histochem Cytochem. 1984; 32: 30-6
• Display abstract

• The fixation dependence of immunocytochemically demonstrable nerve growth factor (NGF) was investigated. Several commonly used fixation methods have been employed, including buffered formaldehyde, Bouin's fluid, and chloroform-methanol, as well as freezing and cryostat sectioning. The immunostaining technique was an immunoenzyme bridge procedure on either paraffin sections or frozen sections. Of those methods tested, fixation for 1 hr in a buffered formaldehyde appeared to provide optimal preservation and localization of immunoreactive material. Using this method, reaction product was localized in granules of the granular tubule cells of the male mouse submandibular gland. Prolonged fixation in buffered formaldehyde resulted in a diffuse background staining and loss of granule immunoreactivity. In frozen sections and in tissues fixed with either Bouin's solution, chloroform-methanol, or buffered paraformaldehyde-glutaraldehyde increased cytoplasmic background staining and loss of granule immunoreactivity were observed. It was concluded that, for the localization of NGF at the light microscopic level, a brief (1 hr) buffered formaldehyde fixation is optimal.

• Sanes JR
• More nerve growth factors?
• Nature. 1984; 307: 500-500

• Murphy RA, Watson AY, Rhodes JA
• Biological sources of nerve growth factor.
• Appl Neurophysiol. 1984; 47: 33-42

• Ullrich A, Gray A, Wood WI, Hayflick J, Seeburg PH
• Isolation of a cDNA clone coding for the gamma-subunit of mouse nerve growth factor using a high-stringency selection procedure.
• DNA. 1984; 3: 387-92
• Display abstract

• A new approach was taken to isolate a full-length cDNA encoding the gamma-subunit of nerve growth factor (NGF). An oligonucleotide probe was used first to identify kallikrein-related sequences in a cDNA library derived from male mouse submaxillary gland mRNA. Of 10,000 clones, 860 were identified as being kallikrein-related; these were analyzed further by hybridization to synthetic oligonucleotides using a procedure which eliminates the effects of base composition and thus allows distinction between partial and perfect sequence complementarity. Nine clones hybridized with a gamma-subunit-specific probe at high stringency, and the nucleotide sequence of the longest cDNA insert was determined. Regions of homology and variability within the kallikrein gene family are maintained in this protein and probably reflect the similar functions and different substrates for this family of proteins.

• Wion D, Dicou E, Brachet P
• Synthesis and partial maturation of the alpha- and gamma-subunits of the mouse submaxillary gland nerve growth factor in Xenopus laevis oocytes.
• FEBS Lett. 1984; 166: 104-8
• Display abstract

• Sera raised against the alpha-, beta- and gamma-subunits of the mouse 7 S NGF were used to characterize translation products coded by submaxillary gland mRNAs microinjected into Xenopus oocytes. Anti-beta NGF sera did not cross-react with any material. In contrast, the precursors of the alpha- and gamma-subunits, as well as that of renin were identified. Use of tunicamycin, and a comparison of the translation products obtained in oocytes or in the reticulocyte lysate indicated that oocytes achieved the cleavage of signal sequences, the glycosylation of the alpha- and gamma-precursors, and the subsequent secretion of the 3 proteins. In the submaxillary gland, however, the mature forms of alpha NGF, gamma NGF and renin are composed of peptides of smaller size than those produced by the oocytes. These latter appear to lack specific proteases involved in the terminal processing of the submaxillary gland proteins.

• Banks BE
• Nerve growth factor--an enigma still?
• Biochem Soc Trans. 1984; 12: 173-6

• Rao AG, Neet KE
• Subunit interactions of 7 S nerve growth factor. Gamma-esterase activity, rates, and conformational changes during reassociation.
• J Biol Chem. 1984; 259: 73-9
• Display abstract

• The gamma-esterase activity of 7 S nerve growth factor (NGF) is depressed relative to free subunit because of constraints within the oligomer. 4 M urea causes a reversible dissociation of 7 S NGF and concomitant increase in esterase activity. The gamma-esterase activity with tosyl-arginyl-methyl-ester increased between 1 and 4 M urea and was inhibited at higher concentrations. Upon dilution of 7 S NGF from 6 M urea, the esterase activity was initially identical with that of the similarly treated gamma-subunit but decayed rapidly (10 min) to approach that of the native 7 S NGF. In the presence of 100 microM EDTA the activity was higher, but still required the same length of time to reach a constant depressed value. Upon dilution of 7 S NGF from 2 M NaCl the rapid decay was not observed and the activity remained constant at about the same level as the equilibrium values for the urea-treated and renatured 7 S NGF. A red shift in the fluorescence maximum and a concomitant increase in quantum yield occurred for the 7 S in 8 M urea. Dilution into buffer resulted in a rapid decay and return to native 7 S fluorescence but not when EDTA was included. Both the decay in esterase activity and in fluorescence intensity upon dilution from urea were first order reactions with a rate constant of about 8 X 10(-3) s-1 suggesting that both methods were measuring a unimolecular renaturation process. Renaturation from NaCl or recombination of isolated subunits was much more rapid, indicating a simple combination of subunits in a native conformation. The circular dichroism spectra of urea and NaCl-treated 7 S NGF were different, and spectra of the renatured species with and without EDTA differed. Reassociation to a conformationally different 7 S NGF probably occurs in the presence of EDTA. The results emphasize the role of zinc in 7 S NGF formation and the influence of conformational changes in the effect of beta-NGF on the activity of the gamma-esterase in 7 S NGF.

• Calissano P, Cattaneo A, Biocca S, Aloe L, Mercanti D, Levi-Montalcini R
• The nerve growth factor. Established findings and controversial aspects.
• Exp Cell Res. 1984; 154: 1-9

• Tischler AS, Slayton VW, Costopoulos DS, Leape LL, DeLellis RA, Wolfe HJ
• Nerve growth factor may function as a survival factor for human neuroblastoma cells in culture.
• Cancer. 1984; 54: 1344-7
• Display abstract

• Cells from two human neuroblastomas were found to exhibit a marked dependency on nerve growth factor (NGF) for survival in primary cultures. Cells from both tumors also responded to NGF by forming processes, but survival was not necessarily associated with process outgrowth. Lines of replicating cells could not be obtained from either tumor. NGF-dependent survival has not previously been reported as a characteristic of NGF-responsive human neuroblastoma cells in primary cultures or in cultures of established cell lines. Our findings suggest that tumors which require NGF for survival might constitute a biologically distinctive subset of neuroblastomas, or that NGF might function as a survival factor for some human neuroblastomas only in suboptimal or deleterious environments.

• Howles PN, Dickinson DP, DiCaprio LL, Woodworth-Gutai M, Gross KW
• Use of a cDNA recombinant for the gamma-subunit of mouse nerve growth factor to localize members of this multigene family near the TAM-1 locus on chromosome 7.
• Nucleic Acids Res. 1984; 12: 2791-805
• Display abstract

• The gamma-subunit of mouse 7S nerve growth factor (gamma-NGF) is a member of a family of closely related serine proteases that includes kallikreins and tamases. We have isolated from a DBA/2J male submaxillary gland cDNA library a clone, pSM676, which codes for gamma-NGF. Sequence analysis of the clone shows that it codes for the C-terminal 138 amino acids of the protein plus 23 bases of the 3'-nontranslated portion of the message. The predicted amino acid sequence agrees with that determined by Thomas et al. (1) for the gamma-subunit of nerve growth factor from Swiss Webster mice except for the single, conservative substitution of glutamate for aspartate at amino acid 175. When used as a probe for Southern blot analysis, pSM676 hybridizes to at least twelve fragments of restricted mouse genomic DNA which correspond to several different serine protease genes. Using mouse-hamster hybrid cell lines and recombinant inbred strains of mice, we have demonstrated that all of the genes which show homology to pSM676 are located on mouse chromosome 7, clustered at or near the Tam-1 locus.

• Amano T
• [Nerve growth factors]
• Nippon Rinsho. 1984; 42: 931-46

• Isackson PJ, Ullrich A, Bradshaw RA
• Mouse 7S nerve growth factor: complete sequence of a cDNA coding for the alpha-subunit precursor and its relationship to serine proteases.
• Biochemistry. 1984; 23: 5997-6002
• Display abstract

• Two synthetic oligonucleotides, one 14-mer and one 15-mer, each containing 32 sequences and corresponding to two regions of the partially determined protein sequence, were utilized to identify three cDNA clones coding for the precursor of the alpha-subunit of 7S mouse nerve growth factor (NGF). This library, containing 860 clones, had been preselected from a much larger one by low-stringency hybridization using a cDNA probe corresponding to one of the large family of glandular kallikreins expressed in the adult male mouse submandibular gland. Partial sequence analysis had previously established the alpha-subunit to be a member of this group, albeit with no demonstrable catalytic activity. Nucleotide sequence analysis of the longest of these clones (2A4) predicted the apparent complete amino acid sequence of the 265-residue precursor. One of the other clones (3F2) contained an A----G substitution at position 565 resulting in a Lys----Glu change at position 160 of the mature sequence. These clones probably represent two different alleles. Several amino acid changes, relative to other serine proteases, are evident, which may account for the apparent lack of enzymatic activity. An Arg----Gln substitution at residue -1 would prevent cleavage of the putative activation peptide, and the deletion of residues 2-5 interrupts the highly conserved Ile/Val-Ile/Val-Gly-Gly N-terminal sequence. An Asp----Tyr substitution in the binding pocket and a Gly----His substitution near the active site serine also probably contribute to the inactive structure. The role of this subunit in NGF function remains obscure.

• Rush RA
• Immunohistochemical localization of endogenous nerve growth factor.
• Nature. 1984; 312: 364-7
• Display abstract

• Nerve growth factor (NGF) has been proposed as a trophic molecule essential for the development of sympathetic and primary sensory neurones. In newborn mice and rats, administration of nerve growth factor results in an increase in the number of surviving neurones, whereas administration of antiserum to NGF decreases neuronal survival. Thus it has been proposed that the factor is produced and secreted by the relevant target tissues to provide trophic support for the ingrowing nerves. The site of synthesis of nerve growth factor is still unknown, and it has been emphasized that a precise physiological role for the molecule cannot be ascribed until the cell types that produce it are known. I report here the use of immunohistochemistry to localize endogenous NGF in the rat iris, a tissue in which there is sound biochemical evidence for the production of NGF activity. Surprisingly, the results reveal that NGF can be detected readily in Schwann cells, but not in smooth muscle cells of the iris when it is sympathetically denervated or cultured.

• Ronne H, Anundi H, Rask L, Peterson PA
• 7S Nerve growth factor alpha and gamma subunits are closely related proteins.
• Biochemistry. 1984; 23: 1229-34
• Display abstract

• The polypeptide composition and partial amino acid sequence of the 7S nerve growth factor (NGF) alpha subunit have been determined. Residues in 76 unique positions corresponding to 35% of the molecule were identified. The sequence shows that the NGF alpha subunit is closely related to the NGF gamma subunit and thus a member of the same protein family as the serine proteases. This finding is unexpected since the NGF alpha subunit is devoid of detectable protease activity. However, the NGF alpha subunit differs in one important respect from the NGF gamma subunit and related serine proteases. The highly conserved amino-terminal activation cleavage structure, common to most serine proteases, has been deleted, and an uncleaved activation peptide remains attached to the amino terminus of the mature NGF alpha subunit. It is suggested that this feature is causally related to the apparent lack of proteolytic activity.

• Bradshaw RA et al.
• Receptors for nerve growth factor in responsive peripheral neurons.
• Birth Defects Orig Artic Ser. 1983; 19: 47-59

• Greene LA et al.
• Genomic and non-genomic actions of nerve growth factor in development.
• Prog Brain Res. 1983; 58: 347-57

• Tsuji S, Arita M, Nagai Y
• GQ1b, a bioactive ganglioside that exhibits novel nerve growth factor (NGF)-like activities in the two neuroblastoma cell lines.
• J Biochem (Tokyo). 1983; 94: 303-6
• Display abstract

• To clarify the role of gangliosides in the morphological and biochemical differentiation of neuronal cell cultures, the model cell culture system represented by two neuroblastoma cell lines, GOTO and NB-1, which were established from adrenal gland and metastatic neck lymph node, respectively, was examined. We found that the total ganglioside fraction from human brain had two remarkable effects on these cell lines, which are similar to those of nerve growth factor (NGF): (a) an increase in the cell number, and (b) an increase in the neurite number and the total length of neurites. In these cases, the genuine effector in total gangliosides could not be ascribed to a possibly contaminating NGF-like protein, but rather to a particular molecular species of the gangliosides, GQ1b, which could completely replace the effector function not only qualitatively but also quantitatively. Our results provide direct evidence for the participation of gangliosides in such functions.

• Riopelle RJ, Haliotis T, Roder JC
• Nerve growth factor receptors of human tumors of neural crest origin: characterization of binding site heterogeneity and alteration by theophylline.
• Cancer Res. 1983; 43: 5184-9
• Display abstract

• Heterogeneous populations of saturable specific high-affinity binding sites for the biologically active subunit of the 7S nerve growth factor complex purified from mouse submaxillary gland (NGF) are detected on human tumor cells of neural crest origin. Detailed studies of the melanoma cell line MeWo demonstrate two populations of binding sites of high affinity with dissociation equilibrium constants of Kd 4 X 10(-11) and 5 X 10(-10) M, respectively. Other cell lines of neural crest origin also show high-affinity binding heterogeneity. Exposure of the cell lines to 1 mM theophylline reversibly reduces the number of available NGF binding sites without influencing the affinities of binding. On the MeWo cell line, this is not related to the stages of the cell cycle or to production and release of a NGF-like molecule by the theophylline-exposed cells. These observations complement earlier studies of theophylline-induced alterations in NK cell sensitivity on the MeWo cell line, providing further evidence for cell surface phenotypic changes induced by a compound that promotes differentiation in melanocytes and melanoma cells. Future studies of cell surface phenomena involved in theophylline-induced NGF binding site disappearance may lead to a better understanding of the NGF receptor and its disappearance from certain embryonic cells of neural crest origin during differentiation.

• Bradshaw RA
• What cloned genes can tell us about nerve growth factor.
• Nature. 1983; 303: 751-751

• Ullrich A, Gray A, Berman C, Dull TJ
• Human beta-nerve growth factor gene sequence highly homologous to that of mouse.
• Nature. 1983; 303: 821-5
• Display abstract

• Nerve growth factor (NGF) is thought to have a profound effect on the development and maintenance of sympathetic and embryonic sensory neurones (see refs 1-3 for review). NGF activity isolated from the male mouse submaxillary gland (MSG) consists of three types of subunits, alpha, beta and gamma, which specifically interact to form a 7S, approximately 130,000-molecular weight (Mr) complex. The 7S complex contains two identical 118-amino acid beta-chains, which are solely responsible for the nerve growth-stimulating activity of NGF. While NGF is found in almost all vertebrates, most research has focused on murine NGF, as the mouse male submaxillary gland contains higher levels of this polypeptide than other tissues. Even so, beta-NGF comprises only approximately 0.1% of the protein in this small gland, which has made the study of this polypeptide difficult. The amino acid sequence of the mouse NGF beta-chain has been determined and some information has been obtained regarding the size of a mouse precursor molecule, pro-beta-NGF, but little was known about the structure and relatedness of beta-NGF from other vertebrates. Here we describe the isolation of mouse beta-NGF complementary DNA (cDNA) and present its nucleotide sequence, which predicts a prepro-beta-NGF molecule of Mr 27,000 (27K) and a pro-beta-NGF molecule of Mr 25K. We have used the mouse beta-NGF cDNA clone to isolate the human beta-NGF gene, the coding regions of which are highly homologous to the mouse prepro-beta-NGF nucleotide and amino acid sequences.

• Lumsden AG, Davies AM
• Earliest sensory nerve fibres are guided to peripheral targets by attractants other than nerve growth factor.
• Nature. 1983; 306: 786-8
• Display abstract

• Recent studies have shown that developing nerve fibres grow directly to their targets and are guided by specific cues, but the nature of these cues and the mechanism of guidance remain unknown. The growth of sympathetic axons towards an artificial source of nerve growth factor (NGF) in vivo and of sensory neurites up a concentration gradient of NGF in vitro has supported the hypothesis that NGF, produced by target tissues, acts as a chemotactic attractant for these nerve fibres during development. Both these studies and those of the influence of NGF or target tissues on neurite growth in vitro were conducted late in development when, following target encounter, the neurones had become dependent on NGF or target for survival. Here we have co-cultured embryonic mouse sensory neurones and their peripheral target tissue at a stage preceding their contact in vivo. Neurites grew directly and exclusively towards their own target but not to regionally inappropriate peripheral tissue. Antiserum to isogeneric NGF did not reduce this outgrowth but did reduce undirected neurite outgrowth which occurred in co-cultures of older neurones with denervated target tissue. These results demonstrate that agents other than NGF guide neurites of NGF-responsive neurones in development.

• Scott J, Selby M, Urdea M, Quiroga M, Bell GI, Rutter WJ
• Isolation and nucleotide sequence of a cDNA encoding the precursor of mouse nerve growth factor.
• Nature. 1983; 302: 538-40
• Display abstract

• Nerve growth factor (NGF) is a polypeptide that enhances survival, nerve fibre outgrowth and neurotransmitter biosynthesis in sympathetic and sensory neurones. Administration of antibodies against NGF to developing animals leads to atrophy of the sympathetic system. NGF is not normally detectable in innervated tissues but ablation of the innervating neurones leads to the production of measurable NGF in the target tissue. After transplantation of the denervated tissue, reinnervation occurs, then NGF decreases to undetectable levels. Thus NGF seems to act as a neurotrophic messenger and its level is regulated by innervating neurones. Because of the minute levels present it is very difficult to study NGF biosynthesis in innervated tissue. However, NGF can be isolated from male mouse submaxillary glands, where it exists in inexplicably high levels. Its amino acid sequence has been determined, and the synthesis of NGF and its larger precursors has been demonstrated in cultured submaxillary glands. We report here the nucleotide sequence of a submaxillary cDNA encoding the mouse NGF precursor (preproNGF). In contrast to previous suppositions the NGF moiety is situated near the carboxyterminus of the polyprotein precursor. It is flanked at the amino-terminus by 187 amino acids which may be cleaved at dibasic residues to generate three peptides; there are only two additional amino acids at the carboxy-terminus.

• Boyle MD, Young M
• Nerve growth factor: activation of the classical complement pathway by specific substitution for component C1-.
• Proc Natl Acad Sci U S A. 1982; 79: 2519-22
• Display abstract

• The interaction of homogeneous preparations of mouse submandibular gland nerve growth factor (NGF) with the classical complement pathway was studied. NGF was found to be capable of carrying out the enzyme activities of the first component (C1-) of the classical complement pathway (i.e., the cleavage of zymogen C4 and C2). NGF would not substitute for any other classical pathway component, C2-C9. The C1(-)-like activity of NGF was inhibited by human C1- inactivator. This interaction of NGF with the complement system may account for the previously described ability of NGF to accelerate the rate of contraction of experimentally induced wounds.

• Mukherjee SP, Mukherjee C
• Similar activities of nerve growth factor and its homologue proinsulin in intracellular hydrogen peroxide production and metabolism in adipocytes. Transmembrane signalling relative to insulin-mimicking cellular effects.
• Biochem Pharmacol. 1982; 31: 3163-72
• Display abstract

• Generation of hydrogen peroxide in adipocyte plasma membrane and its intracellular metabolism and regulatory role have been shown by Mukherjee and co-workers to be a major effector system for insulin [Fedn Proc. 35, 1694 (1976); Archs Biochem. Biophys. 184, 69 (1977); Biochem. Pharmac. 27, 2589 (1978); Fedn Proc. 37, 1689 (1978); and Biochem. Pharmac. 29, 1239 (1980)]. The possible involvement of this mechanism in the action of structurally similar polypeptides having some insulin-like metabolic effects was investigated. The beta-subunit of nerve growth factor (2.5 S NGF, mol. wt 13,500) which has a striking structural homology with proinsulin and has been reported to exert certain insulin-like metabolic effects in its own target tissues (e.g. growing neurites and sympathetic ganglia), and the insulin-derived polypeptides, desalanine-insulin and desoctapeptide-insulin, as well as proinsulin, were examined for their effects on rat adipocytes, employing the technique of formate oxidation. Both NGF and proinsulin caused increased [14C]formate oxidation, showing similar intrinsic activities, up to a maximum of 140-160% of the basal rate; insulin increased the rate to 190-210% of the basal rate. The relative potencies of the hormones toward H2O2 formation and stimulation of the pentose phosphate pathway activity were: insulin (EC50: 2.5 x 10(-11) M), desalanine-insulin (EC50: 2.5 x 10(-10) M), proinsulin (EC50: 8 x 10(-9) M), and NGF (EC50: 10(-9) M). The biologically inactive derivative, desoctapeptide-insulin, did not stimulate glucose oxidation, although it caused a small increase in formate oxidation, with an EC50 of 5 x 10(-7) M, indicating a suboptimal level of H2O2 formation in the elevation of the hexose monophosphate shunt activity. 3-Amino-1,2,4-triazole (50 mM), which irreversibly decomposes the peroxidatic compound II of the catalase: H2O2 complex, inhibited formate oxidation to a greater extent in the hormone-treated cells than in the control cells, whereas sodium azide, an inhibitor of the hemoprotein, catalase, completely inhibited it. The abilities of the polypeptides to stimulate H2O2 formation correlated with their abilities to promote lipogenesis from [U-14C]-D-glucose, as expected of insulin. The cellular GSH/GSSG ratio increased concomitantly with the stimulation of glucose oxidation via the shunt, indicating a tight coupling between these processes. The results confirm that the hydrogen peroxide production is a common basis of the metabolic actions of growth-promoting polypeptide hormones or mitogens beyond their respective receptors.

• Yankner BA, Shooter EM
• The biology and mechanism of action of nerve growth factor.
• Annu Rev Biochem. 1982; 51: 845-68

• Nasi S, Cirillo D, Naldini L, Marchisio PC, Calissano P
• Microtubules and microfilaments in fixed and permeabilized cells are selectively decorated by nerve growth factor.
• Proc Natl Acad Sci U S A. 1982; 79: 820-4
• Display abstract

• A specific antibody against nerve growth factor (NGF) and indirect immunofluorescence microscopy have been used to follow the in vitro binding of NGF to cells made permeable to large molecules. All cells tested, both target (sensory neurons and PCI2 cells) and nontarget (3T3, BKH 2I, C6 glioma cells), revealed a decoration of cytoskeletal structures which on the basis of their form, reactivity with antibodies, and sensitivity to specific drugs may be identified as microtubules (MTs) and microfilaments (MFs). The decoration of either structure depends on the fixation and permeabilization conditions: MFs, in the form of stress fibers, are stained by NGF when the plasma membrane is permeabilized with methanol/acetone; MTs become intensely stained when the plasma membrane is solubilized with a nonionic detergent in the presence of a MT-stabilizing medium. The two procedures do not affect the staining of these structures with specific antibodies. Binding of 125I-labeled NGF to PCI2 cells was not competitively inhibited by a 100-fold excess of several positively charged proteins but it was markedly decreased in the presence of DNase I. 125I-Labeled NGF interacted with MTs and F-actin (fixed with paraformaldehyde) in a range of concentrations similar to that used for their cellular localization with NGF-anti-NGF. Our studies show that the specificity and affinity of NGF binding to MTs and MFs is in the range of that of antibodies against tubulin and actin. The possible relevance of these findings to the mechanism of action of NGF in target cells is discussed.

• Levi-Montalcini R
• Developmental neurobiology and the natural history of nerve growth factor.
• Annu Rev Neurosci. 1982; 5: 341-62

• Riemschneider R, Dembele F, Ghouri IA
• [Nerve growth factors, IX. NGF from human submandibular gland]
• Z Naturforsch [C]. 1982; 37: 1045-7

• Williams R, Gaber B, Gunning J
• Raman spectroscopic determination of the secondary structure of crystalline nerve growth factor.
• J Biol Chem. 1982; 257: 13321-3
• Display abstract

• The secondary structure of crystalline nerve growth factor (NGF) has been measured with 90% confidence to contain between 0 and 11% helix and between 53 and 67% antiparallel beta-strand using the intensity distribution of the amide I region of the Raman spectrum. A comparison of spectra collected from crystals and solutions confirms that the secondary structure of NGF is the same in these two states. The helical content of NGF as determined by Raman differs substantially from predictions based on sequence. The amide I band of NGF is substantially different from that of insulin, indicating that no structural hemology is likely to exist between these two proteins.

• Bruni A, Bigon E, Boarato E, Mietto L, Leon A, Toffano G
• Interaction between nerve growth factor and lysophosphatidylserine on rat peritoneal mast cells.
• FEBS Lett. 1982; 138: 190-2

• Hofmann HD, Unsicker K
• The seminal vesicle of the bull: a new and very rich source of nerve growth factor.
• Eur J Biochem. 1982; 128: 421-6

• Levi-Montalcini R
• Nerve growth factor--target cells. A model of choice in the study of neuron differentiation.
• Boll Ist Sieroter Milan. 1982; 61: 164-9

• Harper GP, Glanville RW, Thoenen H
• The purification of nerve growth factor from bovine seminal plasma. Biochemical characterization and partial amino acid sequence.
• J Biol Chem. 1982; 257: 8541-8
• Display abstract

• Nerve growth factor (NGF), a protein regulating the development and function of certain neural crest derivatives, has been purified from bovine seminal plasma, and extremely rich source of the protein. Around 10 mg of pure NGF (yield, 10-20%) can be isolated from 10 g of lyophilized seminal plasma (around 100 ml of semen). The behavior during purification indicates that, like the NGF in the mouse submandibular gland, bovine NGF exists as a high molecular weight complex that dissociates at extremes of pH to reveal a smaller subunit having NGF biological activity. The isolated low molecular weight form of bovine NGF is a dimer of noncovalently linked polypeptide chains (Mr approximately 15,000 on sodium dodecyl sulfate-polyacrylamide gels), with an isoelectric point of 9.5-10. These properties differ from those of low molecular weight (beta-subunit) mouse NGF, which comprises two noncovalently linked peptide chains of Mr = 13,256 (from sequence studies), and which has an isoelectric point of 9.3. The amino acid sequence of the NH2-terminal 26 residues of bovine NGF has been determined and found to be similar to, but not identical with, that of mouse NGF. Thus, residues 3, 9, and 18, which are threonine, methionine, and valine, respectively, in mouse NGF, are serine, arginine, and isoleucine in bovine NGF.

• Butler RH, Revoltella RP
• Antigenic sites on mouse 2.5S nerve growth factor.
• Mol Immunol. 1982; 19: 323-7
• Display abstract

• Cyanogen bromide and tryptic peptides of mouse 2.5S Nerve Growth Factor (NGF) have been used to inhibit competitively binding of rabbit antiserum anti-NGF to native NGF in a solid-phase radioimmunoassay. The results showed that the larger of the two peptides obtained by cyanogen bromide cleavage (amino-acid residues 10-118) was indistinguishable from NGF in this respect, whereas the smaller N-terminal peptide (residues 1-9) was virtually non-inhibitory. Ten peptides were purified from a tryptic digest of the larger cyanogen bromide fragment. One of them, peptide G7 (residues 58-59, 60-69, 104-114, linked by disulfide bridges), was capable of almost full inhibition of binding, though only when used at a concentration about 100 times higher than that necessary to get the same effect with NGF. One other peptide, G10-H1 (residues 70-74), showed some inhibition at relatively high concentrations, but the majority of peptides, including peptide DE-5 which has been shown to be active in the NGF bioassay, were essentially non-inhibitory. A significant co-operative effect was seen when peptides G7 and G10-H1 were used in conjunction. No such effects were observed with any other combination tested.

• Rao AG, Neet KE
• Tryptophan residues of the gamma subunit of 7S nerve growth factor: intrinsic fluorescence, solute quenching, and N-bromosuccinimide oxidation.
• Biochemistry. 1982; 21: 6843-50
• Display abstract

• The environment of tryptophan residues of the gamma subunit derived from the 7S nerve growth factor has been studied by intrinsic fluorescence, solute quenching, and oxidation with N-bromosuccinimide (NBS). The native protein has a fluorescence emission maximum of 345 nm with a tryptophan quantum yield of 0.10. A red shift in the emission maximum occurs between 3 and 6 M urea; a 20% increase in quantum yield occurred at 7-8 M urea. NBS (20-24 mol of NBS/mol of protein) completely oxidized one tryptophan of the gamma subunit, causing a 70% quenching of the intrinsic fluorescence, a complete loss of esterolytic activity toward synthetic substrates, an inability to combine with alpha and beta subunits to re-form the 7S complex, and a shift of about 12% of the structure from beta strands to random coil as determined by circular dichroism. About 80% of the fluorescing tryptophans are accessible to quenching by acrylamide but not to potassium iodide. These results suggest the presence of an exposed tryptophan contributing greater than or equal to 70% of the native fluorescence, located near a negative charge, critical to the esterase activity and possibly to interaction with the beta subunit to re-form 7S NGF.

• Silverman RE, Bradshaw RA
• Nerve growth factor: subunit interactions in the mouse submaxillary gland 7S complex.
• J Neurosci Res. 1982; 8: 127-36
• Display abstract

• Sedimentation velocity analyses of the isolated subunits of mouse 7S nerve growth factor (NGF) and their binary complexes have been performed. The 2.5S (beta) form of NGF binds 2 alpha subunits independent of the presence of gamma subunits. Two gamma subunits are also complexed by 2.5S NGF; however, this interaction is dependent on the presence of a carboxyl terminal arginine residue on the beta polypeptide chain. The partial loss of this residue in 2.5S NGF preparations results in a ternary complex(es) with a reduced sedimentation coefficient(s). The two binary complexes formed (alpha 2-beta and beta-gamma 2) show markedly different pH stability profiles. The alpha and gamma subunits do not form a demonstrable complex, but apparently can interact in the ternary complex to render it more stable than the sum of the alpha-beta and beta-gamma interactions.

• Burton LE, Shooter EM
• The molecular basis of the heterogeneity of the gamma subunit of 7 S nerve growth factor.
• J Biol Chem. 1981; 256: 11011-7
• Display abstract

• The arginine esteropeptidase gamma subunit of the 7 S nerve growth factor complex contains six different molecular species which arise by post-translational modifications. An endopeptidase cleavage of the larger of the two peptide chains in two-chain gamma species produces the three-chain species and introduces a new COOH-terminal lysine residue. The charge heterogeneity of the various species arises from the removal, by a carboxypeptidase B-like enzyme in the submaxillary gland, of this lysine residue and/or the original COOH-terminal arginine residue of the shorter Mr = 10,000 chain. These latter interconversions of the gamma species have been reproduced in vitro using carboxypeptidase B. The significant decrease in the proportion of two-chain gamma species in saliva compared to submaxillary gamma subunit suggests that the endopeptidase cleavage can occur during secretion of saliva; however, the subcellular localization of the enzyme has not been ascertained.

• Thomas KA, Silverman RE, Jeng I, Baglan NC, Bradshaw RA
• Electrophoretic heterogeneity and polypeptide chain structure of the gamma-subunit of mouse submaxillary 7 S nerve growth factor.
• J Biol Chem. 1981; 256: 9147-55
• Display abstract

• The gamma-subunit of the high molecular weight form of mouse submaxillary gland nerve growth factor is known to be electrophoretically heterogeneous. The protein is a mixture of molecules containing either two or three chains, attached covalently by disulfide bonds, that arise from one or two endoproteolytic cleavages. Analysis of the amino acid composition and partial sequence of the resultant fragments has defined the location of the two cleavage sites. Further heterogeneity is generated by partial removal of the carboxyl-terminal basic residues located at the internal cleavage sites, presumably by an endogenous exopeptidase(s) with carboxypeptidase B specificity. Permutations of these endo- and exoproteolytic cleavages generate six possible microheterogeneous forms, which can account for the five major bands observed on electrophoresis at pH 7.05 and the three principal forms seen on isoelectric focusing.

• Wen BG
• [Nerve growth factor: its distribution, chemistry and function (author's transl)]
• Sheng Li Ke Xue Jin Zhan. 1981; 12: 132-8

• Thomas KA, Baglan NC, Bradshaw RA
• The amino acid sequence of the gamma-subunit of mouse submaxillary gland 7 S nerve growth factor.
• J Biol Chem. 1981; 256: 9156-66
• Display abstract

• The complete amino acid sequence of the gamma-subunit of mouse submaxillary gland 7 S nerve growth factor has been determined from analyses of the peptides generated by cyanogen bromide, trypsin, and chymotrypsin from the naturally occurring fragments. All peptides were sequenced automatically in a spinning-cup sequenator using Polybrene to minimize extractive losses by the solvents employed thoughout the degradation cycles. The gamma-subunit, a serine protease with arginine specificity, contains 233 amino acid residues and shares sequence homology with other proteases of this family. The five disulfide bonds of the gamma-subunit are a subset of the six disulfides present in bovine trypsin, as judged by the location of the half-cystine residues in the primary structure. An N-linked carbohydrate side chain is attached to Asn-78 in at least a majority of tee gamma-molecules.

• Barklis E, Perez-Polo JR
• S-180 cells secrete nerve growth factor protein similar to 7S-nerve growth factor.
• J Neurosci Res. 1981; 6: 21-36
• Display abstract

• The nerve growth factor protein (NGF) has been identified by biological assay and rocket immunoelectrophoresis in media conditioned by monolayers of mouse S-180 sarcoma cells, a transformed cell line of salivary gland origin. By utilization of a purification procedure designed to enrich for acid-dissociable, high-molecular-weight complexes similar to the 7S-NGF isolated from male mouse submaxillary gland, it has been determined that a significant portion of mouse sarcoma NGF is initially present as a complex at neutral pH with a molecular weight indistinguishable from that of 7S-NGF. At pH 4.0 the mouse sarcoma NGF complex dissociates, and the active subunit can be isolated as a species with a molecular weight of less than 40,000. The induced dissociation at pH 4.0 of the mouse sarcoma NGF high-molecular-weight complex, as well as the complex's behavior in isoelectric focusing and sucrose gradient sedimentation is consistent with the hypothesis that 7S-NGF is packaged as a subunit containing protein intracellularly prior to secretion into the extracellular space. Moreover, the stability of the mouse sarcoma NGF complex at dilute concentrations is similar to that reported for the purified 7S-NGF complex.

• Ohi T, Imura H, Furukawa S, Hayashi K
• [Radioimmunoassay of nerve growth factor and its state in human serum (author's transl)]
• No To Shinkei. 1981; 33: 595-601

• Aloe L, Cozzari C, Levi-Montalcini R
• The submaxillary salivary glands of the African rodent Praomys (mastomys) natalensis as the richest available source of the nerve growth factor.
• Exp Cell Res. 1981; 133: 475-80

• Rubin JS, Bradshaw RA
• Isolation and partial amino acid sequence analysis of nerve growth factor from the guinea pig prostate.
• J Neurosci Res. 1981; 6: 451-64
• Display abstract

• Nerve growth factor (NGF) has been purified from the guinea pig prostate using a modification of the Bocchini-Angeletti method for isolating 2.5S NGF from mouse submaxillary glands. As with the mouse preparation, guinea pig prostate NGF appears to migrate as a high molecular weight entity at physiological pH. Following dissociation, NGF, active in neurite proliferation assays and similar in size to mouse 2.5S NGF, can be isolated by chromatography on a column of carboxymethyl-cellulose at pH 4.8. Based on gel filtration, SDS-polyacrylamide gel analysis, and amino-terminal sequence studies, this material consists of two, noncovalently linked, identical polypeptide chains each with a molecule weight of about 13,000. The amino-terminal third of the polypeptide chain is at 90% identical to the corresponding region of the murine molecule, confirming the homology of the guinea pig prostate protein to NGFs obtained from different tissues in other species. However, in contrast to the mouse preparation, the putative high molecular weight form of guinea pig NGF does not contain a subunit with arginine esteropeptidase activity. Although there is an abundance of this enzymatic activity in the homogenate, it does not appear to be associated with the fractions containing NGF. This apparent difference in the mouse and guinea pig material is of interest because the mouse gamma subunit, possessing the arginine esteropeptidase activity, has been alleged to participate in the processing of a precursor of the beta NGF polypeptide chain.

• Hofmann HD, Drenckhahn D
• Distribution of nerve growth factor in the submandibular gland of the male and female mouse. A re-examination by use of an improved immunohistochemical procedure.
• Cell Tissue Res. 1981; 221: 77-83
• Display abstract

• Nerve growth factor (NGF) was localized in the mouse submandibular gland by means of indirect immunofluorescence applied to 0.5 micron-thick sections of freeze-dried, plastic-embedded tissue. The antibody to NGF (IgG-fraction) was raised in rabbits immunized with pure 2.5 S NGF from submandibular glands of adult male mice. In the male gland anti-NGF bound selectively to the secretory granules was present in the cells of the granular ducts. Immunoreactive granules extended from the perinuclear region toward the apical pole. In the female gland immunoreactive cells and granules were considerably less abundant than in males. Immunofluorescence was confined to individual secretory cells located in the wall of the granular striated duct. In the present study no support was found for the hypothesis suggesting that immunoreactive NGF is formed within the secretory granules during their transport from the perinuclear region to the apical pole.

• Chapman CA, Banks BE, Vernon CA, Walker JM
• The isolation and characterisation of nerve growth factor from the prostate gland of the guinea-pig.
• Eur J Biochem. 1981; 115: 347-51
• Display abstract

• Nerve growth factor from the guinea-pig prostate gland has been completely purified and characterised. Its chemical and biological properties in vitro are compared with those of nerve growth factor from other sources.

• Harper GP, Thoenen H
• Target cells, biological effects, and mechanism of action of nerve growth factor and its antibodies.
• Annu Rev Pharmacol Toxicol. 1981; 21: 205-29

• Murphy RA, Watson AY, McCarthy M, Papastavros M, Neutra M, Forssmann WG
• Submandibular glands in mice with muscular dystrophy: studies with nerve growth factor.
• Anat Rec. 1981; 200: 177-94
• Display abstract

• Experiments have been carried out to examine the submandibular glands in mice with hereditary muscular dystrophy. Radioimmunoassay data confirm biological studies which show that submandibular glands in mice with muscular dystrophy contain less nerve growth factor (NGF) than glands of normal animals. Male dystrophics have half as much submandibular NGF as unafflicted mice, while females have only 10% of control levels. Gel filtration and electrophoretic studies detect no differences in the molecular properties of NGF in gland extracts from normal and dystrophic mice. Furthermore, NGF from both sources show equal activity in the sensory ganglion bioassay. Together, these results suggest that NGF deficits in submandibular glands of dystrophic mice are not due to measurement artifacts arising from alterations in the structure of the molecule. Morphological studies have uncovered a cytological basis for chemical deficits within submandibular glands of dystrophic mice. Stereological analysis of light and electron microscopic sections revealed that growth factor containing granular tubule cells (GTC) take up a smaller portion of the total gland volume, are smaller in size, and contain fewer secretory granules than comparable cells in glands from controls. Furthermore, the ultrastructure of GTC in dystrophic animals suggests that the cells are less active in producing secretory protein than GTC in glands from normal animals. These results are consistent with the idea that growth factor deficits arise from cellular abnormalities in the granular tubule segment of the gland.

• Vinores SA, Koestner A
• The effect of nerve growth factor on undifferentiated glioma cells.
• Cancer Lett. 1980; 10: 309-18
• Display abstract

• Nerve growth factor (NGF) treatment of rats bearing implanted cells of an anaplastic glioma clone (F-98) led to a decreased tumor growth rate and increased survival time. Similar, but less pronounced, effects were observed if the cells were pretreated with NGF for 24 h before implantation. NGF also reduced the in vitro growth rate of F-98 and contributed to cellular maturation. Several well differentiated cell lines and clones were not visibly affected by NGF using the above parameters. We concluded that NGF has little or no effect on differentiated glioma cells, but significantly affects undifferentiated glioma cells by retarding their growth and inducing maturation.

• Ebendal T, Olson L, Seiger A, Hedlund KO
• Nerve growth factors in the rat iris.
• Nature. 1980; 286: 25-8
• Display abstract

• Nerve growth factor (NGF) activity was not detected by bioassay in irides killed immediately after excision but NGF appeared within 24 h in living irides placed in culture or grafted to a host eye. Furthermore, sensory and, although less effective, sympathetic denervation of irides in situ led within 10 days to the appearance of NGF activity. In addition, freezing and thawing released a parasympathetic neuronotrophic factor activity from irides.

• Sabesan MN
• Secondary structural and "active site" homologies between nerve growth factor and insulin.
• J Theor Biol. 1980; 83: 469-76

• Sabesan MN, Harper ET
• Are aromatic residues essential at the "active sites" of peptide hormones?
• J Theor Biol. 1980; 83: 457-67

• Young M, Koroly MJ
• Nerve growth factor zymogen. Stoichiometry of the active-site serine and role of zinc(II) in controlling autocatalytic self-activation.
• Biochemistry. 1980; 19: 5316-21
• Display abstract

• Mouse submandibular gland nerve growth factor (NGF) is a 116 000 molecular weight protease with a high degree of specificity for certain lysyl and arginyl bonds. Thie protein can activate plasminogen and it is also a member of the general class of serine proteases [Orenstein, N. S., Dvorak, H. A., Blanchard, M. H., & Young, M. (1978) Proc. Natl. Acad. Sci, U.S.A. 75, 5497]. As isolated, NGF is an enzymically inactive zymogen. Upon dilution from high to very low protein concentrations or upon treatment with EDTA, the zymogen undergoes autocatalytic activation. Atomic absorption spectroscopy measurements reveal that NGF contains 1 g-atom of tightly bound Zn(II) per mol. Reaction of the fully autoactivated protease with [3H]DFP yields 1 mol of labeled serine per mol of enzyme. All results indicate that as long as Zn(II) remains bound to the zymogen, autocatalytic activation is inhibited. Removal of this ion, by dilution of the protein or by chelation, initiates autoactivation. The physiologic purpose of this unusual reaction is not known but it may be that Zn(II) serves to act as a control ion which keeps the protein in an inactive form (the zymogen) until it recognizes its naturally occurring substrate.

• Harper GP, Thoenen H
• Nerve growth factor: biological significance, measurement, and distribution.
• J Neurochem. 1980; 34: 5-16

• Aloe L, Levi-Montalcini R
• Comparative studies on testosterone and L-thyroxine effects on the synthesis of nerve growth factor in mouse submaxillary salivary glands.
• Exp Cell Res. 1980; 125: 15-22

• Thoenen H, Barde YA
• Physiology of nerve growth factor.
• Physiol Rev. 1980; 60: 1284-335

• Harper GP, Pearce FL, Vernon CA
• The production and storage of nerve growth factor in vivo by tissues of the mouse, rat, guinea pig, hamster, and gerbil.
• Dev Biol. 1980; 77: 391-402

• Palmer TE, Neet KE
• Subunit interactions of nerve growth factor: sedimentation analysis of the dissociation of the 7 S oligomer promoted by salt and ethylenediaminetetraacetate.
• Arch Biochem Biophys. 1980; 205: 412-21

• Crain SM, Peterson ER, Leibman M, Schulman H
• Dependence on nerve growth factor of early human fetal dorsal root ganglion neurons in organotypic cultures.
• Exp Neurol. 1980; 67: 205-14

• Furukawa S, Hayashi K, Ohi T, Imura H
• Molecular feature of the nerve growth factor in human serum.
• Life Sci. 1980; 26: 837-44

• Li AK, Koroly MJ, Schattenkerk ME, Malt RA, Young M
• Nerve growth factor: acceleration of the rate of wound healing in mice.
• Proc Natl Acad Sci U S A. 1980; 77: 4379-81
• Display abstract

• Earlier studies have shown that removal of the submandibular glands of mice retards the rate of contraction of experimentally induced wounds and that communal licking of wounds accelerates contraction in intact animals [Hutson, J. M., Niall, M., Evans, D. & Fowler, R. (1979) Nature (London) 279, 793-795]. In the light of the observation that nerve growth factor (NGF) is secreted in high concentrations in mouse saliva, we have studied the effect of topically applied high molecular weight nerve growth factor (HMW-NGF) upon the rate of wound contraction in sialoadenectomized animals. Results show that HMW-NGF significantly accelerates the rate of wound contraction and that this phenomenon is probably dependent upon the enzymic activity of the protein. Neither diisopropyl fluorophosphate-inactivated NGF nor 2.5S NGF [isolated according to Bocchini, V. & Angeletti, P. U. (1969) Proc. Natl. Acad. Sci. USA 64, 787-794] displays this biological activity. Thus, it may be that one of the physiological roles of NGF in saliva is to promote wound healing by the licking process.

• Palmer TE, Neet KE
• Subunit interactions in 7S nerve growth factor. Gamma-esterase activity as a probe of the dissociation of the 7S oligomer promoted by salt and EDTA.
• J Biol Chem. 1980; 255: 5170-6

• Vinores SA, Perez-Polo JR
• The effect of nerve growth factor and antibodies to nerve growth factor on ethylnitrosourea carcinogenesis in mice.
• J Cancer Res Clin Oncol. 1980; 98: 59-63
• Display abstract

• The specific induction of neural tumors by the carcinogen, ethylnitrosourea (ENU), can be enhanced by reducing the in vivo nerve growth factor (NGF) levels in mice using IgG directed against the biologically active subunit of NGF (anti-NGF). This effect is reversible, confirming that the altered endogenous NGF levels do return to normal following injection with anti-NGF. Correspondingly, no neural tumors were observed when in vivo NGF levels were elevated by administering exogenous NGF with ENU. The higher physiological levels of NGF in control mice when compared to control rats might explain why fetal administration of ENU to rats results in a greater percentage of neural tumors. This would suggest that the long studied maturation effect that NGF has on developing neural cels of the peripheral nervous system may also influence neural oncogenesis.

• Meyer WJ, Schochet SS, Perez-Polo JR, Werrbach-Perez K, Davis A, Haggard ME
• Cerebral neuroblastoma with elevated nerve growth factor.
• Bull Cancer. 1980; 67: 333-6
• Display abstract

• Cerebral neuroblastoma is often difficult to differentiate by clinical criteria from other central nervous system neoplasm. This report is of a young child with cerebral neuroblastoma and elevated nerve growth factor. This case illustrates the need for further studies into the usefulness of nerve growth factor in differentiating cerebral neuroblastoma from other central nervous system tumors.

• Bradshaw RA, Rubin JS
• Polypeptide growth factors: some structural and mechanistic considerations.
• J Supramol Struct. 1980; 14: 183-99
• Display abstract

• Polypeptide growth factors ae substances that stimulate an increase in cell size and/or cell number during embryonic development. In some cases, they have a similar effect on tissues in the mature organism where they function as "maintenance" factors to sustain cell viability. While their profound impact on cell behavior is well recognized, their relationship to other regulators of cell function has remained generally ill-defined. However, the developing appreciation of their hormone-like behavior suggests that they may be conveniently grouped with many other endocrine agents to form a broader group of secondary hormones. The utility of the classification is illustrated by the insulin-related family of molecules. It also serves to emphasize the similarities in function shared by many of these substances including trophic stimulation and modulation of gene expression. Internalization, though, appears to be another common feature. However, whether the uptake of the growth factor mediates an intracellular action or is designed solely to regulate responsiveness at the cell surface and/or degradation remains an important unanswered question. A brief review of two growth factors (nerve growth factor and epidermal growth factor) serves to outline the possible functions that may be served by this endocytotic process.

• Dunn MF, Pattison SE, Storm MC, Quiel E
• Comparison of the zinc binding domains in the 7S nerve growth factor and the zinc-insulin hexamer.
• Biochemistry. 1980; 19: 718-25

• Young M
• Proteolytic activity of nerve growth factor: a case of autocatalytic activation.
• Biochemistry. 1979; 18: 3050-5
• Display abstract

• Nerve growth factor is a highly specific protease that can convert plasminogen to plasmin and that can hydrolyze certain synthetic N-substituted arginine esters (e.g., Nalpha=p-toluenesulfonyl-L-arginine methyl ester (TAME); N.S. Orenstein et al. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 5497). Hydrolysis of TAME is characterized by a lag phase of lower velocity which precedes development of the steady-state maximal velcity. Kinetic analyses indicate that this behavior stems from autocatalytic activation of a nerve growth factor (NGF)-zymogen by NGF. As isolated from the mouse submandibular gland at high concentration, NGF is largely enzymically inactive. Upon high dilution, the protein undergoes autocatalytic activation with concomitant generation of full enzymic activity. The biologic significance of this unusual property of NGF is not clear, but it may serve to prevent expression of enzymic activity until the protein reaches its target cell(s) or until it recognizes its physiological substrate.

• Levi-Montalcini R
• Trophic, tropic and transforming effects of the nerve growth factor on its target cells.
• Bull Mem Acad R Med Belg. 1979; 134: 217-28

• Levi-Montalcini R, Calissano P
• The nerve-growth factor.
• Sci Am. 1979; 240: 68-77

• Harper GP et al.
• Guinea pig prostate is a rich source of nerve growth factor.
• Nature. 1979; 279: 160-2

• Vinores SA, Perez-Polo JR
• Immunochemotherapy against a nerve growth factor producing tumor (S-180) using anti-NGF IgG as a carrier for adriamycin.
• Bull Cancer. 1979; 66: 386-386

• Walker P, Weichsel ME Jr, Fisher DA, Guo SM, Fisher DA
• Thyroxine increases nerve growth factor concentration in adult mouse brain.
• Science. 1979; 204: 427-9
• Display abstract

• The effects of thyroxine and propylthiouracil on nerve growth factor concentrations in cerebral cortex, cerebellum, and brainstem of adult male mice were assessed by using a sensitive radioimmunoassay for the beta-subunit of mouse nerve growth factor. Thyroxine administration significantly increased the concentration of nerve growth factor in all three brain areas compared to control values, whereas propylthiouracil was without effect. These results suggest that thyroid hormones stimulate nerve growth factor synthesis in the mature central nervous system, and raise the possibility that the influence of thyroid hormones on central nervous system development might be mediated or influenced by nerve growth factor.

• Perkins MS, Margules DL, Ward IL
• Nerve growth factor: intense polydipsia of long duration produced by ventral diencephalic application.
• Brain Res. 1979; 161: 351-5

• Wiche G
• In vitro synthesis of nerve growth factor related glioma C6 cell polypeptides.
• Biochem Biophys Res Commun. 1979; 89: 620-6

• Bradshaw RA
• Nerve growth factor.
• Annu Rev Biochem. 1978; 47: 191-216

• Partlow LM, Wallace LJ, Ellis ME
• Salivary secretion of a substance immunologically identical to the alpha-subunit of 7S nerve growth factor.
• Proc Soc Exp Biol Med. 1978; 158: 342-9

• Carstairs JR, Edwards DC, Pearce FL, Vernon CA, Walter SJ
• Immunogenic contaminants in mouse nerve-growth factor.
• Eur J Biochem. 1977; 77: 311-7
• Display abstract

• Experiments are described that confirm the presence of gamma-globulin in standard preparations of mouse nerve growth factor (7-S complex and beta subunit) and show that antibodies to this protein are present in horse antisera to the growth factor. These antibodies may be partially removed by adsorption with soluble antigen and totally removed by affinity chromatography on columns of insolubilised antigen. These procedures do not affect the potency of the antiserum in vitro or in vivo. Contaminating globulin may be removed from samples of the isolated beta subunit by gel filtration to give an immunochemically pure preparation which will permit more meaningful studies on nerve growth factor and its antiserum.

• Hayashi K, Furukaka S
• [Nerve growth factor (author's transl)]
• Tanpakushitsu Kakusan Koso. 1977; 22: 700-14

• Bradshaw R, Young M
• Nerve growth factor-recent developments and perspectives.
• Biochem Pharmacol. 1976; 25: 1445-9

• Argos P
• Prediction of the secondary structure of mouse nerve growth factor and its comparison with insulin.
• Biochem Biophys Res Commun. 1976; 70: 805-11

• Witzke DJ, Kaye MP
• Myocardial ultrastructural changes induced by administration of nerve growth factor.
• Surg Forum. 1976; 27: 295-7

• Harper GP, Pearce FL, Vernon CA
• Production of nerve growth factor by the mouse adrenal medulla.
• Nature. 1976; 261: 251-3

• Hogue-Angeletti RA, Frazier WA, Jacobs JW, Niall HD, Bradshaw RA
• Purification, characterization, and partial amino acid sequence of nerve growth factor from cobra venom.
• Biochemistry. 1976; 15: 26-34
• Display abstract

• The nerve growth factor (NGF) from Naja naja (cobra) venom has been purified and its structure compared to the NGF from mouse submaxillary gland. A two-step purification procedure has been devised, consisting of a gel filtration step in 1 M acetic acid followed by chromatography of the active pool on carboxymethylcellulose at pH 5. The molecular weight of the native protein was found to be 28000, and this value was reduced by approximately one-half under denaturing conditions. These values are comparable to those obtained for mouse 2.5S or betaNGF. Tryptic peptide maps of S-[14C]carboxymethyl NGF gave the number of labeled peptides expected for a structure composed of two identical or very similar subunits. Thus, the quaternary structures of mouse and cobra NGF are the same. Cyanogen bromide (CNBr) treatment of Naja naja NGF produced three fragments, of which two were purified to homogeneity. These fragments and the whole protein were analyzed in the automated protein Sequencer. The amino-terminal CNBr fragment of the protein was also subjected to digestion by thermolysin and the resultant peptides were purified and characterized. These data plus those from the characterization of the tryptic peptides provided the basis of the construction of a tentative primary structure of Naja naja NGF which is approximately 60% identical with mouse NGF.

• Mobley WC, Schenker A, Shooter EM
• Characterization and isolation of proteolytically modified nerve growth factor.
• Biochemistry. 1976; 15: 5543-52

• Wlodawer A, Hodgson KO, Shooter EM
• Crystallization of nerve growth factor from mouse submaxillary glands.
• Proc Natl Acad Sci U S A. 1975; 72: 777-9
• Display abstract

• Crystals of the nerve growth factor protein were grown by vapor diffusion from ethanol solution. The crystals are hexagonal, belonging to space group P622 (or its enantiomorph) with a equals 56.1 A, c equals 181.4 A, and V equals 494,400 A. The unit cell contains six molecules of dimeric protein and thus has one monomer per asymmetric unit. The diffraction pattern extends to at least 2.7 A, indicating that this crystal form is suitable for structural analysis to near-atomic resolution.

• DiNicola AF
• Nerve-growth factor and lipoatrophic diabetes.
• Am J Clin Nutr. 1975; 28: 1344-5

• Glass RE, Banthorpe DV
• Properties of nerve growth factor from the venom of Bothrops atrox.
• Biochim Biophys Acta. 1975; 405: 23-6
• Display abstract

• A glycoprotein fraction islated in poor yield (approx. 0.04%) from the venom of Bothrops atrox contained nerve growth factor. The material had biological activity, stability, the property of anomalous adsorption onto surfaces, apparent molecular weight and sub-unit structure that were similar to those for nerve growth factor that had been previously purified from the venom of Vipera russelli. Antiserum raised against nerve growth factor from Vipera russelli showed definite cross-reactivity with either the material from Bothrops atrox or a nerve growth factor-containing fraction from the venom of Ancistrodon piscivorus piscivorus, while antiserum against nerve growth factor from mouse had little effect on any of these preparations.

• Radeva V, Taxi J
• [Influence of nerve growth factor (NGF) on formation of synapses in newt embryonic neural tube]
• Arch Anat Microsc Morphol Exp. 1975; 64: 135-47
• Display abstract

• Under the influence of the nerve growth factor (NGF) a general stimulation of the synthetic processes, especially protein synthesis, occurs in the neuroblasts. The morphological counterpart is an hypertrophy of the nucleoli and spiralization of nucleolonema, an increase of the number of ribosomes with the persistence of a specific organization for each stage of development, then the proliferation of the granular endoplasmic reticulum and the modification of the predominant shape of mitochondria, which becomes annular, their outer surface being largely increased. The nutrient stores are quickly exhausted, with an accelerated disintegration of yolk droplets. There is an obvious increase in the number of non-synaptic contacts (puncta adherentia) between processes of neuroblasts and neighbouring nervous structures, and also an acceleration of formation of typical synaptic zones, indicating that NGF stimulates the functional maturation of the nervous system.

• Radeva V, Radev S, Roussev G
• The effect of nerve growth factor in the early stages of nerve tissue formation.
• Agressologie. 1974; 15: 449-53

• Bradshaw RA, Hogue-Angeletti RA, Frazier WA
• Nerve growth factor and insulin: evidence of similarities in structure, function, and mechanism of action.
• Recent Prog Horm Res. 1974; 30: 575-96

• Banks BE, Banthorpe DV, Charlwood KA, Pearce FL, Vernon CA, Edwards DC
• Distribution and immunology of mammalian nerve growth factor.
• Nature. 1973; 246: 503-4

• Weis JS, Weis P
• Nerve growth factor versus insulin.
• Science. 1973; 180: 1301-2

• Valenzuela SA
• [Endocrinous aspects of the physiology of salivary glands. 2. Nerve growth factor (NGF) and its hormonal relation]
• Odontol Chil. 1972; 20: 11-4

• Kumar S, Steward JK, Waghe M
• Nerve-growth factor in human tumours.
• Lancet. 1972; 2: 234-234

• Frazier WA, Angeletti RH, Bradshaw RA
• Nerve growth factor and insulin.
• Science. 1972; 176: 482-8

• Arancibia S
• [Endocrine aspects of the physiology of the salivary glands. II. Nerve growth factor (NGF) and its hormonal relation]
• Rev Med Chil. 1972; 100: 1126-8

• Shooter EM, Varon S
• The biological activities of the subunits of the 7S nerve growth factor protein.
• UCLA Forum Med Sci. 1971; 14: 269-72

• Angeletti RH, Bradshaw RA
• Nerve growth factor from mouse submaxillary gland: amino acid sequence.
• Proc Natl Acad Sci U S A. 1971; 68: 2417-20

• Angeletti RH
• The role of the tryptophan residues in the activity of the nerve growth factor.
• Biochim Biophys Acta. 1970; 214: 478-82

• Nomura J
• [Studies on nerve growth factor]
• Tanpakushitsu Kakusan Koso. 1969; 14: 274-8

• Greene LA, Shooter EM, Varon S
• Enzymatic activities of mouse nerve growth factor and its subunits.
• Proc Natl Acad Sci U S A. 1968; 60: 1383-8

• Yamauchi T
• [The effect of nerve growth factor (NGF) and F-fraction extracted from the bovine submaxillary gland, on the nervous system]
• Seishin Shinkeigaku Zasshi. 1967; 69: 757-69

• Levi-Montalcini R
• The nerve growth factor: its mode of action on sensory and sympathetic nerve cells.
• Harvey Lect. 1966; 60: 217-59

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