NORMAL GENOMIC

• Chan JY, Li L, Miao J, Cai DQ, Lee KK, Chui YL
• Differential expression of a novel gene BRE (TNFRSF1A modulator/BRCC45) inresponse to stress and biological signals.
• Mol Biol Rep. 2010; 37: 363-8
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• Stress-responsive genes play critical roles in many biological functionsthat includes apoptosis, survival, differentiation and regeneration. Wehave identified a novel stress-responsive gene called BRE which interactswith TNF-receptor-1 and blocks the apoptotic effect of TNF-alpha. BREenhances tumor growth in vivo and is up-regulated in hepatocellular andesophageal carcinomas. BRE also regulates the ubiquitination of the DNArepair complex BRCC, and the synthesis of steroid hormones. Here, weexamined BRE-mRNA in cells after treatments with UV and ionizing radiation(IR). UV and IR treatment alone suppressed BRE-mRNA levels by more than90% at 24 h, while hydroxyurea, fluorodeoxyuridine, aphidicolin, knowninhibitors of S-phase DNA synthesis, had no significant effect. BREprotein expression was unaltered in cells treated with TNF-alpha,Interleukin-1 and Dexamethasone, while a threefold increase was observedfollowing chorionic gonadotropin exposure. Although BRE plays a regulatoryrole in many different pathways, yet its expression is apparently undervery stringent control.

• Hamilton DK
• Evidence is found in many domains.
• HERD. 2008; 1: 5-6

• Sharif O, Bolshakov VN, Raines S, Newham P, Perkins ND
• Transcriptional profiling of the LPS induced NF-kappaB response inmacrophages.
• BMC Immunol. 2007; 8: 1-1
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• BACKGROUND: Exposure of macrophages to bacterial products such aslipopolysaccharide (LPS) results in activation of the NF-kappaBtranscription factor, which orchestrates a gene expression programme thatunderpins the macrophage-dependent immune response. These changes includethe induction or repression of a wide range of genes that regulateinflammation, cell proliferation, migration and cell survival. Thisprocess is tightly regulated and loss of control is associated withconditions such as septic shock, inflammatory diseases and cancer. Tostudy this response, it is important to have in vitro model systems thatreflect the behaviour of cells in vivo. In addition, it is necessary tounderstand the natural differences that can occur between individuals. Inthis report, we have investigated and compared the LPS response inmacrophage derived cell lines and peripheral blood mononuclear cell (PBMC)derived macrophages. RESULTS: Gene expression profiles were determinedfollowing LPS treatment of THP-1 cells for 1 and 4 hours. LPSsignificantly induced or repressed 72 out of 465 genes selected as beingknown or putative NF-kappaB target genes, which exhibited 4 temporalpatterns of expression. Results for 34 of these genes, including severalgenes not previously identified as LPS target genes, were validated usingreal time PCR. A high correlation between microarray and real time PCRdata was found. Significantly, the LPS induced expression profile of THP-1cells, as determined using real time PCR, was found to be very similar tothat of human PBMC derived macrophages. Interestingly, some differenceswere observed in the LPS response between the two donor PBMC macrophagepopulations. Surprisingly, we found that the LPS response in U937 cellswas dramatically different to both THP-1 and PBMC derived macrophages.CONCLUSION: This study revealed a dynamic and diverse transcriptionalresponse to LPS in macrophages, involving both the induction andrepression of gene expression in a time dependent manner. Moreover, wedemonstrated that the LPS induced transcriptional response in the THP-1cell line is very similar to primary PBMC derived macrophages. Therefore,THP-1 cells represent a good model system for studying the mechanisms ofLPS and NF-kappaB dependent gene expression.

• Kulman JD, Harris JE, Xie L, Davie EW
• Proline-rich Gla protein 2 is a cell-surface vitamin K-dependent proteinthat binds to the transcriptional coactivator Yes-associated protein.
• Proc Natl Acad Sci U S A. 2007; 104: 8767-72
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• Proline-rich Gla protein 2 (PRGP2) is one of four known vertebratetransmembrane gamma-carboxyglutamic acid (Gla) proteins. Members of thisprotein family are broadly expressed in fetal and adult human tissues andshare a common architecture consisting of a predicted propeptide and Gladomain, a single-pass transmembrane segment, and tandemPro/Leu-Pro-Xaa-Tyr (PY) motifs near their C termini. Using a methodologydeveloped for the regulated expression of enzymatically biotinylatedproteins in mammalian cells, we demonstrate that PRGP2 undergoesgamma-glutamyl carboxylation in a manner that is both dependent upon thepresence of a proteolytically cleavable propeptide and sensitive towarfarin, a vitamin K antagonist that is widely used as an antithromboticagent. When expressed at physiologically relevant levels, the majority ofPRGP2 is present in the gamma-glutamyl carboxylated, propeptide-cleaved(mature) form. We additionally demonstrate, by Western blotting and flowcytometry, that mature PRGP2 is predominantly located on the cell surfacewith the Gla domain exposed extracellularly. In a yeast two-hybrid screenthat used the C-terminal cytoplasmic region of PRGP2 as bait, weidentified the WW domain-containing transcriptional coactivatorYes-associated protein (YAP) as a binding partner for PRGP2. In GSTpull-down experiments, both PRGP2 PY motifs and both YAP WW domains wereessential for complex formation, as were residues proximal to the coresequence of the first PY motif. These findings suggest that PRGP2 may beinvolved in a signal transduction pathway, the impairment of which may bean unintended consequence of warfarin therapy.

• Kim BH et al.
• Inhibitory effect of chroman carboxamide on interleukin-6 expression inresponse to lipopolysaccharide by preventing nuclear factor-kappaBactivation in macrophages.
• Eur J Pharmacol. 2006; 543: 158-65
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• 6-Hydroxy-7-methoxychroman-2-carboxylic acid (3-nitrophenyl)amide(CP-1158) is a synthetic chroman carboxamide with trolox-like chemicalstructure. In the present study, CP-1158 was found to inhibit interleukin(IL)-6 production in lipopolysaccharide (LPS)-stimulated macrophages RAW264.7. The CP-1158 attenuated LPS-induced synthesis of IL-6 transcript butalso inhibited LPS-induced IL-6 promoter activity. Further, CP-1158attenuated LPS-induced syntheses of tumor necrosis factor (TNF)-alpha,IL-1beta, interferon-inducible protein (IP)-10 and macrophage inflammatoryprotein (MIP)-1beta transcripts. Nuclear factor (NF)-kappaB has beenevidenced to play a major mechanism in LPS-induced expression of IL-6 orother inflammatory cytokines. CP-1158 prevented LPS-induced nucleartranslocation of NF-kappaB complex and subsequently inhibited DNA bindingactivity of NF-kappaB complex as well as NF-kappaB transcriptionalactivity in macrophages RAW 264.7. However, CP-1158 did not affectLPS-induced phosphorylation and degradation of inhibitory kappaB(IkappaB). In another experiment, CP-1158 inhibited IL-6 promoter activityelicited by expression vectors encoding NF-kappaB p50 or p65 subunit.Taken together, CP-1158 inhibited LPS-induced expression of inflammatorycytokines including IL-6, targeting NF-kappaB activating pathwaydownstream IkappaB degradation, and thus could provide ananti-inflammatory potential of chroman carboxamide.

• Fotia AB, Ekberg J, Adams DJ, Cook DI, Poronnik P, Kumar S
• Regulation of neuronal voltage-gated sodium channels by theubiquitin-protein ligases Nedd4 and Nedd4-2.
• J Biol Chem. 2004; 279: 28930-5
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• Nedd4 and Nedd4-2 are ubiquitin-protein ligases known to regulate a numberof membrane proteins including receptors and ion transporters. Regulationof the epithelial Na(+) channel by Nedd4 and Nedd4-2 is mediated viainteractions between the PY motifs of the epithelial sodium channelsubunits and the Nedd4/Nedd4-2 WW domains. This example serves as a modelfor the regulation of other PY motif-containing ion channels by Nedd4 andNedd4-2. We found that the carboxyl termini of the six voltage-gated Na(+)(Na(v)) channels contain typical PY motifs (PPXY), and a further Na(v)contains a PY motif variant (LPXY). Not only did we demonstrate byFar-Western analysis that Nedd4 and Nedd4-2 interact with the PYmotif-containing Na(v) channels, but we also showed that these channelshave conserved WW domain binding specificity. We further showed that thecarboxyl termini fusion proteins of one central nervous system and oneperipheral nervous system-derived Na(+) channel (Na(v)1.2 and Na(v)1.7,respectively) are readily ubiquitinated by Nedd4-2. In Xenopus oocytes,Nedd4-2 strongly inhibited the activities of all three Na(v)s (Na(v)1.2,Na(v)1.7, and Na(v)1.8) tested. Interestingly, Nedd4 suppressed theactivity of Na(v)1.2 and Na(v)1.7 but was a poor inhibitor of Na(v)1.8.Our results provide evidence that Nedd4 and Nedd4-2 are likely to be keyregulators of specific neuronal Na(v) channels in vivo.

• Ludes-Meyers JH, Kil H, Bednarek AK, Drake J, Bedford MT, Aldaz CM
• WWOX binds the specific proline-rich ligand PPXY: identification ofcandidate interacting proteins.
• Oncogene. 2004; 23: 5049-55
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• WWOX, the gene that maps to common chromosomal fragile site FRA16D, isfrequently affected by aberrations in multiple types of cancers. WWOXencodes a 46 kDa protein that contains two WW domains and a short-chainoxidoreductase (SDR) domain. We recently demonstrated that ectopicexpression of WWOX inhibits xenograft tumor growth of tumorigenic breastcancer cells. Little is known of the biochemical function(s) of WWOX. TheSDR domain is predicted to be involved in sex-steroid metabolism and theWW domains are likely involved in protein-protein interactions. In thisreport, we identify the specific proline-rich ligand for WWOX as PPXY andshow that the amino-terminal WW domain is responsible for thisinteraction. Using the WWOX WW domains as a probe, we screenedhigh-density protein arrays and identified five candidate-bindingpartners. The binding to one of these candidates, small membrane proteinof the lysosome/late endosome (SIMPLE), was further analysed, and weobserved that a specific PPSY motif in the SIMPLE amino-acid sequence wasrequired to interact with the amino-terminal WW domain of WWOX. Inaddition, immunofluorescence staining demonstrated that endogenous WWOXand SIMPLE co-localize to perinuclear compartments of MCF-7 human breastcancer cells. These studies demonstrate that WWOX contains a Group I WWdomain that binds known cellular proteins containing the specific ligandPPXY. Identification and characterization of WWOX interacting proteinswill lead to an understanding of the biological functions of WWOX innormal and tumor cells.

• Huang Y, Krein PM, Muruve DA, Winston BW
• Complement factor B gene regulation: synergistic effects of TNF-alpha andIFN-gamma in macrophages.
• J Immunol. 2002; 169: 2627-35
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• Complement factor B (Bf) plays an important role in activating thealternative complement pathway. The inflammatory cytokines, in particularTNF-alpha and IFN-gamma, are critical in the regulation of Bf geneexpression in macrophages. In this study, we investigated the mechanismsof Bf gene regulation by TNF-alpha and IFN-gamma in murine macrophages.Northern analysis revealed that Bf mRNA expression was synergisticallyup-regulated by TNF-alpha and IFN-gamma in MH-S cells. Truncations of the5' Bf promoter identified a region between -556 and -282 bp that mediatedTNF-alpha responsiveness as well as the synergistic effect of TNF-alphaand IFN-gamma on Bf expression. Site-directed mutagenesis of aNF-kappaB-binding element in this region (-433 to -423 bp) abrogatedTNF-alpha responsiveness and decreased the synergistic effect of TNF-alphaand IFN-gamma on Bf expression. EMSAs revealed nuclear protein binding tothis NF-kappaB cis-binding element on TNF-alpha stimulation. Supershiftanalysis revealed that both p50 and p65 proteins contribute to inductionof Bf by TNF-alpha. An I-kappaB dominant negative mutant blocked Bfinduction by TNF-alpha and reduced the synergistic induction by TNF-alphaand IFN-gamma. In addition, the proteasome inhibitor MG132, which blocksNF-kappaB induction, blocked TNF-alpha-induced Bf promoter activity andthe synergistic induction of Bf promoter activity by TNF-alpha andIFN-gamma. LPS was found to induce Bf promoter activity through the sameNF-kappaB cis-binding site. These findings suggest that a NF-kappaBcis-binding site between -433 and -423 bp is required for TNF-alpharesponsiveness and for TNF-alpha- and IFN-gamma-stimulated synergisticresponsiveness of the Bf gene.

• Means TK, Pavlovich RP, Roca D, Vermeulen MW, Fenton MJ
• Activation of TNF-alpha transcription utilizes distinct MAP kinasepathways in different macrophage populations.
• J Leukoc Biol. 2000; 67: 885-93
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• Stimulation of macrophages by lipopolysaccharide (LPS) leads to the rapidactivation of MAP kinases (MAPK) and the subsequent induction of cytokinegene expression. We sought to determine whether LPS-inducible cytokinegenes were differentially regulated in macrophages derived from differenttissues. Our studies revealed that PD98059, an inhibitor of theextracellular-regulated kinase (ERK) pathway, blocked LPS-inducedactivation of tumor necrosis factor alpha (TNF-alpha) gene expression in amurine cell line derived from alveolar macrophages but not in anonpulmonary macrophage cell line. These findings were confirmed usingprimary murine alveolar and peritoneal macrophages. This suggests that theTNF-alpha promoter contains MAPK-dependent and -independent regulatoryelements that are used in a cell type-specific manner. We also found thatdifferences in MAPK-regulated signaling were not mediated by NF-KB, LITAF,Egr-1, CREB, or ATF2/ c-Jun. Together, these studies demonstrate thattranscriptional activation of the TNF-alpha gene requires the ERKsignaling cascade in selected macrophage populations.

• Maehara K, Hasegawa T, Isobe KI
• A NF-kappaB p65 subunit is indispensable for activating manganesesuperoxide: dismutase gene transcription mediated by tumor necrosisfactor-alpha.
• J Cell Biochem. 2000; 77: 474-86
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• Expression of the manganese superoxide dismutase (Mn-SOD) is induced bytumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), andlipopolysaccharide (LPS). Recently, a TNF-responsive element (TNFRE) wasidentified within the second intron of the murine Mn-SOD gene. The 5'CCAAT/enhancer binding protein (C/EBP)-related region within the TNFRE wasresponsive to TNF, whereas the 3' NF-kappaB-related region alone was not.This report describes the minimal promoter region of the Mn-SOD gene andinvestigates the cis-acting elements and trans-acting factors responsiblefor TNF-alpha-induced Mn-SOD gene expression. Reporter plasmidtransfection studies demonstrated that inducible transcription factorsenhanced the transcriptional activity of the Mn-SOD gene through theintronic enhancer region. Electrophoretic mobility shift assaysdemonstrated that after TNF-alpha stimulation, p50 and p65 NF-kappaBsubunits bound specifically to the newly identified NF-kappaBtranscription factor-binding site, distinct from the previously describedNF-kappaB site, within the intronic enhancer region. In addition,site-directed mutagenesis and cotransfection studies demonstrated that theNF-kappaB p65 subunit enhanced the transcriptional activity of the Mn-SODgene through the newly identified NF-kappaB site. These results show thata NF-kappaB p65 subunit is mainly involved in the molecular mechanismscontrolling TNF-alpha-mediated Mn-SOD gene transcription.

• Mori K, Stone S, Khaodhiar L, Braverman LE, DeVito WJ
• Induction of transcription factor interferon regulatory factor-1 byinterferon-gamma (IFN gamma) and tumor necrosis factor-alpha (TNF alpha)in FRTL-5 cells.
• J Cell Biochem. 1999; 74: 211-9
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• While it is well known that interferon-gamma (IFN gamma) and tumornecrosis factor-alpha (TNF alpha) play a role in the regulation of thyroidgrowth and differentiated functions, the cellular and molecular mechanismsinvolved in mediating the effects of IFN gamma and TNF alpha on thyroidfunction are unknown. In the present study, we used FRTL-5 rat thyroidcells to examine the effects of IFN gamma and TNF alpha on gene expressionof transcription factor interferon regulatory factor-1 (IRF-1), which isinvolved in mediating the effects of these cytokines in a number of celltypes. Northern blot analysis of FRTL-5 mRNA showed a single IRF-1 mRNA at2.2 Kb. In quiescent FRTL-5 cells, IRF-1 mRNA levels were low butdetectable by Northern analysis. Incubation of FRTL-5 cells with IFN gammaor TNF alpha resulted in a dose- and time-dependent increase in IRF-1 mRNAlevels. We have shown that TNF-alpha and IFN-gamma act synergistically toblock the TSH-induced increase in type I 5'-deiodinase(5'D-I) activity and5'D-I gene expression in FRTL-5 rat thyroid cells. Incubation of FRTL-5cells with IFN gamma and TNF alpha in combination, however, did notsynergistically increase IRF-1 mRNA levels. Electrophoretic mobility shiftassay (EMSA) revealed that IFN gamma induced the formation of a singlecomplex to a IFN gamma activation site (GAS) probe in a dose dependentmanner. Several lines of evidence suggest that TNF alpha activatestranscription factor nuclear factor-kappa B (NF kappa B) throughactivation of protein kinase C (PKC) or the hydrolysis of sphingomyelin toceramide in a number of cell types. Here we demonstrate that hydrolysis ofsphingomyelin to ceramide by sphingomyelinase (SMase), but not activationof PKC by 12-O-tetradecanoylphorbol 13-acetate (TPA), was involved in theactivation of NF kappa B in FRTL-5 cells. Similarly, hydrolysis ofsphingomyelin to ceramide, but not activation of PKC, resulted in anincreased in IRF-1 mRNA levels in FRTL-5 cells. The present datademonstrate that IFN gamma and TNF alpha increase IRF-1 mRNA levels inFRTL-5 cells through activation of GAS and NF kappa B binding proteins,respectively. Thus, our results suggest that upregulation of IRF-1 mayplay a role in mediating the effects of IFN gamma and TNF alpha on thyroidfunction. Our results also suggest that the induction of IRF-1 mRNA by IFNgamma and TNF alpha is not the cellular mechanism involved in thesynergistic effect of these cytokines on thyroid function.

• Bodner SM et al.
• Cloning and chromosomal localization of the gene encoding human cyclinD-binding Myb-like protein (hDMP1).
• Gene. 1999; 229: 223-8
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• The murine transcription factor murine cyclin D-binding Myb-like protein(mDmp1) arrests the cell cycle in G1 phase, through an activity that canbe overridden by direct interaction with the D-type cyclins. Here, wedescribe the identification, sequence, chromosomal localization, andexpression of the human cognate, hDMP1. The hDMP1 cDNA contains a 2280bpopen reading frame that shares a high degree of identity with the mDmp1coding region. The 4.4kb hDMP1 messenger RNA is ubiquitously expressed innormal human tissues, with highest levels in testis and substructureswithin the brain. By use of fluorescence in situ hybridization with ahuman genomic P1 probe, we assigned hDMP1 to chromosome 7, band q21. Thischromosomal region is frequently deleted as part of the 7q-minus andmonosomy 7 abnormalities of human acute myeloid leukemia (AML) andmyelodysplastic syndrome (MDS). We analyzed hDMP1 copy number byfluorescence in situ hybridization in leukemic blasts from nine patientswith abnormalities of the long arm of chromosome 7, and in each case oneallele of the hDMP1 gene was deleted. Functional analysis of the mDmp1protein has shown that it negatively regulates cell proliferation, whichsuggests that this gene is a candidate suppressor of malignanttransformation. Further study will be needed to determine whethergene-specific mutations implicate hDMP1 as a tumor suppressor in acuteleukemias with deletions of the long arm of chromosome 7 or in other typesof human malignancy.

• Mayer H, Salzer U, Breuss J, Ziegler S, Marchler-Bauer A, Prohaska R
• Isolation, molecular characterization, and tissue-specific expression of anovel putative G protein-coupled receptor.
• Biochim Biophys Acta. 1998; 1395: 301-8
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• We isolated a 40 kDa integral membrane protein (p40) from humanerythrocyte ghosts by affinity chromatography, using a C-terminal peptideof stomatin, and obtained partial sequences which enabled us to isolatetwo full-length cDNAs from human bone marrow and fetal brain cDNAlibraries. The cDNA sequences were identical and encoded a novel putativeG protein-coupled receptor (399 amino acids). Northern and RNA dot blotanalyses demonstrated that the major 4.8 kb-transcript is predominantlyexpressed in brain. In situ hybridization studies of tissue sectionsrevealed high expression in neurons of the brain and spinal cord, inthymocytes, megakaryocytes, and macrophages.

• Raabe T, Bukrinsky M, Currie RA
• Relative contribution of transcription and translation to the induction oftumor necrosis factor-alpha by lipopolysaccharide.
• J Biol Chem. 1998; 273: 974-80
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• The synthesis of tumor necrosis factor-alpha has been suggested to beregulated at both the transcriptional and translational levels in responseto stimulation by bacterial lipopolysaccharide, although the relativecontribution of these two mechanisms has not been quantitativelyevaluated. Here, using the murine monocytic cell line RAW 264.7 as a modelsystem, we show that steady-state TNF-alpha mRNA levels increaseapproximately 77-fold following treatment with lipopolysaccharide for 2 hand to a maximum of 164-fold after 8 h as measured by an RNase protectionassay. The TNF-alpha gene transcription rate increases approximately5-fold following exposure to lipopolysaccharide for 2 h as measured by anuclear run-on assay. TNF-alpha mRNA stability did not change in thepresence of lipopolysaccharide. A ribosomal sedimentation assay and an RNAtransfection assay revealed that the translation rate of endogenous aswell as transiently transfected TNF-alpha mRNAs increases onlyapproximately 2-3-fold after stimulation with lipopolysaccharide for 2 h.Taken together, these results suggest that the large increase in the levelof secreted TNF-alpha protein in RAW 264.7 cells is due primarily toactivation of TNF-alpha gene transcription.

• Rocchigiani M et al.
• Human FIGF: cloning, gene structure, and mapping to chromosome Xp22.1between the PIGA and the GRPR genes.
• Genomics. 1998; 47: 207-16
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• We report the identification, structural characterization, and mapping ofthe human FIGF gene. FIGF is the human homologue of mouse figf(c-fos-induced growth factor), a new member of the platelet-derived growthfactor/vascular endothelial growth factor (PDGF/VEGF) family. It codes fora secreted factor with mitogenic and morphogenic activity on fibroblastcells. The predicted amino acid sequence of FIGF is 84% identical to thatof the mouse protein, and it is highly conserved (up to 40%) in thedimerization domain with respect to the VEGF members of the family. The2.5-kb mRNA of FIGF was detected in adult lung and heart tissues. The genespans about 50 kb and is organized into seven exons and six introns. TheFIGF promoter contains an optimal AP-1-binding site and lacks a canonicalTATA box. Fluorescence in situ hybridization mapped FIGF to chromosomalregion Xp22.1. The subsequent identification of YAC positive clones fromthis region allowed us to refine the map and localize FIGF centromeric tothe phosphatidylinositol glycan complementation class A (PIGA) gene andtelomeric to the gastrin-releasing peptide receptor (GRPR) gene. FIGF andPIGA genes lie next to each other in a head-to-tail orientation, with theFIGF polyadenylation signal about 12 kb from the PIGA transcriptionalstart site.

• Galarneau L, Drouin R, Belanger L
• Assignment of the fetoprotein transcription factor gene (FTF) to humanchromosome band 1q32.11 by in situ hybridization.
• Cytogenet Cell Genet. 1998; 82: 269-70

• Bootcov MR et al.
• MIC-1, a novel macrophage inhibitory cytokine, is a divergent member ofthe TGF-beta superfamily.
• Proc Natl Acad Sci U S A. 1997; 94: 11514-9
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• Macrophages play a key role in both normal and pathological processesinvolving immune and inflammatory responses, to a large extent throughtheir capacity to secrete a wide range of biologically active molecules.To identify some of these as yet not characterized molecules, we have useda subtraction cloning approach designed to identify genes expressed inassociation with macrophage activation. One of these genes, designatedmacrophage inhibitory cytokine 1 (MIC-1), encodes a protein that bears thestructural characteristics of a transforming growth factor beta (TGF-beta)superfamily cytokine. Although it belongs to this superfamily, it has nostrong homology to existing families, indicating that it is a divergentmember that may represent the first of a new family within this grouping.Expression of MIC-1 mRNA in monocytoid cells is up-regulated by a varietyof stimuli associated with activation, including interleukin 1beta, tumornecrosis factor alpha (TNF-alpha), interleukin 2, and macrophagecolony-stimulating factor but not interferon gamma, or lipopolysaccharide(LPS). Its expression is also increased by TGF-beta. Expression of MIC-1in CHO cells results in the proteolytic cleavage of the propeptide andsecretion of a cysteine-rich dimeric protein of Mr 25 kDa. Purifiedrecombinant MIC-1 is able to inhibit lipopolysaccharide -inducedmacrophage TNF-alpha production, suggesting that MIC-1 acts in macrophagesas an autocrine regulatory molecule. Its production in response tosecreted proinflammatory cytokines and TGF-beta may serve to limit thelater phases of macrophage activation.

• Polyak K, Xia Y, Zweier JL, Kinzler KW, Vogelstein B
• A model for p53-induced apoptosis.
• Nature. 1997; 389: 300-5
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• The inactivation of the p53 gene in a large proportion of human cancershas inspired an intense search for the encoded protein's physiological andbiological properties. Expression of p53 induces either a stable growtharrest or programmed cell death (apoptosis). In human colorectal cancers,the growth arrest is dependent on the transcriptional induction of theprotein p21WAF1/CIP1 , but the mechanisms underlying the development ofp53-dependent apoptosis are largely unknown. As the most well documentedbiochemical property of p53 is its ability to activate transcription ofgenes, we examined in detail the transcripts induced by p53 expressionbefore the onset of apoptosis. Of 7,202 transcripts identified, only 14(0.19%) were found to be markedly increased in p53-expressing cellscompared with control cells. Strikingly, many of these genes werepredicted to encode proteins that could generate or respond to oxidativestress, including one that is implicated in apoptosis in plant meristems.These observations stimulated additional biochemical and pharmacologicalexperiments suggesting that p53 results in apoptosis through a three-stepprocess: (1) the transcriptional induction of redox-related genes; (2) theformation of reactive oxygen species; and (3) the oxidative degradation ofmitochondrial components, culminating in cell death.

• White AM, Yoshimura T, Smith AW, Westwick J, Watson ML
• Airway inflammation induced by recombinant guinea pig tumor necrosisfactor-alpha.
• Am J Physiol. 1997; 273: 52430-52430
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• We have cloned and expressed recombinant guinea pig tumor necrosisfactor-alpha (gpTNF-alpha) and examined its inflammatory activities aftertracheal instillation in guinea pigs. A 1,071-bp cDNA, including theregion encoding the full-length 234-amino acid gpTNF-alpha protein, wascloned from concanavalin A-stimulated guinea pig splenocytes. The154-amino acid protein corresponding to secreted gpTNF-alpha was expressedas a fusion protein in Escherichia coli, purified by affinitychromatography, and cleaved to yield a 17-kDa protein. gpTNF-alpha had acytotoxic effect on WEHI 164 cells and was detected by goat anti-murinetumor necrosis factor-alpha (TNF-alpha) antibody in Western blots.Intratracheal instillation of gpTNF-alpha (50-150 ng) caused pronouncedand dose-dependent airway eosinophilia. Incubation of gpTNF-alpha withrabbit anti-murine TNF-alpha sera or heating the gpTNF-alpha beforeinstillation reduced bronchoalveolar lavage (BAL) eosinophils to nearcontrol levels. Maximum BAL eosinophilia was observed at 24 h, buteosinophil numbers remained significantly above vehicle-treated animalsfor 72 h. Hence, gpTNF-alpha elicits a pronounced and protractedeosinophil accumulation in the guinea pig lung.

• Everett LM, Li A, Devaraju G, Caperell-Grant A, Bigsby RM
• A novel estrogen-enhanced transcript identified in the rat uterus bydifferential display.
• Endocrinology. 1997; 138: 3836-41
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• Estrogen exerts its physiological effects in the uterus by inducing acascade of transcriptional events; however, the number of genes known tobe directly activated by estrogen in the uterus is small. In this study,immature ovariectomized rats were treated with estrogen or vehicle, and 3h later the uterine horns were flushed to extract epithelial RNA. This RNAwas used in the differential display technique to search forestrogen-responsive genes. Products of reverse transcriptase-PCR, madewith pairs of arbitrary and oligo-deoxythymidine primers, were separatedon denaturing polyacrylamide gels; candidate bands were excised andreamplified to produce probes for use in Northern blot analysis andscreening of a lambda gt10 complementary DNA library made from rat uterus.A novel estrogen-enhanced transcript, designated EET-1, was identifiedfrom a differential display band, and the estrogen sensitivity of itsexpression was verified in Northern analysis. Characterization of EET-1expression in the uterus showed that estrogen treatment resulted in arapid and transient increase in EET-1 messenger RNA; steady state levelspeaked between 2-3 h, returning to basal levels by 6 h. This increase wasnot abolished by pretreatment with cycloheximide, indicating thatinduction of EET-1 is a primary response to estrogen. Induction wasspecific to estrogen when extracts of whole uterus were examined; in theepithelium, there was also a slight response to progesterone. Expressionof the gene was found in all organs surveyed; however, hormonal regulationwas observed only in tissues of the reproductive tract and in the kidney.Analysis of cloned EET-1 complementary DNA revealed a 2008-base sequencethat showed 61% identity with a reported transcript that encodes a proteinthat plays a role in phorbol ester-induced regulation of the tumornecrosis factor-alpha gene. Potential casein kinase-2 and protein kinase Cphosphorylation sites and a cysteine-rich region were identified in theamino acid sequence deduced from EET-1. Thus, it appears that EET-1represents a primary estrogen response gene that may code for aphosphorylated protein involved in gene regulation through a proteinkinase C-activated pathway.

• Neville LF, Abdullah F, Whiteford M, Ovadia P, Feuerstein GZ, Rabinovici R
• Application of differential display to isolate novel genes ininterleukin-2-induced adult respiratory distress syndrome.
• Ann N Y Acad Sci. 1996; 796: 257-71

• Wedlock DN, Aldwell FE, Buddle BM
• Molecular cloning and characterization of tumor necrosis factor alpha(TNF-alpha) from the Australian common brushtail possum, Trichosurusvulpecula.
• Immunol Cell Biol. 1996; 74: 151-8
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• Immune responses in the Australian common brushtail possum (Trichosurusvulpecula) and in particular the role of cytokines are poorly understood.We have undertaken to isolate cytokine genes using reversetranscriptase-polymerase chain reaction (RT-PCR) and in this studydescribe the molecular cloning of TNF-alpha. Primers were designed fromconsensus sequences at the N-terminus end of eutherian mammalian TNF-alphaand the possum cDNA, derived from spleen RNA, identified by RT-PCR. Thecomplete cDNA encoding possum TNF-alpha was amplified from lymphocyte RNAby 5' and 3' rapid amplification of cDNA ends (RACE). The nucleotidesequence of the protein coding region of this cDNA shared 66-69% identitywith other mammalian TNF-alpha genes. The predicted protein of 233 aminoacids shared 56-58% identity with eutherian mammalian TNF-alpha wasexpressed in both Saccharomyces cerevisiae and Escherichia coli byconstructing expression plasmid derivatives of the vectors pYES2 andpGEX-2T respectively. Cell extracts prepared from transformants and thepurified GST/TNF-alpha fusion protein exhibited cytotoxic activity on theTNF-alpha-sensitive murine fibroblast L929 cells and stimulatedproliferation of possum thymocyte cells. The induction of possum TNF-alphamRNA in alveolar macrophages was analysed by RT-PCR using possum-specificTNF-alpha primers. Macrophages cultured in the presence of LPS showedenhanced transcription of TNF-alpha mRNA. This is the first report of thecloning and sequence analysis of the cDNA encoding a marsupial cytokinegene.

• Takashiba S, Shapira L, Amar S, Van Dyke TE
• Cloning and characterization of human TNF alpha promoter region.
• Gene. 1993; 131: 307-8
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• We report the sequence of a 1.2-kb human tumor necrosis factor alpha (TNFalpha) promoter region, which was cloned using PCR. The sequence hasseveral variations from two previous reports and exhibits many potentialDNA-binding sites specific to mammalian gene regulatory proteins inducibleby lipopolysaccharides.

• Tran Van Nhieu J, Misset B, Lebargy F, Carlet J, Bernaudin JF
• Expression of tumor necrosis factor-alpha gene in alveolar macrophagesfrom patients with the adult respiratory distress syndrome.
• Am Rev Respir Dis. 1993; 147: 1585-9
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• The adult respiratory distress syndrome (ARDS) is a complex syndrome inwhich pathogenesis is multifactorial. TNF-alpha, known to be pivotal intissue damage, has been shown to have high levels in blood and alveolarfluid in ARDS. The identification of the cells responsible for thisproduction in the alveolar milieu has not yet been reported. In order toevaluate the TNF-alpha gene expression in ARDS we have analyzed by in situhybridization, using RNA probes, alveolar macrophages (AM) obtained by BALfrom seven patients with ARDS, eight patients with miscellaneousrespiratory diseases, and three control patients. In freshly collected AMfrom patients with ARDS, 66 +/- 14.5% cells expressed the TNF-alpha genewithout in vitro stimulation. This TNF-alpha expression does not resultfrom the BAL procedure itself since only a few unstimulated control AMcontained TNF-alpha mRNA transcripts. TNF-alpha expression in AM is notrestricted to patients with ARDS since it has also been observed inmiscellaneous respiratory diseases; however, this expression is a constantfeature in ARDS. These results demonstrated the major role of AM in theintra-alveolar production of TNF-alpha, and they point out the necessityin ARDS for a specific intra-alveolar therapy.

• Hanna Z et al.
• The Vin-1 gene, identified by provirus insertional mutagenesis, is thecyclin D2.
• Oncogene. 1993; 8: 1661-6
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• The Vin-1 gene was initially identified as a gene whose expression isaltered by the integration of proviruses in the Vin-1 common site ofintegration in retrovirus-induced rodent T-cell leukemias. We have nowisolated the Vin-1 cDNA. Sequencing of the Vin-1 cDNA and Vin-1 exonsrevealed that the proviruses are integrated at the 5' end of the Vin-1gene in an inverse transcriptional orientation. The sequence of the Vin-1gene is identical to that of the recently identified G1-phase cyclin D2gene. The human homolog of the Vin-1/cyclin D2 gene (CCND2) was mapped tochromosome 12, band p13.3, by in situ hybridization, confirming previousmapping data. Our results strongly support a role of the cyclin D2 gene inoncogenesis and thereby implicate altered cell cycle regulation intransformation.

• Briggs JA, Burrus GR, Stickney BD, Briggs RC
• Cloning and expression of the human myeloid cell nuclear differentiationantigen: regulation by interferon alpha.
• J Cell Biochem. 1992; 49: 82-92
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• The human myeloid cell nuclear differentiation antigen (MNDA) is a proteinof 406 amino acids that is expressed specifically in granulocytes,monocytes and earlier stage cells of these lineages. Degenerateoligonucleotides that could encode regions of MNDA amino acid sequencewere used to amplify the MNDA cDNA sequence using the polymerase chainreaction. The amplified cDNA product was sequenced to confirm that itencoded the MNDA protein. It was then used as a probe to isolate fiveclones from a human bone marrow lambda gt10 cDNA library. A clonecontaining a 1,672 base pair cDNA insert was sequenced and found to encodethe entire MNDA open reading frame, as well as 5' and 3' untranslatedregions. The primary structure of the MNDA contains extensive regions ofsequence similarity with the protein products of the interferon-induciblegenes: 204 and interferon regulatory factor 2. In addition, a 12-basesequence matching the interferon-stimulated response element consensussequence [GAAAN(N)GAAA] is located in the 5' untranslated region of theMNDA cDNA. The 1.8 kb MNDA mRNA was detected only in cells that expressthe antigen and the level of MNDA mRNA was elevated in cells treated witheither recombinant or natural interferon alpha. The MNDA mRNA was notinduced by interferon alpha in cells that do not exhibit a constitutivelevel of the MNDA mRNA. The MNDA contains sequence motifs found in generegulatory proteins. The expression and the primary structure of the MNDAindicates that it plays a role in the granulocyte/monocyte cell-specificresponse to interferon.

• McDonald JA
• Applications of cell and molecular biology to pneumonology.
• Schweiz Med Wochenschr. 1991; 121: 89-95
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• I briefly review comments I made concerning recent advances in techniquesof cell and molecular biology, including molecular cloning andcharacterization of genes, gene therapy, and newer applications of thesetechniques to the study of lung disease during the 1990 annual meeting ofthe Swiss Society of Pneumonology. Recent preliminary findings from my ownlaboratory utilizing the example of immunohistochemical and in situhybridization techniques to study connective tissue and cytokine growthfactor expression and remodeling in human pulmonary fibrosis are alsodiscussed.

• Young AJ, Hay JB, Chan JY
• Primary structure of ovine tumor necrosis factor alpha cDNA.
• Nucleic Acids Res. 1990; 18: 6723-6723

• Yamamoto R, Wang A, Vitt CR, Lin LS
• Histidine-15: an important role in the cytotoxic activity of human tumornecrosis factor.
• Protein Eng. 1989; 2: 553-8
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• The amino acids that are required for the cytotoxic activity ofrecombinant human tumor necrosis factor-alpha (TNF) were investigated bychemical modification and oligonucleotide-directed site-specificmutagenesis. TNF contains three histidine residues, located at positions15, 73 and 78. The histidine-specific reagent diethylpyrocarbonate (DEP)was used to chemically modify TNF. The chemical inactivation of the invitro cytotoxic activity of this lymphokine (using murine L929 targetcells) was found to be time- and dose-dependent. Inactivated TNF failed tocompete with fully bioactive [125I]TNF for human MCF-7 target cellreceptors. Mutant polypeptides of TNF were genetically engineered byoligonucoleotide-directed site-specific mutagenesis. The cytotoxicity of adouble histidine mutant, in which histidine-73 and histidine-78 werereplaced with glutamine, was not altered and was chemically inactivated byDEP. Substituting glutamine for histidine-15 resulted in 10-15% of thewild-type bioactivity. Replacing histidine-15 with either asparagine,lysine or glycine resulted in a biologically inactive molecule. The datashow that the histidine residue at position 15 is an amino acid that isrequired for the cytotoxic activity of TNF.

• Soma G et al.
• Biological activities of novel recombinant tumor necrosis factor havingN-terminal amino acid sequences derived from cytotoxic factors produced byTHP-1 cells.
• J Biol Response Mod. 1988; 7: 587-95
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• Eight species of novel recombinant tumor necrosis factor-S (rTNF-SAMgroup) were constructed in which N-terminal amino acid sequences werebased on that of TNF-S from THP-1 cells with higher basicity thanconventional rTNF-alpha. Two of this rTNF-SAM group, denoted as rTNF-SAM1and rTNF-SAM2, showed more cytocidal activity on A549 lung carcinoma cellsand G401 Wilm's tumor cells than did rTNF-alpha. In addition to these celllines, rTNF-SAM1 revealed strong cytocidal activity on T24 bladdercarcinoma cells, which are resistant to rTNF-alpha. Moreover, possiblecachectin activity of rTNF-SAM2 seemed to be lower than that ofconventional rTNF-alpha, suggesting that rTNF-SAM2 has less side effects.Actually, toxicity as expressed by LD50 value of rTNF-SAM2 as well asothers of the rTNF-SAM group was significantly lower than that ofconventional rTNF-alpha. Thus, newly constructed rTNF-SAM1 and rTNF-SAM2should be more promising antitumor reagents for clinical use, since theywere shown to be superior to conventional rTNF-alpha both in antitumoreffect and in less side effects.

• Sastre L et al.
• A partial genomic DNA clone for the alpha subunit of the mouse complementreceptor type 3 and cellular adhesion molecule Mac-1.
• Proc Natl Acad Sci U S A. 1986; 83: 5644-8
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• A genomic clone coding for the alpha subunit of the mouse complementreceptor type 3 and the cellular adhesion molecule Mac-1 has been isolateddirectly from a genomic library using synthetic oligonucleotide probesbased on the amino-terminal amino acid sequence of the protein. Theidentity of the clone has been established by DNA sequencing and in vitrotranslation of hybrid-selected mRNA. The gene is present in a single copyin the murine genome. The region containing the amino-terminal exon hasbeen sequenced. RNA gel blotting shows that the Mac-1 alpha-subunit mRNAis 6 kilobases in length. Mac-1 alpha-subunit mRNA is present inmacrophages but not T lymphoma or L cells. During gammainterferon-stimulated maturation of the mouse premyelocytic cell line M1,Mac-1 alpha-subunit mRNA is induced. This corresponds with the tissuedistribution of the Mac-1 alpha subunit, showing expression is regulatedat least partially at the message level.