Cancer Res, 1997 Aug 1, 57(15), 3135 - 9 Characterization of a high copy number amplification at 6q24 in pancreatic cancer identifies c-myb as a candidate oncogene; Wallrapp C et al.; In a recent study designed to identify chromosomal aberrations in pancreatic cancer tissues using comparative genomic hybridization, a high copy number amplification on 6q was detected . To identify the most likely candidate oncogene, the extension of the amplification in pancreatic cancer tissues and cell lines was determined by Southern blot analysis . Exon trapping was performed with DNA from a yeast artificial chromosome clone containing the complete minimally amplified region . Only fragments from two genes, namely, the c-myb oncogene and a novel gene, were shown to be amplified . The c-myb proto-oncogene was amplified in 10% of the pancreatic carcinoma tissues and in the pancreatic cancer cell line PC2 . Interestingly, the c-myb oncogene was overexpressed not only in the amplified samples but also in the majority of the examined pancreatic cancer tissues and cell lines, suggesting that amplification is only one of the mechanisms leading to overexpression . In contrast, the novel gene, which was called human eRF3b (eukaryotic release factor 3b), seems to be only coamplified with c-myb . Genetic alterations of c-myb were mainly found in advanced tumors, indicating a possible correlation to tumor progression and aggressive tumor phenotypes. J Nutr, 1997 Aug, 127(8), 1445 - 50 Cellular glutathione peroxidase knockout mice express normal levels of selenium-dependent plasma and phospholipid hydroperoxide glutathione peroxidases in various tissues; Cheng WH et al.; Selenium-dependent cellular glutathione peroxidase (GPX1) knockout {GPX1(-)} mice were derived from 129/SVJ x C57BL/6 hybrid mice by microinjecting C57BL/6 blastocysts with recombinant embryonic stem cells carrying a target mutation in the GPX1 gene . Experiment 1 was conducted to determine the effects of the GPX1 knockout on the susceptibility of mice to dietary vitamin E and Se deficiency and on the expression of the Se-dependent plasma glutathione peroxidase (GPX3) and phospholipid hydroperoxide glutathione peroxidase (GPX4), and the Se-independent glutathione S-transferase (GST) . Eleven GPX1(-) and 11 control mice (5 wk old, six males and five females) were fed a Se-deficient, Torula yeast basal diet (0.02 mg Se/kg, no supplemental vitamin E) or the basal diet supplemented with 0.5 mg Se/kg (as Na2SeO3) for 13 wk . Experiment 2 was conducted to determine the effect of the GPX1 knockout on the total Se concentration in the liver of Se-adequate mice . Six GPX1(-) and four control mice (5 wk old, half males and females) were fed the basal diet supplemented with 0.2 mg Se/kg and 15 mg of all-rac-alpha-tocopheryl acetate/kg for 5 wk . There was no difference in body weight gain or apparent susceptibility to dietary vitamin E and Se deficiency between the GPX1(-) and control mice . Knockout of GPX1 resulted in almost complete abolishment of GPX1 activity in various tissues, but had no effect on the GPX3 or GPX4 mRNA level and activity or the GST activity in several tissues at either level of dietary Se . The liver total Se concentration in the Se-adequate GPX1(-) mice was only 42% of that in the controls (P < 0 . 0001) . These results indicate that GPX1 is expressed independently of GPX3 or GPX4 and represents approximately 60% of the total hepatic Se in Se-adequate mice. J Biol Chem, 1997 Aug 1, 272(31), 19538 - 46 Disassembly of RanGTP-karyopherin beta complex, an intermediate in nuclear protein import; Floer M et al.; We previously showed that RanGTP forms a 1:1 complex with karyopherin beta that renders RanGTP inaccessible to RanGAP (Floer, M., and Blobel, G . (1996) J . Biol . Chem . 271, 5313-5316) and karyopherin beta functionally inactive (Rexach, M., and Blobel, G . (1995) Cell 83, 683-692) . Recycling of both factors for another round of function requires dissociation of the RanGTP-karyopherin beta complex . Here we show using BIAcoreTM, a solution binding assay, and GTP hydrolysis and exchange assays, with yeast proteins, that karyopherin beta and RanGTP are recycled efficiently in a reaction that involves karyopherin alpha, RanBP1, RanGAP, and the C terminus of the nucleoporin Nup1 . We find that karyopherin alpha first releases RanGTP from karyopherin beta in a reaction that does not require GTP hydrolysis . The released RanGTP is then sequestered by RanBP1, and the newly formed karyopherin alphabeta binds to the C terminus of Nup1 . Finally, RanGTP is converted to RanGDP via nucleotide hydrolysis when RanGAP is present . Conversion of RanGTP to RanGDP can also occur via nucleotide exchange in the presence of RanGEF, an excess of GDP, and if RanBP1 is absent . Additional nucleoporin domains that bind karyopherin alphabeta stimulate recycling of karyopherin beta and Ran in a manner similar to the C terminus of Nup1. J Biol Chem, 1997 Aug 1, 272(31), 19518 - 24 Molecular cloning of a novel human CC chemokine secondary lymphoid-tissue chemokine that is a potent chemoattractant for lymphocytes and mapped to chromosome 9p13; Nagira M et al.; By searching the Expressed Sequence Tag (EST) data base, we identified partial cDNA sequences potentially encoding a novel human CC chemokine . We determined the entire cDNA sequence which encodes a highly basic polypeptide of 134 amino acids total with a putative signal peptide of 23 amino acids . The predicted mature protein of 111 amino acids has the four canonical cysteine residues and shows 21-33% identity to other human CC chemokines, but has a unique carboxyl-terminal extension of about 30 amino acids which contains two extra cysteine residues . The mRNA was expressed strongly in tissues such as the lymph nodes, Appendix, and spleen . The recombinant protein, which was produced by the baculovirus system and purified to homogeneity, was a highly efficient chemoattractant for certain human T cell lines and a highly potent one for freshly isolated peripheral blood lymphocytes and cultured normal T cells expanded by phytohemagglutinin and interleukin 2 . Unlike most other CC chemokines, however, this novel chemokine was not chemotactic for monocytes or neutrophils, suggesting that it is specific for lymphocytes . From these results, we designated this novel CC chemokine as SLC from secondary lymphoid-tissue chemokine . SLC fused with the secreted form of alkaline phosphatase (SLC-SEAP) was used to characterize the SLC receptor . Binding of SLC-SEAP to freshly isolated lymphocytes was blocked by SLC (IC50, 0.12 nM) but not by any other CC chemokine so far tested, suggesting that resting lymphocytes express a class of receptors highly specific for SLC . By using somatic cell hybrids, radiation hybrids, and selected yeast and bacterial artificial chromosome clones, we mapped the SLC gene (SCYA21) at chromosome 9p13 and between chromosomal markers, D9S1978(WI-8765) and AFM326vd1, where the gene for another novel CC chemokine termed ELC from EBI1-ligand chemokine (SCYA19) also exists . Collectively, SLC is a novel CC chemokine specific for lymphocytes and, together with ELC, constitutes a new group of chemokines localized at chromosome 9p13. J Biol Chem, 1997 Aug 1, 272(31), 19418 - 24 Phosphorylation by p34cdc2 protein kinase regulates binding of the kinesin-related motor HsEg5 to the dynactin subunit p150; Blangy A et al.; The kinesin-related motor HsEg5 is essential for centrosome separation, and its association with centrosomes appears to be regulated by phosphorylation of tail residue threonine 927 by the p34(cdc2) protein kinase . To identify proteins able to interact with the tail of HsEg5, we performed a yeast two-hybrid screen with a HsEg5 stalk-tail construct as bait . We isolated a cDNA coding for the central, alpha-helical region of human p150(Glued), a prominent component of the dynactin complex . The interaction between HsEg5 and p150(Glued) was enhanced upon activation of p34(CDC28), the budding yeast homolog of p34(cdc2), provided that HsEg5 had a phosphorylatable residue at position 927 . Phosphorylation also enhanced the specific binding of p150(Glued) to the tail domain of HsEg5 in vitro, indicating that the two proteins are able to interact directly . Immunofluorescence microscopy revealed co-localization of HsEg5 and p150(Glued) during mitosis but not during interphase, consistent with a cell cycle-dependent association between the two proteins . Taken together, these results suggest that HsEg5 and p150(Glued) may interact in mammalian cells in vivo and that p34(cdc2) may regulate this interaction . Furthermore, they imply that the dynactin complex may functionally interact not only with dynein but also with kinesin-related motors. J Biol Chem, 1997 Aug 1, 272(31), 19236 - 41 p110delta, a novel phosphatidylinositol 3-kinase catalytic subunit that associates with p85 and is expressed predominantly in leukocytes; Chantry D et al.; We have identified a novel p110 isoform of phosphatidylinositol 3-kinase from human leukocytes that we have termed p110delta . In addition, we have independently isolated p110delta from a mouse embryo library on the basis of its ability to interact with Ha-RasV12 in the yeast two-hybrid system . This unique isoform contains all of the conserved structural features characteristic of the p110 family . Recombinant p110delta phosphorylates phosphatidylinositol and coimmunoprecipitates with p85 . However, in contrast to previously described p110 subunits, p110delta is expressed in a tissue-restricted fashion; it is expressed at high levels in lymphocytes and lymphoid tissues and may therefore play a role in phosphatidylinositol 3-kinase-mediated signaling in the immune system. J Biol Chem, 1997 Aug 1, 272(31), 19099 - 102 A novel interaction between adrenergic receptors and the alpha-subunit of eukaryotic initiation factor 2B; Klein U et al.; The alpha-subunit of eukaryotic initiation factor 2B (eIF-2B), a guanine nucleotide exchange protein that functions in regulation of translation, was observed to associate with the carboxyl-terminal cytoplasmic domains of the alpha2A- and alpha2B-adrenergic receptors in a yeast two-hybrid screen of a cDNA library prepared from 293 cells . This protein association was confirmed in vitro by affinity chromatography and was shown to be specific for a subset of G protein-coupled receptors, including the alpha2A-, alpha2B-, alpha2C-, and beta2-adrenergic receptors, but not the vasopressin (V2) receptor . Association of these proteins in vivo was confirmed by specific co-immunoprecipitation of eIF-2Balpha with full-length beta2-adrenergic receptors expressed in transfected 293 cells and by fluorescence microscopy showing co-localization of these proteins in intact cells . Remarkably, eIF-2Balpha co-localized with receptors exclusively in regions of the plasma membrane that are in contact with the extracellular medium, but failed to associate with membranes making cell-cell contacts . Overexpression of eIF-2Balpha in 293 cells caused a small (approximately 15%) but significant enhancement of beta2-adrenergic receptor-mediated activation of adenylyl cyclase, without affecting forskolin or V2 receptor-mediated activation . These observations suggest a new role for a previously identified guanine nucleotide exchange protein in membrane biology and cell signaling. Mol Cell Biol, 1997 Aug, 17(8), 4707 - 17 The product of the murine homolog of the Drosophila extra sex combs gene displays transcriptional repressor activity; Denisenko ON et al.; The heterogeneous nuclear ribonucleoprotein K protein represents a novel class of proteins that may act as docking platforms that orchestrate cross-talk among molecules involved in signal transduction and gene expression . Using a fragment of K protein as bait in the yeast two-hybrid screen, we isolated a cDNA that encodes a protein whose primary structure has extensive similarity to the Drosophila melanogaster extra sex combs (esc) gene product, Esc, a putative silencer of homeotic genes . The cDNA that we isolated is identical to the cDNA of the recently positionally cloned mouse embryonic ectoderm development gene, eed . Like Esc, Eed contains six WD-40 repeats in the C-terminal half of the protein and is thought to repress homeotic gene expression during mouse embryogenesis . Eed binds to K protein through a domain in its N terminus, but interestingly, this domain is not found in the Drosophila Esc . Gal4-Eed fusion protein represses transcription of a reporter gene driven by a promoter that contains Gal4-binding DNA elements . Eed also represses transcription when recruited to a target promoter by Gal4-K protein . Point mutations within the eed gene that are responsible for severe embryonic development abnormalities abolished the transcriptional repressor activity of Eed . Results of this study suggest that Eed-restricted homeotic gene expression during embryogenesis reflects the action of Eed as a transcriptional repressor . The Eed-mediated transcriptional effects are likely to reflect the interaction of Eed with multiple molecular partners, including K protein. J Immunol, 1997 Aug 1, 159(3), 1255 - 64 Identification of a STAT6 domain required for IL-4-induced activation of transcription; Lu B et al.; Tyrosine phosphorylation of STAT6 in response to IL-4 results in the formation of STAT6 homodimers that bind specific DNA elements . Although binding sites for STAT6 have been shown to be important for the function of several IL-4-inducible promoters, the role of STAT6 in this activation has not been defined . To determine whether STAT6 is a transcriptional activator, different portions of the carboxyl terminus of STAT6 were fused to the yeast Gal4 protein DNA binding domain . Analysis of these chimeric Gal4-STAT6 proteins demonstrates that a 140-amino-acid proline-rich region of the carboxyl terminus of STAT6 contains a region that activates transcription . Truncation mutants of STAT6 that lack this domain cannot activate transcription and are capable of repressing transcription stimulated by a wild-type STAT6 protein . Strikingly, the ability of IL-4 to induce transcription from the Ig germline epsilon promoter is suppressed by overexpression of a carboxyl-terminal deletion mutant of STAT6 . These studies demonstrate that the carboxyl terminus of STAT6 contains an activating domain required for the induction of genes by IL-4. J Immunol, 1997 Aug 1, 159(3), 1140 - 9 A novel human CC chemokine PARC that is most homologous to macrophage-inflammatory protein-1 alpha/LD78 alpha and chemotactic for T lymphocytes, but not for monocytes; Hieshima K et al.; By searching the expressed sequence tag (EST) database, we identified partial cDNA sequences encoding a polypeptide with significant sequence identity to the human CC chemokine macrophage-inflammatory protein-1 alpha (MIP-1 alpha)/LD78 alpha . We determined the complete cDNA sequence that contained a reading frame of 89 amino acids with 61% identity to human MIP-1 alpha/LD78 alpha . The mRNA was expressed constitutively at high levels in human lung and at low levels in some lymphoid tissues . Furthermore, the mRNA was strongly induced in several human cell lines, including monocytic U937 cells, by PMA . From these results, we designated this novel CC chemokine as PARC from pulmonary and activation-regulated chemokine . In situ hybridization analyses showed that alveolar macrophages, follicular dendritic cells in the germinal centers of regional lymph nodes, and peripheral blood monocytes stimulated with LPS express PARC mRNA . Using the human CC chemokine yeast artificial chromosome contig that we constructed recently, we mapped the PARC gene (SCYA18) within one of the two subregions of the CC chemokine gene cluster at chromosome 17q11.2 . To investigate its biologic activity, the PARC protein was expressed in insect cells . PARC was chemotactic for both activated (CD3+) T cells and nonactivated (CD14-) lymphocytes, but not for monocytes or granulocytes . Binding analysis using PARC fused with alkaline phosphatase-(His)6 showed the presence of a single class of receptors for PARC on lymphocytes with a Kd of 1.9 nM and 590 sites/cell . Thus, PARC is a novel CC chemokine with a close phylogenic relationship with MIP-1 alpha/LD78 alpha, but with a highly selective activity on lymphocytes. Nucleic Acids Res, 1997 Aug 1, 25(15), 3159 - 63 A branch point consensus from Arabidopsis found by non-circular analysis allows for better prediction of acceptor sites; Tolstrup N et al.; Little knowledge exists about branch points in plants; it has even been claimed that plant introns lack conserved branch point sequences similar to those found in vertebrate introns . A putative branch point consensus sequence for Arabidopsis thaliana resembling the well known metazoan consensus sequence has been proposed, but this is based on search of sequences similar to those in yeast and metazoa . Here we present a novel consensus sequence found by a non-circular approach . A hidden Markov model with a fixed A nucleotide was trained on sequences upstream of the acceptor site . The consensus found by the Markov model shares features with the metazoan consensus, but differs in its details from the consensus proposed earlier . Despite the fact that branch point consensus sequences in plants are weak, we show that a prediction scheme incorporating them leads to a substantial improvement in the recognition of true acceptor sites; the false positive rate being reduced by a factor of 2 . We take this as an indication that the consensus found here is the genuine one and that the branch point does play a role in the proper recognition of the acceptor site in plants. Nucleic Acids Res, 1997 Aug 1, 25(15), 3135 - 42 Cellular distribution of mammalian DNA topoisomerase II is determined by its catalytically dispensable C-terminal domain; Adachi N et al.; Mammalian cells express two genetically distinct isoforms of DNA topoisomerase II, designated topoisomerase IIalphaand topoisomerase IIbeta . We have recently shown that mouse topoisomerase IIalpha can substitute for the yeast topoisomerase II enzyme and complement yeast top2 mutations . This functional complementation allowed functional analysis of the C-terminal domain (CTD) of mammalian topoisomerase II, where the amino acid sequences are divergent and species-specific, in contrast to the highly conserved N-terminal and central domains . Several C-terminal deletion mutants of mouse topoisomerase IIalpha were constructed and expressed in yeast top2 cells . We found that the CTD of topoisomerase IIalphais dispensable for enzymatic activity in vitro but is required for nuclear localization in vivo . Interestingly, the CTD of topoisomerase IIbetawas also able to function as a signal for nuclear targeting . We therefore examined whether the CTD alone is sufficient for nuclear localization in vivo . The C-terminal region was fused to GFP (green fluorescent protein) and expressed under the GAL1 promoter in yeast cells . As expected, GFP signal was exclusively detected in the nucleus, irrespective of the CTD derived from either topoisomerase IIalphaor IIbeta . Surprisingly, when the upstream sequence of each CTD was added nuclear localization of the GFP signal was found to be cell cycle dependent: topoisomerase IIalpha-GFP was seen in the mitotic nucleus but was absent from the interphase nucleus, while topoisomerase IIbeta-GFP was detected predominantly in the interphase nucleus and less in the mitotic nucleus . Our results suggest that the catalytically dispensable CTD of topoisomerase II is sufficient as a signal for nuclear localization and that yeast cells can distinguish between the two isoforms of mammalian topoisomerase II, localizing each protein properly. J Virol, 1997 Aug, 71(8), 6194 - 9 Interaction of the UV-damaged DNA-binding protein with hepatitis B virus X protein is conserved among mammalian hepadnaviruses and restricted to transactivation-proficient X-insertion mutants; Sitterlin D et al.; We carried out a comparative analysis of several proposed host protein partners of the human hepatitis B virus X protein (HBx) using both the GAL4- and the LexA-based yeast two-hybrid system . We showed that the interaction of HBx with the UV-damaged DNA-binding protein (UVDDB) is positive in both yeast systems, detectable in cotransfected human cells, conserved by rodent hepadnavirus X proteins (known to transactivate in human cells), and tightly correlated with the transactivation proficiency of X-insertion mutants . Taken together, our results strongly suggest that UVDDB is involved in X-mediated transactivation. J Neurosci, 1997 Aug 1, 17(15), 5687 - 96 Characterization of guanylate kinase-associated protein, a postsynaptic density protein at excitatory synapses that interacts directly with postsynaptic density-95/synapse-associated protein 90; Naisbitt S et al.; The structure of central synapses is poorly understood at the molecular level . A recent advance came with the identification of the postsynaptic density-95 (PSD-95)/synapse-associated protein 90 family of proteins as important mediators of the synaptic clustering of certain classes of ion channels . By yeast two-hybrid screening, a novel protein termed guanylate kinase-associated protein (GKAP) has been isolated that binds to the GK-like domain of PSD-95 () . Here we present a detailed characterization of GKAP expression in the rat brain and report the cloning of a novel GKAP splice variant . By Northern blot, GKAP mRNAs (4, 6.5, and 8 kB) are expressed predominantly in the rat brain . By in situ hybridization, GKAP is expressed widely in neurons of cortex and hippocampus and in the Purkinje and granule cells of the cerebellum . On brain immunoblots, two prominent bands of 95 and 130 kDa are detected that correspond to products of short and long N-terminal splice variants of GKAP . Two independent GKAP antibodies label somatodendritic puncta in neocortical and hippocampal neurons in a pattern consistent with synaptic elements . Immunogold electron microscopy reveals GKAP to be predominantly postsynaptic and present at asymmetric synapses and in dendritic spines . The distribution of GKAP immunogold particles is uniform in the lateral plane of the PSD but peaks in the perpendicular axis approximately 20 nm from the postsynaptic membrane . In cultured hippocampal neurons GKAP immunoreactive puncta colocalize with the AMPA receptor subunit Glu receptor 1 but not with the GABAA receptor subunits beta2 and beta3 . Thus GKAP is a widely expressed neuronal protein localized specifically in the PSD of glutamatergic synapses, consistent with its direct interaction with PSD-95 family proteins. Gene, 1997 Jul 31, 194(2), 277 - 82 Isolation of a mouse cDNA encoding mSTI1, a stress-inducible protein containing the TPR motif; Blatch GL et al.; We report the isolation and sequencing of the complete 2079-bp cDNA fragment encoding mSTI1, a murine stress-inducible protein . The predicted ORF encodes a protein of 543 amino acids (aa) and Mr 62,582 . The predicted protein has significant homology to stress-inducible proteins from humans (IEF SSP 3521), soybean (GMSTI), yeast (STI1) and a parasite, Leishmania donovani (LSIP) . All of these proteins contain 34-aa repeat motifs, termed tetratricopeptide repeats (TPRs), that are proposed to be involved in intra- and intermolecular protein interactions . mSTI1 has ten potential TPR motifs, a putative nuclear localization signal (NLS), six potential phosphorylation sites for casein kinase II and a central proline-rich region . Western analysis detected a protein of approx . 63 kDa in all the major mouse organs and in mouse, monkey and human cell lines. Nature, 1997 Jul 31, 388(6641), 492 - 5 Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells; Grawunder U et al.; Mutation of the XRCC4 gene in mammalian cells prevents the formation of the signal and coding joints in the V(D)J recombination reaction, which is necessary for production of a functional immunoglobulin gene, and renders the cells highly sensitive to ionizing radiation . However, XRCC4 shares no sequence homology with other proteins, nor does it have a biochemical activity to indicate what its function might be . Here we show that DNA ligase IV co-immunoprecipitates with XRCC4 and that these two proteins specifically interact with one another in a yeast two-hybrid system . Ligation of DNA double-strand breaks in a cell-free system by DNA ligase IV is increased fivefold by purified XRCC4 and seven- to eightfold when XRCC4 is co-expressed with DNA ligase IV . We conclude that the biological consequences of mutating XRCC4 are primarily due to the loss of its stimulatory effect on DNA ligase IV: the function of the XRCC4-DNA ligase IV complex may be to carry out the final steps of V(D)J recombination and joining of DNA ends. J Cell Biol, 1997 Jul 28, 138(2), 283 - 90 A novel Rab9 effector required for endosome-to-TGN transport; Diaz E et al.; Rab9 GTPase is required for the transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network in living cells, and in an in vitro system that reconstitutes this process . We have used the yeast two-hybrid system to identify proteins that interact preferentially with the active form of Rab9 . We report here the discovery of a 40-kD protein (p40) that binds Rab9-GTP with roughly fourfold preference to Rab9-GDP . p40 does not interact with Rab7 or K-Ras; it also fails to bind Rab9 when it is bound to GDI . The protein is found in cytosol, yet a significant fraction (approximately 30%) is associated with cellular membranes . Upon sucrose density gradient flotation, membrane- associated p40 cofractionates with endosomes containing mannose 6-phosphate receptors and the Rab9 GTPase . p40 is a very potent transport factor in that the pure, recombinant protein can stimulate, significantly, an in vitro transport assay that measures transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network . The functional importance of p40 is confirmed by the finding that anti-p40 antibodies inhibit in vitro transport . Finally, p40 shows synergy with Rab9 in terms of its ability to stimulate mannose 6-phosphate receptor transport . These data are consistent with a model in which p40 and Rab9 act together to drive the process of transport vesicle docking. Biochim Biophys Acta, 1997 Jul 25, 1327(2), 204 - 12 Cloning and molecular characterization of a voltage-dependent anion-selective channel (VDAC) from Drosophila melanogaster; Ryerse J et al.; A full length voltage-dependent anion-selective channel (VDAC) cDNA was cloned from Drosophila melanogaster by expression library screening using an antibody against an insect VDAC protein . The cDNA clone (denoted DmVDAC) is 1082 base pairs (bp) in length and contains an open reading frame (bp 62-907) encoding a 282 amino acid protein which has a predicted molecular mass of 30550 Da, a predicted pI of 6.98 and no cysteines . Hydrophobicity analysis suggests 15 or 16 membrane-spanning domains . The DmVDAC amino acid sequence has variable homology with VDACs from other species ranging from 62% identity with a human VDAC to 23% identity with a Dictyostelium discoideum VDAC . DmVDAC has 92% identity with the 38 conserved residues in a VDAC consensus sequence . DmVDAC was expressed in VDAC-null yeast but failed to rescue viability . DmVDAC has 88% identity at the amino acid level and 99% identity at the nucleic acid level with a recently reported D . melanogaster VDAC sequence (A . Messina et al., FEBS Lett . 384 (1996) 9-13) . Homology analyses with the Messina and other VDAC sequences indicate that the amino acid differences are due to minor errors in the Messina sequence . Southern blots and chromosomal in situ hybridizations suggest a single VDAC gene occurs in the fly with a locus at 32B on the left arm of the second chromosome. Cell, 1997 Jul 25, 90(2), 373 - 83 Identification and characterization of an IkappaB kinase; Regnier CH et al.; Activation of the transcription factor NF-kappaB by tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the NF-kappaB-inducing kinase (NIK) . In a yeast two-hybrid screen for NIK-interacting proteins, we have identified a protein kinase previously known as CHUK . Overexpression of CHUK activates a NF-kappaB-dependent reporter gene . A catalytically inactive mutant of CHUK is a dominant-negative inhibitor of TNF-, IL-1-, TRAF-, and NIK-induced NF-kappaB activation . CHUK associates with the NF-kappaB inhibitory protein, IkappaB-alpha, in mammalian cells . CHUK specifically phosphorylates IkappaB-alpha on both serine 32 and serine 36, modifications that are required for targeted degradation of IkappaB-alpha via the ubiquitin-proteasome pathway . This phosphorylation of IkappaB-alpha is greatly enhanced by NIK costimulation . Thus, CHUK is a NIK-activated IkappaB-alpha kinase that links TNF- and IL-1-induced kinase cascades to NF-kappaB activation. J Biol Chem, 1997 Jul 25, 272(30), 18966 - 73 The murine voltage-dependent anion channel gene family . Conserved structure and function; Sampson MJ et al.; Voltage-dependent anion channels (VDACs) are pore-forming proteins found in the outer mitochondrial membrane of all eucaryotes . VDACs are the binding sites for several cytosolic enzymes, including the isoforms of hexokinase and glycerol kinase . VDACs have recently been shown to conduct ATP when in the open state, allowing bound kinases preferential access to mitochondrial ATP and providing a possible mechanism for the regulation of adenine nucleotide flux . Two human VDAC cDNAs have been described previously, and we recently reported the isolation of mouse VDAC1 and VDAC2 cDNAs, as well as a third novel VDAC cDNA, designated VDAC3 . In this report we describe the structural organization of each mouse VDAC gene and demonstrate that, based on conserved exon/intron boundaries, the three VDAC isoforms belong to a single gene family . The 5'-flanking region of each VDAC gene was shown to have transcription promoter activity by transient expression in cultured cells . The promoter region of each VDAC isoform lacks a canonical TATA box, but all are G+C-rich, a characteristic of housekeeping gene promoters . To examine the conservation of VDAC function, each mouse VDAC was expressed in yeast lacking the endogenous VDAC gene . Both VDAC1 and VDAC2 are able to complement the phenotypic defect associated with the mutant yeast strain . VDAC3, however, is only able to partially complement the mutant phenotype, suggesting an alternative physiologic function for the VDAC3 protein. J Biol Chem, 1997 Jul 25, 272(30), 18608 - 13 ATP-binding properties of human Hsp90; Scheibel T et al.; Hsp90 is one of the most abundant proteins in the cytosol of eukaryotic cells . Under physiological conditions Hsp90 has been shown to play a major role in several specific signaling pathways, including maturation of various kinases and maintenance of steroid receptors in an activable state . It is well established that the level of Hsp90 increases severalfold under stress conditions, and it has been shown that the chaperone function of Hsp90 is ATP-independent . Although yeast Hsp90 does not bind ATP, as determined by a number of methods monitoring tight binding, ATP-dependent functions of Hsp90 in the presence of co-factors and elevated temperatures are still under discussion . Here, we have reinvestigated ATP-binding properties and ATPase activity of human Hsp90 under various conditions . We show that human Hsp90 does not bind ATP tightly and does not exhibit detectable ATPase activity . However, using electron spin resonance spectroscopy, weak binding of spin-labeled ATP analogues with half-maximal binding at 400 microM ATP was detected . The functional significance of this weak interaction remains enigmatic. J Biol Chem, 1997 Jul 25, 272(30), 18522 - 5 Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity; Ju H et al.; Endothelial nitric-oxide synthase (eNOS) and caveolin-1 are associated within endothelial plasmalemmal caveolae . It is not known, however, whether eNOS and caveolin-1 interact directly or indirectly or whether the interaction affects eNOS activity . To answer these questions, we have cloned the bovine caveolin-1 cDNA and have investigated the eNOS-caveolin-1 interaction in an in vitro binding assay system using glutathione S-transferase (GST)-caveolin-1 fusion proteins and baculovirus-expressed bovine eNOS . We have also mapped the domains involved in the interaction using an in vivo yeast two-hybrid system . Results obtained using both in vitro and in vivo protein interaction assays show that both N- and C-terminal cytosolic domains of caveolin-1 interact directly with the eNOS oxygenase domain . Interaction of eNOS with GST-caveolin-1 fusion proteins significantly inhibits enzyme catalytic activity . A synthetic peptide corresponding to caveolin-1 residues 82-101 also potently and reversibly inhibits eNOS activity by interfering with the interaction of the enzyme with Ca2+/calmodulin (CaM) . Regulation of eNOS in endothelial cells, therefore, may involve not only positive allosteric regulation by Ca2+/CaM, but also negative allosteric regulation by caveolin-1. Proc Natl Acad Sci U S A, 1997 Jul 22, 94(15), 7862 - 7 Direct involvement of the ubiquitin-conjugating enzyme Ubc9/Hus5 in the degradation of IkappaBalpha; Tashiro K et al.; The NF-kappaB/Rel proteins are sequestered in the cytoplasm in association with IkappaBalpha . In response to external signals, IkappaBalpha is phosphorylated, multi-ubiquitinated, and degraded by proteasomes, thereby releasing NF-kappaB/Rel proteins to migrate to the nucleus . We have cloned a mouse ubiquitin-conjugating enzyme (mE2), which associates with IkappaBalpha . mE2 is homologous to the yeast Ubc9/Hus5 ubiquitin-conjugating enzyme . A transdominant-negative mutant of mE2 had no effect on phosphorylation of IkappaBalpha, but delayed its degradation . Correspondingly, tumor necrosis factor-alpha-inducible NF-kappaB activity was diminished . We propose that mE2 is directly involved in the ubiquitin conjugation of IkappaBalpha, a pivotal step in its degradation pathway. Proc Natl Acad Sci U S A, 1997 Jul 22, 94(15), 7831 - 6 Association of the mammalian helicase MAH with the pre-mRNA splicing complex; Molnar GM et al.; Conversion of pre-mRNAs into mature mRNAs includes several consecutive enzymatic modification steps that are carried out in the spliceosomes . Helicases have been shown to contribute to these catalytic processes both in yeast and in mammalian cells . Our results identify the mammalian protein MAH (matrix-associated helicase) as a new helicase present in the spliceosome complex . Sequence comparison describes MAH as the first higher eukaryotic member of the helicase superfamily I, with demonstrated enzymatic activity . Because MAH does not bind small nuclear ribonucleoproteins (snRNPs), it appears to be a non-snRNP binding factor of the splicing complex . In conclusion, our data suggest the involvement of MAH in processing of pre-mRNAs in mammalian cells. FEBS Lett, 1997 Jul 21, 412(1), 102 - 6 Interaction of Fas(Apo-1/CD95) with proteins implicated in the ubiquitination pathway; Becker K et al.; Fas(Apo-1/CD95), a receptor belonging to the tumor necrosis factor receptor family, induces apoptosis when triggered by Fas ligand . Upon its activation, the cytoplasmic domain of Fas binds several proteins which transmit the death signal . We used the yeast two-hybrid screen to isolate Fas-associated proteins . Here we report that the ubiquitin-conjugating enzyme UBC9 binds to Fas at the interface between the death domain and the membrane-proximal region of Fas . This interaction is also seen in vivo . UBC9 transiently expressed in HeLa cells bound to the co-expressed cytoplasmic segment of Fas . FAF1, a Fas-associated protein that potentiates apoptosis (Chu et al . (1996) Proc . Natl . Acad . Sci . USA 92, 11894-11898), was found to contain sequences similar to ubiquitin . These results suggest that proteins related to the ubiquitination pathway may modulate the Fas signaling pathway. J Exp Med, 1997 Jul 21, 186(2), 209 - 20 Potential immunocompetence of proteolytic fragments produced by proteasomes before evolution of the vertebrate immune system; Niedermann G et al.; To generate peptides for presentation by major histocompatibility complex (MHC) class I molecules to T lymphocytes, the immune system of vertebrates has recruited the proteasomes, phylogenetically ancient multicatalytic high molecular weight endoproteases . We have previously shown that many of the proteolytic fragments generated by vertebrate proteasomes have structural features in common with peptides eluted from MHC class I molecules, suggesting that many MHC class I ligands are direct products of proteasomal proteolysis . Here, we report that the processing of polypeptides by proteasomes is conserved in evolution, not only among vertebrate species, but including invertebrate eukaryotes such as insects and yeast . Unexpectedly, we found that several high copy ligands of MHC class I molecules, in particular, self-ligands, are major products in digests of source polypeptides by invertebrate proteasomes . Moreover, many major dual cleavage peptides produced by invertebrate proteasomes have the length and the NH2 and COOH termini preferred by MHC class I . Thus, the ability of proteasomes to generate potentially immunocompetent peptides evolved well before the vertebrate immune system . We demonstrate with polypeptide substrates that interferon gamma induction in vivo or addition of recombinant proteasome activator 28alpha in vitro alters proteasomal proteolysis in such a way that the generation of peptides with the structural features of MHC class I ligands is optimized . However, these changes are quantitative and do not confer qualitatively novel characteristics to proteasomal proteolysis . The data suggest that proteasomes may have influenced the evolution of MHC class I molecules. Gene, 1997 Jul 18, 194(1), 107 - 13 A compositional map of the cen-q21 region of human chromosome 21; De Sario A et al.; A compositional map of the centromere and of the subcentromeric region of the long arm of human chromosome 21 was established by determining the GC levels (GC is the molar fraction of guanine+cytosine in DNA) of 11 YACs (yeast artificial chromosomes) covering this 13-14 Mb region which extends from the alpha-satellite sequences of the C(entromeric) band q11.1, through R(everse) band q11.2, to the proximal part of G(iemsa) band q21 . The entire region is made up of GC-poor, or L, isochores with only one GC-rich H1 isochore, at least 2 Mb in size, located in band q21 . The almost identical GC levels of the centromeric alpha-satellite repeats (38.5%), of R band q11.2 (39%), and of G bands (38-40%) provide a direct demonstration that base composition cannot be the only cause of the cytogenetic differences between C, G, and the majority of R bands, namely the H3- R bands (which do not contain the GC-richest H3 isochores) . The results obtained also show that isochores may be as long as 6 Mb, at least in the GC-poor regions of the genome, and support previous observations suggesting that YACs from isochore borders are unstable and/or difficult to clone . Genes and CpG islands are very rare in the GC-poor region investigated, as expected from the fact that their concentration is proportional to the GC levels of the isochores in which they are contained. Biochim Biophys Acta, 1997 Jul 17, 1353(1), 39 - 49 Human gene for the RNA polymerase II seventh subunit (hsRPB7): structure, expression and chromosomal localization; Schoen TJ et al.; The human gene for the seventh largest subunit of RNA polymerase II complex, hsRPB7 was cloned, sequenced and mapped . This complex is an integral part of the transcription-coupled DNA repair mechanism and has been shown to be involved in several human genetic diseases and implicated in many others . The hsRPB7 gene consists of 8 exons and spans approximately 5.1 kb . Southern blots of genomic and cloned DNA suggest that hsRPB7 is coded for by a single gene . Using human radiation hybrids and YACs, the gene was localized to 11q13.1, within 70 kb of marker D11S1765 . The sequence of the 5' flanking region does not contain a TATA element, but does contain several Sp1 binding sites, an AP-1 site and a novel inverted polymorphic GATA tandem repeat . This novel GATA repeat can be used for linkage analysis . The hsRPB7 gene seems to be highly conserved among eukaryotic species, showing general sequence conservation to yeast and Drosophila . Northern blot analysis reveals a high degree of tissue-specific expression . For example, adult retina, brain and kidney exhibit a relatively high level of expression . A moderate level of expression is observed in heart, lung, testis, cornea, retinal pigmented epithelium/choroid and placenta with a lower level of expression in the uterus, small intestine and skeletal muscle . A very low level of expression was observed in stomach and liver . Comparison between four fetal and adult tissues also demonstrate a surprising level of developmental specificity . Expression in fetal retina is considerably lower than fetal brain but similar to adult retina. EMBO J, 1997 Jul 16, 16(14), 4267 - 75 Binding of mitochondrial precursor proteins to the cytoplasmic domains of the import receptors Tom70 and Tom20 is determined by cytoplasmic chaperones; Komiya T et al.; We have reconstituted the early steps of precursor targeting to mitochondria in a defined and soluble system consisting of the cytosolic domains of the yeast mitochondrial import receptors Tom20 and Tom70, precursor to bovine adrenal adrenodoxin (which has a cleavable targeting signal) and rat liver cytosolic chaperones hsp70 and mitochondrial import-stimulating factor (MSF) . The Tom70 domain only bound the precursor in the presence of MSF, yielding a precursor-MSF-Tom70 complex; ATP hydrolysis by MSF released MSF and generated a precursor-Tom70 complex whose formation was inhibited by an excess of a functional presequence peptide, but not by 150 mM NaCl . In the presence of the Tom20 domain, ATP caused transfer of the precursor from the precursor-MSF-Tom70 complex to Tom20 . The Tom20 domain alone only bound the precursor in the presence of hsp70; hsp70 itself was not incorporated into the resulting complex . Formation of the Tom20-precursor complex was inhibited by excess presequence peptide or by 150 mM NaCl . Similar results were obtained with the ADP/ATP carrier and porin precursors, which both lack a cleaved targeting signal . Correct targeting of a precursor to mitochondrial import receptors thus requires cytosolic chaperones, irrespective of the presence or absence of a cleavable presequence. EMBO J, 1997 Jul 16, 16(14), 4238 - 49 Complementation of null CF mice with a human CFTR YAC transgene; Manson AL et al.; We have made transgenic mice carrying a 320 kb YAC with the intact human cystic fibrosis transmembrane regulator (CFTR) gene . Mice that only express the human transgene were obtained by breeding with Cambridge null CF mice . One line has approximately two copies of the intact YAC . Mice carrying this transgene and expressing no mouse cftr appear normal and breed well, in marked contrast to the null mice, where 50% die by approximately 5 days after birth . The chloride secretory responses in these mice are as large or larger than in wild-type tissues . Expression of the transgene is highly cell type specific and matches that of the endogenous mouse gene in the crypt epithelia throughout the gut and in salivary gland tissue . However, there is no transgene expression in some tissues, such as the Brunner's glands, where it would be expected . Where there are differences between the mouse and human pattern of expression, the transgene follows the mouse pattern . We have thus defined a cloned fragment of DNA which directs physiological levels of expression in many of the specific cells where CFTR is normally expressed. Genomics, 1997 Jul 15, 43(2), 191 - 201 Three genes encoding zinc finger proteins on human chromosome 6p21.3: members of a new subclass of the Kruppel gene family containing the conserved SCAN box domain; Lee PL et al.; Five genes encoding zinc finger proteins of the Cys2His2 (or Kruppel) family were identified by direct cDNA hybridization to YACs 753H12 and 638D7, which encompass a region of human chromosome 6p21.3 extending from just centromeric of the microsatellite marker D6S306 to telomeric of D6S1260 . The genes span a distance of approximately 1750 kb . The complete cDNA sequence, genomic structure, and tissue distribution of three of the zinc finger proteins, LD65/ZNF165, ZNF192 (previously called LD5-1), and ZNF193, are described . The three zinc finger proteins do not contain either Kruppel-associated box (KRAB) A or KRAB B domain, present in about one-third of all Kruppel-type zinc finger proteins (E . J . Bellefroid et al., 1991, Proc . Natl . Acad . Sci . USA 88: 3608-3612) . The three zinc finger proteins do contain the conserved SCAN box domain (A . J . Williams et al., 1995, J . Biol . Chem . 270: 22143-22152) . SCAN boxes are found in eight other genes in the GenBank database, five of which are also in the Kruppel family of zinc finger proteins lacking KRAB A and B domains and thereby define a new subclass of zinc finger proteins . In addition, three polymorphisms were identified in ZNF192, one of the zinc finger proteins . One of the three polymorphisms, Pro163Leu, is the second proline in a proline cluster (PEPP) in a region separating the SCAN box from the zinc finger motifs. Genomics, 1997 Jul 15, 43(2), 171 - 82 A complete YAC contig and cosmid interval map covering the entirety of human Xq21.33 to Xq22.3 from DXS3 to DXS287; Kendall E et al.; We have produced a physical map that covers the entirety of Xq21.33 to Xq22.3, from DXS3 to DXS287, approximately 15-17 Mb of the proximal long arm of the human X chromosome . This region has already been shown to contain a number of genes involved in genetic disorders, some of which have yet to be cloned . The physical map consists of a contig of 420 yeast artificial chromosome (YAC) clones ordered with respect to 142 DNA markers, approximately one probe every 110 kb . Forty-three YACs from across the contig have been used to isolate 2019 cosmids that have been mapped into 87 intervals, and 667 of these clones are positive for at least 1 single-copy marker . These YACs and cosmids have been used to confirm data from other published contigs that map to the region . The physical map described here constitutes the first step toward a complete transcriptional map of the region. Genomics, 1997 Jul 15, 43(2), 130 - 40 Physical and transcription map in the region 14q24.3: identification of six novel transcripts; Roux AF et al.; The region of chromosome 14q24 has been of particular interest as it is known to contain one of the early-onset Alzheimer disease genes (AD3) . Other genes of medical interest, such as arrhythmogenic right ventricular cardiomyopathy, have been mapped to this region by linkage analysis or chromosome rearrangements . We have focused on the region of a balanced translocation (2;14)(p25;q24) . Members of a family with this translocation all have anterior polar cataracts, suggesting the presence of a gene involved in lens integrity at the vicinity of the breakpoint . The chromosome 14 breakpoint has been defined between the short tandem repeats D14S289 and D14S277, a region of overlap for yeast artificial chromosomes (YACs) 888b2 and 934d4 . We have extended the study of the region to 2 Mb on chromosome 14 and present a physical map of this region, including several sequence-tagged sites . New probes were generated using several end clones and inter-Alu PCR fragments from YACs . cDNA selection was used to identify transcribed sequences . Mapping and alignment of 17 nonoverlapping cDNAs completed by sequence and expression pattern analysis suggested that a minimum of eight putative transcription units is present in this region: six of these units correspond to five new genes and one member of a new gene family. Genes Dev, 1997 Jul 15, 11(14), 1864 - 72 U2AF65 recruits a novel human DEAD box protein required for the U2 snRNP-branchpoint interaction; Fleckner J et al.; Splicing of mRNA precursors (pre-mRNAs) comprises a series of ATP-dependent steps, the first of which is the stable binding of U2 snRNP at the pre-mRNA branchpoint . The basis of ATP use for the interaction between U2 snRNP and the branchpoint is unclear, and, in particular, none of the known mammalian factors required for this step have the sequence characteristics of proteins that hydrolyze ATP . Entry of U2 snRNP into the spliceosome is initiated by interaction of the essential splicing factor U2AF65 with the pre-mRNA polypyrimidine tract . In this report we identify a new region of U2AF65 required for function, and use this information to clone a human 56-kD U2AF65 associated protein (UAP56) . We show that UAP56 is an essential splicing factor, which is recruited to the pre-mRNA dependent on U2AF65, and is required for the U2 snRNP-branchpoint interaction . The sequence of UAP56 indicates it is a member of the DEAD box family of RNA-dependent ATPases, which mediate ATP hydrolysis during several steps of yeast pre-mRNA splicing . Our results reveal a new function of U2AF65: to position a DEAD box protein required for U2 snRNP binding at the pre-mRNA branchpoint region. FEMS Microbiol Lett, 1997 Jul 15, 152(2), 327 - 32 Regulation of chitin synthase activity in the dimorphic fungus Benjaminiella poitrasii by external osmotic pressure; Deshpande MV et al.; The effects of changes in external osmotic pressure on chitin synthase activity of a dimorphic fungus, Benjaminiella poitrasii, have been investigated . Mycelial and yeast cells incubated in medium of low osmolality (distilled water, 0 mOsm) for 10 min had 2-3-fold higher specific activities of native chitin synthase in mixed membrane preparations than cells that had been subjected to a high osmolality medium (1.2 M sorbitol in distilled water, 1612 mOsm) . Cells suspended in media of different osmolalities for 10 min were also affected in the extent of germ tube formation . Germ tube formation was highest in cells incubated in low osmolality medium . The addition of protein phosphatase inhibitors (cyclosporin A, 1.2 micrograms/ml; cantharidin, 20 microM) abolished the effect of hypo-osmotic stress on chitin synthase activation of yeast mixed membrane preparations . The presence of protein kinase inhibitors (genistein, 40 micrograms/ml; H-7, 100 microM) and a Ca2+ channel blocker (verapamil, 50 microM) reduced chitin synthase activity to 50-60% of that observed in cells under hypo-osmotic shock . These inhibitors also inhibited germ tube formation . This suggests that chitin synthase activity and yeast hyphal morphogenesis are both subject to regulation by osmotic pressure, phosphorylation and calcium. Biochem J, 1997 Jul 15, 325 ( Pt 2), 423 - 8 Fatty acyl-CoA-acyl-CoA-binding protein complexes activate the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum; Fulceri R et al.; We previously reported that fatty acyl-CoA esters activate ryanodine receptor/Ca2+ release channels in a terminal cisternae fraction from rabbit skeletal muscle {Fulceri, Nori, Gamberucci, Volpe, Giunti and Benedetti (1994) Cell Calcium 15, 109-116} . Skeletal muscle cytosol contains a high-affinity fatty acyl-CoA-binding protein (ACBP) {Knudsen, Hojrup, Hansen, H.O., Hansen, H.F . and Roepstorff (1989) Biochem . J . 262, 513-519} . We show here that palmitoyl-CoA (PCoA) in a complex with a molar excess of bovine ACBP causes a discrete Ca2+ efflux or allows Ca2+ release from the Ca2+-preloaded terminal cisternae fraction by sub-optimal caffeine concentrations . Both effects were abolished by elevating the free {Mg2+} in the system, which inhibits the Ca2+ release channel activity . Sensitization towards caffeine was a function of both the concentration of the complex and the {PCoA}-to-{ACBP} ratio . In all experimental conditions the calculated free {PCoA} was no more than 50 nM, and such concentrations by themselves were inactive on Ca2+ release channels . The KD for PCoA binding was approx . 2 nM for bovine and yeast ACBP, and slightly higher (8 nM) for rat ACBP . The PCoA-rat ACBP complex behaved in the same manner as the PCoA-bovine ACBP complex, whereas the ester complexed with yeast ACBP was more active in activating/sensitizing Ca2+ efflux . A non-hydrolysable analogue of PCoA bound to (bovine) ACBP also sensitized the Ca2+ release channel towards caffeine . These findings indicate that fatty acyl-CoA-ACBP complexes either interact directly with one or more components in the terminal cisternae membranes or, through interaction with the component(s), donate the fatty acyl-CoA esters to high-affinity binding sites of the membrane, thus affecting (and possibly regulating) Ca2+ release channel activity. Blood, 1997 Jul 15, 90(2), 526 - 34 The Ig heavy chain gene is frequently involved in chromosomal translocations in multiple myeloma and plasma cell leukemia as detected by in situ hybridization; Nishida K et al.; Chromosome rearrangement of 14q32.33 has recurrently occurred with variable partner sites, including 11q13.3, 8q24.1, 18q21.3, and 6p21.1 in multiple myeloma (MM) . To assess the actual incidence of 14q32.33 translocation and to elucidate its implication in the pathogenesis of MM, we studied 42 patients with MM, plasma cell leukemia, or plasmacytoma and 5 with monoclonal gammopathy with undetermined significance (MGUS) by G-banding and molecular cytogenetic methods . Using double-color fluorescence in situ hybridization (DCFISH) with 2 Ig heavy chain (IgH) gene probes, a yeast artificial chromosome (YAC) clone containing variable region, and a phage clone containing gamma constant region, 14q32.33 translocation was detected as split signals of the IgH gene in 31 patients with plasma cell malignancies and 3 with MGUS . In contrast, of 40 patients who were assessed by G-banding, 3 (7.5%) showed the 14q+ chromosome . DCFISH detected a split of the IgH gene on interphase nuclei in 34 (73.9%) of 46 patients analyzed, whereas on metaphase spreads, it was in 22 (51.2%) of 43 patients analyzed . Interphase DCFISH was particularly useful to detect 14q32.33 translocation in 17 (65.4%) of 26 patients with normal karyotypes . Donor sites were identified in 11 of 22 patients demonstrated as carrying 14q32.33 translocation by metaphase FISH . Chromosome t(11;14)(q13.3; q32.33) was detected in 5 patients, t(8;14)(q24.1;q32.33) in 2, t(14;18)(q32.33;q21.3) in 2, and t(7;14)(q32.1;q32.33) in 1 . A complex 14q32.33 translocation involving 3q and 16q24 was detected in 1 patient . Myeloma cells with t(7;14) showed myelomonocytoid surface antigen . Because rearrangements of 14q32.33 were closely associated with translocation of proto-oncogenes into the IgH gene, our findings indicate that 14q32.33 translocation with various partner chromosomes is a critical event in the pathogenesis of MM and MGUS. J Immunol, 1997 Jul 15, 159(2), 952 - 63 Ligation of L-selectin through conserved regions within the lectin domain activates signal transduction pathways and integrin function in human, mouse, and rat leukocytes; Steeber DA et al.; L-selectin is a cell surface adhesion receptor that mediates leukocyte rolling along vascular endothelium at sites of inflammation and lymphocyte attachment to endothelial cells within peripheral lymphoid tissues . Since ligation of L-selectin through its ligand recognition region may mimic physiologic ligand binding, a new panel of mAbs that engaged a conserved ligand-binding region within the lectin domains of human, mouse, and rat L-selectin were generated using L-selectin-deficient mice . Indeed, appropriate ligation of L-selectin generated transmembrane signals that resulted in immediate intercellular adhesion following cell-cell contact of lymphocytes, neutrophils, and L-selectin cDNA-transfected cells . Ab binding to only some epitopes within the lectin domain of L-selectin induced adhesion, while mAb binding to numerous other epitopes or other domains of L-selectin had no effect . PPME (yeast polyphosphomonoester core polysaccharide), a complex carbohydrate that mimics the natural L-selectin ligand, also induced potent intercellular adhesion . The induction of intercellular adhesion required cellular energy, an intact cytoskeleton, and cytoplasmic kinase activity as well as the membrane proximal and cytoplasmic domains of L-selectin . Therefore, ligation of L-selectin through specific ligand-binding epitopes identified by the mAbs generated in this study can generate transmembrane signals . Signaling through L-selectin may enhance leukocyte-endothelial cell interactions by serving as an activation/priming step during rolling, thereby promoting subsequent firm cell-cell adhesion in the presence of inflammatory mediators. Nucleic Acids Res, 1997 Jul 15, 25(14), 2752 - 8 The leptin receptor promoter controls expression of a second distinct protein; Bailleul B et al.; The leptin receptor (OB-R) is a single membrane- spanning protein that mediates the weight-regulatory effects of leptin (OB protein) . Several mRNA splice variants have been described which either encode OB-R proteins with cytoplasmic domains of different length or the OB-R and B219/OBR variants, which have different 5'-untranslated regions . Here we report evidence for the synthesis of a human mRNA splice variant of the OB-R gene that potentially encodes a novel protein, leptin receptor gene-related protein (OB-RGRP), which displays no sequence similarity to the leptin receptor itself . This OB-RGRP transcript contains the first two OB-R gene 5'-untranslated exons, but then is alternatively spliced to two novel exons which were mapped to a yeast artificial chromosome containing the leptin receptor gene . First identified by analysis of a large human expressed sequence tag database, the OB-RGRP transcript has now also been found in human and mouse tissues by the use of PCR . Preliminary experiments suggest that OB-RGRP and the OB-R variants share similar patterns of expression that are distinct from that of the B219/OBR variant . OB-RGRP is highly homologous to putative open reading frames in both yeast and Caenorhabditis elegans , suggesting a phylogenetically conserved role for this novel protein. FEBS Lett, 1997 Jul 14, 411(2-3), 383 - 8 RAP1-like binding activity in islet cells corresponds to members of the Sp1 family of transcription factors; Simon B et al.; Deletion and mutational analyses of the gastrin promoter have identified a binding site for the yeast transcription factor RAP1 relevant for transcriptional activation in islet cells . We here report that the mammalian transcription factors binding to this site in islet cells are the Sp transcription factor members Sp1 and Sp3 . Furthermore, functional analyses revealed Sp1- and Sp3-mediated transcriptional activation of gastrin . These data reveal that the zinc finger proteins Sp1 and Sp3 do have similar binding specificities as the multifunctional yeast RAP1 protein. Biochim Biophys Acta, 1997 Jul 12, 1347(1), 69 - 74 Modification of the N-terminal cysteine of plasma cholesteryl ester transfer protein selectively inhibits triglyceride transfer activity; Kotake H et al.; An invariant cysteine residue is found at the N-terminus of cholesteryl ester transfer protein (CETP) isolated from plasma of humans, rabbits and cynomolgus monkeys . We previously reported the expression of recombinant rabbit cholesteryl ester transfer protein in yeast (Kotake et al., J . Lipid Res . 1996; 37: 599-605) . The recombinant CETP secreted into the medium contains an altered N-terminal sequence but was fully capable of facilitating both cholesteryl ester (CE) and triglyceride (TG) transfer between lipoproteins . We investigated the importance of the conserved N-terminal cysteine of plasma CETP in the lipid transfer activity by chemical modification of the free sulfhydryl groups of the recombinant CETP and CETP from human and rabbit plasma . The unmodified forms of these CETPs had similar specific activities of CE and TG transfer . Neither 5,5'-dithiobis-(2-nitrobenzoate) nor N-ethyl maleimide altered the lipid transfer activity . In contrast, p-chloromercuriphenyl sulfonate selectively inhibited the TG transfer activity of both human and rabbit plasma CETP . The TG and CE transfer activities of the recombinant CETP, which lacks the N-terminal cysteine residue, was not affected . These results demonstrate that the N-terminal cysteine residue of both human and rabbit plasma CETP is free and is likely to be involved in the construction of a critical part of the active site of CETP that can determine the selectivity of the lipid molecule for the transfer reaction. Cell, 1997 Jul 11, 90(1), 121 - 9 Developmentally regulated mitochondrial fusion mediated by a conserved, novel, predicted GTPase; Hales KG et al.; The Drosophila melanogaster fuzzy onions (fzo) gene encodes the first known protein mediator of mitochondrial fusion . During Drosophila spermatogenesis, mitochondria in early postmeiotic spermatids aggregate, fuse, and elongate beside the growing flagellar axoneme . fzo mutant males are defective in this developmentally regulated mitochondrial fusion and are sterile . fzo encodes a large, novel, predicted transmembrane GTPase that becomes detectable on spermatid mitochondria late in meiosis II, just prior to fusion, and disappears soon after fusion is complete . Missense mutations that alter conserved residues required for GTP binding in other GTPases inhibit the fusogenic activity of Fzo in vivo but do not affect its localization . Fzo has homologs of unknown function in mammals, nematodes, and yeast. J Biol Chem, 1997 Jul 11, 272(28), 17542 - 50 ArgBP2, a multiple Src homology 3 domain-containing, Arg/Abl-interacting protein, is phosphorylated in v-Abl-transformed cells and localized in stress fibers and cardiocyte Z-disks; Wang B et al.; Arg and c-Abl represent the mammalian members of the Abelson family of protein-tyrosine kinases . A novel Arg/Abl-binding protein, ArgBP2, was isolated using a segment of the Arg COOH-terminal domain as bait in the yeast two-hybrid system . ArgBP2 contains three COOH-terminal Src homology 3 domains, a serine/threonine-rich domain, and several potential Abl phosphorylation sites . ArgBP2 associates with and is a substrate of Arg and v-Abl, and is phosphorylated on tyrosine in v-Abl-transformed cells . ArgBP2 is widely expressed in human tissues and extremely abundant in heart . In epithelial cells ArgBP2 is located in stress fibers and the nucleus, similar to the reported localization of c-Abl . In cardiac muscle cells ArgBP2 is located in the Z-disks of sarcomeres . These observations suggest that ArgBP2 functions as an adapter protein to assemble signaling complexes in stress fibers, and that ArgBP2 is a potential link between Abl family kinases and the actin cytoskeleton . In addition, the localization of ArgBP2 to Z-disks suggests that ArgBP2 may influence the contractile or elastic properties of cardiac sarcomeres and that the Z-disk is a target of signal transduction cascades. Gene, 1997 Jul 9, 193(2), 163 - 72 Molecular characterization of the C-3 DNA puff gene of Rhynchosciara americana; Penalva LO et al.; We have mapped a region of about 33 kb which includes the transcription unit of the C-3 DNA puff gene of Rhynchosciara americana . The C-3 TU and a region extending approximately 800 bp upstream of the C-3 promoter were characterized . The TU is composed of three exons and produces a 1.1-kb mRNA whose level in salivary glands increases with the expansion of the C-3 puff . The C-3 messenger appears to undergo rapid deadenylation resulting in an RNA of about 0.95 kb which can still be observed in gland cells 15 h after the puff has regressed . The 1.1-kb mRNA codes for a 32.4-kDa, predominantly alpha-helical polypeptide with three conserved parallel coiled-coil stretches . The aa composition and structure of this polypeptide suggests that it is secreted and contributes to the formation of the cocoon in which the larvae pupate . The region upstream of the promoter contains several A-rich sequences with similarity to the ACS of yeast which might have a role in the initiation of replication/amplification. Eur J Pharmacol, 1997 Jul 9, 330(2-3), 221 - 9 Inhibition of brain cyclooxygenase-2 activity and the antipyretic action of nimesulide; Taniguchi Y et al.; The antipyretic action and the mechanism of action of 4-nitro-2-phenoxymethanesulfonanilide (nimesulide), a new nonsteroidal antiinflammatory drug, were investigated in yeast-induced febrile rats . Yeast-injected rats developed marked fever and exhibited an approximately 7-fold increase in brain levels of prostaglandin E2 and an approximately 2-fold increase in the expression of cyclooxygenase-2 mRNA despite an almost unchanged expression of cyclooxygenase-1 mRNA . Nimesulide produced a dose dependent antipyretic action, which was stronger than that of indomethacin and ibuprofen, and decreased dose dependently the increased brain prostaglandin E2 levels, whereas it did not influence the expression of cyclooxygenase-2 mRNA . It inhibited markedly the enhanced brain cyclooxygenase activity, primarily cyclooxygenase-2, in vivo and dose dependently increased brain cyclooxygenase activity in vitro . These results suggest that the marked antipyretic action of nimesulide is primarily mediated through the selective inhibition of the activity of brain cyclooxygenase-2 induced under febrile conditions. Biochem Biophys Res Commun, 1997 Jul 9, 236(1), 130 - 4 Evidence for a mammalian Nim1-like kinase pathway acting at the G0-1/S transition; Baldin V et al.; In fission yeast the Nim1 kinase phosphorylates and inactivates the cdc2-inhibitory Weel tyrosine kinase . In addition, Nim1 is necessary for an efficient cellular response to nutritional starvation leading to cell cycle arrest in G1 . Given the remarkable evolutionary conservation of the cell cycle regulatory mechanism we have investigated the effect of Nim1 expression on the control of the mammalian cell cycle using a plasmid microinjection approach . In synchronised IMR90 human fibroblasts, expression of Nim1 strongly inhibited entry into S-phase . This effect was dependent on the catalytic activity of Nim1 and did not require its regulatory domain . Furthermore we show that co-expression of human Wee1 kinase reverted the inhibitory effect, indicating that Nim1 was acting in a Wee1-dependent manner . These results provide evidence for the existence of a Nim1-like kinase pathway acting at the G0-1/S transition in human cells. Biochem Biophys Res Commun, 1997 Jul 9, 236(1), 59 - 65 The amino-terminal region of amyloid precursor protein is responsible for neurite outgrowth in rat neocortical explant culture; Ohsawa I et al.; We have previously shown that secreted forms of beta-amyloid precursor protein (APP(s)s) promote neurite outgrowth in embryonic rat neocortical explant culture . To determine the region of APP(s) responsible for its biological activity, we produced both amino- and carboxyl-terminal regions of APP(s) using a yeast expression system . The purified fragment corresponding to the amino-terminal region (NAPP) enhanced neurite outgrowth of neocortical explants, but the carboxyl-terminal region fragment did not . The neurite-promoting activity of full length APP(s) and NAPP was blocked by the antibody, 22C11, specific for the amino-terminal region, and the 16-mer peptide of epitope for 22C11 also enhanced neurite outgrowth . However, the 17-mer peptide which contains RERMS sequence did not enhance the neurite outgrowth, but promoted the survival of neocortical neurons in dissociated culture . These findings suggested that the amino-terminal region is responsible for the neurite-promoting activity of APP(s)s. Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7373 - 7 Evolution of foraging behavior in Drosophila by density-dependent selection; Sokolowski MB et al.; One of the rare examples of a single major gene underlying a naturally occurring behavioral polymorphism is the foraging locus of Drosophila melanogaster . Larvae with the rover allele, forR, have significantly longer foraging path lengths on a yeast paste than do those homozygous for the sitter allele, fors . These variants do not differ in general activity in the absence of food . The evolutionary significance of this polymorphism is not as yet understood . Here we examine the effect of high and low animal rearing densities on the larval foraging path-length phenotype and show that density-dependent natural selection produces changes in this trait . In three unrelated base populations the long path (rover) phenotype was selected for under high-density rearing conditions, whereas the short path (sitter) phenotype was selected for under low-density conditions . Genetic crosses suggested that these changes resulted from alterations in the frequency of the fors allele in the low-density-selected lines . Further experiments showed that density-dependent selection during the larval stage rather than the adult stage of development was sufficient to explain these results . Density-dependent mechanisms may be sufficient to maintain variation in rover and sitter behavior in laboratory populations. Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7326 - 30 Identification of an early endosomal protein regulated by phosphatidylinositol 3-kinase; Patki V et al.; Phosphatidylinositol 3-kinases (PI 3-kinases) have been implicated in membrane trafficking in the secretory and endocytic pathways of yeast and mammalian cells, but the molecular mechanisms by which these lipid kinases operate are not known . Here we identify a protein of 170 kDa that is rapidly released from cell membranes in response to wortmannin, a potent inhibitor of mammalian PI 3-kinases . The amino acid sequence of peptides from p170 reveal its identity to early endosomal antigen (EEA) 1, an endosomal antigen with homology to several yeast proteins genetically implicated in membrane trafficking . Immunofluorescence analysis of 3T3-L1 adipocytes with antisera against p170/EEA1 reveal a punctate peripheral pattern that becomes diffuse in response to wortmannin . In vitro, p170/EEA1 binds specifically to liposomes containing PIns(3)P, suggesting that the effect of wortmannin on cells is due to inhibition of PIns(3)P production . Thus, p170/EEA1 may define a family of proteins that mediate the regulatory effects of 3'-phosphoinositides on membrane trafficking in yeast and mammalian cells. Biochemistry, 1997 Jul 8, 36(27), 8224 - 30 The QM protein associates with ribosomes in the rough endoplasmic reticulum; Loftus TM et al.; QM is a human cDNA originally isolated as a transcript elevated in a nontumorigenic Wilms' tumor microcell hybrid, relative to the tumorigenic parental cell line . Homologs of this gene have been identified from a large number of diverse eukaryotic species which demonstrate a high degree of conservation . The functional importance implied by this strong conservation is supported by the observation that the disruption of the yeast homolog is lethal . In spite of its apparent importance, the function of the encoded protein remains elusive . Indirect immunofluorescent cell staining of cultured human, G401 cells with an antibody to the QM protein shows a punctate staining pattern in the cytoplasm with much of the signal in a perinuclear pattern . Subcellular fractionation demonstrated an association of QM protein with the rough endoplasmic reticulum . It was possible to disrupt this association by washing microsomal membranes with 1M NaCl, suggesting a peripheral association . Proteolytic latency studies showed the protein to be exposed on the cytoplasmic face of the membrane . In situ cross-linking followed by diagonal SDS gel analysis indicates that QM exists as a member of a large protein complex . In agreement with this, QM was found to copurify with the ribosome complex . Incubation with 1 M NaCl was found to disrupt this association while having no effect on the association of core ribosomal proteins. Science, 1997 Jul 4, 277(5322), 88 - 91 Identification of maize histone deacetylase HD2 as an acidic nucleolar phosphoprotein; Lusser A et al.; The steady state of histone acetylation is established and maintained by multiple histone acetyltransferases and deacetylases, and this steady state affects chromatin structure and function . The identification of a maize complementary DNA encoding the chromatin-bound deacetylase HD2 is reported . This protein was not homologous to the yeast RPD3 transcriptional regulator . It was expressed throughout embryo germination in correlation with the proliferative activity of cells . Antibodies against recombinant HD2-p39 immunoprecipitated the native enzyme complex, which was composed of phosphorylated p39 subunits . Immunofluorescence microscopy and sequence homologies suggested nucleolar localization . HD2 is an acidic nucleolar phosphoprotein that might regulate ribosomal chromatin structure and function. J Biol Chem, 1997 Jul 4, 272(27), 17209 - 15 Genetic evidence for a tyrosine kinase cascade preceding the mitogen-activated protein kinase cascade in vertebrate G protein signaling; Wan Y et al.; The signal transduction pathway from heterotrimeric G proteins to the mitogen-activated protein kinase (MAPK) cascade is best understood in the yeast mating pheromone response, in which a serine/threonine protein kinase (STE20) serves as the critical linking component . Little is known in metazoans on how G proteins and the MAPK cascade are coupled . Here we provide genetic and biochemical evidence that a tyrosine kinase cascade bridges G proteins and the MAPK pathway in vertebrate cells . Targeted deletion of tyrosine kinase Csk in avian B lymphoma cells blocks the stimulation of MAPK by Gq-, but not Gi-, coupled receptors . In cells deficient in Bruton's tyrosine kinase (Btk), Gi-coupled receptors failed to activate MAPK, while Gq-coupled receptor-mediated stimulation is unaffected . Taken together with our previous data on tyrosine kinases Lyn and Syk, the Gq-coupled pathway requires tyrosine kinases Csk, Lyn, and Syk, while the Gi-coupled pathway requires tyrosine kinases Btk and Syk to feed into the MAPK cascade in these cells . The central role of Syk is further strengthened by data showing that Syk can bind to purified Lyn, Csk, or Btk. J Biol Chem, 1997 Jul 4, 272(27), 17166 - 70 Evidence for a direct interaction between insulin receptor substrate-1 and Shc; Kasus-Jacobi A et al.; Insulin receptor substrate-1 (IRS-1) and Shc are two proteins implicated in intracellular signal transduction . They are activated by an increasing number of extracellular signals, mediated by receptor tyrosine kinases, cytokine receptors, and G protein-coupled receptors . In this study we demonstrate that Shc interacts directly with IRS-1, using the yeast two-hybrid system and an in vitro interaction assay . Deletion analysis of the proteins to map the domains implicated in this interaction shows that the phosphotyrosine binding domain of Shc binds to the region of IRS-1 comprising amino acids 583-661 . An in vitro association assay, performed with or without activation of tyrosine kinases, gives evidence that tyrosine phosphorylation of IRS-1 and Shc drastically improves the interaction . Site-directed mutagenesis on IRS-1 583-693 shows that the asparagine, but not the tyrosine residue of the N625GDY628motif domain, is implicated in the IRS-1-Shc-phosphotyrosine binding interaction . Mutation of another tyrosine residue, Tyr608, also induced a 40% decrease in the interaction . This study, describing a phosphotyrosine-dependent interaction between IRS-1 and Shc, suggests that this association might be important in signal transduction. J Biol Chem, 1997 Jul 4, 272(27), 17070 - 7 Characterization of the WW domain of human yes-associated protein and its polyproline-containing ligands; Chen HI et al.; We had previously identified the WW domain as a novel globular domain that is composed of 38-40 semiconserved amino acids and is involved in mediating protein-protein interaction . The WW domain is shared by proteins of diverse functions including structural, regulatory, and signaling proteins in yeast, nematode, and mammals . Functionally it is similar to the Src homology 3 domain in that it binds polyproline ligands . By screening a 16-day mouse embryo expression library, we identified two putative ligands of the WW domain of Yes kinase-associated protein which we named WW domain-binding proteins 1 and 2 . These proteins interacted with the WW domain via a short proline-rich motif with the consensus sequence of four consecutive prolines followed by a tyrosine . Herein, we report the cDNA cloning and characterization of the human orthologs of WW domain-binding proteins 1 and 2 . The products encoded by these cDNA clones represent novel proteins with no known function . Furthermore, these proteins show no homology to each other except for a proline-rich motif . By fluorescence in situ hybridization on human metaphase chromosomes, we mapped the human genes for WW domain-binding proteins 1 and 2 to chromosomes 2p12 and 17q25, respectively . In addition, using site-directed mutagenesis, we determined which residues in the WW domain of Yes kinase-associated protein are critical for binding . Finally, by synthesizing peptides in which the various positions of the four consecutive proline-tyrosine motif and the five surrounding residues were replaced by all possible amino acid residues, we further elucidated the binding requirements of this motif. J Biol Chem, 1997 Jul 4, 272(27), 16955 - 61 Structural and functional complementation of an inactive Bcl-2 mutant by Bax truncation; Ottilie S et al.; Interactions among proteins in the Bcl-2 family regulate the onset of programmed cell death . Previous work has shown that the death-inhibiting family members Bcl-2 and Bcl-xL form heterodimers with the death-promoting homologue Bax and that certain site-directed mutants of Bcl-2 and Bcl-xL lose both biological activity and the ability to bind Bax . To better understand the structural basis of heterodimer formation, we have used a yeast two-hybrid assay to screen for mutants of Bax that regain the ability to bind to these inactive Bcl-2(G145A) and Bcl-xL(G138A) mutants . This screen identified a series of C-terminally truncated Bax molecules that contain complete BH3 (Bcl-2 homology domain 3) domains but that have lost BH1 and BH2 sequences . These results indicate that while the Bcl-2 and Bcl-xL mutants fail to bind full-length Bax, they still retain a binding site for the critical BH3 domain . This suggests that conformational constraints in full-length Bax regulate its ability to bind to other Bcl-2 family members . Furthermore, we demonstrate that the normally inert Bcl-2(G145A) mutant effectively blocks apoptosis induced by a C-terminally truncated Bax molecule, but does not block apoptosis induced by wild-type Bax . This demonstrates that cell protection can be effected by directly binding pro-apoptotic members of the Bcl-2 family. Oncogene, 1997 Jul 3, 15(1), 7 - 15 Cloning and characterization of APS, an adaptor molecule containing PH and SH2 domains that is tyrosine phosphorylated upon B-cell receptor stimulation; Yokouchi M et al.; Stimulation of B lymphocytes through their antigen receptor (BCR) results in rapid increases in tyrosine phosphorylation of a number of proteins, which leads to a cascade of biochemical changes that initiates B cell proliferation and differentiation or growth inhibition . A novel cDNA, designed APS, encoding an adaptor protein with a Pleckstrin homology (PH) domain, Src homology 2 (SH2) domain, and a tyrosine phosphorylation site was cloned from a B cell cDNA library using a yeast two hybrid system . APS is structurally similar to SH2-B, an SH2 protein that potentially binds to the immunoreceptor tyrosine-based activation motif (ITAM) as well as Lnk which is postulated to be a signal transducer that links T-cell receptor to phospholipase Cgamma, Grb2 and phosphatidylinositol 3-kinase . APS expressed only in human Burkitt's lymphoma cells among cell lines we examined and tyrosine phosphorylated in response to BCR stimulation . APS bound to Shc irrespective of stimulation and bound to Grb2 after stimulation, suggesting that it plays a role in linkage from BCR to Shc/Grb2 pathway . These results indicate that APS, SH2-B and Lnk form a new adaptor family that links immune receptors to signaling pathways involved in tyrosine-phosphorylation. Oncogene, 1997 Jul 3, 15(1), 1 - 6 A giant protein that stimulates guanine nucleotide exchange on ARF1 and Rab proteins forms a cytosolic ternary complex with clathrin and Hsp70; Rosa JL et al.; We have recently identified an unusually large human protein that stimulates guanine nucleotide exchange on ARF1 and Rab proteins (EMBO J 15: 4262-4273, 1996) . This protein, designated p532 based on its predicted molecular weight (EMBO J 15: 5738, 1996), contains multiple structural domains including two regions of seven internal repeats highly related to the cell cycle regulator RCC1, a guanine nucleotide exchange factor for the small GTP-binding protein Ran, seven beta-repeat domains characteristic of the beta subunit of heterotrimeric G proteins, three putative SH3 binding sites, a putative leucine-zipper and a carboxy-terminal HECT domain characteristic of E3 ubiquitin-protein ligases . Some of these domains are known to be involved in protein-protein interactions, suggesting the existence of p532-interacting proteins . To identify some of these proteins, we used the carboxy-terminal RCC1-like domain (RLD) of p532 in the yeast two-hybrid system . We report here the isolation of a clone that encodes the last 654 amino acid residues of the clathrin heavy chain . This interaction involves amino acid residues 1315-1557 of the clathrin heavy chain and the carboxy, but not the amino-terminal RLD of p532 . p532 has been located in the cytosolic fraction as well as in the Golgi apparatus . The interaction between p532 and clathrin only occurs in the cytosol and is mediated by the formation of an ATP-dependent ternary complex with the heat shock protein, Hsp70 . These observations suggest that p532 is involved in membrane transport processes. Anim Behav, 1997 Jul, 54(1), 109 - 19 Why do animals repeat displays? Payne RJH, Pagel M. Both agonistic and sexual animal displays often involve more than one performance of some specific display action . Since repetition is energetically costly there must be good reasons why a signaller should carry out such repetitive actions, rather than simply displaying once . We briefly review three different 'reasons' which arise from three different receiver assessment rules: when assessment is based on the average magnitude of all display actions so far, the reason for the repetition is to improve the accuracy of the estimate (model A); when the assessment is based solely on the action of greatest magnitude so far, the repetition is to replace the signal with one of greater magnitude (model B); when the assessment is based on the cumulative sum of all display actions so far, the repetition is to augment that sum (model C) . We discuss how to characterize each case from an understanding of its expected optimal behaviour as predicted by formal models . For model A the mean magnitude of display actions should stay constant and the contest duration should depend on relative qualities . In models B and C the encounter duration depends only on the weaker participant . In model B each display action is greater than the previous, but only a small number of steps are expected . In model C the magnitude of display actions can either escalate, stay constant, or even decrease . The displays of cichlid fish, the roaring contests of red deer, Cervus elaphusthe calling of Blanchard's cricket frogs, Acris crepitans blanchardiand the pheromonal exchanges of yeast gametes are used as illustrative examples. Inflamm Res, 1997 Jul, 46(7), 265 - 71 SH3 domain-mediated interactions involving the phox components of the NADPH oxidase; Wilson L et al.; OBJECTIVE AND DESIGN: To derive a model describing the SH3 domain-mediated assembly of the activated NADPH oxidase . MATERIALS: Recombinant SH3 domain and Pro-rich fusion proteins were used to investigate potential co-associations . METHODS: Interactions were assessed using biotinylated overlay assays and the yeast two hybrid system . Association with p47phox from cell lysates was examined by immunoblot analysis . RESULTS: The association between p47- and p22phox involves the SH3 domains of p47phox functioning in tandem . The Prorich motif in p47phox interacts with both p40phox and the COOH-terminal SH3 domain of p67phox . CONCLUSIONS: In the resting cell, the Pro-rich motif of p47phox interacts with the SH3 domain of p40phox, which in turn associates with p67phox . Upon activation, the p47-p40phox regulatory complex dissociates, permitting the association of p47phox with the COOH-terminal SH3 domain of p67phox . This complex translocates to the plasma membrane and associates with cytochrome b558, via interaction of the tandem SH3 domains of p47phox with the p22phox Pro-rich motif. Plant J, 1997 Jul, 12(1), 39 - 48 Possible involvement of differential splicing in regulation of the activity of Arabidopsis ANP1 that is related to mitogen-activated protein kinase kinase kinases (MAPKKKs); Nishihama R et al.; Three types of Arabidopsis cDNA (cANP1, cANP2 and cANP3) have been isolated that encode putative protein kinases, designated ANP1, ANP2 and ANP3 . These kinases exhibit a high degree of homology to NPK1, a tobacco protein that is a member of the family of mitogen-activated protein kinase kinase kinases (MAPKKKs), which appears to function in the proliferation of tobacco cells . The predicted amino acid sequences of the kinase domains in the amino-terminal halves of the ANPs were more than 80% identical to that of NPK1, while the kinase-unrelated regions in the carboxy-terminal halves exhibited relatively low homology . Two species of cANP1 were identified, ANP1L cDNA (cANP1L) and ANP1S cDNA (cANP1S), which were derived from a single ANP1 gene: the former had an intron-like sequence in the coding region for the kinase-unrelated region, while the latter did not include such an intron-like sequence . cANP1L encoded a putative protein with both kinase and kinase-unrelated domains, resembling NPK1, whereas cANP1S encoded only the amino-terminal kinase domain because the intron-like sequence was absent, with resulting elimination of most of the kinase-unrelated region . Genetic analysis with mutant yeast cells showed that over-expression of cANP1L or of cANP1S activated the mating pheromone-responsive signal pathway which is mediated by a MAP kinase cascade . Moreover, the extent of such activation by cANP1S was greater than that by cANP1L . These results predict that differential splicing of the intron-like sequence in the ANP1 transcript might be at least one of the molecular mechanisms involved in the generation of active ANP1 protein kinase. Pediatr Pulmonol, 1997 Jul, 24(1), 57 - 60 Massive hemoptysis as the presenting manifestation in a child with histoplasmosis; Shaffer JP et al.; A previously healthy and asymptomatic 7-year-old white boy presented with a history of two episodes of hemoptysis productive of bright red blood in the 5 days preceding admission . After admission he developed massive hemoptysis that, on bronchoscopy, was noted to be emanating from the right lower lobe . An emergency right lower lobe resection was done . Pathological examination revealed hilar adenopathy and peripheral lesions with caseating granulomas containing yeast, morphologically consistent with Histoplasma capsulatum. Steroids, 1997 Jul, 62(7), 508 - 15 Two transcription activation functions in the amino terminus of the mouse estrogen receptor that are affected by the carboxy terminus; Gandini O et al.; To determine the characteristics of the N-terminal transactivation domain (AF-1) of the mouse estrogen receptor (ER), we constructed a number of deletion mutants . Wild-type and mutant receptors were expressed in yeast cells and assayed for their ability to transactivate an estrogen-responsive reporter plasmid (ERE-CYCl-LacZ) that contained a single estrogen response element of the vitellogenin A2 gene promoter . Deletion of the N-terminal 121 amino acids from the mouse ER resulted in a 50% reduction in transactivation activity compared with the full-length wild-type ER . Deletion of the first 150 amino acids resulted in loss of 90% transactivation activity . An ER deletion mutant lacking residues 121-154 retained full transcriptional activity, suggesting that this region plays a significant transacting role only when the first portion is deleted . A point mutation was introduced in the C-terminal region at Met-521 in order to study the possible interaction between the C-terminal ligand-binding domain and the N-terminal AF-1 region . This mutant ER, M521G, exhibited 150% of the transcriptional activity of the wild-type ER . An M521G mutant lacking the N-terminal 121 amino acids retained full transactivation activity, whereas, M521G lacking 150 amino acids resulted in only 10% of wild-type activity . These results suggest that residues 121-154 might interact with the C terminus to affect transcription . In summary, multiple N-terminal regions in the ER were identified that function in transactivation . Furthermore, a point mutation in the C-terminal portion of the ER may change the conformation of the ER ligand-binding domain, producing a more stable receptor/ligand complex that increases transcriptional activity . These data suggest that the N- and C-terminal portions of the ER interact in a cooperative manner to activate transcription from target genes. Genome Res, 1997 Jul, 7(7), 716 - 24 Characterization of Alu repeats that are associated with trinucleotide and tetranucleotide repeat microsatellites; Yandava CN et al.; The association of subclasses of Alu repetitive elements with various classes of trinucleotide and tetranucleotide microsatellites was characterized as a first step toward advancing our understanding of the evolution of microsatellite repeats . In addition, information regarding the association of specific classes of microsatellites with families of Alu elements was used to facilitate the development of genetic markers . Sequences containing Alu repeats were eliminated because unique primers could not be designed . Various classes of microsatellites are associated with different classes of Alu repeats . Very abundant and poly(A)-rich microsatellite classes (ATA, AATA) are frequently associated with an evolutionarily older subclass of Alu repeats, AluSx, whereas most of GATA and CA microsatellites are associated with a recent Alu subfamily, AluY . Our observations support all three possible mechanisms for the association of Alu repeats to microsatellites . Primers designed using a set of sequences from a particular microsatellite class showed higher homology with more sequences of that class than probes designed for other classes . We developed an efficient method of prescreening GGAA and ATA microsatellite clones for Alu repeats with probes designed in this study . We also showed that Alu probes labeled in a single reaction (multiplex labeling) could be used efficiently for prescreening of GGAA clones . Sequencing of these prescreened GGAA microsatellites revealed only 5% Alu repeats . Prescreening with primers designed for ATA microsatellite class resulted in the reduction of the loss of markers from approximately 50% to 10% . The new Alu probes that were designed have also proved to be useful in Alu-Alu fingerprinting. Genome Res, 1997 Jul, 7(7), 673 - 92 A physical map of human chromosome 7: an integrated YAC contig map with average STS spacing of 79 kb; Bouffard GG et al.; The construction of highly integrated and annotated physical maps of human chromosomes represents a critical goal of the ongoing Human Genome Project . Our laboratory has focused on developing a physical map of human chromosome 7, a approximately 170-Mb segment of DNA that corresponds to an estimated 5% of the human genome . Using a yeast artificial chromosome (YAC)-based sequence-tagged site (STS)-content mapping strategy, 2150 chromosome 7-specific STSs have been established and mapped to a collection of YACs highly enriched for chromosome 7 DNA . The STSs correspond to sequences generated from a variety of DNA sources, with particular emphasis placed on YAC insert ends, genetic markers, and genes . The YACs include a set of relatively nonchimeric clones from a human-hamster hybrid cell line as well as clones isolated from total genomic libraries . For map integration, we have localized 260 STSs corresponding to Genethon genetic markers and 259 STSs corresponding to markers orders by radiation hybrid (RH) mapping on our YAC contigs . Analysis of the data with the program SEGMAP results in the assembly of 22 contigs that are "anchored" on the Genethon genetic map, the RH map, and/or the cytogenetic map . These 22 contigs are ordered relative to one another, are (in all but 3 cases) oriented relative to the centromere and telomeres, and contain > 98% of the mapped STSs . The largest anchored YAC contig, accounting for most of 7p, contains 634 STSs and 1260 YACs . An additional 14 contigs, accounting for approximately 1.5% of the mapped STSs, are assembled but remain unanchored on either the genetic or RH map . Therefore, these 14 "orphan" contigs are not ordered relative to other contigs . In our contig maps, adjacent STSs are connected by two or more YACs in > 95% of cases . With 2150 mapped STSs, our map provides an average STS spacing of approximately 79 kb . The physical map we report here exceeds the goal of 100-kb average STS spacing and should provide an excellent framework for systematic sequencing of the chromosome. Ann Clin Biochem, 1997 Jul, 34 ( Pt 4), 384 - 8 New enzymatic assay with urea amidolyase for determining potassium in serum; Kimura S et al.; We developed a new simple assay for potassium ion in serum using urea amidolyase (UAL) from yeast sp . The method is based on activation of the enzyme by potassium ion . We eliminated endogenous ammonium ion by use of glutamate dehydrogenase (GLDH), and then monitored the production of ammonium ion by UAL, urea, ATP, bicarbonate and magnesium ions . Ammonium ion was produced proportional to the potassium ion concentration and was determined by adding GLDH to produce NADP+ in the presence of 2-oxoglutarate and NADPH . We monitored the change of absorbance at 340 nm . The inhibitory effect of calcium ion to this assay was eliminated by adding glycoletherdiamine-N, N, N', N'-tetraacetic acid to the reaction . The within-assay coefficients of variation (CV) of this method were 0.9-1.55% (n = 10) at 3.32-6.18 mmol/L . Day-to-day CVs ranged from 1.49% to 2.46% . The analytical recovery was 96-108% . The correlation coefficient between the values obtained by our method (y) and those by the ion-selective electrode (ISE) method (x) was 0.994 (y = 1.032x-0.166 mmol/L, Syx = 0.110, n = 100) . The presence of bilirubin, haemoglobin or other ions did not affect this assay, confirming the usefulness of this assay for clinical purposes. J Protein Chem, 1997 Jul, 16(5), 545 - 51 On the classification and evolution of protein modules; Hegyi H et al.; Our efforts to classify the functional units of many proteins, the modules, are reviewed . The data from the sequencing projects for various model organisms are extremely helpful in deducing the evolution of proteins and modules . For example, a dramatic increase of modular proteins can be observed from yeast to C . elegans in accordance with new protein functions that had to be introduced in multicellular organisms . Our sequence characterization of modules relies on sensitive similarity search algorithms and the collection of multiple sequence alignments for each module . To trace the evolution of modules and to further automate the classification, we have developed a sequence and a module alerting system that checks newly arriving sequence data for the presence of already classified modules . Using these systems, we were able to identify an unexpected similarity between extracellular C1Q modules with bacterial proteins. Dig Dis Sci, 1997 Jul, 42(7), 1530 - 6 Dietary supplementation of nucleotides and arginine promotes healing of small bowel ulcers in experimental ulcerative ileitis; Sukumar P et al.; We previously showed that intravenous total parenteral nutrition supplemented with nucleosides and nucleotides (NS/NT) promoted ulcer healing in rats with indomethacin-induced ileitis . The present study evaluated whether dietary NT supplementation would similarly affect ulcer healing in this model . Female Lewis rats were randomized into either control or experimental groups receiving yeast RNA containing NT or arginine, glutamine, fish oil, guar gum, or a combination of yeast RNA+arginine diets . Ileitis was induced by two doses of indomethacin (7.5 mg/kg) administered subcutaneously 24 hr apart . Ulcer number and length were determined at 4, 8, and 14 days after induction of ileitis . Ileal villous and crypt length, crypt-villous ratio, and bromodeoxyuridine (BrdU) labeling were studied in the control and yeast RNA-supplemented diet groups . Ileal ulceration was present in all groups at 4 and 8 days and was almost healed by 14 days . Rats receiving yeast RNA, arginine, and yeast RNA + arginine diets showed a significant decrease in ulcer number (56%, 28%, and 34%, respectively) and length (67%, 41%, and 48%, respectively) compared to controls at 8 but not at 4 days . Glutamine, fish oil, and guar gum had no effect on ulcer healing at 4, 8, or 14 days . Among the histological parameters, a significant decrease in crypt length in the yeast RNA-supplemented group at 8 days suggested an acceleration of the healing process and restoration to a near-normal crypt-villous architecture . We conclude that the yeast RNA, arginine, and yeast RNA + arginine diets accelerated ulcer healing, as indicated by decreased ulcer number and length . We postulate that the underlying mechanism(s) contributing to ulcer healing may be related, in part, to increased cell proliferation. Microbiology, 1997 Jul, 143 ( Pt 7), 2237 - 45 Cloning and disruption of the ornithine decarboxylase gene of Ustilago maydis: evidence for a role of polyamines in its dimorphic transition; Guevara-Olvera L et al.; The gene encoding ornithine decarboxylase (ODC) from Ustilago maydis was cloned . A conserved PCR product amplified from U . maydis DNA was synthesized and used to screen a genomic library of the fungus . Alignment of its deduced protein sequence with those of other cloned ODCs showed a high degree of homology . Gene replacement was obtained by removal of a central part of the gene and insertion of the hygromycin resistance cassette . The null mutant thus obtained displayed no ODC activity and behaved as a polyamine auxotroph . This result is evidence that a single ODC gene exists in the fungus, and that U . maydis utilizes the ODC pathway as the only mechanism for polyamine biosynthesis . When grown in polyamine-containing media, the null mutant accumulated a polyamine pool which further sustained its normal rate of growth in polyamine-free media for approximately 12-16 h . When putrescine concentrations lower than 0.5 mM were employed, the mutant grew at a normal rate but was unable to engage in the dimorphic transition . Under conditions favourable for mycelial growth, the mutant grew with a yeast-like morphology in liquid media, and formed smooth colonies consisting of yeast cells on solid media . Reversion to normal dimorphic phenotype required high concentrations of putrescine or spermidine . These results are evidence that concentrations of polyamines higher than those necessary to sustain vegetative growth are required for the dimorphic transition in U . maydis. Mol Biol Cell, 1997 Jul, 8(7), 1343 - 60 Evidence for a recycling role for Rab7 in regulating a late step in endocytosis and in retention of lysosomal enzymes in Dictyostelium discoideum; Buczynski G et al.; The mammalian small molecular weight GTPase Rab7 (Ypt7 in yeast) has been implicated in regulating membrane traffic at postinternalization steps along the endosomal pathway . A cDNA encoding a protein 85% identical at the amino acid level to mammalian Rab7 has been cloned from Dictyostelium discoideum . Subcellular fractionation and immunofluorescence microscopy indicated that Rab7 was enriched in lysosomes, postlysosomes, and maturing phagosomes . Cell lines were generated that overexposed Rab7 wild-type (WT), Rab7 Q67L (constitutively active form), and Rab7 T22N (dominant negative form) proteins . The Rab7 T22N cell line internalized fluid phase markers and latex beads (phagocytosis) at one-third the rate of control cells, whereas Rab7 WT and Rab7 Q67L cell lines were normal in uptake rates but exocytosed fluid phase faster than control cells . In contrast, fluid phase markers resided in acidic compartments for longer periods of time and were more slowly exocytosed from Rab7 T22N cells as compared with control cells . Light microscopy indicated that Rab7-expressing cell lines contained morphologically altered endosomal compartments . Compared with control cells, Rab7 WT- and Rab7 Q67L-expressing cells contained a reduced number of vesicles, the size of postlysosomes (> 2.5 microns) and an increased number of smaller vesicles, many of which were nonacidic; in control cells, > 90% of the smaller vesicles were acidic . In contrast, Rab7 T22N cells contained an increased proportion of large acidic vesicles relative to nonacidic vesicles . Radiolabel pulse-chase experiments indicated that all of the cell lines processed and targeted lysosomal alpha-mannosidase normally, indicating the lack of a significant role for Rab7 in the targeting pathway; however, retention of mature lysosomal hydrolases was affected in Rab7 WT and Rab7 T22N cell lines . Contrary to the results observed for the fluid phase efflux experiments, Rab7 T22N cells oversecreted alpha-mannosidase, whereas Rab7 WT cells retained this hydrolase as compared with control cells . These data support a model that Rab7 may regulate retrograde transport of lysosomal enzymes and the V-type H(+)-ATPase from postlysosomes to lysosomes coupled with the efficient release of fluid phase from cells. Mol Biol Cell, 1997 Jul, 8(7), 1261 - 71 Syntaxin 6 functions in trans-Golgi network vesicle trafficking; Bock JB et al.; The specific transfer of vesicles between organelles is critical in generating and maintaining the organization of membrane compartments within cells . Syntaxin 6 is a recently discovered member of the syntaxin family, whose constituents are required components of several vesicle trafficking pathways . To better understand the function of syntaxin 6, we generated a panel of monoclonal antibodies that specifically recognize different epitopes of the protein . Immunoelectron microscopy shows syntaxin 6 primarily on the trans-Golgi network (TGN), where is partially colocalizes with the TGN adapter protein AP-1 on clathrin-coated membranes . Additional label is present on small vesicles in the vicinity of endosome-like structures . Immunoprecipitation of syntaxin 6 revealed that it is present in a complex or complexes with alpha-soluble NSF attachment protein, vesicle-associated membrane protein 2, or cellubrevin and a mammalian homologue of VPS45, which is a member of the sec1 family implicated in Golgi to prevacuolar compartment trafficking in yeast . We show that mammalian VPS45 is found in multiple tissues, is partially membrane associated, and is enriched in the Golgi region . Converging lines of evidence suggest that syntaxin 6 mediates a TGN trafficking event, perhaps targeting to endosomes in mammalian cells. Eur J Cell Biol, 1997 Jul, 73(3), 198 - 204 Phylogenetic analysis of membrane trafficking proteins: a family reunion and secondary structure predictions; Terrian DM et al.; The realization that a highly conserved family of membrane proteins are localized to transport vesicles and selectively interact with proteins anchored at appropriate target sites of membrane fusion inspired a simple and compelling explanation of how proteins might be transferred and segregated within the cell, the "SNARE hypothesis" . This model holds that vesicle and target membrane proteins (designated as v-SNARE and t-SNARE proteins, respectively) wind around one another to form a three-stranded coiled coil structure, termed the prefusion complex . While the molecular topology of the prefusion complex has not been established, the concept that phylogenetically diverse SNARE proteins may become interlocked in a stable coiled coil is particularly attractive, because such a tertiary fold would only be permitted between strictly matched binding partners . For this reason, we have performed a phenetic analysis of all known SNARE sequences to assess the evolutionary and structural relatedness of these ancient protein families . Our phylogenetic analysis and consensus structure predictions revealed that syntaxin and SNAP-25 homologs are significantly related and constitute a superfamily of t-SNARE proteins that fall naturally into four major classes with distinct architectural motifs . The synaptobrevins sorted into three different classes of v-SNARE proteins . Comparison of the consensus structure predictions within each lineage or class of SNARE proteins strongly implied that coiled coil domains may not be required for fusion complex assembly in simple eukaryotic cells . It is our hypothesis that SNARE proteins in the late secretory pathway of mammalian cells may have elaborated more complex secondary structures (coiled coils), at about the time metazoan organisms diverged from yeast, that provide a sterically rigid foundation for positioning a conserved binding domain, the amphipathic alpha-helix. EMBO J, 1997 Jul 1, 16(13), 3995 - 4006 Transcriptional regulation in endoderm development: characterization of an enhancer controlling Hnf3g expression by transgenesis and targeted mutagenesis; Hiemisch H et al.; The hepatic nuclear factor 3gamma (Hnf3g) is a member of the winged helix gene family of transcription factors and is thought to be involved in anterior-posterior regionalization of the primitive gut . In this study, cis-regulatory elements essential for the expression of Hnf3g in vivo have been characterized . To this end, a 170 kb yeast artificial chromosome (YAC) carrying the entire Hnf3g locus was isolated and modified with a lacZ reporter gene . The two mouse lines carrying the unfragmented Hnf3g-lacZ YAC showed tissue-specific, copy number-dependent and position-independent expression, proving that 170 kb of the Hnf3g locus contain all elements important in the regulation of Hnf3g . Cis-regulatory elements necessary for expression of Hnf3g were identified in a three-step procedure . First, DNase I hypersensitive site mapping was used to delineate important chromatin regions around the gene required for tissue-specific activation of Hnf3g . Second, plasmid-derived transgenes and gene targeting of the endogenous Hnf3g gene locus were used to demonstrate that the 3'-flanking region of the gene is necessary and sufficient to direct reporter gene expression in liver, pancreas, stomach and small intestine . Third, a binding site for HNF-1alpha and beta, factors expressed in organs derived from the endoderm such as liver, gut and pancreas, was identified in this 3'-enhancer and shown to be crucial for enhancer function in vitro . Based on its expression pattern we inferred that HNF-1beta is a likely candidate for directly activating Hnf3g gene expression during development. EMBO J, 1997 Jul 1, 16(13), 3935 - 43 ZEB, a vertebrate homolog of Drosophila Zfh-1, is a negative regulator of muscle differentiation; Postigo AA et al.; A number of genes, spanning the evolutionary scale from yeast to mammals, that are involved in spatial and temporal patterning during development contain zinc finger and homeodomain motifs . One such zinc finger/homeodomain protein is Drosophila Zfh-1, a member of the zfh family of Drosophila genes, which is expressed in muscle precursors and is critical for the proper development of muscle . Here we demonstrate that a vertebrate homolog of Zfh-1 (ZEB) is a negative regulator of muscle differentiation . We show that ZEB binds to a subset of E boxes in muscle genes and functions by actively repressing transcription . One target of this repression is the members of the MEF-2 family, which synergize with proteins of the myogenic basic helix-loop-helix family (bHLH) (myoD, myf-5, myogenin and MRF-4) to induce myogenic differentiation . As muscle differentiation proceeds, myogenic bHLH proteins accumulate to levels sufficient to displace ZEB from the E boxes, releasing the repression and allowing bHLH proteins to further activate transcription . This mechanism of active transcriptional repression distinguishes ZEB from other negative regulators of myogenesis (Id, Twist and I-mfa) that inhibit muscle differentiation by simply binding and inactivating myogenic factors . The relative affinity of ZEB versus myogenic bHLH proteins varies for E boxes in different genes such that ZEB would be displaced from different genes at distinct times as myogenic bHLH proteins accumulate during myogenesis, thus providing a mechanism to regulate temporal order of gene expression. EMBO J, 1997 Jul 1, 16(13), 3898 - 911 Proteolytic processing regulates receptor specificity and activity of VEGF-C; Joukov V et al.; The recently identified vascular endothelial growth factor C (VEGF-C) belongs to the platelet-derived growth factor (PDGF)/VEGF family of growth factors and is a ligand for the endothelial-specific receptor tyrosine kinases VEGFR-3 and VEGFR-2 . The VEGF homology domain spans only about one-third of the cysteine-rich VEGF-C precursor . Here we have analysed the role of post-translational processing in VEGF-C secretion and function, as well as the structure of the mature VEGF-C . The stepwise proteolytic processing of VEGF-C generated several VEGF-C forms with increased activity towards VEGFR-3, but only the fully processed VEGF-C could activate VEGFR-2 . Recombinant 'mature' VEGF-C made in yeast bound VEGFR-3 (K{D} = 135 pM) and VEGFR-2 (K{D} = 410 pM) and activated these receptors . Like VEGF, mature VEGF-C increased vascular permeability, as well as the migration and proliferation of endothelial cells . Unlike other members of the PDGF/VEGF family, mature VEGF-C formed mostly non-covalent homodimers . These data implicate proteolytic processing as a regulator of VEGF-C activity, and reveal novel structure-function relationships in the PDGF/VEGF family. Mol Biochem Parasitol, 1997 Jul, 87(1), 49 - 60 The chromosomal organization of the Plasmodium falciparum var gene family is conserved; Thompson JK et al.; The var gene family of Plasmodium falciparum encodes the protein PfEMP1 which is located on the surface of infected erythrocytes and is the receptor that mediates binding to ligands on endothelial cells . This family of proteins is responsible for antigenic variation and differences in binding phenotype to ligands such as CD36 and ICAM1 . We have compared the organization of the var gene family in three in vitro cloned lines of P . falciparum and show that most var genes are located in the subtelomeric region of each chromosome closely linked to the repetitive sequence rep20 . While most chromosomes possess var genes in the subtelomeric region, in each in vitro cloned line there are some chromosomes that have deleted subtelomeric repetitive regions which include var genes . Comparison of the location of var genes in a field isolate showed that it does not have any detectable subtelomeric deletions as all chromosomes contain var genes and rep20 sequences . We have detected three chromosomes (4, 7 and 12) that contain var gene loci in more stable central regions and the position of these genes on chromosome 4 in the cloned lines analysed is conserved . The location of most of the var gene family in the