Matrix Biol, 1998 Oct, 17(6), 401 - 12 Two-hybrid analysis reveals multiple direct interactions for thrombospondin 1; Aho S et al.; The yeast two-hybrid system was used to reveal the interactions between proteins residing within the cutaneous basement membrane zone and other gene products expressed in cultured human keratinocytes . The proteins of interest included type VII collagen, the predominant component of anchoring fibrils, and laminin 5, a component of anchoring filaments . Although the two-hybrid system was not able to verify a direct interaction between the type VII collagen NC1 domain and the short arm of Lam(beta)3, the type VII collagen NC1 domain (tVII/NC1) and the laminin 5 beta3 chain globular domain VI (lam5/beta3) cDNAs, when used as baits, detected four overlapping cDNA clones encoding thrombospondin 1 (TSP1) . The overlapping region of these cDNAs encodes amino acids 400-459, a segment included within a 70 kDa chymotryptic fragment known to bind type V collagen, laminin-1 and other matrix components . The type VII collagen NC1/TSP1 interaction was confirmed by exchanging the vectors, and the interacting domain was mapped by testing a set of both 5' and 3' deletion constructs . The central region of TSP1, when used as a bait in two-hybrid system, showed strong binding to the fibronectin (FN) type III-like repeats 4-7 of type VII collagen NC1 domain . The TSP1 bait also interacted with laminin 5 beta3 chain domain V/III, and the TSP1/laminin 5 beta3 chain interaction was verified by a GST-fusion protein interaction assay . The transcripts encoding TSP1, TSP2, Lam(beta)3 and type VII collagen were abundant in cultured foreskin keratinocytes, and the expression of TSP1 and TSP2 in a wide variety of adult and fetal tissues was confirmed by PCR analysis of multiple tissue cDNA panels . Furthermore, TSP1 type I repeats showed self interaction, and recognized a clone for extracellular matrix protein fibrillin-2 . In addition, clones encoding angiogenesis related protein Jagged1 and a platelet enzyme phospholipase scramblase were identified . Thus, the results indicate several previously undetected interactions of TSP1, which is known to be highly expressed during embryonic development, tissue remodeling and wound healing. Eur J Biochem, 1998 Nov 1, 257(3), 529 - 37 Identification of an essential gene encoding a class-V unconventional myosin in Drosophila melanogaster; MacIver B et al.; Class-V myosins are a unique type of myosin motor with roles in intracellular transport . The mouse dilute gene was the first member of this class to be cloned, with mutations resulting in lightening of the coat colour or neurogenic defects leading to early death . Further examples of class-V myosins have been described in yeast, chicken and rat . Here, we report the cloning of the first class-V myosin from Drosophila . We show that expression of this myosin is predominantly in the adult germ line and early embryo and that the transcript is localised in the oocyte during oogenesis . Genetic and in situ hybridisation experiments have determined that this gene is located in the 43C region . We have evidence that it maps to a mutation in this region with an embryonic lethal phenotype. Chemosphere, 1998 Dec, 37(14-15), 3035 - 45 Immunotoxic effects of organotin compounds in Tapes philippinarum; Cima F et al.; One of the most harmful groups of coastal pollutants is the organotin compounds (OTCs) which have severe effects on both aquatic organisms and mammals including humans . The immunotoxic effects of OTCs were studied in the cultivated clam Tapes philippinarum by determining the immunosuppressant role on in vitro yeast phagocytosis at low doses (0.01, 0.05, 0.1 microM) . The phagocytic index was significantly reduced in an irreversible non-lethal manner depending on concentration and lipophilic affinity . The order of inhibition was TBT > or = DBT > MBT for butyltins and TPTC > TPTA > or = TPTH for triphenyltins. Biochim Biophys Acta, 1998 Nov 26, 1443(1-2), 155 - 68 Cloning of a third member of the D52 gene family indicates alternative coding sequence usage in D52-like transcripts; Nourse CR et al.; D52 proteins are emerging as signalling molecules which may be regulators of cell proliferation . Having previously reported the existence of the human D52 gene family, comprising the hD52 and hD53 genes expressed in human breast carcinoma, we report the identification of a novel human gene hD54 (TPD52L2), which represents a third D52 gene family member . In situ mapping placed the hD54 gene on human chromosome 20q13.2-q13.3, a localization distinct from those of both hD52 and hD53 genes . The identified hD54 cDNAs predicted three hD54 isoforms, suggesting that alternatively-spliced transcripts may be produced from D52-like genes . This was confirmed by directly sequencing reverse transcriptase-polymerase chain reaction (RT-PCR) products amplified from D52-like gene transcripts expressed in developing and adult rat tissues, and by performing sequence analyses of the expressed sequence tag divisions of nucleotide databases . Alternative splicing of sequences encoding two regions, termed ins2 and ins3, was identified in one or more D52-like genes, with these alternative splicing events being differentially regulated . The functional consequences of alternative splicing were examined by characterizing the protein-protein interactions mediated by a truncated hD53 isoform within the yeast two-hybrid system . This hD53 isoform displayed altered interaction capabilities with respect to those of full-length hD53, suggesting that alternative splicing within the D52 gene family functions in part to alter the protein-protein interaction capabilities of encoded isoforms. Biochim Biophys Acta, 1998 Dec 8, 1436(1-2), 127 - 50 Structure and function of phosphoinositide 3-kinases; Wymann MP et al.; Phosphoinositide kinases (PI3Ks) play an important role in mitogenic signaling and cell survival, cytoskeletal remodeling, metabolic control and vesicular trafficking . Here we summarize the structure-function relationships delineating the activation process of class I PI3Ks involving various domains of adapter subunits, Ras, and interacting proteins . The resulting product, PtdIns(3,4,5)P3, targets Akt/protein kinase B (PKB), Bruton's tyrosine kinase (Btk), phosphoinositide-dependent kinases (PDK), integrin-linked kinase (ILK), atypical protein kinases C (PKC), phospholipase Cgamma and more . Surface receptor-activated PI3Ks function in mammals, insects, nematodes and slime mold, but not yeast . While many members of the class II family have been identified and characterized biochemically, it is presently unknown how these C2-domain containing PI3Ks are activated, and which PI substrate they phosphorylate in vivo . PtdIns 3-P is produced by Vps34p/class III PI3Ks and operates via the PtdIns 3-P-binding proteins early endosomal antigen (EEA1), yeast Vac1p, Vps27p, Pip1p in lysosomal protein targeting . Besides the production of D3 phosphorylated lipids, PI3Ks have an intrinsic protein kinase activity . For trimeric GTP-binding protein-activated PI3Kgamma, protein kinase activity seems to be sufficient to trigger mitogen-activated protein kinase (MAPK) . Recent disruption of PI3K genes in slime mold, Caenorhabditis elegans, Drosophila melanogaster and mice further underlines the importance of PI3K signaling systems and elucidates the role of PI3K signaling in multicellular organisms. J Biol Chem, 1998 Dec 11, 273(50), 33876 - 83 Regulation of insulin-stimulated GLUT4 translocation by Munc18c in 3T3L1 adipocytes; Thurmond DC et al.; Insulin stimulates glucose transporter (GLUT) 4 vesicle translocation from intracellular storage sites to the plasma membrane in 3T3L1 adipocytes through a VAMP2- and syntaxin 4-dependent mechanism . We have observed that Munc18c, a mammalian homolog of the yeast syntaxin-binding protein n-Sec1p, competed for the binding of VAMP2 to syntaxin 4 . Consistent with an inhibitory function for Munc18c, expression of Munc18c, but not the related Munc18b isoform, prevented the insulin stimulation of GLUT4 and IRAP/vp165 translocation to the plasma membrane without any significant effect on GLUT1 trafficking . As expected, overexpressed Munc18c was found to co-immunoprecipitate with syntaxin 4 in the basal state . However, these complexes were found to dissociate upon insulin stimulation . Furthermore, endogenous Munc18c was predominantly localized to the plasma membrane and its distribution was not altered by insulin stimulation . Although expression of enhanced green fluorescent protein-Munc18c primarily resulted in a dispersed cytosolic distribution, co-expression with syntaxin 4 resulted in increased localization to the plasma membrane . Together, these data suggest that Munc18c inhibits the docking/fusion of GLUT4-containing vesicles by blocking the binding of VAMP2 to syntaxin 4 . Insulin relieves this inhibition by inducing the dissociation of Munc18c from syntaxin 4 and by sequestering Munc18c to an alternative plasma membrane binding site. J Biol Chem, 1998 Dec 11, 273(50), 33741 - 9 Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins . Involvement of basic leucine zipper transcription factors; Yoshida H et al.; When unfolded proteins accumulate in the endoplasmic reticulum (ER), transcription of glucose-regulated proteins (GRPs) representing ER-resident molecular chaperones is markedly induced via the unfolded protein response (UPR) pathway . In contrast to recent progress in the analysis of yeast UPR, both cis-acting elements and transactivators responsible for mammalian UPR have remained obscure . Here, we analyzed the promoter regions of human GRP78, GRP94, and calreticulin genes and identified a novel element designated the ER stress response element (ERSE) . ERSE, with a consensus of CCAATN9CCACG, was shown to be necessary and sufficient for induction of these GRPs . Using yeast one-hybrid screening, we isolated a human cDNA encoding a basic leucine zipper (bZIP) protein, ATF6, as a putative ERSE-binding protein . When overexpressed in HeLa cells, ATF6 enhanced transcription of GRP genes in an ERSE-dependent manner, whereas CREB-RP, another bZIP protein closely related to ATF6, specifically inhibited GRP induction . Endogenous ATF6 constitutively expressed as a 90-kDa protein was converted to a 50-kDa protein in ER-stressed cells, which appeared to be important for the cellular response to ER stress . These results suggest that, as in yeast, bZIP proteins are involved in mammalian UPR, acting through newly defined ERSE. J Biol Chem, 1998 Dec 11, 273(50), 33556 - 60 Interaction of cytosolic adaptor proteins with neuronal apolipoprotein E receptors and the amyloid precursor protein; Trommsdorff M et al.; Apolipoprotein E, alpha2-macroglobulin, and amyloid precursor protein (APP) are involved in the development of Alzheimer's disease . All three proteins are ligands for the low density lipoprotein (LDL) receptor-related protein (LRP), an abundant neuronal surface receptor that has also been genetically linked to Alzheimer's disease . The cytoplasmic tails of LRP and other members of the LDL receptor gene family contain NPxY motifs that are required for receptor endocytosis . To investigate whether these receptors may have functions that go beyond ligand internalization, e.g . possible roles in cellular signaling, we searched for proteins that might interact with the cytoplasmic tails of the receptors . A family of adaptor proteins containing protein interaction domains that can interact with NPxY motifs has previously been described . Using yeast 2-hybrid and protein coprecipitation approaches in vitro, we show that the neuronal adaptor proteins FE65 and mammalian Disabled bind to the cytoplasmic tails of LRP, LDL receptor, and APP, where they can potentially serve as molecular scaffolds for the assembly of cytosolic multiprotein complexes . FE65 contains two distinct protein interaction domains that interact with LRP and APP, respectively, raising the possibility that LRP can modulate the intracellular trafficking of APP . Tyrosine-phosphorylated mammalian Disabled can recruit nonreceptor tyrosine kinases, such as src and abl, to the cytoplasmic tails of the receptors to which it binds, suggesting a molecular pathway by which receptor/ligand interaction on the cell surface could generate an intracellular signal. J Biol Chem, 1998 Dec 11, 273(50), 33524 - 32 P-CIP1, a novel protein that interacts with the cytosolic domain of peptidylglycine alpha-amidating monooxygenase, is associated with endosomes; Chen L et al.; The cytosolic domain of the peptide processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) contains signals that direct its trafficking in the secretory and endosomal pathways . Using the yeast two-hybrid system, Alam et al . (Alam, M . R., Caldwell, B . D., Johnson, R . C., Darlington, D . N., Mains, R . E., and Eipper, B . A . (1996) J . Biol . Chem . 271, 28636) identified three proteins that interact with a fragment of the PAM cytosolic domain containing these targeting signals . We cloned the rat and human cDNAs encoding PAM COOH-terminal interactor protein-1 (P-CIP1) . Both cDNAs contain an open reading frame that encodes a novel protein of 435 amino acids . The P-CIP1 protein is highly conserved from rat to human (85% identity) but does not display significant homology to proteins in the GenBank data base . In vitro, P-CIP1 interacts with the cytosolic domain of wild type PAM-1, but does not interact with mutant PAM-1 proteins that fail to target correctly when expressed in endocrine cells . P-CIP1 contains multiple consensus serine/threonine phosphorylation sites and a region predicted to form a coiled-coil at the COOH terminus . When expressed in endocrine cells or fibroblasts, P-CIP1 is distributed in a punctate pattern in the perinuclear area but does not significantly overlap the distribution of transfected wild type PAM-1 . The distribution of P-CIP1 displays significant overlap with the distribution of the secretory carrier membrane proteins, internalized Texas Red-conjugated transferrin, and Rab11 . The data suggest that P-CIP1 associates with vesicles in the recycling endosomal pathway, and may play a role in regulating the trafficking of integral membrane PAM. Blood, 1998 Dec 1, 92(11), 4031 - 5 Molecular cytogenetic characterization of a critical region in bands 7q35-q36 commonly deleted in malignant myeloid disorders; Dohner K et al.; Loss of chromosome 7 (-7) or deletion of the long arm (7q-) are recurring chromosome abnormalities in myeloid leukemias . The association of -7/7q- with myeloid leukemia suggests that these regions contain novel tumor suppressor gene(s), whose loss of function contribute to leukemic transformation or tumor progression . Based on chromosome banding analysis, two critical regions have been identified, one in band q22 and another in bands q32-q35 . Presently there are no data available on the molecular delineation of the distal critical region . In this study we analyzed bone marrow and blood samples from 13 patients with myeloid leukemia (de novo myelodysplastic syndrome {MDS}, n = 3; de novo acute myeloid leukemia {AML}, n = 9; therapy-related (t-) AML, n = 1) which, on chromosome banding analysis, exhibited deletions (n = 12) or in one case a balanced translocation involving bands 7q31-qter using fluorescence in situ hybridization (FISH) . As probes we used representative clones from a contig map of yeast artificial chromosome (YAC) clones that spans chromosome bands 7q31.1-qter . In the 12 cases with loss of 7q material, we identified a commonly deleted region of approximately 4 to 5 megabasepairs in size encompassing the distal part of 7q35 and the proximal part of 7q36 . Furthermore, the breakpoint of the reciprocal translocation from the patient with t-AML was localized to a 1,300-kb sized YAC clone that maps to the proximal boundary of the commonly deleted region . Interestingly, in this case both homologs of chromosome 7 were affected: one was lost (-7) and the second exhibited the t(7q35) . The identification and delineation of translocation and deletion breakpoints provides the first step toward the identification of the gene(s) involved in the pathogenesis of 7q35-q36 aberrations in myeloid disorders. Development, 1999 Jan, 126(1), 97 - 107 The DAF-3 Smad binds DNA and represses gene expression in the Caenorhabditis elegans pharynx; Thatcher JD et al.; Gene expression in the pharyngeal muscles of Caenorhabditis elegans is controlled in part by organ-specific signals, which in the myo-2 gene target a short DNA sequence termed the C subelement . To identify genes contributing to these signals, we performed a yeast one-hybrid screen for cDNAs encoding factors that bind the C subelement . One clone recovered was from daf-3, which encodes a Smad most closely related to vertebrate Smad4 . We demonstrated that DAF-3 binds C subelement DNA directly and specifically using gel mobility shift and DNase1 protection assays . Mutation of any base in the sequence GTCTG interfered with binding in the gel mobility shift assay, demonstrating that this pentanucleotide is a core recognition sequence for DAF-3 binding . daf-3 is known to promote formation of dauer larvae and this activity is negatively regulated by TGFbeta-like signaling . To determine how daf-3 affects C subelement enhancer activity in vivo, we examined expression a gfp reporter controlled by a concatenated C subelement oligonucleotide in daf-3 mutants and other mutants affecting the TGFbeta-like signaling pathway controlling dauer formation . Our results demonstrate that wild-type daf-3 can repress C subelement enhancer activity during larval development and, like its dauer-promoting activity, daf-3's repressor activity is negatively regulated by TGFbeta-like signaling . We have examined expression of this gfp reporter in dauer larvae and have observed no daf-3-dependent repression of C activity . These results suggest daf-3 directly regulates pharyngeal gene expression during non-dauer development. J Immunol, 1998 Dec 1, 161(11), 6022 - 9 Human CD5 signaling and constitutive phosphorylation of C-terminal serine residues by casein kinase II; Calvo J et al.; CD5 is a lymphocyte surface glycoprotein with a long cytoplasmic domain suitable for phosphorylation and signal transduction, which is involved in the modulation of Ag-specific receptor-mediated activation and differentiation signals . In this study, we use Jurkat T cell transfectants of CD5 cytoplasmic tail mutants to reveal phosphorylation sites relevant to signal transduction . Our results show that casein kinase II (CKII) is responsible for the constitutive phosphorylation of CD5 molecules at a cluster of three serine residues located at the extreme C terminus (S458, S459, and S461) . Furthermore, the yeast two-hybrid system demonstrates the specific association between the C-terminal regions of the CD5 cytoplasmic tail and the regulatory beta subunit of CKII . We demonstrate that CKII associates with and phosphorylates the C-terminal region of CD5, a conserved domain known to be relevant for the generation of second lipid messengers, and thereby enables at least one component of its signaling function. Virus Res, 1998 Sep, 57(1), 1 - 9 Insertion of a bovine SMT3B gene in NS4B and duplication of NS3 in a bovine viral diarrhea virus genome correlate with the cytopathogenicity of the virus; Qi F et al.; Bovine viral diarrhea virus (BVDV) infection in cattle can lead to the development of a fatal syndrome called mucosal disease (MD) . The induction of MD requires co-existence of two biotypes of BVDV: cytopathic (cp) and non-cytopathic (ncp) . Studies have shown that in some cases, cp viruses are generated from ncp viruses by cellular gene insertions, duplications or rearrangements in the viral genome . Cellular ubiquitin gene is the most frequently reported insert in BVDV 1 . Here we report the finding of a novel cellular gene insertion (termed cSNCINS) in the NS4B gene accompanied by the duplication of NS3 in a cpBVDV genome . The amino acid sequence of the insert is identical to that of the human SMT3B protein and is 98% similar to that of the human SMT3A protein . Both SMT3B and SMT3A proteins are homologues of the yeast SMT3 protein . The cSNCINS protein is encoded by an open reading frame located on a 1150-bp bovine endometrial cDNA fragment . Our results indicate that the cSNCINS is a bovine homologue of the human SMT3B gene and that insertion of the BSMT3B gene together with duplication of NS3 in the viral genome may account for the cytopathogenicity of this virus. J Biomol Struct Dyn, 1998 Oct, 16(2), 413 - 24 Dynamics of protein-protein docking: cytochrome c and cytochrome c peroxidase revisited; Castro G et al.; The dynamics of the docking step in the electron transfer reaction between yeast cytochrome c peroxidase and iso-1-cytochrome c has been studied using the Brownian dynamics method . In particular we have calculated the bimolecular rate constant at which a specific complex, the xray crystalline complex, can form in solution by translational and rotational diffusion in a field of force . Complexation criteria have been assessed based on the simultaneous alignment of three atom-atom contacts, as well as alternative criteria . The proteins are able to align one or two contacts at remarkably high rates, in fact, at rates approaching the diffusion-controlled limit for two spheres reactive over their entire surfaces . Three contacts may align, and hence the specific complex may dock, at rates on the order of 10(8) M(-1) s(-1), which is quite representative of the experimental association rate constant for ET-competent complex(es) . The formation of the specific complex is strongly influenced by the favorable electrostatic interaction between these proteins . It is striking that a specific protein-protein complex can form within one order of magnitude as fast as two spherical proteins can touch at any orientation . It remains plausible that the high ET tunneling rate in this system can take place through a single highly favorable specific complex using a single high efficiency pathway . Still the contribution from a nonspecific set of complexes is not ruled out, particularly considering the marginal reproduction of the ionic strength dependence in the formation of the xray complex. Pediatrics, 1998 Dec, 102(6), 1480 - 2 Non-aspergillus allergic bronchopulmonary mycosis in a pediatric patient with cystic fibrosis; Gondor M et al.; This article describes a child with cystic fibrosis (CF) and allergic bronchopulmonary mycosis caused by Tricosporon beigelii . An 11-year-old boy with CF failed to respond to conventional treatment for a pulmonary exacerbation . Bronchial washings contained copious budding yeast forms, subsequently identified as T beigelii . Total serum immunoglobulin E was elevated and precipitating antibodies to T beigelii were positive . Together these findings support the diagnosis of allergic bronchopulmonary mycosis . The patient improved with antifungal therapy and systemic glucocorticoid therapy . The pathologic potential of yeast in the airways of patients with CF is unclear . The diagnosis of non-Aspergillus allergic bronchopulmonary mycosis requires a high degree of suspicion and has potentially important implications for the management of patients with CF. Genetics, 1998 Dec, 150(4), 1595 - 603 Physical mapping of duplicated genomic regions of two chromosome ends in rice; Wu J et al.; Two genomic regions duplicated in distal ends of the short arms of chromosomes 11 and 12 in rice (Oryza sativa L.) were characterized by YAC ordering with 46 genetic markers . Physical maps covering most of the duplicated regions were generated . Thirty-five markers, including 21 rice cDNA clones, showed the duplicated loci arrayed strictly in the same order along the two specific genomic regions . Regardless of their different genetic distances, the two duplicated segments may have a similar and minimum physical size with an expected length of about 2.5 Mb . However, differences of RFLP frequency for the duplicated DNA copies and recombination frequency for a given homoeologous area between the two regions were observed, indicating that these changes in genome organization occurred after the duplication . Our results establish a good model system for resolving the relationships between gene duplication, expression of duplicated genes, and the frequency of meiotic recombination in small chromosomal regions. Endocrinology, 1998 Dec, 139(12), 4911 - 9 Interaction of insulin receptor substrate-1 (IRS-1) with phosphatidylinositol 3-kinase: effect of substitution of serine for alanine in potential IRS-1 serine phosphorylation sites; Delahaye L et al.; Serine and threonine phosphorylation has been shown to down-regulate insulin signaling at multiple steps, including the receptor and downstream molecules such as insulin receptor substrate-1 (IRS-1) . To further address the mechanism of this regulation at the level of IRS-1, we constructed a double serine mutant of IRS-1: S662A/S731A-IRS-1 . The serines 662 and 731 mutated to alanine are surrounding tyrosines Y658 and Y727, respectively . These tyrosines are comprised in YXXM motifs, which are potential binding sites for the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase . In a first series of experiments using the yeast two-hybrid system, we show that IRS-1 interacts with p85alpha, and this interaction depends on tyrosine phosphorylation, as shown with the IRS-1 mutant F18 and 3Y-IRS-1 . F18-IRS-1 contains 18 potential tyrosine phosphorylation sites mutated to phenylalanine; three of them, i.e . Y608, 628, and 658, which are potential binding sites for p85alpha, have been added back in the 3Y-IRS-1 mutant . The tyrosine phosphorylation of IRS-1, which is required for the interaction with p85alpha, is thought to occur via endogenous yeast kinases that phosphorylate IRS-1 at least on these PI 3-kinase-binding sites . Next, we show that not only p85alpha but also p55PIK, another regulatory subunit of PI 3-kinase, interacts with IRS-1 in yeast . Interestingly, for both regulatory subunits their interaction with IRS-1 is up-regulated by mutating serines 662 and 731 on IRS-1 . In a previous study we found that insulin-stimulated PI 3-kinase activity was increased not only in the presence of S662A/S731A-IRS-1 but also under resting conditions compared with the activity seen with WT-IRS-1 . Here we demonstrate in 293-EBNA cells overexpressing S662A/S731A-IRS-1 that insulin-stimulated protein kinase B activity is not augmented, whereas without insulin treatment, basal activity is increased compared with that in cells overexpressing wild-type IRS-1 . In conclusion, we have shown that 1) potential serine phosphorylation sites on IRS-1, which are adjacent to YXXM binding motifs for PI 3-kinase, negatively regulate binding of IRS-1 to PI 3-kinase regulatory subunits; and 2) these modulations affect protein kinase B activity. J Biol Chem, 1998 Dec 4, 273(49), 32883 - 8 Inhibition of Hsp70 ATPase activity and protein renaturation by a novel Hsp70-binding protein; Raynes DA et al.; A cDNA that codes for an Hsp70-interacting protein (HspBP1) was isolated from a human heart cDNA library using the yeast two-hybrid system . The derived amino acid sequence is unique and therefore represents a new regulator of Hsp70 . Northern blots of RNA from human tissues indicate that HspBP1 mRNA has a size of approximately 1.7 kilobase pairs and is present in all tissues analyzed but is most abundant in heart and skeletal muscle . Western blot analysis revealed a protein of approximately 40 kilodaltons detected in cell extracts . The ATPase domain of Hsp70 demonstrated binding to HspBP1 . Further experiments showed binding of HspBP1 to Hsp70 and Hsc70 in a total heart extract . HspBP1 (8 microM) inhibited approximately 90% of the Hsp40-activated Hsp70 ATPase activity . HspBP1 prevented ATP binding to Hsp70, and therefore this is the likely mechanism of inhibition . Hsp40-activated ATPase activity is essential for the renaturation activity of Hsp70; therefore, the effects of HspBP1 on renaturation of luciferase in a reticulocyte lysate and a defined system were examined . HspBP1 inhibited renaturation with half-maximal inhibition at 2 microM . These data indicate that we have identified a novel Hsp70-interacting protein that inhibits Hsp70 chaperone activity. J Biol Chem, 1998 Dec 4, 273(49), 32528 - 34 Oxygenation cascade in conversion of n-alkanes to alpha,omega-dioic acids catalyzed by cytochrome P450 52A3; Scheller U et al.; Purified recombinant cytochrome P450 52A3 and the corresponding NADPH-cytochrome P450 reductase from the alkane-assimilating yeast Candida maltosa were reconstituted into an active alkane monooxygenase system . Besides the primary product, 1-hexadecanol, the conversion of hexadecane yielded up to five additional metabolites, which were identified by gas chromatography-electron impact mass spectrometry as hexadecanal, hexadecanoic acid, 1, 16-hexadecanediol, 16-hydroxyhexadecanoic acid, and 1, 16-hexadecanedioic acid . As shown by substrate binding studies, the final product 1,16-hexadecanedioic acid acts as a competitive inhibitor of n-alkane binding and may be important for the metabolic regulation of the P450 activity . Kinetic studies of the individual sequential reactions revealed high Vmax values for the conversion of hexadecane, 1-hexadecanol, and hexadecanal (27, 23, and 69 min-1, respectively), whereas the oxidation of hexadecanoic acid, 1, 16-hexadecanediol, and 16-hydroxyhexadecanoic acid occurred at significantly lower rates (9, 9, and 5 min-1, respectively) . 1-Hexadecanol was found to be the main branch point between mono- and diterminal oxidation . Taken together with data on the incorporation of 18O2-derived oxygen into the hexadecane oxidation products, the present study demonstrates that a single P450 form is able to efficiently catalyze a cascade of sequential mono- and diterminal monooxygenation reactions from n-alkanes to alpha, omega-dioic acids with high regioselectivity. J Biol Chem, 1998 Dec 4, 273(49), 32437 - 45 Identification and characterization of a membrane protein (y+L amino acid transporter-1) that associates with 4F2hc to encode the amino acid transport activity y+L . A candidate gene for lysinuric protein intolerance; Torrents D et al.; We have identified a new human cDNA (y+L amino acid transporter-1 (y+LAT-1)) that induces system y+L transport activity with 4F2hc (the surface antigen 4F2 heavy chain) in oocytes . Human y+LAT-1 is a new member of a family of polytopic transmembrane proteins that are homologous to the yeast high affinity methionine permease MUP1 . Other members of this family, the Xenopus laevis IU12 and the human KIAA0245 cDNAs, also co-express amino acid transport activity with 4F2hc in oocytes, with characteristics that are compatible with those of systems L and y+L, respectively . y+LAT-1 protein forms a approximately 135-kDa, disulfide bond-dependent heterodimer with 4F2hc in oocytes, which upon reduction results in two protein bands of approximately 85 kDa (i.e . 4F2hc) and approximately 40 kDa (y+LAT-1) . Mutation of the human 4F2hc residue cysteine 109 (Cys-109) to serine abolishes the formation of this heterodimer and drastically reduces the co-expressed transport activity . These data suggest that y+LAT-1 and other members of this family are different 4F2 light chain subunits, which associated with 4F2hc, constitute different amino acid transporters . Human y+LAT-1 mRNA is expressed in kidney >> peripheral blood leukocytes >> lung > placenta = spleen > small intestine . The human y+LAT-1 gene localizes at chromosome 14q11.2 (17cR approximately 374 kb from D14S1350), within the lysinuric protein intolerance (LPI) locus (Lauteala, T., Sistonen, P . , Savontaus, M . L., Mykkanen, J., Simell, J., Lukkarinen, M., Simmell, O., and Aula, P . (1997) Am . J . Hum . Genet . 60, 1479-1486) . LPI is an inherited autosomal disease characterized by a defective dibasic amino acid transport in kidney, intestine, and other tissues . The pattern of expression of human y+LAT-1, its co-expressed transport activity with 4F2hc, and its chromosomal location within the LPI locus, suggest y+LAT-1 as a candidate gene for LPI. Mol Gen Genet, 1998 Oct, 260(1), 92 - 101 Chromosome landing at the barley Rar1 locus; Lahaye T et al.; The barley Rar1 gene is an essential component of the race-specific, Mla-12-specified powdery mildew resistance reaction . As part of a map-based cloning strategy designed to isolate Rar1, five barley yeast artificial chromosomes (YACs) have been identified, ranging in size from 300 to 1100 kb . PCR-based YAC end-specific markers have been established and were employed to construct a local YAC contig . Four out of five YAC clones were found to be non-colinear with the source DNA . High-resolution genetic mapping of the YAC ends demonstrated that the set of five overlapping YAC clones encompasses the barley Rar1 gene. Hokkaido Igaku Zasshi, 1998 Jul, 73(4), 407 - 17 {Clinical and pathological significance of p53 gene mutation in human cerebral glioblastoma multiforme}; Matsumoto R; Cerebral glioblastomas multiforme (GMs), the most malignant human primary brain tumor, molecular genetically comprise two subsets characterized by p53 tumor suppressor gene status . One is characterized by p53 mutation and another is with wild-type p53 and overexpression of epidermal growth factor receptor (EGFR) . We analyzed the p53 status by a highly reliable yeast-based assay, overexpression of EGFR by immunohistochemistry, and their relations to clinical outcome along with other several clinical parameters, in a total of 42 patients with GMs . We found p53 mutations in 18 cases (43%) of 42 GMs and overexpression of EGFR in 16 cases (43%) of 37 GMs . The tumors with p53 mutation were associated with younger onset ages and longer survival . We also analyzed their survival and radiation sensitivity relative to p53 status, EGFR and other prognostic factors, including onset age, extent of surgery, post-operative Karnofsky performance score (KPS), and location of tumor . Log-rank tests showed that younger onset ages (below 50 years; p < 0.0111), p53 mutation (p < 0.0005), superficial tumor location (p < 0.0026) and post operatives KPS (over 80%; p < 0.0147) correlated with longer survival . Multivariate Cox regression analysis identified the absence of p53 mutation (p < 0.0335) and the deep tumor location (p < 0.0297) as the independent, adverse prognostic factors . Tumor response to radiation was assessed by progression-free period (time to progression; TTP) . A significant correlation was found between TTP and survival (p < 0.0001) . Log-rank tests and multivariate Cox regression analysis showed that younger onset ages (below 50 years; p < 0.0145), p53 mutation (p < 0.0001), radical resection (p < 0.0198) and post operative KPS (over 80%; p < 0.0001) correlated with radiation sensitivity, and identified the absence of p53 mutation (p < 0.0146) as the sole independent hazard for radiation response . In conclusion, p53 mutation confers radiation sensitivity in GMs, which results in patients' longer survival. Int J Syst Bacteriol, 1998 Oct, 48 Pt 4, 1425 - 9 Trichosporon japonicum sp . nov . isolated from the air; Sugita T et al.; A yeast strain isolated from the air in Japan was found to represent a new species, and was named Trichosporon japonicum . This species produced arthroconidia and appressoria . T . japonicum formed a cluster with the appressorium-forming species Trichosporon inkin and Trichosporon ovoides in a phylogenetic tree constructed using small-subunit rDNA sequences . However, they had low relatedness to each other in DNA-DNA hybridization experiments . T . japonicum is distinguished from both T . inkin and T . ovoides by its ability to assimilate inulin, and its inability to assimilate L-rhamnose . JCM 8357T is the type strain of the species. Genomics, 1998 Dec 1, 54(2), 267 - 77 IFI60/ISG60/IFIT4, a new member of the human IFI54/IFIT2 family of interferon-stimulated genes; de Veer MJ et al.; We report the cloning and sequencing of a full-length cDNA encoding a new member of the human IFI54 (HGMW-approved symbol IFIT2) gene family, designated IFI60 (HGMW-approved symbol IFIT4) . The upstream regulatory region of IFI60 shows conservation in structure with that of the IFI54 and IFI56 (HGMW-approved symbol IFIT1) genes, each containing two interferon-stimulated response elements upstream of a conserved TATA box . We have established a partial gene map of the IFI54 gene family by analysis of YAC library clones . All four members of the human family are clustered together at chromosome 10q23.3 . It is proposed that the four members of the IFI54 gene family evolved by a series of duplication events from a common gene of origin . Genomics, 1998 Dec 1, 54(2), 256 - 66 Genomic organization of a 225-kb region in Xq28 containing the gene for X-linked myotubular myopathy (MTM1) and a related gene (MTMR1); Kioschis P et al.; MTM1 is responsible for X-linked recessive myotubular myopathy, which is a congenital muscle disorder linked to Xq28 . MTM1 is highly conserved from yeast to humans . A number of related genes also exist . The MTM1 gene family contains a consensus sequence consisting of the active enzyme site of protein tyrosine phosphatases (PTPs), suggesting that they belong to a new family of PTPs . Database searches revealed homology of myotubularin and all related peptides to the cisplatin resistance-associated alpha protein, which implicates an as yet unknown function . In addition, homology to the Sbf1 protein (SET binding factor 1), involved in the oncogenic transformation of fibroblasts and differentiation of myoblasts, was also evident . We describe 225 kb of genomic sequence containing MTM1 and the related gene, MTMR1, which lies 20 kb distal to MTM1 . Although there is only moderate conservation of the exons, the striking similarity in the gene structures indicates that these two genes arose by duplication . Calculations suggest that this event occurred early in evolution long before separation of the human and mouse lineages . So far, mutations have been identified in the coding sequence of only 65% of the patients analyzed, indicating that the remaining mutations may lie in noncoding regions of MTM1 or possibly in MTMR1 . Knowledge of the genomic sequence will facilitate mutation analyses of the coding and noncoding sequences of MTM1 and MTMR1 . Genomics, 1998 Dec 1, 54(2), 191 - 9 The human gene encoding the lectin-type oxidized LDL receptor (OLR1) is a novel member of the natural killer gene complex with a unique expression profile; Yamanaka S et al.; LOX-1 is an endothelial receptor for oxidized low-density lipoprotein that plays essential roles in atherogenesis . LOX-1 has the highest homology with C-type lectin receptors expressed on natural killer cells . In the present study, we cloned and characterized the human LOX-1 gene (HGMW-approved symbol OLR1) . The gene structure of LOX-1 resembles that of the natural killer cell receptors . Fluorescence in situ hybridization and analyses of a yeast artificial chromosome contig revealed that the human LOX-1 gene is located in the natural killer gene complex on chromosome 12p12-p13, where the genes of the natural killer cell receptors cluster . In contrast, the expression pattern of LOX-1 is different from that of the natural killer cell receptors; LOX-1 is expressed in vascular-rich organs, but not in lymphocytes . A 1753-bp fragment of the 5' flanking region of the LOX-1 gene had a functional promoter activity . This region contains binding sites for several transcription factors, including the STAT family and NF-IL6, and the expression of LOX-1 was upregulated by several cytokines . These results demonstrate that the human LOX-1 gene is a new member of the natural killer gene complex with a unique expression profile . J Cell Biochem, 1998 Dec 15, 71(4), 467 - 78 Binding of CDK9 to TRAF2; MacLachlan TK et al.; CDK9 has been recently shown to have increased kinase activity in differentiated cells in culture and a differentiated tissue-specific expression in the developing mouse . In order to identify factors that contribute to CDK9's differentiation-specific function, we screened a mouse embryonic library in the yeast two-hybrid system and found a tumor necrosis factor signal transducer, TRAF2, to be an interacting protein . CDK9 interacts with a conserved domain in the TRAF-C region of TRAF2, a motif that is known to bind other kinases involved in TRAF-mediated signaling . Endogenous interaction between the two proteins appears to be specific to differentiated tissue . TRAF2-mediated signaling may incorporate additional kinases to signal cell survival in myotubes, a cell type that is severely affected in TRAF2 knockout mice. Proc Natl Acad Sci U S A, 1998 Nov 24, 95(24), 14266 - 71 Targeting cyclin-dependent kinases in Drosophila with peptide aptamers; Kolonin MG et al.; Two-hybrid technology provides a simple way to isolate small peptide aptamers that specifically recognize and strongly bind to a protein of interest . These aptamers have the potential to dominantly interfere with specific activities of their target proteins and, therefore, could be used as in vivo inhibitors . Here we explore the ability to use peptide aptamers as in vivo inhibitors by expressing aptamers directed against cell cycle regulators in Drosophila . We expressed two peptide aptamers, each of which specifically recognizes one of the two essential cyclin-dependent kinases (Cdks), DmCdk1 and DmCdk2, in Drosophila . Expression of each Cdk aptamer during organogenesis caused adult eye defects typical of those caused by cell cycle inhibition . Co-overexpression of DmCdk1 or DmCdk2 resulted in suppression of the eye phenotypes, indicating that each aptamer interacts with a Cdk target in vivo and suggesting that these peptides disrupt normal eye development by inhibiting Cdk function . Moreover, the specificity of each aptamer for one of the two Cdks as determined in two-hybrid assays was retained in Drosophila . Combined, our results demonstrate that peptide aptamers generated by yeast two-hybrid methods can serve as inhibitory reagents to target specific proteins in vivo. Cell, 1998 Nov 13, 95(4), 521 - 30 A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK; Takekawa M et al.; The stress-responsive p38 and JNK MAPK pathways regulate cell cycle and apoptosis . A human MAPKKK, MTK1 (= MEKK4), mediates activation of both p38 and JNK in response to environmental stresses . Using a yeast two-hybrid method, three related proteins, GADD45alpha (= GADD45), GADD45, (= MyD118), and GADD45gamma, were identified that bound to an N-terminal domain of MTK1 . These proteins activated MTK1 kinase activity, both in vivo and in vitro . The GADD45-like genes are induced by environmental stresses, including MMS, UV, and gamma irradiation . Expression of the GADD45-like genes induces p38/JNK activation and apoptosis, which can be partially suppressed by coexpression of a dominant inhibitory MTK1 mutant protein . We propose that the GADD45-like proteins mediate activation of the p38/JNK pathway, via MTK1/ MEKK4, in response to environmental stresses. FEBS Lett, 1998 Nov 6, 438(3), 183 - 9 Characterization of the cullin and F-box protein partner Skp1; Ng RW et al.; Skp1 interacts with cullins, F-box containing proteins, and forms a complex with cyclin A-Cdk2 in mammalian cells . Skp1 is also involved in diverse biological processes like degradation of key cell cycle regulators, glucose sensing, and kinetochore function . However, little is known about the structure and exact function of Skp1 . Here we characterized the interaction between Skp1 and the F-box protein Skp2 . We show that Skp1 can bind to Skp2 in vitro using recombinant proteins, and in vivo using the yeast two-hybrid system . Deletion analysis of Skp1 indicated that most of the Skp1 protein is required for binding to Skp2 . In mammalian cell extracts, a large portion of Skp1 appears to associate with proteins other than Skp2 . Biochemical analysis indicated that Skp1 is likely to be a flexible, non-spherical protein, and is capable of forming dimers. Nucleic Acids Res, 1998 Dec 1, 26(23), 5277 - 87 DNA binding and dimerisation determinants of Antirrhinum majus MADS-box transcription factors; West AG et al.; Members of the MADS-box family of transcription factors are found in eukaryotes ranging from yeast to humans . In plants, MADS-box proteins regulate several developmental processes including flower, fruit and root development . We have investigated the DNA-binding mechanisms used by four such proteins in Antirrhinum majus, SQUA, PLE, DEF and GLO . SQUA differs from the characterised mammalian and yeast MADS-box proteins as it can efficiently bind two different classes of DNA-binding site . SQUA induces bending of these binding sites and the sequence of the site plays a role in determining the magnitude of these bends . Similarly, PLE and DEF/GLO induce DNA bending although the direction of the resulting bends differ . Finally, we demonstrate that the MADS-box and I-domains are sufficient for homodimer formation by SQUA . However, the K-box in SQUA can also act as an oligomerisation motif and in the full-length protein, the K-box plays a different role in mediating dimerisation in the context of SQUA homodimers or heterodimers with PLE . Together these results contribute significantly to our understanding of the function of SQUA and other plant MADS-box proteins at the molecular level. Eur J Biochem, 1998 Oct 15, 257(2), 522 - 7 The metabolic pathway of visual pigment chromophore formation in Drosophila melanogaster--all-trans (3S)-3-hydroxyretinal is formed from all-trans retinal via (3R)-3-hydroxyretinal in the dark; Seki T et al.; Carotenoid-depleted fruit flies, Drosophila melanogaster, were reared on yeast/glucose medium containing lipid-depleted white corn grits and cholesterol . After rearing for more than a year, the yield of flies remained constant and the content of 3-hydroxyretinal in a head was three logarithmic units less than that of normal flies reared on medium containing yellow corn grits . When all-trans retinal was supplied as the sole source of retinoids, the flies formed and accumulated all-trans 3-hydroxyretinal in the dark . To examine the metabolic pathway to produce (3S)-3-hydroxyretinal in Drosophila, all-trans retinal was supplemented for two hours to carotenoid-depleted flies in the dark, and the subsequent changes in the composition of 3-hydroxyretinal enantiomers were analyzed using a chiral column on HPLC . The results indicated initial formation of (3R)-3-hydroxyretinal followed by isomerization into the 3S enantiomer . In another set of experiments, the membrane fraction was obtained from the head homogenate of retinoid-depleted flies and an in vitro assay of 3-hydroxyretinal formation from retinal was performed . The 3-hydroxyretinal produced was the 3R enantiomer, supporting the result obtained from the in vivo experiment whereby (3S)-3-hydroxyretinal is produced from retinal via (3R)-3-hydroxyretinal . Addition of NADPH enhanced 3-hydroxyretinal formation and the presence of carbon monoxide inhibited it, suggesting that hydroxylation at the C3 position of retinal occurred via the monooxygenase activity of cytochrome P-450. Eur J Morphol, 1998 Aug, 36 Suppl, 137 - 41 Molecular characterization of the cation-chloride cotransporter family; Moore-Hoon ML et al.; Na-K-2Cl cotransporters, Na-Cl cotransporters and K-Cl cotransporters have recently been shown to constitute a new gene family of membrane transport proteins . At least five distinct members of this family of cation-chloride cotransporters have been found in vertebrates and several as yet functionally uncharacterized sequences have been identified in insects, worms and yeast . The relationships among the various known members of this gene family is briefly reviewed along with our current knowledge about the topological structure of these proteins in the plasma membrane. Pharmacogenetics, 1998 Oct, 8(5), 365 - 73 Comparisons between in-vitro and in-vivo metabolism of (S)-warfarin: catalytic activities of cDNA-expressed CYP2C9, its Leu359 variant and their mixture versus unbound clearance in patients with the corresponding CYP2C9 genotypes; Takahashi H et al.; To study whether an in-vitro model for three different genotypes of human CYP2C9*3 polymorphism would be useful for predicting the in-vivo kinetics of (S)-warfarin in patients with the corresponding genotypes, the intrinsic clearance (Cl(int) or Vmax/Km) for (S)-warfarin 7-hydroxylation obtained from recombinant human CYP2C9*1 {wild-type (wt)} and CYP2C9*3 (Leu359/Leu) expressed in yeast and the mixture of equal amounts of these were compared with the in-vivo unbound oral CI (CI(po,u)) of (S)-warfarin obtained from 47 Japanese cardiac patients with the corresponding CYP2C9 genotypes . The in-vitro study revealed that the recombinant CYP2C9*1 (wt/wt), 2C9*3 (Leu359/Leu) and their mixture (Ile359/Leu) possessed a mean Km of 2.6, 10.4 and 6.6 microM and Vmax of 280, 67 and 246 pmol/min/nmol P450, respectively . Thus, the mean in-vitro Cl(int) obtained from recombinant CYP2C9*3 (Leu359/Leu) and the mixture (Ile359/Leu) of 2C9*3 and 2C9*1 were 94% and 65% lower than that obtained from CYP2C9*1 (wt/wt) (6.7 versus 38 versus 108 ml/min/micromol P450, respectively) . The in-vivo study showed that the median Cl(po,u) for (S)-warfarin obtained from patients with homozygous (Leu359/Leu, n = 1) and heterozygous (Ile359/Leu, n = 4) CYP2C9*3 mutations were reduced by 90% (62 ml/min) and 66% (212 ml/min, P < 0.05) compared with that obtained from those with homozygous 2C9*1 (625 ml/min, n = 42) . Consequently, there was a significant correlation (r = 0.99, P < 0.05) between the in-vitro Cl(int) for (S)-warfarin 7-hydroxylation and the in-vivo Cl(po,u) for (S)-warfarin in relation to the CYP2C9*3 polymorphism . In conclusion, the in-vitro model for human CYP2C9*3 polymorphism using recombinant cytochrome P450 proteins would serve as a useful means for predicting changes in in-vivo kinetics for (S)-warfarin and possibly other CYP2C9 substrates in relation to CYP2C9*3 polymorphism. Crit Rev Oral Biol Med, 1998, 9(4), 415 - 48 Protein N-glycosylation: molecular genetics and functional significance; Kukuruzinska MA et al.; Protein N-glycosylation is a metabolic process that has been highly conserved in evolution . In all eukaryotes, N-glycosylation is obligatory for viability . It functions by modifying appropriate asparagine residues of proteins with oligosaccharide structures, thus influencing their properties and bioactivities . N-glycoprotein biosynthesis involves a multitude of enzymes, glycosyltransferases, and glycosidases, encoded by distinct genes . The majority of these enzymes are transmembrane proteins that function in the endoplasmic reticulum and Golgi apparatus in an ordered and well-orchestrated manner . The complexity of N-glycosylation is augmented by the fact that different asparagine residues within the same polypeptide may be modified with different oligosaccharide structures, and various proteins are distinguished from one another by the characteristics of their carbohydrate moieties . Furthermore, biological consequences of derivatization of proteins with N-glycans range from subtle to significant . In the past, all these features of N-glycosylation have posed a formidable challenge to an elucidation of the physiological role for this modification . Recent advances in molecular genetics, combined with the availability of diverse in vivo experimental systems ranging from yeast to transgenic mice, have expedited the identification, isolation, and characterization of N-glycosylation genes . As a result, rather unexpected information regarding relationships between N-glycosylation and other cellular functions--including secretion, cytoskeletal organization, proliferation, and apoptosis--has emerged . Concurrently, increased understanding of molecular details of N-glycosylation has facilitated the alignment between N-glycosylation deficiencies and human diseases, and has highlighted the possibility of using N-glycan expression on cells as potential determinants of disease and its progression . Recent studies suggest correlations between N-glycosylation capacities of cells and drug sensitivities, as well as susceptibility to infection . Therefore, knowledge of the regulatory features of N-glycosylation may prove useful in the design of novel therapeutics . While facing the demanding task of defining properties, functions, and regulation of the numerous, as yet uncharacterized, N-glycosylation genes, glycobiologists of the 21st century offer exciting possibilities for new approaches to disease diagnosis, prevention, and cure. Ophthalmic Surg Lasers, 1998 Nov, 29(11), 926 - 9 The effect of vehicle on corneal penetration of triturated ketoconazole and itraconazole; Guzek JP et al.; BACKGROUND AND OBJECTIVES: Triturated (crushed and suspended) ketoconazole has been recommended for the treatment of fungal keratitis when commercial antifungal eyedrops are unobtainable . The authors evaluated the in vivo corneal stromal concentration with different vehicles in the eyes of adult rabbits . MATERIALS AND METHODS: Ketoconazole and itraconazole tablets were triturated to 20 mg/ml in four vehicles: polyvinyl alcohol (PVA), boric acid, olive oil, and balanced salt solution (BSS) . Six eyes (deepithelialized for better penetration) received one drop every 15 minutes for 2 hours . A yeast overlay bioassay of extracts determined the stromal concentration . RESULTS: Itraconazole in BSS, olive oil, PVA, and boric acid produced inhibition zones of 17.3, 15.6, 15.4, and 13.2 mm, respectively . Ketoconazole produced inhibition zones of 35.9, 39.4, 41.8, and 44.7 mm, respectively . From a standard curve, the concentrations of ketoconazole in tissue were 512, 773, 1221, and 1492 micrograms/g, respectively . CONCLUSION: The vehicle that is used to triturate antifungals affects the tissue concentration . This may have an impact on fungal keratitis therapy. FEBS Lett, 1998 Oct 23, 437(3), 287 - 92 Cloning of the V-ATPase subunit G in plant: functional expression and sub-cellular localization; Rouquie D et al.; A 13-kDa tobacco plasma membrane protein was isolated from two-dimensional electrophoresis gels . After microsequencing, RT-PCR techniques and cDNA library screening allowed for the cloning of two cDNAs . These cDNAs encoded for the subunit G of the vacuolar H+-ATPase, the first one identified in plants . Analysis of mRNA distribution showed a maximum level in the leaves and in the stem of the apical part of the tobacco plant . Heterologous functional complementation of the yeast mutant (deltavma10::URA3) was achieved with the two cDNAs . After fractionation of microsomal membranes on linear sucrose gradient, Western blots were performed using antibodies against recombinant protein and three peaks were identified: one which comigrated with the tonoplast marker and the others at slightly higher density corresponding to endoplasmic reticulum and to plasma membrane fractions. Oncogene, 1998 Nov 12, 17(19), 2473 - 84 The BCL-6 POZ domain and other POZ domains interact with the co-repressors N-CoR and SMRT; Huynh KD et al.; Virtually all diffuse large cell lymphomas and a significant fraction of follicular lymphomas contain translocations and/or point mutations in the 5' non-coding region of the putative oncogene BCL-6, that are presumed to deregulate its expression . BCL-6 encodes a Cys2-His2 zinc finger transcriptional repressor with a POZ domain at its amino-terminus . The POZ (or BTB) domain, a 120-amino-acid motif, mediates homomeric and, in some proteins, heteromeric POZ-POZ interactions . In addition, the POZ domain is required for transcriptional repression of several proteins, including BCL-6 . Using a yeast two-hybrid screen, we identified N-CoR and SMRT as BCL-6 interacting proteins . Both N-CoR and SMRT, which were originally identified as co-repressors for the unliganded nuclear thyroid hormone and retinoic acid receptors, are components of large complexes containing histone deacetylases . We show that the interaction between BCL-6 and these co-repressors is also detected in the more physiologically relevant mammalian two-hybrid assay . The POZ domain is necessary and sufficient for interaction with these co-repressors . BCL-6 and N-CoR co-localize to punctate regions of the nucleus . Furthermore, when BCL-6 is bound to its consensus recognition sequence in vivo, it can interact with N-CoR and SMRT . We find, in vitro, that POZ domains from a variety of other POZ domain-containing proteins, including the transcriptional repressor PLZF, as well as ZID, GAGA and a vaccinia virus protein, SalF17R, also interact with varying affinities with N-CoR and SMRT . We find that BCL-6 POZ domain mutations that disrupt the interaction with N-CoR and SMRT no longer repress transcription . In addition, these mutations no longer self associate suggesting that self interaction is required for interaction with the co-repressors and for repression . More recently N-CoR has also been implicated in transcriptional repression by the Mad/Mxi proteins . Our demonstration that N-CoR and SMRT interact with the POZ domain containing proteins indicates that these co-repressors are likely involved in the mediation of repression by multiple classes of repressors and may explain, in part, how POZ domain containing repressors mediate transcriptional repression. Br J Cancer, 1998 Nov, 78(10), 1356 - 60 Absence of somatic alterations of the EB1 gene adenomatous polyposis coli-associated protein in human sporadic colorectal cancers; Jais P et al.; The human EB1 gene product was recently found, by a yeast two-hybrid screening, to be associated with the carboxy terminus of the APC (adenomatous polyposis coli) protein, the product of a tumour-suppressor gene thought to act as a gatekeeper in colorectal carcinogenesis . Because virtually all of the APC mutations result in the synthesis of carboxy-terminal truncated proteins, mutant APC proteins are expected to lose their ability to interact with EB1 gene product . Thus, the interaction between APC and EB1 proteins may be important for the tumour-suppressor activity of APC protein, and raises the hypothesis that EB1 is also involved in sporadic colorectal tumorigenesis . To investigate this hypothesis, somatic mutations in the entire coding sequence of EB1 cDNA were searched by reverse transcriptase single-strand conformational polymorphism (SSCP) analysis in 21 sporadic colorectal cancers and seven adenomas . None of these tumours contained somatic mutation, whereas a silent cDNA variant was identified in 14% of alleles . Furthermore, to investigate whether EB1 locus was included within a region subjected to losses of heterozygosity, four polymorphism markers surrounding EB1 locus were surveyed . Only one out of 28 colorectal tumours contained a loss of heterozygosity at the D20S107 marker . In conclusion, the present findings strongly suggest that EB1 gene is not involved in somatic colorectal carcinogenesis. J Biol Chem, 1998 Nov 27, 273(48), 32102 - 10 Complexity of trypanosomatid endocytosis pathways revealed by Rab4 and Rab5 isoforms in Trypanosoma brucei; Field H et al.; Small G proteins of the Rab family are responsible for vesicle fusion and control flux during intracellular transport . Rab5 is important in endosome maturation and Rab4 in recycling of endocytic material . Three Rab5 isoforms identified so far in mammals and three in the yeast genome suggest that conservation of multiple Rab5 isoforms is required for sophisticated regulation of endocytosis . Trypanosoma brucei homologues of Rab5 and Rab4 (TbRab5A and TbRab4) have been identified . Here we report cloning of a second Rab5 homologue, TbRab5Bp . The TbRAB5A and -5B genes are not linked in the genome, and phylogenetic reconstruction indicates that multiple Rab5 isoforms in yeast, mammals, and trypanosomes evolved independently . Northern blots demonstrate that TbRab5A, -5B, and TbRab4 messages are expressed in bloodstream form (BSF) and procyclic forms of the parasite even though endocytosis is not very active in the latter form . mRNA levels of TbRab5A and -4 are constitutive . Multiple-sized TbRab5B messages at very low abundance are detected, with greater expression in BSF . Also, the TbRab5B mRNA has a large 3'-untranslated region suggestive of potentially complex regulation, and therefore TbRab5Bp may be an important regulator of differential endocytosis levels between BSF and procyclic stage parasites . Affinity purified antibodies raised to C-terminal peptide sequences of all three TbRab proteins recognized small vesicular cytoplasmic structures, which for TbRab5Ap and -5Bp are predominantly near the flagellar pocket . TbRab5Bp colocalizes with invariant surface glycoprotein 100 (ISG100), a protein entering the endocytotic pathway in BSF parasites, whereas in procyclic cells populations of vesicles stained with both TbRab5Ap and -5Bp substantially overlap; TbRab5 proteins are therefore components of the endocytotic pathway . TbRab4p localizes to vesicular structures throughout the cytoplasm, with some overlap with TbRab5Bp, but the majority occupying a different compartment to the TbRab5s . Therefore the trypanosome endosomal system has been functionally dissected for the first time; these reagents provide a unique opportunity for manipulation of the protozoan endosomal system to further our understanding of drug uptake mechanisms and virulence. J Biol Chem, 1998 Nov 27, 273(48), 31962 - 70 Cadmium-regulated genes from the nematode Caenorhabditis elegans . Identification and cloning of new cadmium-responsive genes by differential display; Liao VH et al.; The transition metal cadmium is a pervasive and persistent environmental contaminant that has been shown to be both a human toxicant and carcinogen . To inhibit cadmium-induced damage, cells respond by increasing the expression of genes encoding stress-response proteins . In most cases, the mechanism by which cadmium affects the expression of these genes remains unknown . It has been demonstrated in several instances that cadmium activates gene transcription through signal transduction pathways, mediated by protein kinase C, cAMP-dependent protein kinase, or calmodulin . A codicil is that cadmium should influence the expression of numerous genes . To investigate the ability of cadmium to affect gene transcription, the differential display technique was used to analyze gene expression in the nematode Caenorhabditis elegans . Forty-nine cDNAs whose steady-state levels of expression change 2-6-fold in response to cadmium exposure were identified . The nucleotide sequences of the majority of the differentially expressed cDNAs are identical to those of C . elegans cosmids, yeast artificial chromosomes, expressed sequence tags, or predicted genes . The translated amino acid sequences of several clones are identical to C . elegans metallothionein-1, HSP70, collagens, and rRNAs . In addition, C . elegans homologues of pyruvate carboxylase, DNA gyrase, beta-adrenergic receptor kinase, and human hypothetical protein KIAA0174 were identified . The translated amino acid sequences of the remaining differentially expressed cDNAs encode novel proteins. EMBO J, 1998 Nov 16, 17(22), 6689 - 700 Rescue of the embryonic lethal hematopoietic defect reveals a critical role for GATA-2 in urogenital development; Zhou Y et al.; Mutations resulting in embryonic or early postnatal lethality could mask the activities of any gene in unrelated and temporally distinct developmental pathways . Targeted inactivation of the transcription factor GATA-2 gene leads to mid-gestational death as a consequence of hematopoietic failure . We show here that a 250 kbp GATA-2 yeast artificial chromosome (YAC) is expressed strongly in both the primitive and definitive hematopoietic compartments, while two smaller YACs are not . This largest YAC also rescues hematopoiesis in vitro and in vivo, thereby localizing the hematopoietic regulatory cis element(s) to between 100 and 150 kbp 5' to the GATA-2 structural gene . Introducing the YAC transgene into the GATA-2(-/-) genetic background allows the embryos to complete gestation; however, newborn rescued pups quickly succumb to lethal hydroureternephrosis, and display a complex array of genitourinary abnormalities . These findings reveal that GATA-2 plays equally vital roles in urogenital and hematopoietic development. Biochem J, 1998 Dec 1, 336 ( Pt 2), 437 - 42 Genomic organization, 5'-flanking region and chromosomal localization of the human glutathione transferase A4 gene; Desmots F et al.; We have isolated and characterized a human glutathione transferase A4 (hGSTA4) subunit gene from a yeast artificial chromosome containing several other glutathione transferase alpha genes and pseudogenes . The homodimeric protein hGSTA4-4, is involved in the detoxification of 4-hydroxynonenal and other reactive electrophiles produced by oxidative metabolism, and may have a significant role in protecting intracellular components from oxidative damage . The hGSTA4 gene spans nearly 18 kb, contains seven exons, maps onto chromosome 6p12, and lies in close proximity to the 7SK small nuclear RNA gene in a head-to-tail orientation . The intron/exon borders conform to the standard rules, an open reading frame is present beginning at position 154 in exon 2, and the stop codon is at position 822 in exon 7 . The transcription initiation site has been determined by primer extension analysis and is located 135 bp upstream of intron 1 . Isolation and sequencing of the hGSTA4 gene 5'-flanking region revealed it to be devoid of TATA or CCAAT boxes but it does contain an initiator element overlapping the transcription start site, a GC box and putative binding sites for transcription factors AP1, STAT, GATA1 and NF-kappaB . Reverse transcription-PCR analysis revealed that hGSTA4 mRNA was present in all the tissues tested, although in low amounts, suggesting that this subunit may be ubiquitously expressed. J Gen Virol, 1998 Nov, 79 ( Pt 11), 2717 - 27 Association of simian immunodeficiency virus Nef with the T-cell receptor (TCR) zeta chain leads to TCR down-modulation; Bell I et al.; The Nef protein of simian immunodeficiency virus (SIV) is dispensable for replication in established T-cell lines but essential for high level virus replication in the adult host, though the mechanism by which Nef contributes to this has remained unclear . We demonstrate here that SIV Nef binds to the zeta chain of the T-cell receptor (TCR) . SIV Nef proteins that interact with TCR zeta in a yeast two-hybrid system also reduce T-cell surface expression levels of TCR alphabeta, CD3 and CD4 . These findings are the first demonstration that Nef can bind directly to a component of the TCR-CD3 complex and modulate its surface expression. Trends Genet, 1998 Oct, 14(10), 417 - 22 Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits; Lupski JR; Molecular medicine began with Pauling's seminal work, which recognized sickle-cell anemia as a molecular disease, and with Ingram's demonstration of a specific chemical difference between the hemoglobins of normal and sickled human red blood cells . During the four decades that followed, investigations have focused on the gene--how mutations specifically alter DNA and how these changes affect the structure and expression of encoded proteins . Recently, however, the advances of the human genome project and the completion of total genome sequences for yeast and many bacterial species, have enabled investigators to view genetic information in the context of the entire genome . As a result, we recognize that the mechanisms for some genetic diseases are best understood at a genomic level . The evolution of the mammalian genome has resulted in the duplication of genes, gene segments and repeat gene clusters . This genome architecture provides substrates for homologous recombination between nonsyntenic regions of chromosomes . Such events can result in DNA rearrangements that cause disease. Mol Cell Biol, 1998 Dec, 18(12), 7584 - 9 Maturation of human cyclin E requires the function of eukaryotic chaperonin CCT; Won KA et al.; Cyclin E, a partner of the cyclin-dependent kinase Cdk2, has been implicated in positive control of the G1/S phase transition . Whereas degradation of cyclin E has been shown to be exquisitely regulated by ubiquitination and proteasomal action, little is known about posttranscriptional aspects of its biogenesis . In a yeast-based screen designed to identify human proteins that interact with human cyclin E, we identified components of the eukaryotic cytosolic chaperonin CCT . We found that the endogenous CCT complex in yeast was essential for the maturation of cyclin E in vivo . Under conditions of impaired CCT function, cyclin E failed to accumulate . Furthermore, newly translated cyclin E, both in vitro in reticulocyte lysate and in vivo in human cells in culture, is efficiently bound and processed by the CCT . In vitro, in the presence of ATP, the bound protein is folded and released in order to become associated with Cdk2 . Thus, both the acquisition of the native state and turnover of cyclin E involve ATP-dependent processes mediated by large oligomeric assemblies. Mol Cell Biol, 1998 Dec, 18(12), 7106 - 18 Role for a YY1-binding element in replication-dependent mouse histone gene expression; Eliassen KA et al.; Expression of the highly conserved replication-dependent histone gene family increases dramatically as a cell enters the S phase of the eukaryotic cell cycle . Requirements for normal histone gene expression in vivo include an element, designated alpha, located within the protein-encoding sequence of nucleosomal histone genes . Mutation of 5 of 7 nucleotides of the mouse H3.2 alpha element to yield the sequence found in an H3.3 replication-independent variant abolishes the DNA-protein interaction in vitro and reduces expression fourfold in vivo . A yeast one-hybrid screen of a HeLa cell cDNA library identified the protein responsible for recognition of the histone H3.2 alpha sequence as the transcription factor Yin Yang 1 (YY1) . YY1 is a ubiquitous and highly conserved transcription factor reported to be involved in both activation and repression of gene expression . Here we report that the in vitro histone alpha DNA-protein interaction depends on YY1 and that mutation of the nucleotides required for the in vitro histone alpha DNA-YY1 interaction alters the cell cycle phase-specific up-regulation of the mouse H3.2 gene in vivo . Because all mutations or deletions of the histone alpha sequence both abolish interactions in vitro and cause an in vivo decrease in histone gene expression, the recognition of the histone alpha element by YY1 is implicated in the correct temporal regulation of replication-dependent histone gene expression in vivo. Hum Mol Genet, 1998 Dec, 7(13), 2079 - 88 LCR-dependent gene expression in beta-globin YAC transgenics: detailed structural studies validate functional analysis even in the presence of fragmented YACs; Peterson KR et al.; Yeast artificial chromosome (YAC) transgenesis is associated with a high frequency of deletions in the integrated transgenes . To determine the impact of these rearrangements on the ability to derive structure-function relationships using YACs, transgenic mice were generated with 248 or 155 kb beta-globin locus YACs . The transgenics were examined for structural integrity of the YAC using an approach of structural analysis that unambiguously demonstrates intactness of YAC transgene copies . Globin gene expression per copy of each integrated transgene and the profiles of globin gene expression during development were determined . Diverse deletion patterns were observed in one or more integrated YACs in all the 248 and most of the 155 kb transgenic lines we analyzed . However, when the structure of the major regulatory element of the beta-globin locus, the locus control region, was preserved, the genes of the beta-globin locus functioned normally and globin transgenes of both the 248 and 155 kb beta-YACs were expressed in a position-independent, copy number-dependent manner . Furthermore, the globin genes of both beta-YACs displayed normal developmental regulation . We conclude that YACs can be used for analysis of structure-function relationships of large genes or multigene loci in spite of the tendency for rearrangements and deletions of the integrated transgenes . However, detailed structural evidence for integrity and continuity of locus sequences is required for correct interpretation of functional data. J Clin Microbiol, 1998 Dec, 36(12), 3683 - 5 Critical evaluation of 4-week incubation for fungal cultures: is the fourth week useful? Labarca JA, Wagar EA, Grasmick AE, Kokkinos HM, Bruckner DA. To determine the benefit of a 4-week incubation for mycology cultures, we evaluated all positive cultures during the fourth week of incubation in a 1-year period . Of 3,855 positive mycology cultures (yeast, 82%; molds, 18%), 62 (1.6%) were positive during the fourth week (yeast, 42%; molds, 58%) . Only 15 of the 62 cultures (24%) were considered clinically relevant (2 isolates from invasive fungal infection and 13 isolates from cutaneous mycosis) . With the exception of those from skin samples, isolates recovered during the fourth week are rarely important for patient care. Clin Cancer Res, 1996 Oct, 2(10), 1673 - 7 13q14 deletions are not primary events in B-cell chronic lymphocytic leukemia: a study of 100 patients using fluorescence in situ hybridization; Avet-Loiseau H et al.; Fluorescence in situ hybridization with a chromosome 12-specific alpha-centromeric probe and a 13q14 yeast artificial chromosome probe was performed on interphase cells from 100 patients with B-cell chronic lymphocytic leukemia . Thirty-one patients exhibited a 13q14 deletion . No correlation was found between 13q14 deletions and clinical stage, sex, or morphology . Sixteen patients had trisomy 12, including 6 (of 12) with an atypical morphology . Trisomy 12 and 13q14 abnormalities were detected concomitantly in three patients only . The analysis of patients with deletions clearly showed that in five cases a significant number of cells retained two signals with the yeast artificial chromosome probe, indicating a genetic heterogeneity among the leukemic population . Our data confirm that the 13q14 deletion is a frequent event, indicate that the concomitant occurrence of 13q14 deletion and trisomy 12 is rare but possible, and show that both abnormalities are secondary events in B-cell chronic lymphocytic leukemia. Clin Cancer Res, 1997 Oct, 3(10), 1873 - 7 Expression of nucleolar protein p120 in human lung cancer: difference in histological types as a marker for proliferation; Uchiyama B et al.; The function of proliferation-associated nucleolar protein p120 is unclear . A recent report that a yeast protein, NOP2, 67% homologous to human p120, is up-regulated during the onset of growth and influences the morphology of the nucleolus supports the notion that this protein could serve as a marker for proliferation in neoplastic cells . Lung cancer is characteristic in that different histological types show different biological features . We attempted to evaluate the levels of p120 expression in resected human lung cancer tissues of different histological types and the relation of p120 expression and cell proliferation using human lung cancer cell lines . When 37 frozen specimens of human lung cancer and normal lung were stained with a p120 monoclonal antibody, the nucleoli of cancer cells were positively stained, whereas a few macrophages in normal lung revealed only weak staining . The labeling index of p120 in squamous cell carcinoma (67.7 +/- 12.4%) was significantly higher than that in adenocarcinoma (35.3 +/- 12.6%) or in large cell carcinoma (30.1 +/- 17.3%; P < 0.01) . In six human lung cancer cell lines and one normal lung fibroblast cell line cultured in vitro, there was a significant correlation between S-phase fraction and p120 mRNA (r = 0.851, P < 0.02)/p120 protein (r = 0.869, P < 0.01) or between doubling time and p120 protein (r = -0.928, P < 0.01) . In the context of the reports that indicate higher {3H}thymidine incorporation and shorter doubling time in the squamous cell carcinoma, these results indicate that p120 can be a marker for proliferation in human lung cancer cells in vivo and in vitro, and that it has an important function in the cell cycle of tumor proliferation. Biochem Biophys Res Commun, 1998 Nov 9, 252(1), 39 - 45 Identification and characterization of the ubiquitously occurring nuclear matrix protein NMP 238; Holzmann K et al.; By systematic comparison of two-dimensional electrophoretic patterns of nuclear matrix proteins an ubiquitously occurring (common) nuclear matrix protein, termed NMP 238, was detected . Localization of the protein in isolated nuclear matrices and in nuclear and cytoplasmic regions of cells was determined by confocal immunofluorescence microscopy . N-terminal protein sequencing, mass spectrometry, and sequencing of a human EST cDNA clone showed identity of the protein with a nuclear protein, termed TIP49, of as yet uncertain function . Expression of the corresponding gene in diverse human and rat cells was confirmed by Northern blotting . The protein displays two nuclear localization signals . Sequence homologies indicate evolutionary related proteins in nematodes, yeast, and archaebacteria . Similarities to the AAA family of proteins and to a subgroup of chaperones suggest that the nuclear matrix protein may play a role in the assembly and ATP-dependent anchorage of proteins . J Biol Chem, 1998 Nov 20, 273(47), 31555 - 64 Identification of domains essential for the assembly of calcium/calmodulin-dependent protein kinase II holoenzymes; Kolb SJ et al.; Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), as isolated from brain, is a multimeric complex composed predominantly of two subunits, alpha and beta, products of unique genes . Little is known about how subunit composition influences holoenzyme structure or how the domain(s) of each subunit interact to form holoenzymes . We show here that holoenzymes composed of only alpha or only beta subunits exhibit different biophysical properties . The S values of alpha and beta are 17.2 and 14.5 S while the Stokes's radii are 85 and 111 A, respectively, indicating their structures are different . C-terminal truncations of the alpha subunit show that amino acids 382-478 are necessary for holoenzyme formation and that amino acids 427-478 contribute to holoenzyme stability . Additionally, the C-terminal domains of both the alpha subunit, alpha315-478, and beta subunit, beta314-542, formed oligomers indicating the sufficiency of the C-terminal domain for multimer formation . Using the yeast two-hybrid system we show, in vivo, that full-length subunits, alpha1-478 and beta1-542, interact with themselves or each other interchangeably . Additionally, the C-terminal domains of the alpha subunit, alpha315-478 and beta subunit, beta314-542 associated with themselves in a manner indistinguishable from their association with full-length alpha or beta subunits . Further studies revealed that the C-terminal domains of the alpha and beta subunits contain information necessary for interaction with beta but not alpha . These data are summarized into a model describing the assembly of CaM kinase II holoenzymes. J Biol Chem, 1998 Nov 20, 273(47), 31519 - 27 Intragenic suppressors of mutant DNA topoisomerase I-induced lethality diminish enzyme binding of DNA; Hann CL et al.; Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology and is the cellular target of the antitumor drug camptothecin (Cpt) . Mutation of several conserved residues in yeast top1 mutants is sufficient to induce cell lethality in the absence of camptothecin . Despite tremendous differences in catalytic activity, the mutant proteins Top1T722Ap and Top1R517Gp cause cell death via a mechanism similar to that of Cpt, i.e . stabilization of the covalent enzyme-DNA intermediate . To establish the interdomainal interactions required for the catalytic activity of Top1p and how alterations in enzyme structure contribute to the cytotoxic activity of Cpt or specific DNA topoisomerase I mutants, we initiated a genetic screen for intragenic suppressors of the top1T722A-lethal phenotype . Nine single amino acid substitutions were defined that map to the conserved central and C-terminal domains of Top1p as well as the nonconserved linker domain of the protein . All reduced the catalytic activity of the enzyme over 100-fold . However, detailed biochemical analyses of three suppressors, top1C273Y,T722A, top1G295V,T722A, and top1G369D,T722A, revealed this was accomplished via a mechanism of reduced affinity for the DNA substrate . The mechanistic implications of these results are discussed in the context of the known structures of yeast and human DNA topoisomerase I. J Biol Chem, 1998 Nov 20, 273(47), 31297 - 307 Retinal targets for calmodulin include proteins implicated in synaptic transmission; Xu XZ et al.; Ca2+ influxes regulate multiple events in photoreceptor cells including phototransduction and synaptic transmission . An important Ca2+ sensor in Drosophila vision appears to be calmodulin since a reduction in levels of retinal calmodulin causes defects in adaptation and termination of the photoresponse . These functions of calmodulin appear to be mediated, at least in part, by four previously identified calmodulin-binding proteins: the TRP and TRPL ion channels, NINAC and INAD . To identify additional calmodulin-binding proteins that may function in phototransduction and/or synaptic transmission, we conducted a screen for retinal calmodulin-binding proteins . We found eight additional calmodulin-binding proteins that were expressed in the Drosophila retina . These included six targets that were related to proteins implicated in synaptic transmission . Among these six were a homolog of the diacylglycerol-binding protein, UNC13, and a protein, CRAG, related to Rab3 GTPase exchange proteins . Two other calmodulin-binding proteins included Pollux, a protein with similarity to a portion of a yeast Rab GTPase activating protein, and Calossin, an enormous protein of unknown function conserved throughout animal phylogeny . Thus, it appears that calmodulin functions as a Ca2+ sensor for a broad diversity of retinal proteins, some of which are implicated in synaptic transmission. J Biol Chem, 1998 Nov 20, 273(47), 31061 - 7 IRS pleckstrin homology domains bind to acidic motifs in proteins; Burks DJ et al.; Using a yeast two-hybrid system, we identified several proteins that interact with the PH domains in IRS-1 and IRS-2, including Lon protease, myeloblast protein, and nucleolin . Although the roles of these molecules in insulin action are not yet known, each protein contained an acidic motif that interacted with the PH domain of IRS-2 . However, only the acidic motif in nucleolin bound to IRS-1, suggesting that the PH domain in IRS-1 and IRS-2 are not identical . Moreover, synthetic peptides based on the acidic motif in Lon protease and myeloblast protein inhibited the binding of nucleolin to the PH domain of IRS-2 but not to the PH domain of IRS-1, confirming the selectivity of these PH domains . The ability to bind acidic motifs may be a specific function of the PH domain in IRS proteins, because the PH domains in betaARK, phospholipase Cgamma, or spectrin did not bind nucleolin . In 32D cells, nucleolin bound to both IRS-1 and IRS-2, and expression of the acidic motif of nucleolin inhibited insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2 . These results suggest that the binding of acidic motifs to the PH domain of IRS-1 and IRS-2 disrupts coupling to the activated insulin receptor . Our results are consistent with the hypothesis that the PH domain in the IRS proteins may ordinarily bind acidic peptide motifs in membrane proteins or other acidic membrane elements that couple IRS proteins to activated membrane receptors. J Biol Chem, 1998 Nov 20, 273(47), 30933 - 8 Bcl3, an IkappaB protein, as a novel transcription coactivator of the retinoid X receptor; Na SY et al.; We have recently shown that the IkappaB protein IkappaBbeta interacted with the retinoid X receptor (RXR) and inhibited the 9-cis-retinoic acid (RA)-dependent transactivations (Na, S.-Y., Kim, H.-J., Lee, S.-K., Choi, H.-S., Na, D . S., Lee, M.-O., Chung, M., Moore, D . D., and Lee, J . W . (1998) J . Biol . Chem . 6, 3212-3215) . Herein, we show that a distinct IkappaB protein Bcl3 also interacts with RXR, as shown in the yeast two-hybrid tests and glutathione S-transferase pull-down assays . The Bcl3 interaction involved two distinct subregions of RXR, i.e . constitutive interactions of the N-terminal ABC domains and 9-cis-RA-dependent interactions of the C-terminal DEF domains . In contrast to IkappaBbeta, Bcl3 did not interact with the AF2 domain of RXR . Bcl3 specifically interacted with the general transcription factors TFIIB, TBP, and TFIIA but not with TFIIEalpha in the GST pull-down assays . TBP and TFIIA, however, were not able to interact with IkappaBbeta . Accordingly, Bcl3 coactivated the 9-cis-RA-induced transactivations of RXR, in contrast to the inhibitory actions of IkappaBbeta . In addition, coexpression of SRC-1 but not p300 further stimulated the Bcl3-mediated enhancement of the 9-cis-RA-induced transactivations of RXR . These results suggest that distinct IkappaB proteins differentially modulate the 9-cis-RA-induced transactivations of RXR in vivo. J Mol Biol, 1998 Nov 20, 284(1), 57 - 69 Higher-order structure and thermal instability of bovine mitochondrial tRNASerUGA investigated by proton NMR spectroscopy; Hayashi I et al.; Although mammalian mitochondrial serine-specific tRNA with the anticodon UGA (tRNASerUGA) appears to possess an almost normal cloverleaf secondary structure, it exhibits an extraordinarily low melting temperature (tm) . An in vitro tRNASerUGA transcript without modified nucleosides had an even lower tm and slightly less hyperchromicity, but its tertiary structure was apparently very similar to that of the native counterpart judging from its aminoacylation activity and the body of experimental evidence so far obtained for canonical tRNAs . The transcript was therefore used to investigate the higher-order structure and thermal instability of tRNASerUGA . 1H-NMR analysis of the transcript showed that it takes a nearly L-shaped tertiary structure with similar tertiary base-pairings to those found in yeast tRNAPhe, which is representative of canonical tRNAs . However, magnesium ion titration revealed that Mg2+ affected the chemical shifts of the tRNASerUGA transcript differently than those of canonical tRNAs so far studied; the former was less sensitive toward Mg2+, especially in the D-arm region . This observation was confirmed by NMR analysis with paramagnetic manganese ion titration . Hill plots derived from the CD spectral changes caused by titration with Mg2+ suggested that the tRNASerUGA transcript had fewer Mg2+ binding sites than those of yeast tRNAPhe as well as its transcript, a finding that was consistent with the NMR data . We thus surmise that the thermal instability of both the transcript and tRNASerUGA itself originated from a reduction in the number of the divalent ion binding sites within the tRNA molecule . These results suggest a new type of thermal instability for mitochondrial tRNA . Oncogene, 1998 Nov 5, 17(18), 2279 - 85 Characterization of a carboxy-terminal BRCA1 interacting protein; Wong AK et al.; There are several lines of evidence indicating that the carboxy-terminal region of the tumor suppressor protein BRCA1 is a functionally significant domain . Using the yeast two-hybrid and in vitro biochemical assays, we show that a protein, CtIP, interacts specifically with the carboxy-terminal segment of human BRCA1 from residues 1602-1863 . A germ line truncation mutation, Y1853ter, that removes the last 11 amino acids from the carboxy-terminus of BRCA1, abolishes not only its transcriptional activation function, but also binding to CtIP . The function of CtIP is unknown, but its reported association with a transcriptional repressor CtBP lends further support that it may have a role in transcription . A sequence based screen of a panel of 89 tumor cell line cDNAs for mutations in the CtIP coding region identified five missense variants . In the pancreatic carcinoma cell line, BxPC3, the non-conservative lysine to glutamic acid change at codon 337 is accompanied with apparent loss of heterozygosity or non-expression of the wild type allele . Thus it is plausible that CtIP may itself be a tumor suppressor acting in the same pathway as BRCA1. Am J Pathol, 1998 Nov, 153(5), 1401 - 9 Subtracted, unique-sequence, in situ hybridization: experimental and diagnostic applications; Davison JM et al.; Nonrandom chromosomal aberrations, particularly in cancer, identify pathogenic biological pathways and, in some cases, have clinical relevance as diagnostic or prognostic markers . Fluorescence and colorimetric in situ hybridization methods facilitate identification of numerical and structural chromosome abnormalities . We report the development of robust, unique-sequence in situ hybridization probes that have several novel features: 1) they are constructed from multimegabase contigs of yeast artificial chromosome (YAC) clones; 2) they are in the form of adapter-ligated, short-fragment, DNA libraries that may be amplified by polymerase chain reaction; and 3) they have had repetitive sequences (eg, Alu and LINE elements) quantitatively removed by subtractive hybridization . These subtracted probes are labeled conveniently, and the fluorescence or colorimetric detection signals are extremely bright . Moreover, they constitute a stable resource that may be amplified through at least four rounds of polymerase chain reaction without diminishing signal intensity . We demonstrate applications of subtracted probes for the MYC and EWS oncogene regions, including 1) characterization of a novel EWS-region translocation in Ewing's sarcoma, 2) identification of chromosomal translocations in paraffin sections, and 3) identification of chromosomal translocations by conventional bright-field microscopy. Cancer Res, 1998 Nov 1, 58(21), 4888 - 94 Caspase-mediated activation of a 36-kDa myelin basic protein kinase during anticancer drug-induced apoptosis; Kakeya H et al.; A novel anticancer drug, cytotrienin A, isolated from Streptomyces sp., induces apoptosis (or programmed cell death) in human promyelocytic leukemia HL-60 cells within 4 h . To elucidate the mechanism of this process, we performed an in-gel kinase assay using myelin basic protein (MBP) as a substrate and found the activation of kinase with an apparent molecular mass of 36 kDa (p36 MBP kinase) . The dose of cytotrienin A required to activate p36 MBP kinase was consistent with that required to induce apoptotic DNA fragmentation in HL-60 cells . This p36 MBP kinase was activated with kinetics distinct from the activation of JNK (c-Jun N-terminal kinase)/stress-activated protein kinase and p38 MAPK (mitogen-activated protein kinase) . Importantly, the p36 MBP kinase was immunologically different from MAPK superfamily molecules such as ERK1, JNK isoforms, and p38 MAPK . In addition, the p36 MBP kinase activation and apoptotic DNA fragmentation were inhibited by antioxidants such as N-acetylcysteine and reduced-form glutathione . The p36 MBP kinase activation was also observed during hydrogen peroxide (H2O2) and okadaic acid-induced apoptosis . Although a specific inhibitor of caspase-3-like proteases (Ac-DEVD-CHO) or a specific inhibitor of caspase-1-like proteases (Ac-YVAD-CHO) did not block the cytotrienin A-, H2O2-, or okadaic acid-induced apoptosis, a broad specificity inhibitor of caspases (Z-Asp-CH2-DCB) strongly inhibited the apoptosis of HL-60 cells . Surprisingly, Z-Asp-CH2-DCB inhibited the activation of p36 MBP kinase induced by cytotrienin A or H2O2, but did not inhibit the activation of JNK/stress-activated protein kinase and p38 MAPK . Taken together, these results indicate that p36 MBP kinase activation is downstream of the activation of Z-Asp-CH2-DCB-sensitive caspases, and reactive oxygen species could be included in the apoptotic events . Moreover, according to the Western blotting using the antibodies against MST1/Krs2 or MST2/Krs1, it is suggested that the p36 MBP kinase is an active proteolytic product of MST1/Krs2 and MST2/Krs1, which are originally cloned by virtue of its homology to the budding yeast Ste20 kinase . Thus, the p36 MBP kinase might be a common component of the diverse signaling pathways leading to apoptosis, and controlling this p36 MBP kinase pathway might be a novel strategy for cancer chemotherapy. Genome, 1998 Oct, 41(5), 720 - 7 Isolation and chromosomal mapping of human glycogen synthase kinase-3 alpha and -3 beta encoding genes; Shaw PC et al.; Glycogen synthase kinase-3 (GSK-3) is a serine-threonine kinase that exists as two isoforms, alpha and beta, encoded by separate genes . Phosphorylation targets include a variety of cytoplasmic and nuclear proteins . Recent studies found that neurofilaments, amyloid precursor protein, and tau proteins are substrates of GSK-3 and that aberrant phosphorylation of these proteins is implicated in pathologies of the nervous system . To analyse the organisation of these two genes, a YAC library was screened by polymerase chain reaction, using primers specific for human GSK-3 alpha and GSK-3 beta cDNA . Two clones, 220 and 285 kb in size, containing the complete GSK-3 alpha coding sequence, and two clones, 365 and 285 kb in size, containing the 5' coding sequence of GSK-3 beta, were isolated . By somatic cell hybrid panel DNA amplification and radiation hybrid mapping, GSK-3 alpha was found to be located at 19q13.2 . On the other hand, by somatic cell hybrid panel DNA amplification and fluorescence in situ hybridisation using the 285-kb YAC clone, GSK-3 beta was mapped to 3q13.3. Mol Cell, 1998 Oct, 2(4), 417 - 25 Pim-1 kinase and p100 cooperate to enhance c-Myb activity; Leverson JD et al.; The pim-1 oncogene is regulated by hematopoietic cytokine receptors, encodes a serine/threonine protein kinase, and cooperates with c-myc in lymphoid cell transformation . Using a yeast two-hybrid screen, we found that Pim-1 protein binds to p100, a transcriptional coactivator that interacts with the c-Myb transcription factor . Pim-1 phosphorylated p100 in vitro, formed a stable complex with p100 in animal cells, and functioned downstream of Ras to stimulate c-Myb transcriptional activity in a p100-dependent manner . Thus, Pim-1 and p100 appear to be components of a novel signal transduction pathway affecting c-Myb activity, linking all three to the cytokine-regulated control of hematopoietic cell growth, differentiation, and apoptosis. J Nutr, 1998 Nov, 128(11), 1956 - 60 Properties of food folates determined by stability and susceptibility to intestinal pteroylpolyglutamate hydrolase action; Seyoum E et al.; The intestinal absorption of folate occurs at the monoglutamyl level, and an important measure of food folate bioavailability is how much folate from the food reaches the intestinal sites in forms that can readily be absorbed . In the absence of protecting agents, e.g., vitamin C and reduced thiols, many labile folates may be lost during cooking and during residence in the acid-peptic milieu of the stomach . On the other hand, the presence of polyglutamyl folate necessitates the action of intestinal hydrolases, which could be affected by food constituents . In this study, we developed an in vitro assay for the determination of an index of food folate availability . The index of folate availability in this study was defined as that proportion of folate that has been identified as monoglutamyl derivatives after tests for stability and susceptibility to an enzymatic hydrolysis . The index of folate availability varied widely among foods . The highest index was for egg yolk (72.2%), followed by cow's livers (55.7%), orange juice (21 . 3%), cabbage (6.0%), lima beans (4.5%) and lettuce (2.9%) . Yeast folate had the lowest index (0.3%) . The availability indices generated by this study correlate with the indices of the bioavailability of the corresponding food folate observed in earlier studies, R2 = 0.529 (P = 0.068) . Additional information is required on the bioavailability of other food products to test the usefulness of this in vitro approach for assessing food folate availability. Genes Dev, 1998 Nov 1, 12(21), 3408 - 18 Faithful anaphase is ensured by Mis4, a sister chromatid cohesion molecule required in S phase and not destroyed in G1 phase; Furuya K et al.; The loss of sister chromatid cohesion triggers anaphase spindle movement . The budding yeast Mcd1/Scc1 protein, called cohesin, is required for associating chromatids, and proteins homologous to it exist in a variety of eukaryotes . Mcd1/Scc1 is removed from chromosomes in anaphase and degrades in G1 . We show that the fission yeast protein, Mis4, which is required for equal sister chromatid separation in anaphase is a different chromatid cohesion molecule that behaves independent of cohesin and is conserved from yeast to human . Its inactivation in G1 results in cell lethality in S phase and subsequent premature sister chromatid separation . Inactivation in G2 leads to cell death in subsequent metaphase-anaphase progression but missegregation occurs only in the next round of mitosis . Mis4 is not essential for condensation, nor does it degrade in G1 . Rather, it associates with chromosomes in a punctate fashion throughout the cell cycle . mis4 mutants are hypersensitive to hydroxyurea (HU) and UV irradiation but retain the ability to restrain cell cycle progression when damaged or sustaining a block to replication . The mis4 mutation results in synthetic lethality with a DNA ligase mutant . Mis4 may form a stable link between chromatids in S phase that is split rather than removed in anaphase. Plant J, 1998 Sep, 15(6), 783 - 9 An Arabidopsis immunophilin, AtFKBP12, binds to AtFIP37 (FKBP interacting protein) in an interaction that is disrupted by FK506; Faure JD et al.; AtFKBP12 is an Arabidopsis cDNA that encodes a protein similar to the mammalian immunophilin, FKBP12 . AtFKBP12 was used as 'bait' in a yeast 2-hybrid system to screen for cDNAs in Arabidopsis encoding proteins that bind to FKBP12 . Two partial cDNAs were recovered encoding the C-terminus of a protein we have called Arabidopsis thaliana FKBP12 interacting protein 37 (AtFIP37) . AtFIP37 is similar to a mammalian protein, FAP48, that also binds to FKBP12 . The interaction between AtFKBP12 and AtFIP37 in the 2-hybrid system, as assessed by histidine auxotrophy and beta-galactosidase activity, was disrupted by FK506, but not by cyclosporin A, a drug that binds to cyclophilin A . AtFIP37 was also shown to bind in vitro to AtFKBP12 in GST-fusion protein binding assays . The binding was abolished by prior incubation of AtFKBP12 with FK506 . These findings indicate that an Arabidopsis FKBP12 ortholog encodes a protein that binds FK506 and that the interaction between AtFKBP12 and AtFIP37 may involve the FK506 binding site of AtFKBP12 . The interaction provides interesting new opportunities for controlling protein:protein interactions in vivo in plants. Crit Rev Eukaryot Gene Expr, 1998, 8(3-4), 225 - 55 Energy-dependent chromatin remodelers: complex complexes and their components; Imbalzano AN; Chromatin structure is dynamically regulated such that it can be modified by a number of factors in response to a variety of signals . One class of factors that can mediate changes in chromatin structure is the ATP-dependent nucleosome remodeling complexes . Genetic and biochemical evidence supports the idea that a family of related multisubunit complexes hydrolyzes ATP in order to facilitate the rearrangement of chromatin structure . These complexes are conserved from yeast to mammals and apparently have diverse functions in modifying chromatin structure; ATP-dependent chromatin remodelers have been implicated in nucleosome deposition, nucleosome assembly, and disruption of nucleosome structure to facilitate transcriptional activation . In addition, individual components of these complexes have been linked to control of cell growth, cell cycle regulation, development, and differentiation, and they may also be targets for viral regulatory proteins . The diversity of subunit functions likely relates to effects on chromatin structure, suggesting that the regulation of chromatin structure by ATP-dependent remodelers is important in many different aspects of cellular metabolism. Insect Biochem Mol Biol, 1998 Oct, 28(10), 791 - 9 Identification and developmental expression of the mitochondrial phosphate transport protein gene from the spruce budworm, Choristoneura fumiferana; Feng QL et al.; Phosphate transport protein (PTP) is a mitochondrial inner membrane protein responsible for the translocation of inorganic phosphate into the mitochondrial matrix . A full length cDNA clone encoding the PTP was isolated from the spruce budworm, Choristoneura fumiferana . The deduced amino acid sequence of the longest ORF of CfPTP cDNA showed high similarity with the amino acid sequences of PTPs cloned from several species . Phylogenetic tree analysis indicated that CfPTP occupied an intermediate position between vertebrates on the one side and yeast and nematodes on the other side . Studies on the developmental expression of CfPTP mRNA showed that higher levels of mRNA were present during the feeding and growing stages than during molting periods. Biochem J, 1998 Nov 15, 336 ( Pt 1), 213 - 22 Cloning of an intracellular protein that binds selectively to mitogenic acidic fibroblast growth factor; Kolpakova E et al.; In addition to its extracellular action, there is evidence that acidic fibroblast growth factor (aFGF) acts inside cells . To identify intracellular proteins interacting with aFGF, we screened a HeLa cell library in the yeast two-hybrid system using pLex-aFGF as a bait . A clone binding to aFGF, but not to the non-mitogenic mutant aFGF-K132E, was isolated and characterized . The insert contained an open reading frame corresponding to a novel protein of 42 kDa . The protein, termed aFGF intracellular binding protein (FIBP), is mainly hydrophilic and does not contain an N-terminal signal sequence . In vitro-translated FIBP bound specifically to a fusion protein of maltose-binding protein and aFGF . FIBP became post-translationally associated with microsomes added to the cell-free protein synthesizing system, and the membrane-associated protein bound aFGF with high efficiency . Immunoblots and fluorescence microscopy demonstrated that the protein is present in nuclei and, to a lesser extent, associated with mitochondria and other cytoplasmic membranes . The possibility is discussed that FIBP may be involved in the mitogenic action of aFGF. Genomics, 1998 Nov 15, 54(1), 159 - 64 A 500-kb YAC and BAC contig encompassing the high-growth deletion in mouse chromosome 10 and identification of the murine Raidd/Cradd gene in the candidate region; Horvat S et al.; The mouse high growth (hg) gene was identified in a selection experiment for rapid growth . It produces a 30-50% increase in weight gain of homozygous individuals without resulting in obesity . Recently, hg was mapped to a deletion around marker D10Mit69 . Here we report a map of yeast artificial chromosome and bacterial artificial chromosome clones spanning the entire region deleted in high-growth mice . The size of the deletion estimated from the clone lengths is approximately 500 kb . Using exon trapping, the murine Raidd/Cradd gene was identified in the hg region, and its cDNA sequence and expression pattern were determined . The human RAIDD/CRADD was mapped using radiation hybrid mapping to human chromosome 12, 2.9 cR distal to marker AFMB311WC5 . The identified Raidd/Cradd gene, which is deleted in high-growth mice, presents a potential candidate for hg . Genomics, 1998 Nov 15, 54(1), 107 - 15 High-resolution genetic, physical, and transcript map of the mnd2 region of mouse chromosome 6; Weber JS et al.; The autosomal recessive mutation mnd2 is responsible for a lethal neuromuscular wasting disorder in the mouse . A high-resolution genetic map of the mnd2 region of mouse chromosome 6 was generated by analysis of 1147 F2 offspring from an intersubspecific cross between strains C57BL/6J-mnd2/+ and CAST/Ei . The results localize mnd2 to the 0.2-cM interval between D6Mit164 and D6Mit128 . A contig of overlapping YAC, BAC, and P1 clones spanning the nonrecombinant interval was constructed . One novel gene isolated from the contig, D6Mm3e, is a new member of the WD repeat gene family . The observed gene order for the five positional candidate genes previously mapped to the region and five newly isolated genes is centromere-Hexokinase II-D6Mm5e-p62 Dok-Aup1-Rhotekin, D6Mm3e-Dynactin 1-Smooth muscle gamma actin-D6Mm4e-beta-adducin-telomere . Seven of these genes are located within the 400-kb nonrecombinant interval for mnd2 . Comparison between wildtype and mutant failed to detect any differences in mRNA size, abundance, or coding sequence for these seven genes . The genes described here are positional candidates for the Parkinson disease susceptibility locus PARK3 that was recently mapped to the corresponding region of human chromosome band 2p13.1 . Genomics, 1998 Nov 15, 54(1), 31 - 8 A 4-Mb high-density single nucleotide polymorphism-based map around human APOE; Lai E et al.; Whole-genome association studies using single-nucleotide polymorphisms (SNPs) are the proposed method of choice for the identification of loci associated with complex diseases . In this report, we address the feasibility of generating high-density SNP maps (with <100-kb spacing) . As a pilot study, we concentrated on a 4-Mb region around the human APOE locus on chromosome 19 . We compared the efficiency of SNP detection using YAC-based versus BAC/PAC-based maps, sequencing individual DNAs versus a pooled DNA sample, and we evaluated three different software applications for polymorphism detection . A total of 121 SNPs (25 in coding regions) were identified . The frequency of SNP detection was 1 SNP/1.1 kb of genomic sequence . From APOE to CALM3 (approximately 2 Mb), the average marker spacing was approximately 30 kb . Fifty-one SNPs were genotyped in five populations, and 10 SNPs showed an allele frequency differential greater than 0.5 between populations . Our results demonstrated that high-density SNP maps can be efficiently generated using existing technologies and that a genome-wide map with 60,000-100,000 SNPs is achievable in a reasonable time frame . Genomics, 1998 Nov 15, 54(1), 5 - 12 A long-range restriction map of deletion interval 6 of the human Y chromosome: a region frequently deleted in azoospermic males; Yen PH; Deletion interval 6 (DI6) of the human Y chromosome, located at the distal end of the long arm euchromatic region, is required for normal spermatogenesis . About 10% of males with idiopathic azoospermia or oligospermia have microdeletions in this region . Six gene families, including RBMY (RNA binding motif, Y chromosome), DAZ (deleted in azoospermia), and four recently isolated genes, have been mapped to this interval . Genes from all of these families show testis-specific expression and are thus candidates for azoospermic factor (AZF) . DI6 is also rich in Y-specific repetitive sequences, which may be responsible for its frequent deletion . To understand the sequence organization of this region, a 5-Mb restriction map was constructed based on YAC clones and was partially verified on genomic DNA . The locations of five gene family members, as well as numerous STSs, were determined . The map shows several inverted and direct repeats several hundred kilobases in size . The restriction map of DI6 will facilitate future mapping of deletion breakpoints in infertile males and elucidation of mechanisms behind frequent deletions . Clin Chem Lab Med, 1998 Aug, 36(8), 511 - 4 The Human Genome Project: from mapping to sequencing; Weissenbach J; Until recently, the "human genome" programs were mainly directed towards the development of maps to identify disease genes . The genetic map comprises about 8000 highly informative second generation markers of the microsatellite type . The density of markers is now sufficient to localize a gene for a monogenic disease with a precision of 1 to 2 million base pairs easily, and to define intervals which contain susceptibility genes for multifactorial disorders . A third generation map based on single nucleotide polymorphisms that can be genotyped using DNA chip technology is in progress . The physical map, based on sets of overlapping yeast artificial chromosomes ordered using sequence-tagged sites, covers over 90% of the genome . However, this physical map cannot serve as a support for sequencing because of the numerous rearrangements that occur in yeast artificial chromosomes . An international network of laboratories has mapped a set of more than 30,000 expressed sequences from cDNAs using whole genome radiation hybrids that enable integration of genes within existing maps . The human genome program is now progressively shifting to massive sequencing, although sequence ready maps are not available for the major part of the human genome . Similarly, our capacity to interpret the available genomic sequence remains limited. Life Sci, 1998, 63(19), 1693 - 9 Inhibitory effects of uridine diphosphate on UDP-glucuronosyltransferase; Yokota H et al.; Inhibitory effects of uridine diphosphate on the enzymatic activity of UDP-glucuronosyltransferase (UGT) were investigated . Pyrimidine nucleotides such as UDP, UTP and cytidine diphosphate reduced the activity of rat purified UGT (phenol UGT) to about 10%, 48% and 46% of the control, respectively, at the same concentration as a donor substrate, UDP-glucuronic acid . Purine nucleotides, uridine monophosphate, glucuronic acid and some UDP-sugars were only slightly inhibitory toward the transferase . Similar effects were observed in the expressed UGT (UGT1A6; corresponding to phenol UGT) in yeast cells and rat liver microsomal membrane-binding UGT, indicating that uracil and diphosphate residues are essential for the UDP inhibition . Interestingly, 2'-deoxy UDP was found to be a less effective inhibitor (about 50% inhibition) than UDP on the purified, the expressed (UGT1A6 and UGT2B1) and microsomal membrane-binding UGTs . These results indicate that not only uracil and diphosphate residues but also 2'-hydroxyl residue of UDP ribose participates in the interactions between UDP and UDP-glucuronosyltransferase. Rev Cubana Med Trop, 1995, 47(1), 65 - 70 {The reactogenicity of Heberbiovac-HB vaccine at different doses}; Diaz Gonzalez M et al.; Reactogenicity was measured after applying the Heberbiovac-HB recombinant vaccine against hepatitis B virus to three groups of children aged 6-9 years . The vaccine was derived from yeast cells, administered at doses of 10, 5, and 2.5 g, with a schedule of 0, 1 and 6 months . The overall observed symptomatology was low (12.2%) in the three groups with 10.7%, 13.5%, and 12.3% for 10, 5, and 2.5 g, respectively . The predominant symptoms and signs were febricula (7.0%), local pain (3.1%), and erythema (1.2%) . No significant differences were found when making a comparison between groups and sexes . An acceptable reactogenicity of the immunogen was confirmed, thus its use is recommended for the protection against hepatitis B virus. Biochim Biophys Acta, 1998 Nov 11, 1414(1-2), 217 - 30 Characterization of glucose transport activity reconstituted from heart and other tissues; Wheeler TJ et al.; We examined several aspects of glucose transport reconstituted in liposomes, with emphasis on transporters of rat heart (mostly GLUT4) compared to those of human erythrocytes (GLUT1), and on effects of agents that modulate transport in intact cells . Several types of samples gave higher recons