J Invest Dermatol, 2000 Sep, 115(3), 477 - 85 Biochemical characterization of S100A2 in human keratinocytes: subcellular localization, dimerization, and oxidative cross-linking; Deshpande R et al.; S100A2 is a calmodulin-like protein of unknown function, whose transcription is positively regulated in response to ErbB and p53 signaling . Expression of S100A2 is markedly increased in the context of ErbB-driven reactive epidermal hyperplasia, and decreased in the context of hypofunctional p53 mutations in carcinoma cell lines and tumors . This bimodal pattern of regulation suggests an important function for S100A2 in keratinocyte differentiation and carcinogenesis . Taking the biochemical approach to the determination of S100A2 function, we have characterized its physical state and subcellular localization in normal human keratinocytes . S100A2 in hypotonic lysates remained soluble after centrifugation at 100 000 x g, indicating that it is not associated with cell membranes . Permeabilization experiments confirmed the lack of membrane association and revealed a digitonin-insoluble nuclear fraction of S100A2, which was confirmed by immunofluorescence microscopy . Pulldown assays of epitope-tagged S100A2 and yeast two-hybrid screening revealed that S100A2 displays a strong propensity to homodimerize . Naturally expressed S100A2 dimers in normal human keratinocytes readily underwent intermolecular disulfide cross-linking unless a strong denaturant was present during cell lysis . Treatment of intact normal human keratinocytes with hydrogen peroxide strongly promoted S100A2 cross-linking . These results demonstrate that native S100A2 is a homodimer that does not depend on disulfide cross-linking for stability, but undergoes intermolecular cross-linking at cysteine residues in response to oxidative stress . Based on these findings, we propose that S100A2 may protect normal keratinocytes against carcinogens by participating in the cellular proof-reading response to oxidative stress. Eur J Biochem, 2000 Sep, 267(17), 5421 - 6 Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity; O'Brien J et al.; We show here the identity of Alamar Blue as resazurin . The 'resazurin reduction test' has been used for about 50 years to monitor bacterial and yeast contamination of milk, and also for assessing semen quality . Resazurin (blue and nonfluorescent) is reduced to resorufin (pink and highly fluorescent) which is further reduced to hydroresorufin (uncoloured and nonfluorescent) . It is still not known how this reduction occurs, intracellularly via enzyme activity or in the medium as a chemical reaction, although the reduced fluorescent form of Alamar Blue was found in the cytoplasm and of living cells nucleus of dead cells . Recently, the dye has gained popularity as a very simple and versatile way of measuring cell proliferation and cytotoxicity . This dye presents numerous advantages over other cytotoxicity or proliferation tests but we observed several drawbacks to the routine use of Alamar Blue . Tests with several toxicants in different cell lines and rat primary hepatocytes have shown accumulation of the fluorescent product of Alamar Blue in the medium which could lead to an overestimation of cell population . Also, the extensive reduction of Alamar Blue by metabolically active cells led to a final nonfluorescent product, and hence an underestimation of cellular activity. J Biol Chem, 2000 Nov 17, 275(46), 36278 - 84 Interferon-inducible Myc/STAT-interacting protein Nmi associates with IFP 35 into a high molecular mass complex and inhibits proteasome-mediated degradation of IFP 35; Chen J et al.; Nmi is an interferon (IFN)-inducible protein homologous to IFN-inducible protein IFP 35 . The homology consists of a novel Nmi/IFP 35 domain (NID) of 90-92 amino acids that is repeated in tandem in each protein and mediates Nmi-Nmi protein interactions and subcellular localization . In a yeast two-hybrid screen with a fragment of Nmi protein containing both NIDs, we identified an interaction between Nmi and IFP 35 . Deletion derivatives of the proteins indicate that both NIDs are required for the interaction between Nmi and IFP 35 . In mammalian cells, Nmi and IFP 35 co-immunoprecipitate and co-localize in large cytoplasmic speckles . Nmi and IFP 35 proteins associate into a high molecular mass complex of 300-400 kDa as determined by native gel electrophoresis and gel filtration . The association of Nmi and IFP 35 into a complex can be demonstrated in multiple cell lines and is not dependent on treatment with IFN . Short term and long term cultures of transfected HEK293 cells suggest that Nmi and IFP 35 proteins stabilize each other through complex formation . IFP 35 appears to be more labile because Nmi was stable in the absence of IFP 35, whereas IFP 35 was degraded in the absence of Nmi . A deletion analysis revealed that Nmi must interact with IFP 35 to prevent its degradation and that the amino terminus of Nmi is required, but not sufficient, for this function . Inhibition of the proteasome, but not other proteases, led to increased levels of IFP 35 . Thus, we have shown that Nmi and IFP 35 associate into a protein complex, that IFP 35 is degraded in a proteasome-mediated process, and that a novel function of Nmi is to prevent IFP 35 degradation . The stabilization of IFP 35 by Nmi may serve to amplify the physiologic effects of IFNs. Blood Cells Mol Dis, 2000 Jun, 26(3), 205 - 10 Exclusion of ZIRTL as candidate gene of juvenile hemochromatosis and refinement of the critical interval on 1q21; Roetto A et al.; Hemochromatosis type 2 (HFE2) or juvenile hemochromatosis (JH) is a rare recessive disorder that causes iron overload, characterized by early onset and severe clinical course . The JH locus maps to chromosome 1q, in a 4-cM region encompassing markers D1S442 and D1S2347 . Recently a gene named ZIRTL has been characterized and mapped to 1q21 . This gene belongs to a family of divalent metal ion-transporting genes that encode for proteins involved in transport of different metals, including iron . Thus, the ZIRTL gene represents a positional and functional candidate for JH . Here we further restrict the candidate region through segregation analysis of two new polymorphic markers and haplotype analysis in JH families . Furthermore, we exclude ZIRTL as a JH candidate gene showing that it maps outside the critical interval and that its genomic sequence is normal in three patients . Genomics, 2000 Aug 15, 68(1), 89 - 92 Human and mouse homologues of the Drosophila melanogaster tweety (tty) gene: a novel gene family encoding predicted transmembrane proteins; Campbell HD et al.; We have cloned cDNA for TTYH1, a human homologue of the Drosophila melanogaster tweety (tty) gene . The 450-residue predicted protein shows 27% amino acid sequence identity (51% similarity) to the Drosophila protein, which contains an additional C-terminal repetitive region . A second Drosophila homologue exhibits 42% identity (65% similarity) to the tty protein . Mouse (Ttyh1), macaque, and Caenorhabditis elegans homologues were also identified, and the complete coding sequence for the mouse gene was determined . The mouse protein is 91% identical to the human protein . Hydrophobicity analysis of the tty-related proteins indicates that they represent a new family of membrane proteins with five potential membrane-spanning regions . The yeast FTR1 and FTH1 iron transporter proteins and the mammalian neurotensin receptors 1 and 2 have a similar hydrophobicity profile, although there is no detectable sequence homology to the tty-related proteins . This suggests that the tweety-related proteins could be involved in transport of iron or other divalent cations or alternatively that they may be membrane-bound receptors . TTYH1 was mapped to chromosome 19q13.4 by FISH and by radiation hybrid mapping using the Stanford G3 panel . Cell Mol Life Sci, 2000 Jun, 57(6), 899 - 913 Structure and function of small heat shock/alpha-crystallin proteins: established concepts and emerging ideas; MacRae TH; Small heat shock/alpha-crystallin proteins are defined by conserved sequence of approximately 90 amino acid residues, termed the alpha-crystallin domain, which is bounded by variable amino- and carboxy-terminal extensions . These proteins form oligomers, most of uncertain quaternary structure, and oligomerization is prerequisite to their function as molecular chaperones . Sequence modelling and physical analyses show that the secondary structure of small heat shock/alpha-crystallin proteins is predominately beta-pleated sheet . Crystallography, site-directed spin-labelling and yeast two-hybrid selection demonstrate regions of secondary structure within the alpha-crystallin domain that interact during oligomer assembly, a process also dependent on the amino terminus . Oligomers are dynamic, exhibiting subunit exchange and organizational plasticity, perhaps leading to functional diversity . Exposure of hydrophobic residues by structural modification facilitates chaperoning where denaturing proteins in the molten globule state associate with oligomers . The flexible carboxy-terminal extension contributes to chaperone activity by enhancing the solubility of small heat shock/alpha-crystallin proteins . Site-directed mutagenesis has yielded proteins where the effect of the change on structure and function depends upon the residue modified, the organism under study and the analytical techniques used . Most revealing, substitution of a conserved arginine residue within the alpha-crystallin domain has a major impact on quaternary structure and chaperone action probably through realignment of beta-sheets . These mutations are linked to inherited diseases . Oligomer size is regulated by a stress-responsive cascade including MAPKAP kinase 2/3 and p38 . Phosphorylation of small heat shock/alpha-crystallin proteins has important consequences within stressed cells, especially for microfilaments. Mol Cell, 2000 Jul, 6(1), 65 - 75 ARK-1 inhibits EGFR signaling in C . elegans; Hopper NA et al.; A screen for synthetic enhancers of sli-1 identified ark-1 (forAck-related tyrosine kinase), a novel inhibitor of let-23 EGFR signaling in C . elegans . An ark-1 mutation synergizes with mutations in other negative regulators of let-23, resulting in increased RAS signaling . Genetic analysis suggests that ARK-1 acts upstream of RAS and is dependent upon SEM-5 . ARK-1 inhibits LET-23-mediated ovulation, a RAS-independent function . ARK-1 physically interacts with SEM-5 in the yeast two-hybrid assay . We find that sem-5 also has a negative function in let-23-mediated ovulation and suggest that this negative function is mediated by the recruitment of inhibitors such as ARK-1. Mol Cell, 2000 Jul, 6(1), 31 - 40 Aven, a novel inhibitor of caspase activation, binds Bcl-xL and Apaf-1; Chau BN et al.; Bcl-x(L), an antiapoptotic Bcl-2 family member, is postulated to function at multiple stages in the cell death pathway . The possibility that Bcl-x(L) inhibits cell death at a late (postmitochondrial) step in the death pathway is supported by this report of a novel apoptosis inhibitor, Aven, which binds to both Bcl-x(L) and the caspase regulator, Apaf-1 . Identified in a yeast two-hybrid screen, Aven is broadly expressed and is conserved in other mammalian species . Only those mutants of Bcl-x(L)that retain their antiapoptotic activity are capable of binding Aven . Aven interferes with the ability of Apaf-1 to self-associate, suggesting that Aven impairs Apaf-1-mediated activation of caspases . Consistent with this idea, Aven inhibited the proteolytic activation of caspases in a cell-free extract and suppressed apoptosis induced by Apaf-1 plus caspase-9 . Thus, Aven represents a new class of cell death regulator. Oncol Rep, 2000 Sep-Oct, 7(5), 995 - 1001 Molecular cloning and characterization of FX3, a novel zinc-finger protein; Xu F et al.; In a screen for cyclin G1 interacting proteins using the yeast two-hybrid system, we identified a cDNA clone encoding a novel zinc-finger protein . In addition to a C-terminal zinc-finger domain, the 18 kDa protein, designated FX3, contains a bipartite nuclear targeting sequence and a number of putative protein kinase phosphorylation sites . The physical interaction of FX3 with cyclin G1 was confirmed in vitro by co-immunoprecipitation . Further studies demonstrated that the zinc-finger domain is not required for the observed interaction between FX3 and cyclin G1 . FX3 is an essential gene expressed in numerous human tissues, with the highest levels observed in the liver. Plant Cell, 2000 Aug, 12(8), 1393 - 407 Identification of a calmodulin-regulated soybean Ca(2+)-ATPase (SCA1) that is located in the plasma membrane; Chung WS et al.; Ca(2)+-ATPases are key regulators of Ca(2+) ion efflux in all eukaryotes . Animal cells have two distinct families of Ca(2+) pumps, with calmodulin-stimulated pumps (type IIB pumps) found exclusively at the plasma membrane . In plants, no equivalent type IIB pump located at the plasma membrane has been identified at the molecular level, although related isoforms have been identified in non-plasma membrane locations . Here, we identify a plant cDNA, designated SCA1 (for soybean Ca(2+)-ATPase 1), that encodes Ca(2+)-ATPase and is located at the plasma membrane . The plasma membrane localization was determined by sucrose gradient and aqueous two-phase membrane fractionations and was confirmed by the localization of SCA1p tagged with a green fluorescent protein . The Ca(2+)-ATPase activity of the SCA1p was increased approximately sixfold by calmodulin (K(1/2) approximately 10 nM) . Two calmodulin binding sequences were identified in the N-terminal domain . An N-terminal truncation mutant that deletes sequence through the two calmodulin binding sites was able to complement a yeast mutant (K616) that was deficient in two endogenous Ca(2+) pumps . Our results indicate that SCA1p is structurally distinct from the plasma membrane-localized Ca(2+) pump in animal cells, belonging instead to a novel family of plant type IIB pumps found in multiple subcellular locations . In plant cells from soybean, expression of this plasma membrane pump was highly and rapidly induced by salt (NaCl) stress and a fungal elicitor but not by osmotic stress. Plant Cell, 2000 Aug, 12(8), 1345 - 55 A new subfamily of sucrose transporters, SUT4, with low affinity/high capacity localized in enucleate sieve elements of plants; Weise A et al.; A new subfamily of sucrose transporters from Arabidopsis (AtSUT4), tomato (LeSUT4), and potato (StSUT4) was isolated, demonstrating only 47% similarity to the previously characterized SUT1 . SUT4 from two plant species conferred sucrose uptake activity when expressed in yeast . The K(m) for sucrose uptake by AtSUT4 of 11.6 +/- 0.6 mM was approximately 10-fold greater than for all other plant sucrose transporters characterized to date . An ortholog from potato had similar kinetic properties . Thus, SUT4 corresponds to the low-affinity/high-capacity saturable component of sucrose uptake found in leaves . In contrast to SUT1, SUT4 is expressed predominantly in minor veins in source leaves, where high-capacity sucrose transport is needed for phloem loading . In potato and tomato, SUT4 was immunolocalized specifically to enucleate sieve elements, indicating that like SUT1, macromolecular trafficking is required to transport the mRNA or the protein from companion cells through plasmodesmata into the sieve elements. Plant Cell, 2000 Aug, 12(8), 1267 - 78 Functional requirement of plant farnesyltransferase during development in Arabidopsis; Yalovsky S et al.; Arabidopsis era1 was identified as an abscisic acid-hypersensitive mutant caused by disruptions or deletions of the gene for the beta subunit (AtFTB) of farnesyltransferase (FTase) . The heterodimeric enzyme catalyzes the covalent attachment of the 15-carbon farnesyl diphosphate to the C terminus of regulatory proteins and is essential for growth in yeast . The first disruption of FTB in a multicellular context revealed several developmental and growth regulatory processes that require the function of FTase . The lack of FTase activity in the Arabidopsis era1-2 FTB deletion mutant resulted in enlarged meristems and organs, supernumerary organs in floral whorls, arrested development of axillary meristems, late flowering, and homeotic transformations of flowers . Complementation of era1-2 with LeFTB, the tomato gene for the beta subunit of FTase, restored a normal phenotype and confirmed that the lesion is in AtFTB alone . The effect of this lesion on control of meristem size and on developmental processes suggests the involvement of regulatory proteins that require farnesylation for their function . At least three distinct processes that require the function of FTase were identified: regulation of cellular differentiation in the meristems, meristem maintenance, and regulation of flower development . Together, these results provide a basis for future studies on the involvement of FTase in specific developmental processes and for structure-function analysis of FTase in vivo. J Biol Chem, 2000 Nov 24, 275(47), 36957 - 65 Human RNase III is a 160-kDa protein involved in preribosomal RNA processing; Wu H et al.; A human RNase III gene encodes a protein of 160 kDa with multiple domains, a proline-rich, a serine- and arginine-rich, and an RNase III domain . The expressed purified RNase III domain cleaves double-strand RNA and does not cleave single-strand RNA . The gene is ubiquitously expressed in human tissues and cell lines, and the protein is localized in the nucleus of the cell . The levels of transcription and translation of the protein do not change during different phases of the cell cycle . However, a significant fraction of the protein in the nucleus is translocated to the nucleolus during the S phase of the cell cycle . That this human RNase III is involved in processing of pre-rRNA, but might cleave at sites different from those described for yeast RNase III, is shown by antisense inhibition of RNase III expression . Inhibition of human RNase III expression causes cell death, suggesting an essential role for human RNase III in the cell . The antisense inhibition technique used in this study provides an effective method for functional analysis of newly identified human genes. Science, 2000 Aug 18, 289(5482), 1202 - 6 Inflammation dampened by gelatinase A cleavage of monocyte chemoattractant protein-3; McQuibban GA et al.; Tissue degradation by the matrix metalloproteinase gelatinase A is pivotal to inflammation and metastases . Recognizing the catalytic importance of substrate-binding exosites outside the catalytic domain, we screened for extracellular substrates using the gelatinase A hemopexin domain as bait in the yeast two-hybrid system . Monocyte chemoattractant protein-3 (MCP-3) was identified as a physiological substrate of gelatinase A . Cleaved MCP-3 binds to CC-chemokine receptors-1, -2, and -3, but no longer induces calcium fluxes or promotes chemotaxis, and instead acts as a general chemokine antagonist that dampens inflammation . This suggests that matrix metalloproteinases are both effectors and regulators of the inflammatory response. Life Sci, 2000 Jul 14, 67(8), 887 - 94 Purification of a novel apolipoprotein H-like milk protein with ribonucleolytic and cell-free translation inhibitory activities; Ye XY et al.; A new whey protein designated apolipoprotein H-like whey protein, with a molecular weight of 62 kDa and an N-terminal amino acid sequence similar to that of apolipoprotein H, was isolated from bovine milk . The isolation procedure involved removal of globulin from acid whey by precipitation with 1.8M (NH4)2SO4, followed by addition of (NH4)2SO4 to attain a concentration of 3.6M . Subsequent steps included chromatography on CM-Sepharose and Mono S and elution of the adsorbed protein of interest with a linear NaCl gradient . The new whey protein displayed some ribonuclease (RNase) activity . It was most active at pH 7.5 with yeast transfer RNA (tRNA) as substrate and showed potent specific ribonucleolytic activity toward poly C . It inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of approximately 63 nM. J Immunol, 2000 Sep 1, 165(5), 2451 - 7 Selective inhibition of IgG-mediated phagocytosis in gelsolin-deficient murine neutrophils; Serrander L et al.; Phagocytosis and the microbicidal functions of neutrophils require dynamic changes of the actin cytoskeleton . We have investigated the role of gelsolin, a calcium-dependent actin severing and capping protein, in peripheral blood neutrophils from gelsolin-null (Gsn-) mice . The phagocytosis of complement opsonized yeast was only minimally affected . In contrast, phagocytosis of IgG-opsonized yeast was reduced close to background level in Gsn- neutrophils . Thus, gelsolin is essential for efficient IgG- but not complement-mediated phagocytosis . Furthermore, attachment of IgG-opsonized yeast to Gsn- neutrophils was reduced ( approximately 50%) but not to the same extent as ingestion ( approximately 73%) . This was not due to reduced surface expression of the Fcgamma-receptor or its lateral mobility . This suggests that attachment and ingestion of IgG-opsonized yeast by murine neutrophils are actin-dependent and gelsolin is important for both steps in phagocytosis . We also investigated granule exocytosis and several steps in phagosome processing, namely the formation of actin around the phagosome, translocation of granules, and activation of the NADPH-oxidase . All these functions were normal in Gsn- neutrophils . Thus, the role of gelsolin is specific for IgG-mediated phagocytosis . Our data suggest that gelsolin is part of the molecular machinery that distinguishes complement and IgG-mediated phagocytosis . The latter requires a more dynamic reorganization of the cytoskeleton. J Biol Chem, 2000 Nov 17, 275(46), 36230 - 7 BRCA1 physically and functionally interacts with ATF1; Houvras Y et al.; BRCA1, a breast and ovarian cancer susceptibility gene, encodes a 220-kDa protein whose precise biochemical function remains unclear . BRCA1 contains an N-terminal RING finger that mediates protein-protein interaction . The C-terminal domain of BRCA1 (BRCT) can activate transcription and interacts with RNA polymerase holoenzyme . Using the yeast two-hybrid system, we identified an interaction between the BRCA1 RING finger and ATF1, a member of the cAMP response element-binding protein/activating transcription factor (CREB/ATF) family . We demonstrate that BRCA1 and ATF1 can physically associate in vitro, in yeast, and in human cells . BRCA1 stimulated transcription from a cAMP response element reporter gene in transient transfections . BRCA1 also stimulated transcription from a natural promoter, that of tumor necrosis factor-alpha, in a manner dependent on the integrity of the cAMP response element . These results implicate BRCA1 in transcriptional activation of ATF1 target genes, some of which are involved in the transcriptional response to DNA damage. Genomics, 2000 Jul 1, 67(1), 28 - 39 Refinement of the smallest commonly deleted segment of chromosome 20 in malignant myeloid diseases and development of a PAC-based physical and transcription map; Wang PW et al.; A deletion of the long arm of chromosome 20, del(20q), is a recurring abnormality in malignant myeloid diseases . In previous studies, we delineated a commonly deleted segment (CDS) of 5 Mb within band 20q12 flanked by D20S206 (proximal) and D20S481 (distal) . We have generated a detailed physical map of P1 artificial chromosome (PAC) clones of this interval as well as a transcriptional map . The contig consists of 81 clones to which 152 markers (27 genes, 45 unique expressed sequence tags (ESTs) or UniGenes, 24 polymorphisms, and 56 sequence-tagged sites) have been mapped . Using PAC clones for fluorescence in situ hybridization analysis of myeloid leukemia cells with reciprocal translocations of 20q, or unbalanced rearrangements leading to loss of 20q, we have narrowed the CDS to an approximately 250-kb interval encompassing two overlapping PACs, P201E16 and P29M7 (between EST AA368224 and D20S481) . This interval is gene-rich and contains 5 characterized genes, 4 UniGenes, and 9 single ESTs . The development of a transcriptional map and the identification of the smallest CDS will facilitate the molecular cloning of a myeloid leukemia suppressor gene on 20q. Genomics, 2000 Jul 1, 67(1), 19 - 27 A novel candidate gene for mouse and human preaxial polydactyly with altered expression in limbs of Hemimelic extra-toes mutant mice; Clark RM et al.; Polydactyly is a common malformation of vertebrate limbs . In humans a major locus for nonsyndromic pre-axial polydactyly (PPD) has been mapped previously to 7q36 . The mouse Hemimelic extra-toes (Hx) mutation maps to a homologous chromosome segment and has been proposed to affect a homologous gene . To understand the molecular changes underlying PPD, we used a positional cloning approach to identify the gene or genes disrupted by the Hx mutation and a closely linked limb mutation, Hammertoe (Hm) . High resolution genetic mapping identified a small candidate interval for the mouse mutations located 1.2 cM distal to the Shh locus . The nonrecombinant interval was completely cloned in bacterial artificial chromosomes and searched for genes using a combination of exon trapping, sample sequencing, and mapping of known genes . Two novel genes, Lmbr1 and Lmbr2, are entirely within the candidate interval we defined genetically . The open reading frame of both genes is intact in mutant mice, but the expression of the Lmbr1 gene is dramatically altered in developing limbs of Hx mutant mice . The correspondence between the spatial and temporal changes in Lmbr1 expression and the embryonic onset of the Hx mutant phenotype suggests that the mouse Hx mutation may be a regulatory allele of Lmbr1 . The human ortholog of Lmbr1 maps within the recently described interval for human PPD, strengthening the possibility that both mouse and human limb abnormalities are due to defects in the same highly conserved gene. J Biol Chem, 2000 Oct 27, 275(43), 33201 - 4 Ligand type-specific interactions of peroxisome proliferator-activated receptor gamma with transcriptional coactivators; Kodera Y et al.; The nuclear peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily and acts as a ligand-dependent transcription factor mediating adipocyte differentiation, cell proliferation and inflammatory processes, and modulation of insulin sensitivity . Members of the 160-kDa protein (SRC-1/TIF2/AIB-1) family of coactivators, CBP/p300 and TRAP220/DRIP205, are shown to interact directly with PPARgamma and potentiate nuclear receptor transactivation function in a ligand-dependent fashion . Because PPARgamma ligands exert partially overlapping but distinct subsets of biological action through PPARgamma binding, we wished to examine whether interactions between PPARgamma and known coactivators were induced to the same extent by different classes of PPARgamma ligand . The natural ligand 15-deoxy-Delta12,14-prostaglandin J(2) induced PPARgamma interactions with all coactivators tested (SRC-1, TIF2, AIB-1, p300, TRAP220/DRIP205) in yeast and mammalian two-hybrid assays, as well as in a glutathione S-transferase pull-down assay . However, under the same conditions troglitazone, a synthetic PPARgamma ligand that acts as an antidiabetic agent, did not induce PPARgamma interactions with any of the coactivators . Our findings suggest that ligand binding may alter PPARgamma structure in a ligand type-specific way, resulting in distinct PPARgamma-coactivator interactions. EMBO J, 2000 Aug 15, 19(16), 4362 - 71 Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes; Lipowsky G et al.; Transport receptors of the importin beta superfamily account for many of the nuclear import and export events in eukaryotic cells . They mediate translocation through nuclear pore complexes, shuttle between nucleus and cytoplasm and co-operate with the RanGTPase system to regulate their interactions with cargo molecules in a compartment-specific manner . We used affinity chromatography on immobilized RanGTP to isolate further candidate nuclear transport receptors and thereby identified exportin 4 as the most distant member of the importin beta family so far . Exportin 4 appears to be conserved amongst higher eukaryotes, but lacks obvious orthologues in yeast . It mediates nuclear export of eIF-5A (eukaryotic translation initiation factor 5A) and possibly that of other cargoes . The export signal in eIF-5A appears to be complex and to involve the hypusine modification that is unique to eIF-5A . We discuss possible cellular roles for nuclear export of eIF-5A. Proc Natl Acad Sci U S A, 2000 Aug 29, 97(18), 10096 - 100 Building a dictionary for genomes: identification of presumptive regulatory sites by statistical analysis; Bussemaker HJ et al.; The availability of complete genome sequences and mRNA expression data for all genes creates new opportunities and challenges for identifying DNA sequence motifs that control gene expression . An algorithm, "MobyDick," is presented that decomposes a set of DNA sequences into the most probable dictionary of motifs or words . This method is applicable to any set of DNA sequences: for example, all upstream regions in a genome or all genes expressed under certain conditions . Identification of words is based on a probabilistic segmentation model in which the significance of longer words is deduced from the frequency of shorter ones of various lengths, eliminating the need for a separate set of reference data to define probabilities . We have built a dictionary with 1,200 words for the 6, 000 upstream regulatory regions in the yeast genome; the 500 most significant words (some with as few as 10 copies in all of the upstream regions) match 114 of 443 experimentally determined sites (a significance level of 18 standard deviations) . When analyzing all of the genes up-regulated during sporulation as a group, we find many motifs in addition to the few previously identified by analyzing the subclusters individually to the expression subclusters . Applying MobyDick to the genes derepressed when the general repressor Tup1 is deleted, we find known as well as putative binding sites for its regulatory partners. Hum Mol Genet, 2000 Aug 12, 9(13), 2029 - 42 Genomic sequence and transcriptional profile of the boundary between pericentromeric satellites and genes on human chromosome arm 10q; Guy J et al.; The organization of centromeric heterochromatin has been established in a number of eucaryotes but remains poorly defined in human . Here we present 1025 kb of contiguous human genomic sequence which links pericentromeric satellites to the RET proto-oncogene in 10q11.2 and is presumed to span the transition from centric heterochromatin to euchromatin on this chromosome arm . Two distinct domains can be defined within the sequence . The proximal approximately 240 kb consists of arrays of satellites and other tandem repeats separated by tracts of complex sequence which have evolved by pericentromeric-directed duplication . Analysis of 32 human paralogues of these sequences indicates that most terminate at or within repeat arrays, implicating these repeats in the interchromosomal duplication process . Corroborative PCR-based analyses establish a genome-wide correlation between the distribution of these paralogues and the distribution of satellite families present in 10q11 . In contrast, the distal approximately 780 kb contains few tandem repeats and is largely chromosome specific . However, a minimum of three independent intrachromosomal duplication events have resulted in >370 kb of this sequence sharing >90% identity with sequences on 10p . Using computer-based analyses and RT-PCR we confirm the presence of three genes within the sequence, ZNF11/33B, KIAA0187 and RET, in addition to five transcripts of unknown structure . All of these transcribed sequences map distal to the satellite arrays . The boundary between satellite-rich interchromosomally duplicated DNA and chromosome-specific DNA therefore appears to define a transition from pericentromeric heterochromatin to euchromatin on the long arm of this chromosome. Hum Mol Genet, 2000 Aug 12, 9(13), 1927 - 35 Copper-dependent trafficking of Wilson disease mutant ATP7B proteins; Forbes JR et al.; We have previously developed a functional assay in yeast for the copper transporter, ATP7B, defective in Wilson disease (WND) . Analysis of WND variant ATP7B proteins revealed that several were able to completely, or nearly completely, complement a mutant yeast strain in which the ATP7B ortholog CCC2 was disrupted, indicating that these ATP7B proteins retained copper transport activity . We analyzed the intracellular localization of these active WND ATP7B variant proteins using transient transfection of Chinese hamster ovary cells and triple-label immunofluorescence microscopy, as a second possible aspect of defective function . Two ATP7B variants, Asp765Asn and Leu776Val, which have normal copper transport activity in yeast, retained partial normal Golgi network localization, but were predominantly mislocalized throughout the cell . Asp765Asn and Leu776Val proteins were capable of only partial copper-dependent redistribution . WND variant protein Arg778Leu, which has defective function in yeast, was extensively mislocalized, presumably to the endoplasmic reticulum . ATP7B variant proteins Gly943Ser, which has nearly normal function in yeast, and CysProCys/Ser (mutation of the conserved CysProCys motif to SerProSer), inactive in yeast, were localized normally but were unable to redistribute in response to copper . Localization data from this study, combined with functional data from our yeast studies, provide a biochemical mechanism that can explain in part the variable biochemical features of WND, in particular the normal holo-ceruloplasmin levels observed in some patients . Our data have direct implications for WND diagnosis, indicating that decreased serum ceruloplasmin concentration is not likely to be observed with certain genetic variants of WND. J Chromatogr B Biomed Sci Appl, 2000 Jun 23, 743(1-2), 195 - 201 Kinetics of phase separation under different process and design parameters in aqueous two-phase systems; Solano-Castillo C et al.; A practical study is presented of the effect of height/diameter (H/D) ratio of settlers on the kinetics of phase separation in aqueous two-phase systems (ATPSs) . Poly(ethylene glycol) (PEG) 1000-phosphate systems with the presence of undiluted and diluted whey and disrupted yeast were used in batch studies . The influence of the biological suspensions on the rate of phase separation was investigated . It was observed that, phase separation time is much faster when disrupted suspension was used . The addition of undiluted suspensions to ATPSs slowed the process of phase separation . When the batch settler with a large cross section area (H/D ratio less than one) was used, the phases separated much faster than in a settler with a H/D ratio greater than one . Conclusions are drawn concerning the characterisation of the process and design parameters involved in the phase separation for the design of appropriate equipment. J Trace Elem Med Biol, 2000 Jun, 14(2), 84 - 7 Effect of supplementation with organic selenium on mercury status as measured by mercury in pubic hair; Seppanen K et al.; The purpose of this study was to evaluate the effect of four months of yeast-based selenium supplementation on selenium and mercury status in subjects with low serum selenium . The study was carried out in Rakvere, Estonia . Pubic hair mercury, serum selenium and blood selenium concentrations in 23 subjects (serum selenium < 90 micrograms/l) were investigated before and after selenium supplementation . Thirteen subjects were randomized into the selenium supplementation group and ten into the placebo group . The selenium supplementation group received daily 100 micrograms of selenomethionine . Selenium supplementation reduced pubic hair mercury level by 34% (p = 0.005) and elevated serum selenium by 73% and blood selenium by 59% in the supplemented group (p < 0.001 for both) . The study indicates that mercury accumulation in pubic hair can be reduced by dietary supplementation with small daily amounts of organic selenium in a short range of time. Eur J Immunol, 2000 Aug, 30(8), 2378 - 87 Engagement of the T lymphocyte antigen receptor regulates association of son-of-sevenless homologues with the SH3 domain of phospholipase Cgamma1; Scholler JK et al.; One mechanism for transducing signals downstream of lymphocyte receptor activation involves the stable association between signaling proteins . To identify protein ligands of the signal activator phospholipase Cgamma1 (PLCgamma1), we screened T cell cDNA libraries with the PLCgamma1-SH3 domain by the yeast two-hybrid assay . We observed association between the PLCgamma1-SH3 domain and the human Ras guanine nucleotide exchange factor son-of-sevenless-2 (hSos2) through a proline-rich domain interaction . Stable and abundant hSos2 / PLCgamma1 and hSos1 / PLCgamma1 complexes were observed in unstimulated T cells . The interaction between these enzymes was augmented following engagement of the T cell antigen receptor (TCR / CD3) . The kinetics of protein complex enhancement correlated with TCR / CD3-induced tyrosine phosphorylation of PLCgamma1; however, those PLCgamma1 molecules in complex with hSos2 were non-phosphorylated after TCR / CD3 stimulation, in contrast to the phosphorylated PLCgamma1 associated with the linker for activation of T cells, LAT . The Grb2 adapter protein was detected in complex with hSos / PLCgamma1, suggesting a regulatory role for Grb2 . SH3 domains from both Grb2 and PLCgamma1, but not RasGAP, bound directly to hSos homologues . The SH2 domain from Grb2 formed an association with the hSos / PLCgamma1 complex, which was enhanced following TCR / CD3 ligation . Together, the data suggest a mechanism for the son-of-sevenless and PLCgamma1 signal transducing enzymes in recruitment to protein complexes with potentially differential signaling consequences in T lymphocytes. Int J Biochem Cell Biol, 2000 Aug, 32(8), 817 - 31 Genetic analysis of homologous DNA recombination in vertebrate somatic cells; Morrison C et al.; The maintenance of genomic stability and the ability to repair induced DNA damage in vertebrate cells require homologues of the yeast RAD52 epistasis group genes . The homologous recombination carried out by the products of these genes is essential and appears to be closely linked to DNA replication . Defects in recombination and associated activities are implicated in human cancer . This review summarises recent biochemical and genetic findings on the roles played by the vertebrate RAD52 group gene products in recombination . We describe the phenotypic analysis of genetically engineered mammalian and chicken mutants of homologous recombination genes. Mech Dev, 2000 Aug, 96(1), 121 - 4 Expression of Cdcrel-1 (Pnutl1), a gene frequently deleted in velo-cardio-facial syndrome/DiGeorge syndrome; Maldonado-Saldivia J et al.; The murine Cdcrel-1 (Pnutl1) gene belongs to the family of septins, which are thought to be involved in cytokinesis in yeast, Drosophila and vertebrates . Recent studies implicate Cdcrel-1 in the regulation of vesicle transport in neurons of the adult brain . The human homologue, hCDCREL-1 maps to chromosome 22q11.2, a region commonly deleted in patients displaying velo-cardio-facial syndrome (VCFS) or DiGeorge syndrome (DGS) . During development, Cdcrel-1 transcripts are expressed from E10.5 on in the nervous system such as the dorsal root ganglia and the cranial ganglia as well as the lateral layer of the neural tube, the area where terminally differentiated neurons are located . Low level expression is found in the mesenchyme of the frontonasal mass and the limb bud mesenchyme of E11.5 and E13.5 murine embryos . At E15.5, expression is detected in the nervous tissue and in the neural layer of the eye . Based on the expression pattern as well as clinical data, Cdcrel-1 may be involved in the etiology of VCFS/DGS. Gene, 2000 Aug 8, 253(2), 271 - 80 Identification and molecular characterization of two novel Trypanosoma cruzi genes encoding polypeptides sharing sequence motifs found in proteins involved in RNA editing reactions; Ouaissi A et al.; We have previously identified a Trypanosoma cruzi cDNA encoding a protein named Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin protein family involved in thiol-disulphide redox reactions . Furthermore, we reported that Tc52 also plays a role in T . cruzi-associated immunosuppression observed during Chagas' disease . Moreover, Tc52 gene targeting deletion strategy allowed us to demonstrate that monoallelic disruption of Tc52 resulted in the alteration of the metacyclogenesis process and the production of less virulent parasites . Sequence analysis of a 7358 bp genomic fragment containing the Tc52 encoding gene revealed two additional open reading frames (ORF-A and C) . The ORFs are likely to have protein coding function by a number of criteria, including reverse transcriptase polymerase chain reaction (RT-PCR), Western blot and immunofluorescence analyses . The deduced amino-acid (aa) sequence of the ORF-A localized upstream of the Tc52 gene revealed that it contains within its N-terminus (aa 1 to 170) four RGG boxes known to act as RNA binding motifs in some proteins that interact with RNA, interspersed with a high density of glycine with regular spacing of tryptophan (WX(9-10)) in which X is often a glycine . Moreover, the C-terminal part of the ORF-C (aa 253-289) contains a motif that is strikingly similar (7-35% identity, 14-46% similarity over 28aa) to a short sequence (RNP1) comprising the consensus sequence RNA binding domain (CS-RBD) found in a number of proteins that interact with RNA . The aa sequence from the ORF-C localized downstream of the Tc52 gene showed significant homology to human adenosine deaminase acting on RNA (hADAT1) that specifically deaminates adenosine 37 to inosine in eukaryotic tRNA(Ala) and to its homologue yeast protein (Tad1p) (22-25% identity and an additional 38-40% similarity over 177aa) . Moreover, highly similar motifs of the deaminase domain are present in the T . cruzi ORF-C . Furthermore, the 5' flanking regions of the genes contained repeat TATA and CAAT nucleotide sequences which resemble the motifs found upstream of the transcription initiation sites in eukaryotic promoters . Therefore, the characterization of novel T . cruzi genes encoding proteins which show similarity to components of RNA processing reactions provides new tools to investigate the gene expression regulation in these parasitic organisms . Moreover, our recent findings on the Tc52 encoding gene underline the interest of genetic manipulation of T . cruzi, not only making it possible to use more closely an in vitro approach to find out how genes function, but also to obtain 'attenuated' strains that could be used in the development of vaccinal strategies. Annu Rev Nutr, 2000, 20, 339 - 63 Mitochondrial uncoupling proteins in energy expenditure; Kozak LP et al.; Four recently discovered homologues of the brown adipose tissue-specific mitochondrial uncoupling protein (UCP1) vary from 29% to 58% in their similarity to UCP1 . Although these homologues share important structural features with UCP1 and like UCP1 can reduce the mitochondrial membrane potential when expressed in yeast, there is no clear evidence that they can function thermogenically in vivo . On the other hand, evidence continues to accumulate indicating that the up-regulation of Ucp1 reduces excessive adiposity. Mol Biol Rep, 2000 Mar, 27(1), 13 - 9 Isolation of replicational cue elements from a library of bent DNAs of Aspergillus oryzae; Kusakabe T et al.; Two fragments that could function as replicational cue elements were isolated from a genomic DNA digest of Aspergillus oryzae on the basis of abnormal behavior in polyacrylamide gel electrophoresis . The vector used in this study contained a scaffold-associated region of the Drosophila melanogaster ftz gene to provide nuclear retention . Neither fragment contained a yeast ARS consensus sequence or an eukaryotic topoisomerase II binding sequence . One of the fragments showed sequence homology with the mitochondrial replication origin of Candida utilis and a portion of mitochondrial DNA of Aspergillus nidulans . This plasmid carrying the cue fragment could also replicate in HeLa and NIH3T3 cells. J Exp Bot, 2000 Feb, 51 Spec No, 439 - 45 Impact of elevated cytosolic and apoplastic invertase activity on carbon metabolism during potato tuber development; Hajirezaei MR et al.; During tuberization in Solanum tuberosum var . Desiree maximal catalytic activities of invertase(s) and sucrose synthase are inversely correlated . During the early stages, invertase activity is high and declines during maturation . The decrease in invertase activity is accompanied by a decrease in the hexose to sucrose ratio and an increase in sucrose synthase activity . This switch is paralleled by the onset of the storage phase as shown by the accumulation of starch and storage proteins . Biochemical and genetic evidence suggests that sucrose synthase activity is positively correlated with sink strength . To explore the possibility of enhancing sink strength in potato tubers by elevating the sucrolytic capacity, transgenic potato plants expressing either cytosolic or apoplastic yeast invertase in their tubers were made . Surprisingly, cytosolic invertase led to a decrease and apoplastic invertase to an increase in tuber yield . To understand the causes of the observed phenotypes, carbon metabolism in tubers of transgenic and control plants was analysed during different stages of tuber development . Both cytosolic and apoplastic invertase resulted in decreased sucrose and elevated glucose contents, indicating that sucrose is accessible in both compartments . Metabolic perturbation, however, was found to be compartment specific . Elevated cytosolic invertase activity led to increased carbon flux towards glycolysis and accumulation of phosphorylated intermediates . The phosphorylated intermediates were not used to build up starch . In contrast, apoplastic invertase does not lead to a significant increase in hexose phosphates compared to untransformed controls . Thus, hexoses originating in the apoplast are not efficiently phosphorylated during potato tuber development, which might be explained by an endocytotic uptake of sucrose and/or hexoses from the apoplast into the vacuole bypassing the cytosolic compartment. J Exp Bot, 2000 Jan, 51(342), 71 - 9 Molecular physiology of zinc transport in the Zn hyperaccumulator Thlaspi caerulescens; Lasat MM et al.; In this manuscript, recent research from this laboratory into physiological and molecular aspects of heavy metal (Zn) transport in the hyperaccumulating plant species, Thlaspi caerulescens is reviewed . This research is aimed at elucidating the processes that underlie the accumulation of extraordinarily high levels of Zn in the T . caerulescens shoot (up to 3% Zn dry wt.) without any associated toxicity symptom . Physiological studies focused on the use of radiotracer flux techniques (65Zn2+) to characterize zinc transport and compartmentation in the root, and translocation and accumulation in the shoot of T . caerulescens in comparison with a related non-accumulator, T . arvense . These studies indicated that Zn transport was stimulated at a number of sites in T . caerulescens, contributing to the hyperaccumulation trait . The transport processes that were stimulated included Zn influx into both root and leaf cells, and Zn loading into the xylem . The 4- to 5-fold stimulation of Zn influx into the root was hypothesized to be due to an increased abundance of Zn transporters in T . caerulescens root cells . Additionally, compartmental analysis (radiotracer wash out or efflux techniques) was used to show that Zn was sequestered in the vacuoles of T . arvense root cells which retarded Zn translocation to the shoot in this non-accumulator species . Molecular studies have focused on the cloning and characterization of Zn transport genes in T . caerulescens . Complementation of a yeast Zn transport-defective mutant with a T . caerulescens cDNA library resulted in the recovery of a cDNA, ZNT1, that encodes a Zn transporter . Sequence analysis of ZNT1 indicated it is a member of a recently discovered micronutrient transport gene family which includes the Arabidopsis Fe transporter, IRT1, and the ZIP Zn transporters . Expression of ZNT1 in yeast allowed for a physiological characterization of this transporter . It was shown to encode a high affinity Zn transporter which can also mediate low affinity Cd transport . Northern analysis of ZNT1 and its homologue in the two Thlaspi species indicated that enhanced Zn transport in T . caerulescens results from a constitutively high expression of the ZNT1 gene in roots and shoots . In T . arvense, ZNT1 is expressed at far lower levels and this expression is stimulated by imposition of Zn deficiency. Nippon Ishinkin Gakkai Zasshi, 2000, 41(3), 177 - 81 Isolation of Candida dubliniensis from the oral cavity of an HIV-positive child in Brazil; Sano A et al.; Candida dubliniensis is a newly-recognized Candida species and an important infectious pathogen, particularly for HIV-positive patients . >From oral smear samples from the radix linguae of 173 HIV-positive children, we obtained four yeast isolates which took a blue-green color on CHROMagar Candida plate at 37 degrees C for 48 hours from one HIV-positive 3-year-old boy in Brazil . The isolates were difficult to grow on potato dextrose agar plate at 42 degrees C, produced abundant chlamydospores on a cornmeal agar plate with Tween 80, and sprouted germ tubes in saline with horse serum, and the antigenic profile by CANDIDA CHECK test was useless . Carbohydrate assimilation tests by ID32C showed no reference code number in the reference book . The isolates were subjected to molecular biological assay of the DNA sequence of the large-subunit ribosomal DNA region (D1/D2) and randomly amplified polymorphic DNA (RAPD) . The DNA sequence agreed with those of standard C . dubliniensis strains, and therefore, the isolates were identified as C . dubliniensis . RAPD band pattern analysis indicated that the clinical isolates might summarize one genotype . Although the child did not present oral lesions, the fungus might be latent for opportunistic infection. Nippon Ishinkin Gakkai Zasshi, 2000, 41(3), 137 - 41 {Pathogenesis and treatment of hypersensitivity pneumonitis}; Ando M; Hypersensitivity pneumonitis (HP) is a granulomatous interstitial lung disease resulting from an immunologic reaction to the repeated inhalation of organic dusts and active chemicals . There are 50 or more groups of HP, but the prevalence varies from country to country, and even within a country, depending on a variety of occupational or environmental inhalants . In Western coutries farmer's lung, bird fancier's disease, humidifier lung, and air-conditioner disease are common, but in Japan summer-type HP is the most prevalent group . Summer-type HP is a house-related illnes induced by Trichosporon asahii and Trichosporon mucoides which contaminate the patients' home environments in hot and humid conditions . The polysaccharide antigen contains mannan backbone attached with short side chains consi-sting of mannose, xylose, and glucuronic acid residues . Both immune complex-mediated and T cell-mediated immune responses to the yeast are involved in the induction and development of the disease . Host factors such as HLA-DQw3 and cigarette smoking also play an important role in the develop-ment or suppression of the disease . An assay for serum anti-Trichosporon antibody by a Triko Kit is very useful for the serodiagnosis, and sanitization by cleaning, disinfecting, and removing from the colonizing location of Trichosporon prevents recurrence of the disease . Summer-type HP induced by Trichosporon is a new type of HP . It can be found in other countries including most Western countries, because Trichosporon asahii and Trichosporon mucoides distribute in the temperate and subtropical areas of the world. Mol Cell Biol, 2000 Sep, 20(17), 6224 - 32 Prothymosin alpha selectively enhances estrogen receptor transcriptional activity by interacting with a repressor of estrogen receptor activity; Martini PG et al.; We find that prothymosin alpha (PTalpha) selectively enhances transcriptional activation by the estrogen receptor (ER) but not transcriptional activity of other nuclear hormone receptors . This selectivity for ER is explained by PTalpha interaction not with ER, but with a 37-kDa protein denoted REA, for repressor of estrogen receptor activity, a protein that we have previously shown binds to ER, blocking coactivator binding to ER . We isolated PTalpha, known to be a chromatin-remodeling protein associated with cell proliferation, using REA as bait in a yeast two-hybrid screen with a cDNA library from MCF-7 human breast cancer cells . PTalpha increases the magnitude of ERalpha transcriptional activity three- to fourfold . It shows lesser enhancement of ERbeta transcriptional activity and has no influence on the transcriptional activity of other nuclear hormone receptors (progesterone receptor, glucocorticoid receptor, thyroid hormone receptor, or retinoic acid receptor) or on the basal activity of ERs . In contrast, the steroid receptor coactivator SRC-1 increases transcriptional activity of all of these receptors . Cotransfection of PTalpha or SRC-1 with increasing amounts of REA, as well as competitive glutathione S-transferase pulldown and mammalian two-hybrid studies, show that REA competes with PTalpha (or SRC-1) for regulation of ER transcriptional activity and suppresses the ER stimulation by PTalpha or SRC-1, indicating that REA can function as an anticoactivator in cells . Our data support a model in which PTalpha, which does not interact with ER, selectively enhances the transcriptional activity of the ER but not that of other nuclear receptors by recruiting the repressive REA protein away from ER, thereby allowing effective coactivation of ER with SRC-1 or other coregulators . The ability of PTalpha to directly interact in vitro and in vivo with REA, a selective coregulator of the ER, thereby enabling the interaction of ER with coactivators, appears to explain its ability to selectively enhance ER transcriptional activity . These findings highlight a new role for PTalpha as a coregulator activity-modulating protein that confers receptor specificity . Proteins such as PTalpha represent an additional regulatory component that defines a novel paradigm enabling receptor-selective enhancement of transcriptional activity by coactivators. Int J Tissue React, 2000, 22(2-3), 85 - 91 Transgenic mouse models to study the role of the macrophage scavenger receptor class A in atherosclerosis; De Winther MP et al.; Several in vivo studies have been performed on the role of the macrophage scavenger receptor class A (SR-A) in atherosclerosis using SR-A knockout mice . The results indicate both an antiatherogenic and a proatherogenic role of SR-A, depending on the nature of the animal model serving as the athero-susceptible background . To study the role of SR-A in a different model, we generated a transgenic mouse model with high level expression of the human SR-A gene using a 180 Kb yeast artificial chromosome (MSR1 transgenic mice) . These mice show increased expression of SR-A according to the natural expression pattern . The MSR1 transgenic mice were crossed onto a low-density lipoprotein receptor deficient background and were fed a high fat diet for 10 weeks . After this period, the size of the atherosclerotic lesions in the proximal aorta was measured . Surprisingly, atherosclerosis was significantly reduced in the MSR1 transgenic mice . In a second study, the effect of SR-A was examined in APOE-3 Leiden mice providing a different athero-susceptible background . To exclude nonmacrophage effects, bone marrow was transplanted from MSR1 mice and wild-type littermates to APOE-3 Leiden transgenic mice . After 8 weeks on a high fat diet, atherosclerosis in the mice that had received MSR1 bone marrow was reduced compared with mice that had received wild-type bone marrow . This difference reached statistical significance when individual cholesterol exposure of the mice was taken into account . Both experiments indicated an antiatherogenic role of the SR-A . This observation cannot be explained easily by SR-A function in foam cell formation because in MSR1 macrophages in vitro foam cell formation is increased . Alternatively, however, SR-A may affect the activation of macrophages . Hence the response to lipopolysaccharide was measured in MSR1-transgenic macrophages . These macrophages showed a reduction in their activation in response to lipopolysaccharide, as measured by nitric oxide production . These data show that an elevated level of SR-A expression reduces atherosclerosis, potentially by modifying the response of macrophages to activation signals in the plaque. Adv Clin Path, 1999 Oct, 3(4), 111 - 8 The applications of FISH in tumor pathology; Tibiletti MG et al.; The current FISH technology was greatly improved during the past 10 years . A large number of cosmids and yeast (YACs), bacterial (BACs), phage P1 derived (PACs) artificial chromosomes have been rapidly mapped and are useful as probes . In parallel, methods were established to specifically "paint" entire chromosomes or chromosome segments . Using these chromosome libraries as probes, complex rearrangements and marker chromosomes can be identified irrespective of their banding pattern . Ripetitive DNA probes specific for each chromosome centromere (alpha satellite sequences), are also available and may be used to identify specific aneuploidies . The use of sensitive digital imaging systems on the basis of "colour" rather than morphology increased the improvement of new FISH techniques . In particular, colour karyotyping results in the differential colour display of all human chromosomes . Another recent development of FISH technology is comparative genome hybridization (CGH), a genome-scanning technique that allows to identify and map chromosomal and subchromosomal gains and losses . FISH techniques may be used to investigate chromosome abnormalities not only on metaphasic chromosomes but also on interphasic nuclei . Any given tissue or cell source, such as sections of frozen tumors, imprinted cells, cultured cells, paraffin-embedded sections may be hybridized . The interphasic FISH may be extremely informative in tumor pathology even if the results are dependent on a good technical quality and adequate controls. Virology, 2000 Aug 15, 274(1), 17 - 25 Selective inhibition of nuclear steroid receptor function by a protein from a human tumorigenic poxvirus; Chen N et al.; The poxvirus molluscum contagiosum (MC) has a worldwide distribution and its prevalence is on the rise . Here we report that the MCV MC013L protein inhibits glucocorticoid and vitamin D, but not retinoid or estrogen, nuclear receptor transactivation . A direct interaction of MC013L with glucocorticoid and vitamin D receptor is supported by yeast two-hybrid, GST pull-down, and far Western blot analyses . Glucocorticoids act as potent inhibitors of keratinocyte proliferation, while vitamin D and retinoids promote and block terminal differentiation, respectively . Therefore, MC013L may promote efficient virus replication by blocking the differentiation of infected keratinocytes . MC013L may be the first member of a new class of poxvirus proteins that directly modulate nuclear receptor-mediated transcription . Genomics, 2000 Aug 1, 67(3), 268 - 72 A sequence-ready BAC clone contig of human chromosome 10p15 spanning the loss of heterozygosity region in glioma; Harada K et al.; Deletion of chromosome 10 is one of the most common chromosomal alterations in glioma . At 10p15, the telomeric region of the short arm of chromosome 10, loss of heterozygosity (LOH) has been frequently observed by microsatellite analysis, suggesting the presence of a tumor suppressor gene . We examined LOH in 34 gliomas on chromosome 10, and frequent LOH on 10p was detected on 10p15, in agreement with deletion mapping studies on chromosome 10 . We then constructed a bacterial artificial chromosome (BAC) clone contig covering the critical region, which spanned the interval between D10S249 and D10S533 on 10p15 . The map contained 68 BAC clones connected by 74 sequenced tag sites (STSs) and covered approximately 2.7 Mb, with one gap . A total of 74 STSs, including 6 microsatellite markers, 29 expressed sequenced tags (ESTs), and 39 BAC end STSs, were physically arranged . Twenty-eight ESTs were mapped in the interval between D10S249 and D10S559 (approximately 1200 kb), and another EST was mapped in the interval between D10S559 and D10S533 (approximately 1300 kb) . This sequence-ready BAC clone contig map will be a basic resource for high-quality sequencing and positional cloning of the putative tumor suppressor gene at 10p15 in glioma . Genomics, 2000 Aug 1, 67(3), 256 - 67 Four DAZ genes in two clusters found in the AZFc region of the human Y chromosome; Saxena R et al.; The DAZ genes are candidate fertility factors that lie within the human Y chromosome's AZFc region, whose deletion is a common cause of spermatogenic failure . The number of DAZ genes has been difficult to determine, in part because the nucleotide sequences of the DAZ genes are nearly identical . Here, fluorescence in situ hybridization and characterization of BAC clones revealed four full-length DAZ genes on the human Y chromosome . They exist in two clusters, each comprising an inverted pair of DAZ genes (3' <-- 5'::5' --> 3') . Analysis of genomic sequences and testicular transcripts suggested that three or four DAZ genes are translated . Each gene contains at least seven tandem copies of a previously described, 2.4-kb repeat unit that encodes 24 amino acids . In addition, two DAZ genes contain tandem copies of a 10.8-kb repeat unit that encodes the RNA-binding domain, which appears to be multimerized in some DAZ proteins . Combining our present results with previous studies, we can reconstruct several steps in the evolution of the DAZ genes on the Y chromosome . In the ancestral Y-chromosomal DAZ gene, amplification of both intragenic repeats began before the human and cynomolgus (Old World) monkey lineages diverged . During subsequent evolution, an inverted duplication of this modified gene occurred . Finally, the resulting two-gene cluster was duplicated, generating the two-cluster/four-gene arrangement found on modern human Y chromosomes . Mol Endocrinol, 2000 Aug, 14(8), 1175 - 86 Role of cyclophilin B in prolactin signal transduction and nuclear retrotranslocation; Rycyzyn MA et al.; The pleiotropic actions of PRL are necessary for mammary growth and differentiation and in vitro lymphoid proliferation . The proximal action of this ligand is mediated by its cell surface receptor via associated networks . PRL action, however, is also associated with the internalization and translocation of this hormone into the nucleus . To delineate the mechanism of this retrotranslocation, a yeast two-hybrid screen was performed and revealed an interaction between PRL and cyclophilin B (CypB) . CypB is a peptidyl prolyl isomerase (PPI) found in the endoplasmic reticulum, extracellular space, and nucleus . The interaction between CypB and PRL was subsequently confirmed in vitro and in vivo through the use of recombinant proteins and coimmunoprecipitation studies . The exogenous addition of CypB potentiated the 3H-thymidine incorporation of PRL-dependent cell lines up to 18-fold . CypB by itself was nonmitogenic and did not potentiate the action of GH or other interleukins . CypB did not alter the affinity of the PRL receptor (PRLr) for its ligand, or increase the phosphorylation of PRLr-associated Jak2 or Stat5a . The potentiation of PRL-action by CypB, however, was accompanied by a dramatic increase in the nuclear retrotranslocation of PRL . A CypB mutant, termed CypB-NT, was generated that lacked the wild-type N-terminal nuclear localization sequence . Although CypB-NT demonstrated levels of PRL binding and PPI activity equivalent to wild-type CypB, it was incapable of mediating the nuclear retrotranslocation of PRL or enhancing PRL-driven proliferation . These studies reveal CypB as an important chaperone facilitating the nuclear retrotransport and action of the lactogenic hormones. Neoplasia, 1999 Apr, 1(1), 16 - 22 Construction of a high-resolution physical map of the approximate 1-Mb region of human chromosome 7q31.1-q31.2 harboring a putative tumor suppressor gene; Zenklusen JC et al.; Reports of frequent loss of heterozygosity (LOH) of markers on human chromosome 7q in malignant myeloid disorders as well as breast, prostate, ovarian, colon, head and neck, gastric, pancreatic, and renal cell carcinomas suggest the presence of a tumor suppressor gene (TSG) . Functional assays have demonstrated that the introduction of an intact copy of human chromosome 7 (hchr7) can restore senescence to immortalized human fibroblast cell lines having LOH of markers within 7q31-q32 and can inhibit the tumorigenic phenotype of a murine squamous cell carcinoma cell line . To facilitate the cloning of the putative TSG, we have constructed a high-resolution physical map of this region of hchr7, specifically that encompassing the markers D7S522 and D7S677 within 7q31.1-q31.2 . By using a lower resolution yeast artificial chromosome-based map as a starting framework, we established complete clone coverage of the implicated critical region in bacterial-artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs) . The resulting BAC/PAC-based contig map has provided suitable clones for the systematic sequencing of the entire interval . In addition, we have already identified 29 clusters of overlapping expressed-sequence tags (ESTs) and 4 known genes contained within these clones . Together, the physical map reported here coupled with the evolving sequence and gene maps should hasten the identification of the putative TSG residing within this region of hchr7. J Cell Sci, 2000 Sep, 113 ( Pt 17), 2927 - 34 Do growth and cell division rates determine cell size in multicellular organisms? Coelho CM, Leevers SJ. Studies in yeast have provided some clues to how cell size might be determined in unicellular eukaryotes; yet little attention has been paid to this issue in multicellular organisms . Reproducible cell sizes might be achieved in the dividing cells of multicellular organisms by the coordination of growth with cell division . Recently, mutations in genes encoding homologues of components of the mammalian insulin/phosphoinositide 3-kinase signalling pathway have been shown to affect organ growth and cell size during Drosophila melanogaster imaginal disc development . The data suggest that signalling through this pathway alters cell size because it primarily affects the growth of these organs (i.e . their increase in mass) and does not have a proportional impact on cell division . These observations are in keeping with the hypothesis that growth and cell division are regulated independently, and that cell size is just a consequence of the rate at which tissues grow and the cells within them divide . However, signalling through this pathway can affect cell cycle phasing and at least influence cell division . These interactions may provide a means of coordinating growth and cell division, such that cells divide only when they are above a minimum size. Arch Biochem Biophys, 2000 Aug 15, 380(2), 228 - 36 A nuclear matrix-associated factor, SAF-B, interacts with specific isoforms of AUF1/hnRNP D; Arao Y et al.; One class of heterogeneous nuclear ribonucleoproteins (hnRNPs), AUF1/hnRNP D, consists of four isoform proteins (p45, p42, p40, and p37) which are generated by alternative splicing . The present study was therefore undertaken to clarify any isoform-specific differences in terms of their functions and nucleocytoplasmic localization . All isoforms primarily localized in the nucleus . However, heterokaryon analysis and a study using RNA polymerase II inhibitor revealed that p40/p37 exhibited a continuous shuttling between the nucleus and cytoplasm . Constant nuclear retention activity was mapped to the p45/p42-specific sequence at the C-terminal region, which is retained by alternative splicing . Using this domain as a probe, we performed a yeast two-hybrid screening and we found that scaffold attachment factor B (SAF-B), a nuclear matrix-associated protein, exhibits protein-protein interaction to this region . Colocalization of p45/p42 and SAF-B was observed as a speckle in the nucleus . Interestingly, p45/p42 isoforms appeared to act as a negative regulator in gene expression by forming a complex with SAF-B . Thus, the present study revealed that the isoform-specific functions of AUF1/hnRNP D are defined by intracellular shuttling capacity . Biochemistry, 2000 Aug 15, 39(32), 10001 - 10 Nonhydrolyzable diubiquitin analogues are inhibitors of ubiquitin conjugation and deconjugation; Yin L et al.; A series of nonhydrolyzable ubiquitin dimer analogues has been synthesized and evaluated as inhibitors of ubiquitin-dependent processes . Dimer analogues were synthesized by cross-linking ubiquitin containing a terminal cysteine (G76C) to ubiquitin containing cysteine at position 11 ((76-11)Ub(2)), 29 ((76-29)Ub(2)), 48 ((76-48)Ub(2)), or 63 ((76-63)Ub(2)) . A head-to-head dimer of cysteine G76C ((76-76)Ub(2)) served as a control . These analogues are mimics of the different chain linkages observed in natural polyubiquitin chains . All analogues showed weak inhibition toward the catalytic domain of UCH-L3 and a UBP pseudogene . In the absence of ubiquitin, isopeptidase T was inhibited only by the dimer linked through residue 29 . In the presence of 0.5 microM ubiquitin, isopeptidase T was inhibited by several of the dimer analogues, with the (76-29)Ub(2) dimer exhibiting a K(i) of 1.8 nM . However, USP14, the human homologue of yeast Ubp6, was not inhibited at the concentrations tested . Some analogues of ubiquitin dimer also acted as selective inhibitors of conjugation and deconjugation of ubiquitin catalyzed by reticulocyte fraction II . (76-76)Ub(2) and (76-11)Ub(2) did not inhibit the conjugation of ubiquitin, while (76-29)Ub(2), (76-48)Ub(2), and (76-63)Ub(2) were potent inhibitors of conjugation . This specificity is consistent with the known ability of cells to form K29-, K48-, and K63-linked polyubiquitin chains . While (76-11)Ub(2), (76-29)Ub(2), and (76-63)Ub(2) inhibited release of ubiquitin from a pool of total conjugates, (76-48)Ub(2) and (76-76)Ub(2) showed no significant inhibition . Isopeptidase T was shown to specifically disassemble two conjugates (assumed to be di- and triubiquitin with masses of 26 and 17 kDa) formed in the reticulocyte lysate system . This activity was inhibited differentially by all dimer analogues . The inhibitor selectivity for deconjugation of the 26 and 17 kDa conjugates was similar to that observed for isopeptidase T . The observations suggest that these two conjugated proteins of the reticulocyte lysate are specific substrates for isopeptidase T in lysates. Recenti Prog Med, 2000 Jul-Aug, 91(7-8), 396 - 401 {Histoplasmosis: not only a tropical disease}; Bregani ER et al.; Histoplasmosis is a fungal infection resulting from inhalation of spores from the fungus Histoplasma capsulatum; it is known to be endemic in various parts of the world, especially in North and Latin America, and can produce a spectrum of illness, from subclinical infection to progressive disseminated disease . The majority of infected persons have an asymptomatic, self-limiting illness . Clinical pneumonia occurs in those with exposure to a large number of infecting spores . Disseminated histoplasmosis usually occurs in immunosuppressed patients or in patients with chronic illness . Diagnosis is best made by visualization of yeast in tissue or by culture . In most cases, amphotericin B is the initial drug of choice, followed by one of the azoles for lifelong maintenance therapy . Itraconazole is the drug of choice for treatment of disseminated histoplasmosis in less severe cases, while fluconazole therapy for histoplasmosis is only moderately effective. J Cell Biol, 2000 Aug 7, 150(3), 681 - 8 The LIS1-related NUDF protein of Aspergillus nidulans interacts with the coiled-coil domain of the NUDE/RO11 protein; Efimov VP et al.; The nudF gene of the filamentous fungus Aspergillus nidulans acts in the cytoplasmic dynein/dynactin pathway and is required for distribution of nuclei . NUDF protein, the product of the nudF gene, displays 42% sequence identity with the human protein LIS1 required for neuronal migration . Haploinsufficiency of the LIS1 gene causes a malformation of the human brain known as lissencephaly . We screened for multicopy suppressors of a mutation in the nudF gene . The product of the nudE gene isolated in the screen, NUDE, is a homologue of the nuclear distribution protein RO11 of Neurospora crassa . The highly conserved NH(2)-terminal coiled-coil domain of the NUDE protein suffices for protein function when overexpressed . A similar coiled-coil domain is present in several putative human proteins and in the mitotic phosphoprotein 43 (MP43) of X . laevis . NUDF protein interacts with the Aspergillus NUDE coiled-coil in a yeast two-hybrid system, while human LIS1 interacts with the human homologue of the NUDE/RO11 coiled-coil and also the Xenopus MP43 coiled-coil . In addition, NUDF coprecipitates with an epitope-tagged NUDE . The fact that NUDF and LIS1 interact with the same protein domain strengthens the notion that these two proteins are functionally related. J Cell Biol, 2000 Aug 7, 150(3), 405 - 16 Identification and characterization of SA/Scc3p subunits in the Xenopus and human cohesin complexes; Losada A et al.; A multisubunit protein complex, termed cohesin, plays an essential role in sister chromatid cohesion in yeast and in Xenopus laevis cell-free extracts . We report here that two distinct cohesin complexes exist in Xenopus egg extracts . A 14S complex (x-cohesin(SA1)) contains XSMC1, XSMC3, XRAD21, and a newly identified subunit, XSA1 . In a second 12.5S complex (x-cohesin(SA2)), XSMC1, XSMC3, and XRAD21 associate with a different subunit, XSA2 . Both XSA1 and XSA2 belong to the SA family of mammalian proteins and exhibit similarity to Scc3p, a recently identified component of yeast cohesin . In Xenopus egg extracts, x-cohesin(SA1) is predominant, whereas x-cohesin(SA2) constitutes only a very minor population . Human cells have a similar pair of cohesin complexes, but the SA2-type is the dominant form in somatic tissue culture cells . Immunolocalization experiments suggest that chromatin association of cohesin(SA1) and cohesin(SA2) may be differentially regulated . Dissociation of x-cohesin(SA1) from chromatin correlates with phosphorylation of XSA1 in the cell-free extracts . Purified cdc2-cyclin B can phosphorylate XSA1 in vitro and reduce the ability of x-cohesin(SA1) to bind to DNA or chromatin . These results shed light on the mechanism by which sister chromatid cohesion is partially dissolved in early mitosis, far before the onset of anaphase, in vertebrate cells. J Biol Chem, 2000 Oct 20, 275(42), 32578 - 84 The effects of changing the site of activating phosphorylation in CDK2 from threonine to serine; Kaldis P et al.; Cyclin-dependent kinases (CDKs) that control cell cycle progression are regulated in many ways, including activating phosphorylation of a conserved threonine residue . This essential phosphorylation is carried out by the CDK-activating kinase (CAK) . Here we examine the effects of replacing this threonine residue in human CDK2 by serine . We found that cyclin A bound equally well to wild-type CDK2 (CDK2(Thr-160)) or to the mutant CDK2 (CDK2(Ser-160)) . In the absence of activating phosphorylation, CDK2(Ser-160)-cyclin A complexes were more active than wild-type CDK2(Thr-160)-cyclin A complexes . In contrast, following activating phosphorylation, CDK2(Ser-160)-cyclin A complexes were less active than phosphorylated CDK2(Thr-160)-cyclin A complexes, reflecting a much smaller effect of activating phosphorylation on CDK2(Ser-160) . The kinetic parameters for phosphorylating histone H1 were similar for mutant and wild-type CDK2, ruling out a general defect in catalytic activity . Interestingly, the CDK2(Ser-160) mutant was selectively defective in phosphorylating a peptide derived from the C-terminal domain of RNA polymerase II . CDK2(Ser-160) was efficiently phosphorylated by CAKs, both human p40(MO15)(CDK7)-cyclin H and budding yeast Cak1p . In fact, the k(cat) values for phosphorylation of CDK2(Ser-160) were significantly higher than for phosphorylation of CDK2(Thr-160), indicating that CDK2(Ser-160) is actually phosphorylated more efficiently than wild-type CDK2 . In contrast, dephosphorylation proceeded more slowly with CDK2(Ser-160) than with wild-type CDK2, either in HeLa cell extract or by purified PP2Cbeta . Combined with the more efficient phosphorylation of CDK2(Ser-160) by CAK, we suggest that one reason for the conservation of threonine as the site of activating phosphorylation may be to favor unphosphorylated CDKs following the degradation of cyclins. J Biol Chem, 2000 Nov 3, 275(44), 34131 - 9 ABC50 interacts with eukaryotic initiation factor 2 and associates with the ribosome in an ATP-dependent manner; Tyzack JK et al.; Eukaryotic initiation factor 2 (eIF2) plays a key role in the process of translation initiation and in its control . Here we demonstrate that highly purified mammalian eIF2 contains an additional polypeptide of apparent molecular mass of 110 kDa . This polypeptide co-purified with eIF2 through five different chromatography procedures . A cDNA clone encoding the polypeptide was isolated, and its sequence closely matched that of a protein previously termed ABC50, a member of the ATP-binding cassette (ABC) family of proteins . Antibodies to ABC50 co-immunoprecipitated eIF2 and vice versa, indicating that the two proteins interact . The presence of ABC50 had no effect upon the ability of eIF2 to bind GDP but markedly enhanced the association of methionyl-tRNA with the factor . Unlike the majority of ABC proteins, which are membrane-associated transporters, ABC50 associates with the ribosome and co-sediments in sucrose gradients with the 40 and 60 S ribosomal subunits . The association of ABC50 with ribosomal subunits was increased by ATP and decreased by ADP . ABC50 is related to GCN20 and eEF3, two yeast ABC proteins that are not membrane-associated transporters and are instead implicated in mRNA translation and/or its control . Thus, these data identify ABC50 as a third ABC protein with a likely function in mRNA translation, which associates with eIF2 and with ribosomes. Radiat Res, 2000 Aug, 154(2), 133 - 9 Influence of ATM function on interactions between telomeres and nuclear matrix; Pandita TK et al.; The ATM (ataxia telangiectasia mutated) gene product has been implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, and cell cycle control . The human ATM protein shows similarity to several yeast and mammalian proteins involved in meiotic recombination and cell cycle progression . Because of the homology of the human ATM gene to the TEL1 and rad3 genes of yeast, it has been suggested that mutations in ATM could lead to defective telomere maintenance . Recently, we have shown that the ATM gene product, which is defective in the cancer-prone disorder ataxia telangiectasia (AT), influences chromosome end associations and telomere length . A possible hypothesis explaining these results is that the defective telomere metabolism in AT cells is due to altered interactions between the telomeres and the nuclear matrix . These interactions were examined in nuclear matrix halos prior to and after irradiation . A difference was observed in the ratio of soluble and matrix-associated telomeric DNA between cells derived from AT and normal individuals . Treatment with ionizing radiation affected the ratio of soluble and matrix-associated telomeric DNA only in the AT cells . To test the hypothesis that the ATM gene product is involved in interactions between telomeres and the nuclear matrix, such interactions were examined in human cells expressing either a dominant-negative effect or complementation of the ATM gene . The phenotype of RKO colorectal tumor cells expressing ATM fragments containing a leucine zipper motif mimics the altered interactions of telomere and nuclear matrix seen in AT cells . Fibroblasts from AT individuals transfected with a wild-type ATM gene had corrected telomere-nuclear matrix interactions . In experiments designed to determine whether there is a link between the altered telomere-nuclear matrix interactions and defective telomere movement and clustering, a significant difference was observed in the ratio of soluble compared to matrix-associated telomeric DNA sequences in meiocytes of Atm(-/-) and control mice . These results suggest that the ATM gene influences the interactions between telomeres and the nuclear matrix and that alterations in telomere chromatin could be at least partly responsible for the pleiotropic phenotypes of the ATM gene . This paper summarizes our recent publications on the influence of inactivation of ATM on the interaction of telomeres with nuclear matrix in somatic and germ cells. Virus Res, 2000 Jun, 68(1), 87 - 92 Dissociation of DDB1-binding and transactivation properties of the hepatitis B virus X protein; Wentz MJ et al.; The hepatitis B virus (HBV) X protein (HBx) is a transactivator encoded by mammalian hepadnaviruses, and is thought to stimulate transcription by interacting with one or more host cell factors . Numerous cellular proteins have been reported to interact with HBx including a component of the nucleotide excision repair complex called ultraviolet damaged DNA binding (UV-DDB, or DDB1) protein . Recent studies have identified a role for DDB1 in transcription, raising the possibility that HBx may acquire its broad transcriptional properties by interacting with DDB1 . A panel of HBx mutant proteins, some of which no longer bind to DDB1, was used to test this hypothesis . Plasmid DNAs encoding HBx wildtype and mutant derivatives were transfected into HepG2 cells, and their ability to transactivate a cotransfected reporter plasmid tested . Results from the transactivation assays in HepG2 cells were then compared with data obtained from HBx-DDB1 binding studies performed in yeast . Several HBx mutant proteins unable to bind DDB1 remained competent for transactivation, indicating that HBx binding to DDB1 is not required for HBx transactivation of the ETS1 promoter . It remains possible that a subset of HBx transactivation function targets an as yet undefined DDB1-specific pathway. FEBS Lett, 2000 Aug 4, 478(3), 253 - 9 PKB/Akt interacts with inosine-5' monophosphate dehydrogenase through its pleckstrin homology domain; Ingley E et al.; The pleckstrin homology (PH) domain of the protooncogenic serine/threonine protein kinase PKB/Akt can bind phosphoinositides . A yeast-based two-hybrid system was employed which identified inosine-5' monophosphate dehydrogenase (IMPDH) type II as specifically interacting with PKB/Akts PH domain . IMPDH catalyzes the rate-limiting step of de novo guanosine-triphosphate (GTP) biosynthesis . Using purified fusion proteins, PKB/Akts PH domain and IMPDH associated in vitro and this association moderately activated IMPDH . Purified PKB/Akt also associated with IMPDH in vitro . We could specifically pull-down PKB/Akt or IMPDH from mammalian cell lysates using glutathione-S-transferase (GST)-IMPDH or GST-PH domain fusion proteins, respectively . Additionally, PKB/Akt and IMPDH could be co-immunoprecipitated from COS cell lysates and active PKB/Akt could phosphorylate IMPDH in vitro . These results implicate PKB/Akt in the regulation of GTP biosynthesis through its interaction with IMPDH, which is involved in providing the GTP pool used by signal transducing G-proteins. FEBS Lett, 2000 Aug 4, 478(3), 227 - 32 Possible His to Asp phosphorelay signaling in an Arabidopsis two-component system; Urao T et al.; We have so far cloned a cDNA encoding a hybrid-type histidine kinase (ATHK1), three cDNAs encoding phosphorelay intermediates (ATHP1-3), and four cDNAs encoding response regulators (ATRR1-4) from Arabidopsis thaliana . To determine which molecules constitute a His to Asp phosphorelay pathway, we examined protein-protein interactions between them using a pairwise yeast two-hybrid analysis, as an initial step . We detected a specific interaction between ATHK1 and ATHP1 . We further examined protein-protein interactions between ATHP1-3 and other histidine kinases . We detected interactions between ETR1 and all ATHPs, and between CKI1 and ATHP1 or ATHP2 . Interestingly, ERS1 could not interact with any ATHPs . We also examined protein-protein interactions between ATHP1-3 and ATRR1-4 . The results indicated that ATHP2 could interact with ATRR4, and that ATHP3 could interact with ATRR1 or ATRR4 . However, ATHP1 could not interact with any ATRRs . On the basis of these results, we discuss the possible phosphorelay networks in an Arabidopsis two-component system. J Biol Chem, 2000 Jul 28, 275(30), 23139 - 45 Truncated form of importin alpha identified in breast cancer cell inhibits nuclear import of p53; Kim IS et al.; Disruption of the function of tumor suppressor proteins occasionally can be dependent on their subcellular localization . In about 40% of the breast cancer tissues, p53 is found in the cytoplasm as opposed to the nucleus, where it resides in normal breast cells . This means that the regulation of subcellular location of p53 is an important mechanism in controlling its function . The transport factors required for the nuclear export of p53 and the mechanisms of their nuclear export have been extensively characterized . However, little is known about the mechanism of nuclear import of p53 . p53 contains putative nuclear localization signals (NLSs) which would interact with a nuclear transport factor, importin alpha . In this report we demonstrate that importin alpha binds to NLSI in p53 and mediates the nuclear import of p53 . Reverse transcriptase-polymerase chain reaction and sequencing analyses showed that a truncated importin alpha deleted the region encoding the putative NLS-binding domain of p53, suggesting that it could not bind to NLSs of p53 proteins . Binding of importin alpha to p53 was confirmed by using yeast two-hybrid assay . When expressed in CHO-K1 cells, the truncated importin alpha predominantly localized to the cytoplasm . In truncated importin alpha expressing cells, p53 preferentially localized to cytoplasmic sites as well . A significant increase in the p21(waf1/cip1) mRNA level and induction of apoptosis were also observed in importin alpha overexpressing cells . These results strongly suggest that importin alpha functions as a component of the NLS receptor for p53 and mediates nuclear import of p53. Plant Cell Physiol, 2000 May, 41(5), 571 - 82 Effects of myosin ATPase inhibitor 2,3-butanedione 2-monoxime on distributions of myosins, F-actin, microtubules, and cortical endoplasmic reticulum in maize root apices; Samaj J et al.; 2,3-Butanedione 2-monoxime (BDM) is a general inhibitor of myosin ATPases of eukaryotic cells, and its effects on animal and yeast cells are well described . Using immunofluorescence and electron microscopy, we have analyzed the impacts of BDM on distributions of plant myosins, actin filaments (AFs), microtubules (MTs), and cortical endoplasmic reticulum (ER) elements in various cell types of maize root apices . Treatment of growing maize roots with BDM altered the typical distribution patterns of unconventional plant myosin VIII and of putative maize homologue(s) of myosin II . This pharmacological agent also induced a broad range of impacts on AFs and on cortical ER elements associated with plasmodesmata and pit fields . BDM-mediated effects on the actomyosin cytoskeleton were especially pronounced in cells of the root transition zone . Additionally, BDM elicited distinct reactions in the MT cytoskeleton; endoplasmic MTs vanished in all cells of the transition zone and cortical MTs assembled in increased amounts preferentially at plasmodesmata and pit-fields . Our data indicate that AFs and MTs interact together via BDM-sensitive plant myosins, which can be considered as putative integrators of the plant cytoskeleton . Morphometric analysis revealed that cell growth was prominently inhibited in the transition zone and the apical part, but not the central part, of the elongation region . Obviously, myosin-based contractility of the actin cytoskeleton is essential for the developmental progression of root cells through the transition zone. Chromosoma, 2000 Jun, 109(3), 190 - 6 A dicentric chromosome of Drosophila melanogaster showing alternate centromere inactivation; Agudo M et al.; Dicentric chromosomes are rarely found, because they interfere with normal cell division causing chromosome instability . By in situ hybridization of region-specific heterochromatic yeast artificial chromosomes we have found that the artificially generated C(1)A chromosome of Drosophila melanogaster has two potential centromeres: one carries all the sequences of the centromere of the Y chromosome and the other carries only a part of the Y centromeric region that is rich in telomere-related sequences . Immunostaining with anti-Bub1 (a kinetochore-specific marker) shows that, in spite of the differences in sequence, both centromeres can be active although as a rule only one at a time . In a small fraction of the chromosomes centromere inactivation is incomplete, giving rise to true dicentric chromosomes . The centromere inactivation is clonally inherited, providing a new example of epigenetic chromosome imprinting and the possibility of genetically dissecting this process . The involvement of telomere-related sequences in centromere function is discussed. Nucleic Acids Res . 2000 Aug 15;28(16):E77. A novel method for constructing gene-targeting vectors; Akiyama K et al.; We developed a simple and rapid method for constructing knockout vectors using inverse-PCR (IPCR) . The method consists of three steps: (i) digestion of a target bacterial artificial chromosome with several restriction enzymes (six-base cutters) followed by self-ligation; (ii) IPCR using circular DNAs as templates and two primers which are oriented in opposite directions; and (iii) cloning into a vector containing a positive selection marker, which results in a typical replacement knockout vector . We successfully targeted three mouse genes including the HPRT gene using this method . Compared with the conventional method, this method requires much less time (no more than 3 weeks) . Notably, this method requires only small amounts of sequence information (several hundred base pairs such as is available from expressed sequence tags) and can be extended to a systematic mass production of targeting vectors applicable to many organisms, including yeast. Trop Med Int Health, 2000 Jun, 5(6), 453 - 8 Microsporidia and Candida spores: their discrimination by Calcofluor, trichrome-blue and methylene-blue combination staining; Schottelius J et al.; Faeces of immunocompromised patients are often contaminated with the chitin-containing spores of microsporidia and Candida, which exclude the use of the chitin-specific fluorescent brightener Calcofluor white M2R for the identification of microsporidian spores . We developed a combination staining of Calcofluor white M2R with modified trichrome-blue staining and subsequent methylene-blue incubation which permits discrimination between these two types of spores . As a basis for diagnosis, a difference in the fluorescence pattern (365-440 nm) is combined with a difference in the light microscopic staining pattern . Under fluorescence conditions microsporidia spores have a spotted, brilliant white Calcofluor fluorescence and can easily be identified, while Candida spores show a reddish purple colour . Under the light microscope microsporidian spores show a light red colour with nonstained vacuole spots or strips in contrast to the yeast spores with their red-brown colour . This combination technique offers a highly specific means for the diagnosis of microsporidia spores in faeces. Plant J, 2000 Aug, 23(3), 423 - 30 FLP-mediated recombination for use in hybrid plant production; Luo H et al.; We have studied the feasibility in Arabidopsis of using a site-specific recombination system FLP/FRT, from the 2 microm plasmid of yeast, for making plant hybrids . Initially, Arabidopsis plants expressing the FLP site-specific recombinase were crossed with plants transformed with a vector containing kanamycin-resistance gene (npt) flanked by FRT sites, which also served to separate the CaMV35S promoter from a promoterless gusA . Hybrid progeny were tested for excision of the npt gene and the positioning of 35S promoter proximal to gusA . GUS activity was observed in the progeny of all crosses, but not in the progeny derived from the self-pollinated homozygous parents . We then induced male sterility in Arabidopsis plants using the antisense expression of a pollen- and tapetum-specific gene, bcp1, flanked by FRT sites . Upon cross-pollination of flowers on the same male-sterile plants with pollen from FLP-containing plants, viable seeds were produced and the progeny hybrid plants developed normally . Molecular analyses revealed that the antisense expression cassette of bcp1 had been excised in these plants . These results show for the first time that a site-specific recombinase can be used to restore fertility in male-sterile plants, providing an alternative method for the production of hybrid seeds and plants. Plant J, 2000 Jul, 23(1), 73 - 83 Cell cycle function of a Medicago sativa A2-type cyclin interacting with a PSTAIRE-type cyclin-dependent kinase and a retinoblastoma protein; Roudier F et al.; In plants multiple A-type cyclins with distinct expression patterns have been isolated and classified into three subgroups (A1-A3), while in animal somatic cells a single type of cyclin A is required for cell-cycle regulation from the S to M phases . We studied the function of an A2-type cyclin from Medicago sativa (Medsa;cycA2) which, in contrast to animal and most plant A-type cyclins, was expressed in all phases of the cell cycle . Using synchronized alfalfa cell cultures and anti-Medsa;CycA2 polyclonal antibodies, we showed that while the mRNA level increased steadily from the late G1 to the G2-M phase, the protein level after a rapid increase in S-phase reached a plateau during the G2 phase . In the yeast two-hybrid system, the Medsa;CycA2 protein interacted with the PSTAIRE-motif-containing cyclin-dependent kinase Cdc2MsA and with the maize retinoblastoma protein . Unexpectedly, the CycA2-associated kinase activity was biphasic: a first activity peak occurred in the S phase while the major one occurred during the G2/M transition, with no apparent dependence upon the actual levels of the Medsa;CycA2 and Cdc2MsA proteins . Immunohistological localization of the cyclin A2 protein by immunofluorescence and immunogold labelling revealed the presence of Medsa;CycA2 in the nucleus of the interphase and prophase cells, while it was undetectable thereafter during mitosis . Together these data suggest that Medsa;CycA2 plays a role both in the S phase and at the G2/M transition. FASEB J, 2000 Aug, 14(11), 1539 - 47 MSH4 acts in conjunction with MLH1 during mammalian meiosis; Santucci-Darmanin S et al.; MSH4 is a meiosis-specific MutS homolog . In yeast, it is required for reciprocal recombination and proper segregation of homologous chromosomes at meiosis I . MLH1 (MutL homolog 1) facilitates both mismatch repair and crossing over during meiosis in yeast . Germ-line mutations in the MLH1 human gene are responsible for hereditary nonpolyposis cancer, but the analysis of MLH1-deficient mice has revealed that MLH1 is also required for reciprocal recombination in mammals . Here we show that hMSH4 interacts with hMLH1 . The two proteins are coimmunoprecipitated regardless of the presence of DNA or ATP, suggesting that the interaction does not require the binding of MSH4 to DNA . The domain of hMSH4 responsible for the interaction is in the amino-terminal part of the protein whereas the region that contains the ATP binding site and helix-turn-helix motif does not bind to hMLH1 . Immunolocalization analysis shows that MSH4 is present at sites along the synaptonemal complex as soon as homologous chromosomes synapse . The number of MSH4 foci decreases gradually as pachynema progresses . During this transition, MLH1 foci begin to appear and colocalize with MSH4 . These results suggest that MSH4 is first required for chromosome synapsis and that this MutS homologue is involved later with MLH1 in meiotic reciprocal recombination. Anticancer Res, 2000 May-Jun, 20(3A), 1723 - 6 Synthesis of 4-(acylaminomethyl)benzamides and their evaluation as potential anticancer agents; Schlitzer M et al.; 4-(acylaminomethyl)benzamides were prepared in two steps from 4-aminomethylbenzoic acid and assayed in the NCl's primary anti-cancer screen . Eight out of 34 compounds showed interesting antiproliferative activity . From these compounds, three were selected for further in vivo testing . In addition, all the compounds were tested against farnesyltransferase and the cell cycle regulating enzymes cdc2 kinase and cdc25 phosphatase . The compounds proved inactive in these assays, as were some selected compounds in an assay searching for possible interference with the ras/raf interaction in a yeast two-hybrid system. J Biol Chem, 2000 Nov 3, 275(44), 34451 - 8 Inhibition of Akt and its anti-apoptotic activities by tumor necrosis factor-induced protein kinase C-related kinase 2 (PRK2) cleavage; Koh H et al.; Akt is stimulated by several growth factors and has a major anti-apoptotic role in the cell . Therefore, we hypothesized that a pathway leading to the inhibition of Akt might be utilized in the process of apoptosis . Accordingly, we used a yeast two-hybrid screening assay to identify the proteins that interact with and possibly inhibit Akt . We found that the C-terminal region of protein kinase C-related kinase 2 (PRK2), containing amino acids 862 to 908, specifically binds to Akt in yeast and mammalian cells . During early stages of apoptosis, the C-terminal region of PRK2 is cleaved from the inhibitory N-terminal region and can bind Akt . The protein-protein interaction between Akt and the PRK2 C-terminal region specifically down-modulates the protein kinase activities of Akt by inhibiting phosphorylation at threonine 308 and serine 473 of Akt . This inhibition of Akt leads to the inhibition of the downstream signaling of Akt in vivo . The PRK2 C-terminal fragment strongly inhibits the Akt-mediated phosphorylation of BAD, a pro-apoptotic Bcl-2 family protein, and blocks the anti-apoptotic activities of Akt in vivo . These results provide direct evidence that the products of protein cleavage during apoptosis inhibit pro-survival signalings, leading to the amplification of pro-apoptotic signalings in the cell. Biochem J, 2000 Aug 15, 350 Pt 1, 19 - 29 RGS14 is a novel Rap effector that preferentially regulates the GTPase activity of galphao; Traver S et al.; In an attempt to elucidate the physiological function(s) of the Ras-related Rap proteins, we used the yeast two-hybrid system and isolated a cDNA encoding a protein that interacts with both Rap1 and Rap2, but not with Ras; the use of Rap2 mutants showed that this interaction is characteristic of a potential Rap effector . This protein was identified as RGS14, a member of the recently discovered family of RGS ('regulators of G-protein signalling') proteins that stimulate the GTPase activity of the GTP-binding alpha subunit of heterotrimeric G-proteins (Galpha) . Deletion analysis, as well as in vitro binding experiments, revealed that RGS14 binds Rap proteins through a domain distinct from that carrying the RGS identity, and that this domain shares sequence identity with the Ras/Rap binding domain of B-Raf and Raf-1 kinases . RGS14 is distinguished from other RGS proteins by its marked preference for Galpha(o) over other Galpha subunits: RGS14 binds preferentially to Galpha(o) in isolated brain membranes, and also interacts preferentially with Galpha(o) (as compared with Galpha(i1)) to stimulate its GTPase activity . In adult mice, RGS14 expression is restricted to spleen and brain . In situ hybridization studies showed that it is highly expressed only in certain areas of mouse brain (such as the CA1 and CA2 regions of the hippocampus), and that this pattern closely resembles that of Rap2, but not Rap1, expression . Double in situ hybridization experiments revealed that certain cells in the hippocampus express both RGS14 and Galpha(o), as well as both RGS14 and Rap2, showing that the interaction of RGS14 with Galpha(o) and Rap2 is physiologically possible . Taken together, these results suggest that RGS14 could constitute a bridging molecule that allows cross-regulation of signalling pathways downstream from G-protein-coupled receptors involving heterotrimeric proteins of the G(i/o) family and those involving the Ras-related GTPase Rap2. J Mol Biol, 2000 Aug 4, 301(1), 1 - 9 Characterisation of the newly identified human Ump1 homologue POMP and analysis of LMP7(beta 5i) incorporation into 20 S proteasomes; Witt E et al.; Biogenesis of mammalian 20 S proteasomes occurs via precursor complexes containing alpha and unprocessed beta subunits . A human homologue of the yeast proteasome maturation factor Ump1 was identified in 2D gels of 16 S precursor preparations and designated as POMP (proteasome maturation protein) . We show that POMP is detected only in precursor fractions and not in fractions containing mature 20 S proteasome . Northern blot experiments revealed that expression of POMP is induced after treatment with interferon gamma . To analyse the role of the beta 5 propeptide for proper maturation and incorporation of the beta 5 subunit into the complex, human T2 cells, which highly express derivatives of the beta 5i subunit (LMP7), were studied . In contrast to yeast, the presence of the beta 5 propeptide is not essential for incorporation of LMP7 into the proteasome complex . Mutated LMP7 subunits either carrying the prosequence of beta 2i (LMP2) or containing a mutation in the active threonine site are incorporated like wild-type LMP7, while a LMP7 derivative lacking the prosequence completely is incorporated to a lesser extent . Although the absence of the prosequence does not affect incorporation of LMP7, its deletion leads to delayed proteasome maturation and thereby to an accumulation of precursor complexes . As a result of the precursor accumulation, an increased amount of the POMP protein can be detected in these cells . Curr Opin Nephrol Hypertens, 2000 Jul, 9(4), 385 - 94 The pathogenesis of autosomal dominant polycystic kidney disease: an update; Somlo S et al.; The identification of PKD1 and PKD2, the two major genes responsible for autosomal dominant polycystic kidney disease, are the seminal discoveries upon which much of the current investigation into the pathogenesis of this common heritable disease is based . A major mechanistic insight was achieved with the discovery that autosomal dominant polycystic kidney disease occurs by a two-hit mechanism requiring somatic inactivation of the normal allele in individual polarized epithelial cells . Most recent advances are focused on the function of the respective protein products, polycystin-1 and polycystin-2 . Indirect evidence supports an interaction between polycystin-1 and -2, albeit it is unlikely that they work in concert in all tissues and at all times . They associate in yeast two hybrid and cotransfection assays and there is a striking similarity in the renal and pancreatic cystic phenotypes of Pkd2-/- and Pkd1del34/del34 mice . Also, the respective homologues of both proteins are expressed in the same sensory neuronal cells in the nematode and the human disease phenotypes remain completely overlapping with the major difference being in relative severity . Mounting evidence supports the hypothesis that polycystin-1 is a cell surface receptor . A close homologue in the sea urchin sperm mediates the acrosome reaction in response to contact with egg-jelly, the nematode homologue functions in mechano- or chemosensation, and the solution structure of the repeated extracellular polycystic kidney disease domains reveals a beta-sandwich fold commonly found in surface receptor molecules . Indirect evidence also supports the initial hypothesis that polycystin-2 is a calcium channel subunit . Several closely related homologues retain the calcium channel signature motif but differ in their predicted interaction domains, and one of these homologues has been shown to be a calcium regulated cation channel . Several important distinctions in polcystin-1 and -2 function have also been discovered . Polycystin-2 has a role in cardiac development that polcystin-1 does not . High level polycystin-2 expression in renal epithelial cells coincides with maturation and elongation of tubules and, unlike polycystin-1, persists into adulthood . In cells in tissue culture, polycystin-2 is expressed exclusively in the endoplasmic reticulum whilst the cellular expression of polycystin-1 remains unknown . Overall, the difficult task of understanding the autosomal dominant polycystic disease process is proceeding apace. J Vector Ecol, 2000 Jun, 25(1), 98 - 101 The effects of flea egg consumption on larval cat flea (Siphonaptera: Pulicidae) development; Lawrence W et al.; Cat flea larvae feeding on the feces of adult fleas that were maintained on cats and were provided with frozen flea eggs ad libitum each consumed an average of 21.7 +/- 3.9 eggs and developed rapidly with 100% adult emergence . In contrast, 93.4% of larvae held individually and provided with only flea feces did not survive to the adult stage . Developing larvae consumed eggs in the presence of yeast and rearing diet . In a second experiment, larvae provided with flea feces and eggs and maintained at 55% RH consumed 26.9 +/- 2.7 eggs per larva, compared to larvae maintained at 75% RH that consumed 20.4 +/- 1.9 eggs per larva. Phytother Res, 2000 Aug, 14(5), 329 - 32 Detection of antifungal activity in Portulaca oleracea by a single-cell bioassay system; Oh KB et al.; The antifungal activity of Portulaca oleracea extracts against hyphal growth of various fungi was evaluated in real time using an automatic single-cell bioassay system . Target organisms were the filamentous fungi Aspergillus and Trichophyton and the yeast Candida . A colony of test fungi was in contact with the assay medium, or assay medium containing plant extract, in sequence . The antifungal activity of each fraction of P . oleracea was evaluated based on the dynamic hyphal growth response curves of test fungi . A crude sample obtained by EtOAc extract showed a specific and marked activity against dermatophytes of the genera Trichophyton . Gene, 2000 Jul 25, 253(1), 87 - 93 Isolation and characterization of a novel PDGF-induced human gene; Nelson SA et al.; Using a differential display RT-PCR strategy to identify novel growth-factor-induced transcripts, we cloned and characterized the human homolog of yeast NOP5/NOP58, whose gene product has been implicated in the execution of early pre-rRNA processing steps . Human NOP5 cDNA was isolated from an M426 fibroblast cDNA library . Determination of the cDNA nucleotide sequence revealed an open reading frame of 1587 nucleotides encoding a predicted gene product of 529 amino acids and mass of 59554Da . The yeast and human NOP5 gene products were found to share 63% homology and 46% identity . NOP5 mRNA was induced within 2h of platelet-derived growth factor (PDGF) treatment of human M426 fibroblasts . Pretreatment with cycloheximide enhanced, while actinomycin blocked induction of the NOP5 transcript . In vitro translational analysis of the cDNA revealed a 60kDa species, consistent with the predicted molecular weight of the gene product . Ubiquitous, but differential NOP5 mRNA expression was revealed after Northern blot analysis of total RNA from several human tissues . Moreover, NOP5 mRNA expression was also demonstrated in cell lines of fibroblast, epithelial, and myeloid origin . A highly charged carboxy terminal domain and consensus phosphorylation sites were identified . The presence of potential regulatory elements, together with growth factor induction and widespread expression is consistent with the hypothesis that the NOP5 gene product may play a role in fundamental cellular growth processes. Gene, 2000 Jul 25, 253(1), 19 - 29 Characterization of the structure and expression of a highly conserved ribosomal protein gene, L9, from pea; Moran DL; The eukaryotic ribosomal protein (RP) L9 is highly conserved in nature, and its gene is expressed to high levels in the actively growing tissues of pea . The transcriptional activity of the gene is highest in root, cambial and shoot meristems and immature tissues of the plant . Promoter deletion analysis using constructs employing the reporter gene gus were stably transferred into tobacco and revealed that the fully functional promoter is found in the first 316bp upstream from the start codon . Transgenic pea plants carrying one of these constructs show that translational efficiency mirrors gene transcription; gene expression appears to be developmentally regulated at the level of transcription . The coding region of the gene shares 80% amino acid homology with Arabidopsis and 76% homology with rice . Comparisons of the gene structure to that of the human, fruit fly, yeast, and Arabidopsis homologues reveal a close relationship in both promoter structure and intron insertion sites with the Arabidopsis gene . A nucleotide sequence alignment of the pea gene with other plant RP genes revealed that a sequence, -TTAGGGTTTT-, was commonly found in the forward and/or the inverted orientation at or near the TATA boxes of the promoters of these genes and may have a role in regulating the coordinate production of the RP genes in plants. J Biol Chem, 2000 Oct 27, 275(43), 33567 - 73 The basic helix-loop-helix transcription factors dHAND and eHAND exhibit dimerization characteristics that suggest complex regulation of function; Firulli BA et al.; dHAND and eHAND are basic helix-loop-helix (bHLH) transcription factors expressed during embryogenesis and are required for the proper development of cardiac and extraembryonic tissues . HAND genes, like the myogenic bHLH genes, are classified as class B bHLH genes, which are expressed in a tissue-restricted pattern and function by forming heterodimers with class A bHLH proteins . Myogenic bHLH genes are shown not to form homodimers efficiently, suggesting that their activity is dependent on their E-protein partners . To identify HIPs (HAND-interacting proteins) that regulate the activity of the HAND genes, we screened an 9.5-10.5-day-old mouse embryonic yeast two-hybrid library with eHAND . Several HIPs held high sequence identity to eHAND, indicating that eHAND could form and function as a homodimer . Based on the high degree of amino acid identity between eHAND and dHAND, it is possible that dHAND could also form homodimers and heterodimers with eHAND . We show using yeast and mammalian two-hybrid assays as well as biochemical pull-down assays that eHAND and dHAND are capable of forming both HAND homo- and heterodimers in vivo . To investigate whether HAND genes form heterodimers with other biologically relevant bHLH proteins, we tested and show HAND heterodimerization with the recently identified Hairy-related transcription factors, HRT1-3 . This finding is exciting, because both HRT and HAND genes are coexpressed in the developing heart and limb and both have been implicated in establishing tissue boundaries and pattern formation . Moreover, competition gel shift analysis demonstrates that dHAND and eHAND can negatively regulate the DNA bindi