Jpn J Cancer Res, 1998 Jul, 89(7), 696 - 702 Significant correlation of nitric oxide synthase activity and p53 gene mutation in stage I lung adenocarcinoma; Fujimoto H et al.; Nitric oxide (NO) and its derivatives can directly cause DNA damage and mutation in vitro and may play a role in the multistage carcinogenic process . It has been reported that NO induces mutation in the p53 tumor suppressor gene; we therefore analyzed the relationship between NO synthase (NOS) activity and p53 gene status in early-stage lung adenocarcinoma . Surgical samples were classified into two categories: 14 lung adenocarcinomas with high NOS activity (>25 pmol/min/g tissue, category A), and 16 with low NOS activity (<25 pmol/min/g tissue, category B) . A yeast functional assay for p53 mutations disclosed a red colony that corresponded to a mutation in the p53 gene in 8 cases (57.1%) in category A and 3 cases (18.8%) in category B, the frequency being significantly higher in the former (P<0.05) . A p53 DNA sequence analysis revealed that 5 of the 8 p53 mutation-positive samples in category A had a G:C-to-T:A transversion, which is reported to be a major target of NO . The mechanism of carcinogenesis of adenocarcinoma is not fully understood, but these results suggest that an excess of endogenously formed NO may induce a p53 gene mutation containing mainly G:C-to-T:A transversion in the early stage of lung adenocarcinoma . Our results suggest that NO has potential mutagenic and carcinogenic activity, and may play important roles in human lung adenocarcinoma. Plant Mol Biol, 1998 Sep, 38(1-2), 145 - 62 The nuclear pore complex; Heese-Peck A et al.; The nuclear pore complex is the largest supramolecular complex that assembles in the eukaryotic cell . This structure is highly dynamic and must disassemble prior to mitosis and reassemble after the event . The directed movement of macromolecules into and out of the nucleus occurs through the nuclear pore complex, a potentially regulatory point for translocation . Using biochemical and genetic approaches, several nuclear pore complex proteins from yeast and vertebrates have been well characterized . Although very little is known about plant nuclear pore proteins, research is providing new information that indicates that plant nuclear pore complexes may have some unique features. Plant Mol Biol, 1998 Sep, 38(1-2), 127 - 44 Sorting of proteins to vacuoles in plant cells; Neuhaus JM et al.; An individual plant cell may contain at least two functionally and structurally distinct types of vacuoles: protein storage vacuoles and lytic vacuoles . Presumably a cell that stores proteins in vacuoles must maintain these separate compartments to prevent exposure of the storage proteins to an acidified environment with active hydrolytic enzymes where they would be degraded . Thus, the organization of the secretory pathway in plant cells, which includes the vacuoles, has a fascinating complexity not anticipated from the extensive genetic and biochemical studies of the secretory pathway in yeast . Plant cells must generate the membranes to form two separate types of tonoplast, maintain them as separate organelles, and direct soluble proteins from the secretory flow specifically to one or the other via separate vesicular pathways . Individual soluble and membrane proteins must be recognized and sorted into one or the other pathway by distinct, specific mechanisms . Here we review the emerging picture of how separate plant vacuoles are organized structurally and how proteins are recognized and sorted to each type. Plant Mol Biol, 1998 Sep, 38(1-2), 49 - 76 The molecular characterization of transport vesicles; Robinson DG et al.; Secretion, endocytosis and transport to the lytic compartment are fundamental, highly coordinated features of the eukaryotic cell . These intracellular transport processes are facilitated by vesicles, many of which are small (100 nm or less in diameter) and 'coated' on their cytoplasmic surface . Research into the structure of the coat proteins and how they interact with the components of the vesicle membrane to ensure the selective packaging of the cargo molecules and their correct targeting, has been quite extensive in mammalian and yeast cell biology . By contrast, our knowledge of the corresponding types of transport vesicles in plant cells is limited . Nevertheless, the available data indicate that a considerable homology between plant and non-plant coat polypeptides exists, and it is also suggestive of a certain similarity in the mechanisms underlying targeting in all eukaryotes . In this article we shall concentrate on three major types of transport vesicles: clathrin-coated vesicles, COP-coated vesicles, and 'dense' vesicles, the latter of which are responsible for the transport of vacuolar storage proteins in maturing legume cotyledons . For each we will summarize the current literature on animal and yeast cells, and then present the relevant data derived from work on plant cells . In addition, we briefly review the evidence in support of the 'SNARE' hypothesis, which explains how vesicles find and fuse with their target membrane. Plant Mol Biol, 1998 Sep, 38(1-2), 1 - 29 The endoplasmic reticulum of plant cells and its role in protein maturation and biogenesis of oil bodies; Galili G et al.; The endoplasmic reticulum (ER) is the port of entry of proteins into the endomembrane system, and it is also involved in lipid biosynthesis and storage . This organelle contains a number of soluble and membrane-associated enzymes and molecular chaperones, which assist the folding and maturation of proteins and the deposition of lipid storage compounds . The regulation of translocation of proteins into the ER and their subsequent maturation within the organelle have been studied in detail in mammalian and yeast cells, and more recently also in plants . These studies showed that in general the functions of the ER in protein synthesis and maturation have been highly conserved between the different organisms . Yet, the ER of plants possesses some additional functions not found in mammalian and yeast cells . This compartment is involved in cell to cell communication via the plasmodesmata, and, in specialized cells, it serves as a storage site for proteins . The plant ER is also equipped with enzymes and structural proteins which are involved in the process of oil body biogenesis and lipid storage . In this review we discuss the components of the plant ER and their function in protein maturation and biogenesis of oil bodies . Due to the large number of cited papers, we were not able to cite all individual references and in many cases we refer the readers to reviews and references therein . We apologize to the authors whose references are not cited. FEBS Lett, 1998 Aug 14, 433(1-2), 63 - 7 Identification of a novel human homolog of the Drosophila dlg, P-dlg, specifically expressed in the gland tissues and interacting with p55; Nakamura H et al.; We have identified a novel human homolog of the Drosophila dlg tumor suppressor gene, termed P-dlg, which has been mapped at chromosome 10q23 . Unlike other human dlg homologs, P-dlg is expressed in placenta and various gland tissues but not in brain . The P-dlg protein is localized at the plasma membrane and cytoplasm, and it is expressed in the gland epithelial cells in normal prostate tissue but not in prostate cancer cell lines . Furthermore, we identified interaction between P-dlg and p55 palmitoylated membrane protein by yeast two-hybrid screening . These findings suggest that P-dlg forms a complex with p55 at the plasma membrane and plays roles in maintaining the structure of epithelial cells and transmitting extracellular signals to the membrane and cytoskeleton, which may negatively regulate cell proliferation. J Biol Chem, 1998 Sep 25, 273(39), 25209 - 15 A novel nuclear receptor heterodimerization pathway mediated by orphan receptors TR2 and TR4; Lee CH et al.; A unique heterodimerization pathway involving orphan receptors TR2 and TR4 is demonstrated . TR2 and TR4 preferentially form heterodimers in solution as well as on DNA elements containing a direct repeat-5 (DR5) . The in vitro interaction between TR2 and TR4 is demonstrated by the yeast and the mammalian two-hybrid interaction assays, the pull-down assay, and the gel mobility shift assay . The in vivo interaction is demonstrated by following the intracellular localization of fusion receptors tagged with a green fluorescent protein . The dimerization is mediated by the ligand binding domains, and the three leucine residues on helix 10 of TR2 are critical for this interaction . In addition, coexpression of these two receptors exerts a much stronger repressive activity on a DR5-containing reporter than expressing either receptor alone . In the developing testis, TR2 and TR4 are coexpressed in the same testicular cell populations and exhibit a parallel pattern of expression along development . The preferential heterodimerization between TR2 and TR4 and their coexistence in specific germ cell populations suggest a physiological role of TR2/TR4 heterodimers in germ cell development. FASEB J, 1998 Sep, 12(12), 1101 - 8 BRE: a modulator of TNF-alpha action; Gu C et al.; A stress-responsive gene highly expressed in brain and reproductive organs (BRE) is down-regulated after UV irradiation, DNA damaging agents, or retinoic acid treatment . The human BRE gene encodes a mRNA of 1.9 kb, which gives rise to a protein of 383 amino acids with a molecular size of 44 kilodaltons . BRE is not homologous to any known gene and its function has not been defined . Here we report that BRE was identified multiple times in a yeast two-hybrid screen of a murine cerebellar cDNA library, using the juxtamembrane domain of the p55 tumor necrosis factor alpha (TNF) receptor . The interaction between the p55 receptor and BRE was verified by an in vitro biochemical assay by using recombinant fusion proteins and by co-immunoprecipitation of transfected mammalian cells . In the yeast two-hybrid assay, BRE specifically interacted with p55 TNF receptor but not with other TNF family members such as the Fas receptor, the p75 TNF receptor, and p75 neurotrophin receptor . Overexpression of BRE inhibited TNF-induced NFkappaB activation, indicating that the interaction of BRE protein with the cytoplasmic region of p55 TNF receptor may modulate signal transduction by TNF-alpha. Clin Exp Immunol, 1998 Sep, 113(3), 423 - 8 Interaction of murine macrophage-membrane proteins with components of the pathogenic fungus Histoplasma capsulatum; Taylor ML et al.; The interaction of macrophage-membrane proteins and histoplasmin, a crude antigen of the pathogenic fungus Histoplasma capsulatum, was studied using murine peritoneal macrophages . Membrane proteins were purified via membrane attachment to polycationic beads and solubilized in Tris-HCl/SDS/DTT/glycerol for protein extraction; afterwards they were adsorbed or not with H . capsulatum yeast or lectin binding-enriched by affinity chromatography . Membrane proteins and histoplasmin interactions were detected by ELISA and immunoblotting assays using anti-H . capsulatum human or mouse serum and biotinylated goat anti-human or anti-mouse IgG/streptavidin-peroxidase system to reveal the interaction . Results indicate that macrophage-membrane proteins and histoplasmin components interact in a dose-dependent reaction, and adsorption of macrophage-membrane proteins by yeast cells induces a critical decrease in the interaction . Macrophage-membrane glycoproteins with terminal D-galactosyl residues, purified by chromatography with Abrus precatorius lectin, bound to histoplasmin; and two bands of 68kD and 180kD of transferred membrane protein samples interacted with histoplasmin components, as revealed by immunoblot assays . Specificity for beta-galactoside residues on the macrophage-membrane was confirmed by galactose inhibition of the interaction between macrophage-membrane proteins and histoplasmin components, in competitive ELISA using sugars, as well as by enzymatic cleavage of the galactoside residues. Planta, 1998 Oct, 206(2), 225 - 33 Functional analysis and cell-specific expression of a phosphate transporter from tomato; Daram P et al.; For a better understanding of the molecular and biochemical processes involved in orthophosphate (Pi) uptake at the root/soil interface, we cloned a Pi-transporter c DNA (LePT1) from a root air-specific cDNA library of tomato (Lycopersicon esculentum Mill.) . The corresponding protein belongs to the growing family of ion transporters with twelve putative transmembrane domains . It is highly homologous to recently isolated Pi transporters from higher plants, yeast and fungi . When expressed in a Pi-uptake-deficient yeast mutant, the L . esculentum phosphate transporter 1 (LePT1) protein exhibits an apparent Km of 31 MicroM . The transporter is still active at submicromolar Pi concentrations and mediates highest Pi uptake at pH 5 . The activity of LePT1 is dependent on the electrochemical membrane potential mediated by the yeast P-type H + - ATPase . Transcript levels of LePT1 in tomato seedlings are detectable in all vegetative organs under Pi-sufficient conditions, with highest concentrations in root hairs . In situ hybridization studies demonstrate cell-specific expression of LePT1 in the tomato root . The LePT1 mRNA is detectable in peripheral cell layers such as rhizodermal and root cap cells . Under Pi-deprivation condition, mRNA levels are also detectable in young stelar tissue . This work presents molecular and biochemical evidence for distinct root cells playing an important role in Pi acquisition at the root/soil interface. Proc Natl Acad Sci U S A, 1998 Sep 15, 95(19), 11324 - 9 Human CUL-1 associates with the SKP1/SKP2 complex and regulates p21(CIP1/WAF1) and cyclin D proteins; Yu ZK et al.; Deregulation of cell proliferation is a hallmark of cancer . In many transformed cells, the cyclin A/CDK2 complex that contains S-phase kinase associated proteins 1 and 2 (SKP1 and SKP2) is highly induced . To determine the roles of this complex in the cell cycle regulation and transformation, we have examined the composition of this complex . We report here that this complex contained an additional protein, human CUL-1, a member of the cullin/CDC53 family . The identification of CUL-1 as a member of the complex raises the possibility that the p19(SKP1)/p45(SKP2)/CUL-1 complex may function as the yeast SKP1-CDC53-F-box (SCF) protein complex that acts as a ubiquitin E3 ligase to regulate the G1/S transition . In mammalian cells, cyclin D, p21(CIP1/WAF1), and p27(KIP1) are short-lived proteins that are controlled by ubiquitin-dependent proteolysis . To determine the potential in vivo targets of the p19(SKP1)/p45(SKP2)/CUL-1 complex, we have used the specific antisense oligodeoxynucleotides against either SKP1, SKP2, or CUL-1 RNA to inhibit their expression . Treatment of cells with these oligonucleotides caused the selective accumulation of p21 and cyclin D proteins . The protein level of p27 was not affected . These data suggest that the human p19(SKP1)/p45(SKP2)/CUL-1 complex is likely to function as an E3 ligase to selectively target cyclin D and p21 for the ubiquitin-dependent protein degradation . Aberrant expression of human p19(SKP1)/p45(SKP2)/CUL-1 complex thus may contribute to tumorigenesis by regulating the protein levels of G1 cell cycle regulators. Proc Natl Acad Sci U S A, 1998 Sep 15, 95(19), 11193 - 8 Mad2 transiently associates with an APC/p55Cdc complex during mitosis; Wassmann K et al.; Activation of the mitotic checkpoint pathway in response to mitotic spindle damage in eukaryotic cells delays the exit from mitosis in an attempt to prevent chromosome missegregation . One component of this pathway, hsMad2, has been shown in mammalian cells to physically associate with components of a ubiquitin ligase activity (termed the anaphase promoting complex or APC) when the checkpoint is activated, thereby preventing the degradation of inhibitors of the mitotic exit machinery . In the present report, we demonstrate that the inhibitory association between Mad2 and the APC component Cdc27 also takes place transiently during the early stages of a normal mitosis and is lost before mitotic exit . We also show that Mad2 associates with the APC regulatory protein p55Cdc in mammalian cells as has been reported in yeast . In contrast, however, this complex is present only in nocodazole-arrested or early mitotic cells and is associated with the APC as a Mad2/p55Cdc/Cdc27 ternary complex . Evidence for a Mad2/Cdc27 complex that forms independent of p55Cdc also is presented . These results suggest a model for the regulation of the APC by Mad2 and may explain how the spindle assembly checkpoint apparatus controls the timing of mitosis under normal growth conditions. EMBO J, 1998 Sep 15, 17(18), 5409 - 17 Complex formation by the Drosophila MSL proteins: role of the MSL2 RING finger in protein complex assembly; Copps K et al.; Drosophila MSL proteins are thought to act within a complex to elevate transcription from the male X chromosome . We found that the MSL1, MSL2 and MSL3 proteins are associated in immunoprecipitations, chromatographic steps and in the yeast two-hybrid system, but that the MLE protein is not tightly complexed in these assays . We focused our analysis on the MSL2-MSL1 interaction, which is postulated to play a critical role in MSL complex association with the X chromosome . Using a modified two-hybrid assay, we isolated missense mutations in MSL2 that disrupt its interaction with MSL1 . Eleven out of 12 mutated residues clustered around the first zinc-binding site of the RING finger domain were conserved in a Drosophila virilis MSL2 homolog . Two pre-existing msl2 alleles, which fail to support male viability in vivo, have lesions in the same region of the RING finger . We tested these in the two-hybrid system and found that they are also defective in interaction with MSL1 . Mutation of the second zinc-binding site had little effect on MSL1 binding, suggesting that this portion of the RING finger may have a distinct function . Our data support a model in which MSL2-MSL1 interaction nucleates assembly of an MSL complex, with which MLE is weakly or transiently associated. Ultrasonics, 1998 Aug, 36(9), 925 - 31 Microparticle manipulation in millimetre scale ultrasonic standing wave chambers; Hawkes JJ et al.; Ultrasonic standing wave chambers with acoustic pathlengths of 1.1 and 0.62 mm have been constructed . The chambers were driven at frequencies over the range 0.66-12.2 MHz . The behaviour of 2 microns diameter latex microparticles and 5 microns diameter yeast in the chambers has been elucidated . One (flow) chamber had a downstream laminar flow expansion section to facilitate observation of concentrated particle bands formed in the ultrasonic field . A second (microscopy) chamber allowed direct observation of band formation in the field and their characterisation by confocal scanning laser microscopy . Clear band formation occurs when the chamber pathlength is a multiple of half wavelengths at the driving frequency, so that the chamber rather than the transducer resonance has the most influence on band formation in this system . Band formation occurred in half-wavelength steps from a position one quarter of a wavelength off the transducer to a band at a similar distance from the reflector . Ordered band formation was preserved by the laminar flow in the expansion chamber, although bands that formed very close to the wall were dissipated downstream . The microscopy chamber provided evidence of significant lateral particle concentration within bands in the pressure nodal planes . The approaches described will be applicable to the manipulation of smaller particles in narrower chambers at higher ultrasonic frequencies. Hum Mol Genet, 1998, 7(10), 1635 - 40 Engineering mammalian chromosomes; Grimes B et al.; Construction of a mammalian artificial chromosome (MAC) will develop our understanding of the requirements for normal chromosome maintenance, replication and segregation while offering the capacity for introducing genes into cells . Construction of MACs with telomere, centromere and replication function has been approached by two methods . The 'top down' strategy uses artificially induced chromosome truncations as a means to define a minimal chromosome that retains the mitotic properties of a normal chromosome . The 'build up' approach has focused on attempts to assemble MAC vectors containing functionally defined telomere repeats together with candidate centromere and replication origin sequences . Here we report on significant advances in both areas, with particular emphasis on two reports showing that stable, low copy number MACs containing a functional centromere can be produced following transfection of naked DNA into the human HT1080 cell line . One approach used a transfection mixture of cloned synthetic alpha-satellite arrays up to 1 Mb in length and unlinked telomeric DNA, in either the presence or absence of random human genomic DNA fragments . In the second approach, MACs were formed from a defined yeast artificial chromosome (YAC) DNA molecule containing 100 kb of highly homo- geneous alphoid DNA retrofitted with human telomere repeats . These results demonstrate for the first time that alpha-satellite DNA can seed de novo centromeres in human cells, indicating that this repetitive sequence family plays an important role in centromere function . The stability of these MACs suggests that they have potential to be developed as gene delivery vectors. J Mol Biol, 1998 Sep 18, 282(2), 421 - 33 NMR structure of the Streptomyces metalloproteinase inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23: another example of an ancestral beta gamma-crystallin precursor structure; Ohno A et al.; The Streptomyces metalloproteinase inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23, is a proteinaceous metalloproteinase inhibitor, and consists of 102 amino acid residues with two disulfide bridges . SMPI specifically inhibits metalloproteinases such as thermolysin . In the present work, the solution structure of SMPI was determined on the basis of 1536 nuclear Overhauser enhancement derived distance restraints and 52 dihedral angle restraints obtained from three-bond spin coupling constants . The final ensemble of 20 NMR structures overlaid onto their mean coordinate with backbone (N, Calpha, C') r.m.s.d . values of 0 . 45(+/-0.11) A and 0.57(+/-0.18) A for residues 6 to 99 and the entire 102 residues, respectively . SMPI is essentially composed of two beta-sheets, each consisting of four antiparallel beta-strands . The structure can be considered as two Greek key motifs with 2-fold internal symmetry, a Greek key beta-barrel . One unique structural feature found in SMPI is in its extension between the first and second strands of the second Greek key motif . Interestingly, this extended segment is known to be involved in the inhibitory activity of SMPI . In the absence of sequence similarity, the SMPI structure shows clear similarity to both domains of the eye lens crystallins, both domains of the calcium sensor protein-S, as well as the single-domain yeast killer toxin . The yeast killer toxin structure was thought to be a precursor of the two-domain beta gamma-crystallin proteins, because of its structural similarity to each domain of the beta gamma-crystallins . SMPI thus provides another example of a single-domain protein structure that corresponds to the ancestral fold from which the two-domain proteins in the beta gamma-crystallin superfamily are believed to have evolved . J Mol Biol, 1998 Sep 11, 282(1), 195 - 208 A model of Cdc25 phosphatase catalytic domain and Cdk-interaction surface based on the presence of a rhodanese homology domain; Hofmann K et al.; Mammalian Cdc25 phosphatase is responsible for the dephosphorylation of Cdc2 and other cyclin-dependent kinases at Thr14 and Tyr15, thus activating the kinase and allowing cell cycle progression . The catalytic domain of this dual-specificity phosphatase has recently been mapped to the 180 most C-terminal amino acids . Apart from a CX3R motif, which is present at the active site of all known tyrosine phosphatases, Cdc25 does not share any obvious sequence similarity with any of those enzymes . Until very recently, the Cdc25 family was the only subfamily of tyrosine phosphates for which no three-dimensional structural data were available . Using the generalized profile technique, a sensitive method for sequence database searches, we found an extended and highly significant sequence similarity between the Cdc25 catalytic domain and similarly sized regions in other proteins: the non-catalytic domain of two distinct families of MAP-kinase phosphates, the non-catalytic domain of several ubiquitin protein hydrolases, the N and C-terminal domain of rhodanese, and a large and heterogeneous groups of stress-response proteins from all phyla . The relationship of Cdc25 to the structurally well-characterized rhodanese spans the entire catalytic domain and served as template for a structural model for human Cdc25a, which is fundamentally different from previously suggested models for Cdc25 catalytic domain organization . The surface positioning of subfamily-specific conserved residues allows us to predict the sites of interaction with Cdk2, a physiological target of Cdc25a . Based on the results of this analysis, we also predict that the budding yeast arsenate resistance protein Acr2 and the ORF Ygr203w encode protein phosphatases with catalytic properties similar to that of the Cdc25 family . Recent determination of the crystal structure of the Cdc25a catalytic domain supports the validity of the model and demonstrates the power of the generalized sequence profile technique in homology-based modeling of the three-dimensional structure of a protein having a weak but significant sequence similarity with a structurally characterized protein . Plant Physiol, 1998 Sep, 118(1), 115 - 23 Prenylcysteine alpha-carboxyl methyltransferase in suspension-cultured tobacco cells Crowell DN, Sen SE, Randall SK. Isoprenylation is a posttranslational modification that is believed to be necessary, but not sufficient, for the efficient association of numerous eukaryotic cell proteins with membranes . Additional modifications have been shown to be required for proper intracellular targeting and function of certain isoprenylated proteins in mammalian and yeast cells . Although protein isoprenylation has been demonstrated in plants, postisoprenylation processing of plant proteins has not been described . Here we demonstrate that cultured tobacco (Nicotiana tabacum cv Bright Yellow-2) cells contain farnesylcysteine and geranylgeranylcysteine alpha-carboxyl methyltransferase activities with apparent Michaelis constants of 73 and 21 &mgr;M for N-acetyl-S-trans, trans-farnesyl-L-cysteine and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine, respectively . Furthermore, competition analysis indicates that the same enzyme is responsible for both activities . These results suggest that alpha-carboxyl methylation is a step in the maturation of isoprenylated proteins in plants. Plant Physiol, 1998 Sep, 118(1), 199 - 207 D-Ribulose-5-phosphate 3-epimerase: cloning and heterologous expression of the spinach gene, and purification and characterization of the recombinant enzyme; Chen YR et al.; We have achieved, to our knowledge, the first high-level heterologous expression of the gene encoding D-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms . The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by DL-alpha-glycerophosphate or ethanol and destabilized by D-ribulose-5-phosphate or 2-mercaptoethanol . Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation . Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis . As deduced from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals . However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions . Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric. J Mol Evol, 1998 Sep, 47(3), 275 - 81 The mouse gene encoding the testis-specific isoform of Poly(A) binding protein (Pabp2) is an expressed retroposon: intimations that gene expression in spermatogenic cells facilitates the creation of new genes; Kleene KC et al.; The gene encoding the testis-specific isoform of mouse poly(A) binding protein (Pabp2) has been isolated and sequenced . Unexpectedly, comparison of the sequence of genomic and cDNAs demonstrated that the Pabp2 gene lacks introns, whereas all other functional Pabp genes in plants, amphibians, and mammals contain introns . Thus, the mouse Pabp2 gene is a retroposon, created by synthesizing a reverse transcriptase copy of a processed mRNA and inserting the copy into the genome . The Pabp2 retroposon is unusual because it is functional: previous work demonstrates that its promoter drives the accumulation of Pabp2 mRNA in meiotic and early haploid spermatogenic cells, and the Pabp2 mRNA encodes a protein whose size and RNA-binding specificities are characteristic of PABP in plants, yeast, and mammals (Kleene et al . 1994) . Two novel factors can be implicated in the retention of function of the Pabp2 retroposon . First, the promoter of the Pabp2 gene is not derived from its intron-containing progenitor, Pabp1 . Second, mRNAs encoding somatic PABP isoform, PABP1, are present at high levels in meiotic and haploid spermatogenic cells . Both features contrast with the phosphoglycerate kinase 2 retroposon, which is believed to compensate for the depletion of the somatic isoform due to X-chromosome inactivation in meiotic spermatogenic cells . We also document that more functional retroposons are expressed in meiotic and haploid spermatogenic cells than in any other tissue and speculate that transcriptional derepression in spermatogenic cells favors the creation of expressed retroposons. Nat Genet, 1998 Sep, 20(1), 46 - 50 Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex; Neubauer G et al.; Many important cell mechanisms are carried out and regulated by multi-protein complexes, for example, transcription and RNA processing machinery, receptor complexes and cytoskeletal structures . Most of these complexes remain only partially characterized due to the difficulty of conventional protein analysis methods . The rapid expansion of DNA sequence databases now provides whole or partial gene sequences of model organisms, and recent advances in protein microcharacterization via mass spectrometry allow the possibility of linking these DNA sequences to the proteins in functional complexes . This approach has been demonstrated in organisms whose genomes have been sequenced, such as budding yeast . Here we report the first characterization of an entire mammalian multi-protein complex using these methods . The machinery that removes introns from mRNA precursors--the spliceosome--is a large multi-protein complex . Approximately half of the components excised from a two-dimensional gel separation of the spliceosome were found in protein sequence databases . Using nanoelectrospray mass spectrometry, the remainder were identified and cloned using public expressed sequence tag (EST) databases . Existing EST databases are thus already sufficiently complete to allow rapid characterization of large mammalian protein complexes via mass spectrometry. Nat Genet, 1998 Sep, 20(1), 37 - 42 A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B; Bashir R et al.; The limb-girdle muscular dystrophies are a genetically heterogeneous group of inherited progressive muscle disorders that affect mainly the proximal musculature, with evidence for at least three autosomal dominant and eight autosomal recessive loci . The latter mostly involve mutations in genes encoding components of the dystrophin-associated complex; another form is caused by mutations in the gene for the muscle-specific protease calpain 3 . Using a positional cloning approach, we have identified the gene for a form of limb-girdle muscular dystrophy that we previously mapped to chromosome 2p13 (LGMD2B) . This gene shows no homology to any known mammalian gene, but its predicted product is related to the C . elegans spermatogenesis factor fer-1 . We have identified two homozygous frameshift mutations in this gene, resulting in muscular dystrophy of either proximal or distal onset in nine families . The proposed name 'dysferlin' combines the role of the gene in producing muscular dystrophy with its C . elegans homology. J Cell Biol, 1998 Sep 7, 142(5), 1269 - 78 Differential membrane localization and intermolecular associations of alpha-dystrobrevin isoforms in skeletal muscle; Peters MF et al.; alpha-Dystrobrevin is both a dystrophin homologue and a component of the dystrophin protein complex . Alternative splicing yields five forms, of which two predominate in skeletal muscle: full-length alpha-dystrobrevin-1 (84 kD), and COOH-terminal truncated alpha-dystrobrevin-2 (65 kD) . Using isoform-specific antibodies, we find that alpha-dystrobrevin-2 is localized on the sarcolemma and at the neuromuscular synapse, where, like dystrophin, it is most concentrated in the depths of the postjunctional folds . alpha-Dystrobrevin-2 preferentially copurifies with dystrophin from muscle extracts . In contrast, alpha-dystrobrevin-1 is more highly restricted to the synapse, like the dystrophin homologue utrophin, and preferentially copurifies with utrophin . In yeast two-hybrid experiments and coimmunoprecipitation of in vitro-translated proteins, alpha-dystrobrevin-2 binds dystrophin, whereas alpha-dystrobrevin-1 binds both dystrophin and utrophin . alpha-Dystrobrevin-2 was lost from the nonsynaptic sarcolemma of dystrophin-deficient mdx mice, but was retained on the perisynaptic sarcolemma even in mice lacking both utrophin and dystrophin . In contrast, alpha-dystrobrevin-1 remained synaptically localized in mdx and utrophin-negative muscle, but was absent in double mutants . Thus, the distinct distributions of alpha-dystrobrevin-1 and -2 can be partly explained by specific associations with utrophin and dystrophin, but other factors are also involved . These results show that alternative splicing confers distinct properties of association on the alpha-dystrobrevins. J Cell Biol, 1998 Sep 7, 142(5), 1195 - 207 Nup154, a new Drosophila gene essential for male and female gametogenesis is related to the nup155 vertebrate nucleoporin gene; Gigliotti S et al.; The Nup154 gene of Drosophila encodes a protein showing similarity with known nucleoporins: rat Nup155 and yeast Nup170 and Nup157 . Hypomorphic mutant alleles of Nup154 affected female and male fertility, allowing investigation of the gene function in various steps of oogenesis and spermatogenesis . Nup154 was required in testes for cyst formation, control of spermatocyte proliferation and meiotic progression . In ovaries, Nup154 was essential for egg chamber development and oocyte growth . In both the male and female germ line, as well as in several other cell types, the Nup154 protein was detected at the nuclear membrane, but was also present inside the nucleus . Intranuclear localization has not previously been described for rat Nup155 or yeast Nup170 and Nup157 . In mutant egg chambers the Nup154 protein accumulated in the cytoplasm, while it was only barely detected at the nuclear envelopes . FG repeats containing nucleoporins detected with mAb414 antibody were also mislocalized to a certain extent in Nup154 mutant alleles . This suggests that Nup154 could be required for localizing other nucleoporins within the nuclear pore complex, as previously demonstrated for the yeast Nup170 . On the other hand, no evident defects in lamin localization were observed, indicating that Nup155 mutations did not affect the overall integrity of the nuclear envelope . However, ultrastructural analyses revealed that in mutant cells the morphology of the nuclear envelope was altered near the nuclear pore complexes . Finally, the multiplicity of phenotypes observed in Nup154 mutant alleles suggests that this gene plays a crucial role in cell physiology. Mol Endocrinol, 1998 Sep, 12(9), 1432 - 40 BOD (Bcl-2-related ovarian death gene) is an ovarian BH3 domain-containing proapoptotic Bcl-2 protein capable of dimerization with diverse antiapoptotic Bcl-2 members; Hsu SY et al.; Using the yeast two-hybrid protein-protein interaction system to search for genes capable of forming dimers with the antiapoptotic protein Mcl-1, we have isolated BOD (Bcl-2-related ovarian death agonist) from an ovarian fusion cDNA library . The three variants of BOD (long, medium, and short) have an open reading frame of 196, 110, and 93 amino acids, respectively; all of them contain a consensus Bcl-2 homology 3 (BH3) domain but lack other BH domains found in channel-forming Bcl-2 family proteins . In the yeast cell assay, BOD interacts with diverse antiapoptotic Bcl-2 proteins {Mcl-1, Bcl-2, Bcl-xL, Bcl-w, Bfl-1, and Epstein-Barr virus (EBV) BHRF-1} but not with different proapoptotic Bcl-2 proteins (BAD, Bak, Bok, and Bax) . After overexpression in mammalian Chinese hamster ovary (CHO) cells, BOD induces apoptosis that can be prevented by the baculoviral caspase inhibitor P35 . The cell-killing activity of BOD is also antagonized in cells cotransfected with the antiapoptotic Bcl-w protein, which showed high affinity for BOD in the two-hybrid assay . Furthermore, mutagenesis studies showed that BOD mutants with alterations in the BH3 domain lose cell-killing ability, suggesting that the BH3 domain is important for the mediation of cell killing by BOD . BOD mRNA is ubiquitously expressed in ovary and multiple other tissues . The BOD gene is also conserved in diverse mammalian species . Identification of BOD expands the group of proapoptotic Bcl-2 proteins that only contains the BH3 domain and allows future elucidation of the intracellular mechanism for apoptosis regulation in ovary and other tissues. Mol Endocrinol, 1998 Sep, 12(9), 1367 - 79 Thyroid hormone receptor does not heterodimerize with the vitamin D receptor but represses vitamin D receptor-mediated transactivation; Raval-Pandya M et al.; The 9,000 Mr calcium-binding protein calbindin-D9k (CaBP9k) is markedly induced by 1,25-dihydroxyvitamin D3 {1,25-(OH)2D3} in mammalian intestine . However, although a vitamin D response element (VDRE) has been reported in the promoter of the rat CaBP9k gene (at -490/-472), the CaBP9k promoter is weakly transactivated by 1,25-(OH)2D3 . Previous studies indicated that when MCF-7 cells are transfected with the rat CaBP9k VDRE ligated to the thymidine kinase promoter and treated with both 1,25-(OH)2D3 and T3 there is an enhancement of the response observed with 1,25-(OH)2D3 alone, suggesting direct cross-talk between thyroid hormone and the vitamin D endocrine system and activation via the formation of vitamin D receptor (VDR)-thyroid hormone receptor (TR) heterodimers . To determine whether the weak response of the rat CaBP9k natural promoter to 1,25-(OH)2D3 could be enhanced by T3, CaBP9k promoter/reporter chloramphenicol acetyltransferase constructs were transfected in MCF-7 cells, and the cells were treated with the two hormones alone or in combination . No induction with T3 alone and no enhancement of reporter activity in the presence of both hormones was observed . To determine whether a lack of effect by T3 was specific for the CaBP9k promoter and to further examine the possibility of cross-talk between the TR- and VDR-signaling pathways, the 1,25-(OH)2D3-responsive rat 24 hydroxylase {24(OH)ase} promoter and the rat osteocalcin VDRE (-457/-430), both fused to reporter genes were similarly examined in MCF-7 cells . Again, no enhancement of the response to 1,25-(OH)2D3 was observed in the presence of T3 . In addition, a similar lack of response to T3 but responsiveness to 1,25-(OH)2D3 was observed when UMR106-01 osteosarcoma cells {which, like MCF-7 cells, express VDR, TR, and the retinoid X receptor (RXR) endogenously} were transfected with a 1,25-(OH)2D3 responsive mouse osteopontin promoter reporter . In vitro DNA binding assays were carried out using purified human VDR, human RXRalpha, and chick T3Ralpha and 24(OH)ase, osteocalcin, osteopontin, and CaBP9k VDRE oligonucleotide probes . No VDR-TR heterodimer binding on any of these VDREs was observed, although, as expected, there was binding by the VDR-RXR complex and strong TR-RXR binding to a consensus thyroid hormone response element . Simultaneous gel retardation assays using similar and lower concentrations of TR with RXR showed strong binding of TR-RXR on a 32P-labeled thyroid response element . Studies using the yeast two-hybrid system also did not provide evidence for the formation of a VDR-TR protein-protein interaction . In addition, in vivo data showed that transfection of TR, in fact, repressed VDR-mediated transcription and that the repression could be reversed by the addition of RXR . Thus, in vitro and in vivo experiments do not support ligand-sensitive transactivation mediated by VDR-TR heterodimer formation but rather suggest that TR expression can repress 1,25-(OH)2D3-induced transcription predominantly by sequestering RXR. Biochemistry, 1998 Sep 8, 37(36), 12513 - 25 Probing the mechanism of proton coupled electron transfer to dioxygen: the oxidative half-reaction of bovine serum amine oxidase; Su Q et al.; Bovine serum amine oxidase (BSAO) catalyzes the oxidative deamination of primary amines, concomitant with the reduction of molecular oxygen to hydrogen peroxide via a ping-pong mechanism . A protocol has been developed for an analysis of chemical and kinetic mechanisms in the conversion of dioxygen to hydrogen peroxide . Steady-state kinetics show that two groups need to be deprotonated to facilitate the oxidative half-reaction . The pH dependence of Vmax/Km(O2) reveals pKa's of 6.2 +/- 0.3 and 7.0 +/- 0.2, respectively . A pKa of 7.2 +/- 0.1 has been obtained from a titration of anaerobically reduced BSAO using UV-vis spectrophotometry . The near identity of the pKa obtained from the reduced enzyme titration with the second pKa from steady-state kinetics suggests that this second pKa arises from the reduced cofactor . The assignment of pKa is supported by the observed pH dependence for formation of the cofactor semiquinone signal, detected by EPR spectroscopy under anaerobic conditions . To address the nature of rate-limiting steps in the oxidative half-reaction, the solvent isotope effect, viscosity effect, and O-18 isotope effect on Vmax/Km(O2) have been determined . The solvent isotope effect is indistinguishable from unity, ruling out a proton transfer as a rate-determining step . Use of glucose as a solvent viscosogen shows no viscosity effect, indicating that binding of oxygen is not in the rate-determining step . The O-18 kinetic isotope effect is independent of pH with an average value of 18(V/K) = 1.0097 +/- 0 . 0010 . This has been compared to calculated equilibrium O-18 isotope effects for various dioxygen intermediate species {Tian and Klinman (1993) J . Am . Chem . Soc . 115, 8891}, leading to the conclusion that either the first electron transfer to dioxygen or the desorption of product peroxide from a Cu(II)-OOH complex could be the rate-limiting step . The distribution of steady-state enzyme species was, therefore, analyzed through a combination of stopped-flow experiments and analysis of DV and D(V/K) for benzylamine oxidation . We conclude that the major species accumulating in the steady state are the oxidized cofactor-substrate Schiff base complex and the reduced, aminoquinol form of cofactor . These data rule out a slow release of product hydroperoxide from the aminoquinone form of enzyme, leading to the conclusion that the first electron transfer from substrate-reduced cofactor to dioxygen is the rate-determining step in the oxidative half-reaction . This step is also estimated to be 40% rate-limiting in kcat . An important mechanistic conclusion from this study is that dioxygen binding is a separate step from the rate-limiting electron-transfer step to form superoxide . On the basis of a recently determined X-ray structure for the active form of a yeast amine oxidase from Hansenula polymorpha {Li et al . (1998) Structure 6, 293}, a hydrophobic space has been identified near the O-2 position of reduced cofactor as the putative dioxygen binding site . Movement of superoxide from this site onto the Cu(II) at the active site may occur prior to further electron transfer from cofactor to superoxide. Cytogenet Cell Genet, 1998, 81(3-4), 259 - 64 High-resolution mapping of the X-linked lymphoproliferative syndrome region by FISH on combed DNA; Monier K et al.; X-linked lymphoproliferative syndrome is an inherited immunodeficiency for which the responsible gene is currently unknown . Several megabase-sized deleted regions mapping to Xq25 have been identified in XLP patients, and more recently a 130-kb deletion has been reported (Lamartine et al., 1996; Lanyi et al., 1996) . To establish a physical map of this deleted region and to identify the XLP gene, two cosmid contigs were established (Lamartine et al., 1996) . However, the physical map of this region is still uncompleted and controversial and three points remain unsolved: (1) the centromeric-telomeric orientation of the whole region, (2) the relative orientation of the two contigs, and (3) the size of the gap between the two contigs . To provide a definitive answer to these questions, high-resolution mapping by fluorescence in situ hybridization on combed DNA and molecular approaches were combined to establish the physical map of the XLP region over 600 kb . Our results identified a gap of 150 kb between the two contigs, established the relative orientation of one contig to the other, and determine the centromeric-telomeric orientation of the whole region . Our results show that the order of the marker over this region is: cen.1D10T7-DF83-DXS982.tel. Cytogenet Cell Genet, 1998, 81(3-4), 247 - 53 Expressed copies of the MN7 (D15F37) gene family map close to the common deletion breakpoints in the Prader-Willi/Angelman syndromes; Buiting K et al.; Approximately 70% of patients with Prader-Willi syndrome or Angelman syndrome have a similar sized de novo deletion of 3-4 Mb in the proximal region of 15q . The distal breakpoints appear to cluster between the P gene (OCA2) and D15S24, whereas two deletion breakpoint clusters have been identified on the proximal side (one centromeric to D15S541 and one between D15S541 and D15S9) . Based on the identification of a gene family in 15q11-->q13 (MN7, D15F37), we have previously proposed that the presence of multiple copies of this sequence may be related to the instability of this region . Using fluorescence in situ hybridization and YAC mapping, we have found that at least one D15F37 locus is centromeric to D15S9 and at least two are between OCA2 and D15S24 . As determined by cDNA cloning and sequence analysis, each of the individual loci is expressed . The close proximity of the D15F37 loci and the deletion breakpoints suggests that the common deletions arise by unequal crossover events at or near these loci. Cytogenet Cell Genet, 1998, 81(3-4), 237 - 46 Comparative mapping of two adjacent regions of MMU19 with their human counterpart on HSA11q13; Fernandes M et al.; High resolution physical maps of two adjacent regions of MMU19 were constructed in order to establish a comparative map between the pericentromeric region of MMU19 and its human counterpart on HSA11q13 . These two physical maps span 2.5 and 0.5 megabases on MMU19 . Long range restriction analysis and YAC contigs have been built, five genes were located on MMU19 and eight new STSs were generated . The 0.5-Mb map which has been positioned close to the centromere of MMU19, based on dual-color FISH experiments and genetic data, includes eight genes (Type I markers), three microsatellites (Type II markers) and five new STSs . The 2.5-Mb map is located more telomeric and contains seven genes, four microsatellites and four new STSs . Gene order and physical distances appear to be similar in human and in mouse in this 2.5-Mb region . Strikingly, the 0.5-Mb region has a similar size in human but gene order is shuffled . The overall comparative map shows that these two regions are inverted on MMU19 when compared with HSA11q13. Genome, 1998 Jun, 41(3), 468 - 70 FISH mapping and inter-Alu fingerprinting define the YAC contig map around the centromeric region of human chromosome 18; Greer WL et al.; Previous reports concerning the location of D18S44 with respect to the centromere have been ambiguous . Also, it has not been possible, based on formerly reported markers, to show that contigs WC18.0 and WC18.1 overlap . However, the data presented here definitively show, using FISH technology, that D18S44 (located on WC18.0) maps to proximal 18q . Furthermore, inter-Alu fingerprinting shows a clear overlap between WC18.0 and WC18.1, thereby establishing a complete contig between D18S44 and markers from WC18.1. Development, 1998 Oct, 125(19), 3801 - 8 A Serum Response Factor homolog is required for spore differentiation in Dictyostelium; Escalante R et al.; A homolog of the Serum Response Factor (SRF) has been isolated from Dictyostelium discoideum and its function studied by analyzing the consequences of its gene disruption . The MADS-box region of Dictyostelium SRF (DdSRF) is highly conserved with those of the human, Drosophila and yeast homologs . srfA is a developmentally regulated gene expressed in prespore and spore cells . This gene plays an essential role in sporulation as its disruption leads to abnormal spore morphology and loss of viability . The mutant spores were round and cellulose deposition seemed to be partially affected . Initial prestalk and prespore cell differentiation did not seem to be compromised in the mutant since the expression of several cell-type-specific markers were found to be unaffected . However, the mRNA level of the spore marker spiA was greatly reduced . Activation of the cAMP-dependent protein kinase (PKA) by 8-Br-cAMP was not able to fully bypass the morphological defects of srfA- mutant spores, although this treatment induced spiA mRNA expression . Our results suggest that DdSRF is required for full maturation of spores and participates in the regulation of the expression of the spore-coat marker spiA and probably other maturation genes necessary for proper spore cell differentiation. Biochem J, 1998 Sep 15, 334 ( Pt 3), 571 - 6 Plant mitochondrial pyruvate dehydrogenase complex: purification and identification of catalytic components in potato; Millar AH et al.; The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv . Romano) tuber mitochondria was purified 40-fold to a specific activity of 5.60 micromol/min per mg of protein . The activity of the complex depended on pyruvate, divalent cations, NAD+ and CoA and was competitively inhibited by both NADH and acetyl-CoA . SDS/PAGE revealed the complex consisted of seven polypeptide bands with apparent molecular masses of 78, 60, 58, 55, 43, 41 and 37 kDa . N-terminal sequencing revealed that the 78 kDa protein was dihydrolipoamide transacetylase (E2), the 58 kDa protein was dihydrolipoamide dehydrogenase (E3), the 43 and 41 kDa proteins were alpha subunits of pyruvate dehydrogenase, and the 37 kDa protein was the beta subunit of pyruvate dehydrogenase . N-terminal sequencing of the 55 kDa protein band yielded two protein sequences: one was another E3; the other was similar to the sequence of E2 from plant and yeast sources but was distinctly different from the sequence of the 78 kDa protein . Incubation of the mPDC with {2-14C}pyruvate resulted in the acetylation of both the 78 and 55 kDa proteins. Gene, 1998 Aug 31, 216(2), 267 - 76 The new gene DmX from Drosophila melanogaster encodes a novel WD-repeat protein; Kraemer C et al.; DmX is a novel gene from Drosophila melanogaster located on the X chromosome in region 5D5/6-E1 . The molecular analysis of the genomic and cDNA sequences of DmX shows that the gene spans appr . 16kb and displays a mosaic structure with 15 exons . The 12kb long DmX transcript is present in Drosophila embryos, larvae and adults of both sexes . The open reading frame of DmX encodes a novel WD-repeat protein, containing at least 30 WD-repeat units . WD-repeat proteins contain a conserved motif of approximately 40 amino acids (aa), usually ending with the dipeptide Trp-Asp (WD) . Homologues of the DmX gene exist in other dipteran species, in Caenorhabditis elegans and human, revealing that DmX is an evolutionarily well conserved gene . The inferred DMX amino acid sequence shows also limited, but significant similarity to a yeast ORF with unknown function . 1998 Elsevier Science B.V. Mutat Res, 1998 Aug 3, 404(1-2), 113 - 7 Radiation induced chromosome aberrations: some biophysical considerations; Chadwick KH et al.; The implications of recent results using FISH chromosome painting and soft X-ray exposures for the mechanisms of chromosome aberration formation are discussed . It is concluded that the evidence in favour of exchange aberrations arising from one radiation induced chromosome break has increased to the point where a 'change in paradigm' from the older breakage-reunion hypothesis needs to be taken seriously into account . A potential role for recombinational repair of DNA double strand breaks, as known in yeast, in the formation of aberrations in mammalian cells is presented and the relationship between DNA repair studies and radiation cytology is emphasized . J Biol Chem, 1998 Sep 11, 273(37), 24095 - 101 Interaction of human suppressor of cytokine signaling (SOCS)-2 with the insulin-like growth factor-I receptor; Dey BR et al.; SOCS (suppressor of cytokine signaling) proteins have been shown to be negative regulators of cytokine receptor signaling via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway . We have cloned a member of this family (hSOCS-2) by utilizing the insulin-like growth factor I receptor (IGF-IR) cytoplasmic domain as bait in a yeast two-hybrid screen of a human fetal brain library . The hSOCS-2 protein interacted strongly with the activated IGF-IR and not with a kinase negative mutant receptor in the two-hybrid assay . Mutation of receptor tyrosines 950, 1250, 1251, and 1316 to phenylalanine or deletion of the COOH-terminal 93 amino acids did not result in decreased interaction of the receptor with hSOCS-2 protein . hSOCS-1 protein also interacted strongly with IGF-IR in the two-hybrid assay . Glutathione S-transferase-hSOCS-2 associated with activated IGF-IR in lysates of mouse fibroblasts overexpressing IGF-IR . Human embryonic kidney cells (293) were transiently transfected with vectors containing IGF-IR and FLAG epitope-tagged hSOCS-2 . After IGF-I stimulation, activated IGF-IR was found in anti-FLAG immunoprecipitates and, conversely, FLAG-hSOCS-2 was found in anti IGF-IR immunoprecipitates . Thus, hSOCS-2 interacted with IGF-IR both in vitro and in vivo . HSOCS-2 mRNA was expressed in many human fetal and adult tissues with particularly high abundance in fetal kidney and adult heart, skeletal muscle, pancreas, and liver . These results raise the possibility that SOCS proteins may also play a regulatory role in IGF-I receptor signaling. Appl Environ Microbiol, 1998 Sep, 64(9), 3202 - 8 Role of endoproteolytic dibasic proprotein processing in maturation of secretory proteins in Trichoderma reesei; Goller SP et al.; Cell extracts of Trichoderma reesei exhibited dibasic endopeptidase activity toward the carboxylic side of KR, RR, and PR sequences . This activity was stimulated by the presence of Ca2+ ions and localized in vesicles of low bouyant density; it therefore exhibited some similarity to yeast Kex2 . Analytical chromatofocusing revealed a single peak of activity . The dibasic endopeptidase activity was strongly and irreversibly inhibited in vitro as well as in vivo by 1 mM p-amidinophenylmethylsulfonyl fluoride (pAPMSF) but not by PMSF at concentrations up to 5 mM . We therefore used pAPMSF to study the role of the dibasic endopeptidase in the secretion of protein by T . reesei . Secretion of xylanase I (proprotein processing sequence -R-R- downward arrow-R- downward arrow-A-) and xylanase II (-K-R- downward arrow-Q-) was strongly inhibited by 1 mM pAPMSF, and a larger, unprocessed enzyme form was detected intracellularly under these conditions . Secretion of cellobiohydrolase II (CBH II; -E-R- downward arrow-Q-) was only slightly inhibited by pAPMSF, and no accumulation of unprocessed precursors was detected . In contrast, secretion of CBH I (-R-A- downward arrow-Q-) was stimulated by pAPMSF addition, and a simultaneous decrease in the concentration of intracellular CBH I was detected . Similar experiments were also carried out with a single heterologous protein, ShBLE, the phleomycin-binding protein from Streptoalloteichus hindustanus, fused to a series of model proprotein-processing sequences downstream of the expression signals of the Aspergillus nidulans gpdA promoter . Consistent with the results obtained with homologous proteins, pAPMSF inhibited the secretion of ShBLE with fusions containing dibasic (RK and KR) target sequences, but it even stimulated secretion in fusions to LR, NHA, and EHA target sequences . Addition of 5 mM PMSF, a nonspecific inhibitor of serine protease, nonspecifically inhibited the secretion of heterologous proteins from fusions bearing the NHA and LR targets . These data point to the existence of different endoproteolytic proprotein processing enzymes in T . reesei and demonstrate that dibasic processing is obligatory for the secretion of the proproteins containing this target. J Environ Pathol Toxicol Oncol, 1998, 17(3-4), 265 - 70 The effects of selenium deficiency, dietary selenium, and vitamin E supplementation on the oxidative status of pig liver; Sobajic SS et al.; The aim of this work was to determine the effect of selenium (Se) deficiency on the porcine liver oxidative stability and to investigate Se content and oxidative status in porcine liver after dietary supplementation with vitamin E (vit E), sodium selenite, and selenized yeast . Experimental animals were fed a basal corn meal, low in Se and vit E, for a 4-week depletion period before being given the experimental diets containing different levels of Se and/or vit E for 5 months . Dietary treatments were the basal diet with no additions (control); the basal diet supplemented with 25 mg of vit E/kg of feed (group I); basal diet + 0.3 mg selenite-Se/kg (group II); basal diet + 0.3 mg selenized yeast-Se/kg (group III); basal diet + 0.1 mg selenite-Se + 10 mg vit E/kg (group IV); and basal diet + 0.3 mg selenite-Se + 25 mg vit E/kg (group V) . The Se content in pig liver samples was 33 to 192% lower in the control group than in all the other groups . Dietary Se from selenized yeast had a more pronounced effect on Se level than dietary sodium selenite . The highest Se content was found in liver samples from the Se + vit E supplemented group (group V) . All the dietary supplementation schemes significantly improved the oxidative status of porcine liver compared with the control group samples . The best results were obtained by simultaneous dietary supplementation with Se + vit E (groups IV and V) > group III > group II > group I. J Environ Pathol Toxicol Oncol, 1998, 17(3-4), 251 - 7 Selenium intake as a modulator of responsiveness to oxidative stress; Jozanov-Stankov O et al.; A deficiency in important components of the endogenous antioxidative defense system (AODS) against the production of reactive oxygen species, including free radicals, results in the accumulation of oxidative damage, inducing oxidative stress . A dietary deficiency in selenium (Se), an important part of AODS, can increase the sensitivity of a living system to oxidative stress . We investigated the effects of Se supplementation, in the form of Se-enriched yeast, on the AODS resistance of red blood cells (RBC) to experimentally induced oxidative stress . We analyzed the alterations in main components of the AODS, such as the amount of reduced (GSH) oxidized glutathione (GSSG), Se-dependent glutathione peroxidase (GSH-Px), Se content, catalase (CAT), and thiobarbituric acid reactive substances (TBARS), in RBC of male Wistar rats exposed to gamma rays and supplemented with Se-enriched yeast (SeY) in drinking water . The results suggested that the increased Se level generally exhibited a protective effect against whole body irradiation, reducing the expenditure of the AODS components in defense . These reductions differed depending on the time observed and the parameter investigated but, generally, SeY supplementation induced a faster restoration of the AODS after this kind of oxidative stress. Int J Cancer, 1998 Sep 25, 78(1), 100 - 5 Delineation of the breakpoint at 18q21.1 in a cell line (Karpas1106) derived from mediastinal B-cell lymphoma by fluorescence in situ hybridization with multiple YAC clones; Tamura A et al.; The breakpoint of the 18q21 translocation of B-cell-non-Hodgkin's lymphoma (NHL) cell line Karpas1106P was delineated by fluorescence in situ hybridization (FISH) . Karpas1106P was derived from mediastinal lymphoblastic B-cell lymphoma and exhibited the immunophenotype characteristic of marginal-zone B-cell lymphoma (MZL): smIg+, pan-B antigen+, CD5-, CD10- and CD23- . The original G-banded karyotype showed a complex translocation containing t(X;18;13)(q28;q21;q12.1) . Double-color FISH (DCFISH) with whole-chromosome-painting (WCP) probes for chromosomes X, 13 and 18, and 18q-specific yeast artificial chromosome (YAC) clones defined t(X;18;13) as ider(X)t(X;18; 13)(q28;q 12.3q21.1;q12.1) . The immunoglobulin-heavy-chain (IgH) gene was not involved in the chromosomal translocation as detected by DCFISH with VH and Cgamma probes . By using contiguous YAC clones mapped from 18q12.3 to q21.1, we identified a YAC clone y852H2 with its breakpoint at 18q21.1 . In Karpas1106P, the distal part of chromosome 18 from the breakpoint (18q21.1-qter) was deleted, showing loss of heterozygosity of this region . In addition, the chromosomal segment 18q21.1 was duplicated and inserted to ider(X)t(X;18; 13) between Xq28 and 13q12.1 with maintaining its original orientation . The DNA sequence of the breakpoint region contained in y852H2 can serve as a candidate locus for further molecular dissection to identify the causative gene of MZL. Virus Res, 1998 Jun, 55(2), 167 - 76 Characterization of the interaction of the human respiratory syncytial virus phosphoprotein and nucleocapsid protein using the two-hybrid system; Slack MS et al.; The interaction between the human respiratory syncytial virus phosphoprotein (P) and nucleocapsid (N) protein has been investigated using the two hybrid system in yeast and in tissue culture cells . Deletion analysis identified two regions in the P protein involved in this interaction . The immediate carboxy-terminal 20 amino acids were essential for interaction with the N protein . Point mutations in this region demonstrated that alteration of two conserved, phosphorylated, serine residues reduced binding to 50% of that of the native protein . The introduction of two proline residues to disrupt the predicted alpha-helical domain in this region dramatically reduced the ability of the mutant P protein to interact with the N protein . A second region which affected the interaction of the two proteins was located adjacent to the essential carboxy-terminal area . Deletion of this second region resulted in an increase in the strength of the interaction between the two proteins . These data shows that the RSV P protein, while having no amino acid sequence identity with the equivalent P protein of other negative strand viruses, is likely to have similar structural and functional features. J Immunol, 1998 Sep 1, 161(5), 2391 - 9 The mannose receptor mediates induction of IFN-alpha in peripheral blood dendritic cells by enveloped RNA and DNA viruses; Milone MC et al.; Peripheral blood dendritic cells (DC) produce IFN-alpha in response to challenge by many enveloped viruses including herpes simplex virus (HSV) and HIV, whereas Sendai virus predominantly stimulates IFN-alpha production by monocytes . Glycosylated viral envelope proteins are known to be important for the induction of IFN-alpha . In this study we demonstrate that stimulation of IFN-alpha synthesis by HSV is inhibited by a number of monosaccharides, including fucose, N-acetylglucosamine, and N-acetylgalactosamine as well as the yeast polysaccharide mannan, supporting a role for lectin(s) in the IFN-alpha stimulation pathway . Furthermore, antiserum to the mannose receptor (MR) also inhibited HSV, vesicular stomatitis virus, and HIV-induced IFN-alpha production, but failed to inhibit the IFN-alpha induced by Sendai virus . We further demonstrated that freshly isolated blood DC and IFN-alpha-producing cells responding to HSV stimulation express the MR . This study therefore implicates the MR as an important receptor for the nonspecific recognition of enveloped viruses by DC and the subsequent stimulation of IFN-alpha production by these viruses . Thus, the MR probably serves as a critical link between innate and adaptive immunity to viruses, especially given the role of the MR in Ag capture by DC and the importance of IFN-alpha in shaping immunity. Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 11020 - 5 Protein kinase CK2 interacts with and phosphorylates the Arabidopsis circadian clock-associated 1 protein; Sugano S et al.; The circadian clock-associated 1 (CCA1) gene encodes a Myb-related transcription factor that has been shown to be involved in the phytochrome regulation of Lhcb1*3 gene expression and in the function of the circadian oscillator in Arabidopsis thaliana . By using a yeast interaction screen to identify proteins that interact with CCA1, we have isolated a cDNA clone encoding a regulatory (beta) subunit of the protein kinase CK2 and have designated it as CKB3 . CKB3 is the only reported example of a third beta-subunit of CK2 found in any organism . CKB3 interacts specifically with CCA1 both in a yeast two-hybrid system and in an in vitro interaction assay . Other subunits of CK2 also show an interaction with CCA1 in vitro . CK2 beta-subunits stimulate binding of CCA1 to the CCA1 binding site on the Lhcb1*3 gene promoter, and recombinant CK2 is able to phosphorylate CCA1 in vitro . Furthermore, Arabidopsis plant extracts contain a CK2-like activity that affects the formation of a DNA-protein complex containing CCA1 . These results suggest that CK2 can modulate CCA1 activity both by direct interaction and by phosphorylation of the CCA1 protein and that CK2 may play a role in the function of CCA1 in vivo. Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10890 - 5 Characterization of a calmodulin kinase II inhibitor protein in brain; Chang BH et al.; Ca2+/calmodulin-dependent protein kinase II (CaM-KII) regulates numerous physiological functions, including neuronal synaptic plasticity through the phosphorylation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors . To identify proteins that may interact with and modulate CaM-KII function, a yeast two-hybrid screen was performed by using a rat brain cDNA library . This screen identified a unique clone of 1.4 kb, which encoded a 79-aa brain-specific protein that bound the catalytic domain of CaM-KII alpha and beta and potently inhibited kinase activity with an IC50 of 50 nM . The inhibitory protein (CaM-KIIN), and a 28-residue peptide derived from it (CaM-KIINtide), was highly selective for inhibition of CaM-KII with little effect on CaM-KI, CaM-KIV, CaM-KK, protein kinase A, or protein kinase C . CaM-KIIN interacted only with activated CaM-KII (i . e., in the presence of Ca2+/CaM or after autophosphorylation) by using glutathione S-transferase/CaM-KIIN precipitations as well as coimmunoprecipitations from rat brain extracts or from HEK293 cells cotransfected with both constructs . Colocalization of CaM-KIIN with activated CaM-KII was demonstrated in COS-7 cells transfected with green fluorescent protein fused to CaM-KIIN . In COS-7 cells phosphorylation of transfected alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors by CaM-KII, but not by protein kinase C, was blocked upon cotransfection with CaM-KIIN . These results characterize a potent and specific cellular inhibitor of CaM-KII that may have an important role in the physiological regulation of this key protein kinase. Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10774 - 8 Equilibrium distributions of microsatellite repeat length resulting from a balance between slippage events and point mutations; Kruglyak S et al.; We describe and test a Markov chain model of microsatellite evolution that can explain the different distributions of microsatellite lengths across different organisms and repeat motifs . Two key features of this model are the dependence of mutation rates on microsatellite length and a mutation process that includes both strand slippage and point mutation events . We compute the stationary distribution of allele lengths under this model and use it to fit DNA data for di-, tri-, and tetranucleotide repeats in humans, mice, fruit flies, and yeast . The best fit results lead to slippage rate estimates that are highest in mice, followed by humans, then yeast, and then fruit flies . Within each organism, the estimates are highest in di-, then tri-, and then tetranucleotide repeats . Our estimates are consistent with experimentally determined mutation rates from other studies . The results suggest that the different length distributions among organisms and repeat motifs can be explained by a simple difference in slippage rates and that selective constraints on length need not be imposed. Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10649 - 54 Deletion of long-range regulatory elements upstream of SOX9 causes campomelic dysplasia; Wunderle VM et al.; Campomelic dysplasia (CD) is a rare, neonatal human chondrodysplasia characterized by bowing of the long bones and often associated with male-to-female sex-reversal . Patients present with either heterozygous mutations in the SOX9 gene or chromosome rearrangements mapping at least 50 kb upstream of SOX9 . Whereas mutations in SOX9 ORF cause haploinsufficiency, the effects of translocations 5' to SOX9 are unclear . To test whether these rearrangements also cause haploinsufficiency by altering spatial and temporal expression of SOX9, we generated mice transgenic for human SOX9-lacZ yeast artificial chromosomes containing variable amounts of DNA sequences upstream of SOX9 . We show that elements necessary for SOX9 expression during skeletal development are highly conserved between mouse and human and reveal that a rearrangement upstream of SOX9, similar to those observed in CD patients, leads to a substantial reduction of SOX9 expression, particularly in chondrogenic tissues . These data demonstrate that important regulatory elements are scattered over a large region upstream of SOX9 and explain how particular aspects of the CD phenotype are caused by chromosomal rearrangements 5' to SOX9. Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10602 - 7 WW domain-mediated interactions reveal a spliceosome-associated protein that binds a third class of proline-rich motif: the proline glycine and methionine-rich motif; Bedford MT et al.; Pre-mRNA splicing requires the bridging of the 5' and 3' ends of the intron . In yeast, this bridging involves interactions between the WW domains in the splicing factor PRP40 and a proline-rich domain in the branchpoint binding protein, BBP . Using a proline-rich domain derived from formin (a product of the murine limb deformity locus), we have identified a family of murine formin binding proteins (FBP's), each of which contains one or more of a special class of tyrosine-rich WW domains . Two of these WW domains, in the proteins FBP11 and FBP21, are strikingly similar to those found in the yeast splicing factor PRP40 . We show that FBP21 is present in highly purified spliceosomal complex A, is associated with U2 snRNPs, and colocalizes with splicing factors in nuclear speckle domains . Moreover, FBP21 interacts directly with the U1 snRNP protein U1C, the core snRNP proteins SmB and SmB', and the branchpoint binding protein SF1/mBBP . Thus, FBP21 may play a role in cross-intron bridging of U1 and U2 snRNPs in the mammalian A complex. Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10596 - 601 The adenomatous polyposis coli-binding protein EB1 is associated with cytoplasmic and spindle microtubules; Berrueta L et al.; The evolutionarily conserved protein EB1 originally was identified by its physical association with the carboxyl-terminal portion of the adenomatous polyposis coli (APC) tumor suppressor protein, an APC domain commonly mutated in familial and sporadic forms of colorectal neoplasia . The subcellular localization of EB1 in epithelial cells was studied by using immunofluorescence and biochemical techniques . EB1 colocalized both to cytoplasmic microtubules in interphase cells and to spindle microtubules during mitosis, with pronounced centrosome staining . The cytoskeletal array detected by anti-EB1 antibody was abolished by incubation of the cells with nocodazole, an agent that disrupts microtubules; upon drug removal, EB1 localized to the microtubule-organizing center . Immunofluorescence analysis of SW480, a colon cancer cell line that expresses only carboxyl-terminal-deleted APC unable to interact with EB1, demonstrated that EB1 remained localized to the microtubule cytoskeleton, suggesting that this pattern of subcellular distribution is not mediated by its interaction with APC . In vitro cosedimentation with taxol-stabilized microtubules demonstrated that a significant fraction of EB1 associated with microtubules . Recent studies of the yeast EB1 homologues Mal3 and Bim1p have demonstrated that both proteins localize to microtubules and are important in vivo for microtubule function . Our results demonstrate that EB1 is a novel component of the microtubule cytoskeleton in mammalian cells . Associating with the mitotic apparatus, EB1 may play a physiologic role connecting APC to cellular division, coordinating the control of normal growth and differentiation processes in the colonic epithelium. Mol Cell Endocrinol, 1998 May 25, 140(1-2), 9 - 14 Imprinting of the mouse Igf2r gene depends on an intronic CpG island; Wutz A et al.; Gametic imprinting is a developmental process that uses cis-acting epigenetic mechanisms to induce parental-specific expression in autosomal and X-linked genes . From the limited knowledge we have of imprinted genes it seems clear that they play a role in regulating the growth and development of the placenta and embryo in utero, and also in the early post-natal phase . The biological function of imprinting in mammals is not yet understood but it may act to regulate the supply of nutrition to the embryo in order to maintain a balance between fetal demands and maternal resources . The molecular basis of imprinting any gene has not yet been disclosed . However . the current model proposes that single or clustered genes should be associated with a specific imprinting element which carries a reversible epigenetic imprinting signal, inherited from one parent and maintained on the same parental chromosome in diploid cells . A common imprinting recognition sequence has not been identified amongst the 20 known imprinted genes, but such an element is thought to exist from observations of mouse transgenes and transgenes of the imprinted H19 gene, which have shown that short DNA sequences can maintain imprinted expression at different chromosome locations . However, it is also clear from gene deletion experiments in the mouse and the analysis of deletions in the human Prader-Willi/Angelman syndromes that imprinted genes are also influenced by distant DNA sequences . We have earlier proposed that CG rich sequences resembling CpG islands, which are associated with many imprinted genes and often subject to parental-specific methylation, could act as a common imprinting element . The mouse insulin-like growth factor type 2 receptor (Igf2r) gene contains a CpG island known as region2, in the second intron . Region2 was proposed as the imprinting element of this gene because it inherited a methylation imprint from the female gamete that was maintained only on the maternal chromosome in diploid cells . We have used YAC (yeast artificial chromosome) transgenes carrying the complete Igf2r locus, to test if imprinting and parental-specific methylation of the mouse Igf2r gene is maintained when transferred to other chromosomal locations and to test whether imprinting is dependent on the intronic CpG island proposed as the imprinting element for this gene . Gametic imprints are epigenetic modifications which are imposed onto the gametic chromosomes and cause parental-specific differences in the expression of a small number of genes in the embryo . As a consequence, correct imposition of the imprints in the parental germlines is a prerequisite for successful development of mammals and any anomaly in the expression of imprinted genes is often accompanied by aberration of embryonic growth . The link between imprinting and growth regulation is best exemplified by the Igf2 and Igf2r genes . Both genes show parental-specific expression patterns in the embryo . Igf2 is a general embryonic mitogen, and mice lacking Igf2 are markedly reduced in size (DeChiara, T.M., Robertson, E.J., Efstratiadis, A., 1991 . Parental imprinting of the mouse insulin-like growth factor II gene . Cell 64, 849-859) . Igf2r acts to fine tune the amount of growth factor, and embryos lacking this gene show overgrowth and die perinatally (Lau, M.M., Stewart, C.E., Liu, Z., Bhatt, H., Rotwein, P., Stewart, C.L., 1994 . Loss of the imprinted IGF2/cation-independent mannose 6-phosphate receptor results in fetal overgrowth and perinatal lethality . Genes Dev . 8, 2953 2963; Wang, Z.Q., Fung, M.R., Barlow, D.P., Wagner, E.F., 1994 . Regulation of embryonic growth and lysosomal targeting by the imprinted Igf2/Mpr gene . Nature 372, 464-467) . The phenomenon of gametic imprinting is predicted to arise by an unusual regulation of the imposition and erasure of epigenetic modifications . There are three events which are required for imprinting: firstly, the imprint must be imposed in one of the gametes before fertilisat J Appl Microbiol, 1998 Mar, 84(3), 368 - 76 Evaluation of the preservative properties of Thymus vulgaris essential oil in topically applied formulations under a challenge test; Manou I et al.; The preservative properties of thyme essential oil (3%) with a known composition were evaluated in two types of final formulations, suitable for use as pharmaceutical or cosmetic vehicles, by means of a standard challenge test proposed by the latest European Pharmacopoeia . The required preservation efficacy criteria were satisfied against the bacterial strains, against the yeast in one of the formulations, but not against the mould strain involved in this study . Interactions between the essential oil compounds and other factors present in the final formulation might have influenced the activity of this essential oil, leading to an incomplete satisfaction of the criteria. Genomics, 1998 Aug 1, 51(3), 417 - 26 Two sequence-ready contigs spanning the two copies of a 200-kb duplication on human 21q: partial sequence and polymorphisms; Potier M et al.; Physical mapping across a duplication can be a tour de force if the region is larger than the size of a bacterial clone . This was the case of the 170- to 275-kb duplication present on the long arm of chromosome 21 in normal human at 21q11.1 (proximal region) and at 21q22.1 (distal region), which we described previously . We have constructed sequence-ready contigs of the two copies of the duplication of which all the clones are genuine representatives of one copy or the other . This required the identification of four duplicon polymorphisms that are copy-specific and nonallelic variations in the sequence of the STSs . Thirteen STSs were mapped inside the duplicated region and 5 outside but close to the boundaries . Among these STSs 10 were end clones from YACs, PACs, or cosmids, and the average interval between two markers in the duplicated region was 16 kb . Eight PACs and cosmids showing minimal overlaps were selected in both copies of the duplication . Comparative sequence analysis along the duplication showed three single-basepair changes between the two copies over 659 bp sequenced (4 STSs), suggesting that the duplication is recent (less than 4 mya) . Two CpG islands were located in the duplication, but no genes were identified after a 36-kb cosmid from the proximal copy of the duplication was sequenced . The homology of this chromosome 21 duplicated region with the pericentromeric regions of chromosomes 13, 2, and 18 suggests that the mechanism involved is probably similar to pericentromeric-directed mechanisms described in interchromosomal duplications . Genomics, 1998 Aug 1, 51(3), 351 - 8 Molecular cloning and characterization of a novel retinoblastoma-binding protein; Fusco C et al.; We describe the isolation and characterization of a novel cDNA encoding a polypeptide that interacts in a yeast two-hybrid system as well as in mammalian cells with the retinoblastoma (RB) protein . This new protein, which we call Rim, consists of 897 amino acids, has two leucine zipper motifs, and has a LECEE sequence previously identified as an RB-binding domain . Rim also has an E1A/CtBP-binding motif and four putative nuclear localization signals . Rim mRNA is expressed ubiquitously at low levels in all human adult tissues tested and at much higher levels in several tumor cell lines . The Rim gene (HGMW-approved symbol RBBP8) is localized on human chromosome 18q11.2 . Genomics, 1998 Aug 1, 51(3), 325 - 31 Human rod monochromacy: linkage analysis and mapping of a cone photoreceptor expressed candidate gene on chromosome 2q11; Wissinger B et al.; We have performed linkage analysis in eight families with rod monochromacy, an autosomal recessively inherited condition with complete color blindness . Significant linkage was found with markers located at the pericentromeric region of chromosome 2 . A maximum lod score of 5.36 was obtained for marker D2S2333 at theta = 0.00 . Mapping of meiotic breakpoints localized the disease gene between markers D2S2187 and D2S2229 . Homozygosity for a number of subsequent markers indicating identity by descent was found in two families and provides evidence for a further refinement of the locus proximal to D2S373 . This defines an interval of approximately 3 cM covering the ACHM2 locus for rod monochromacy . Radiation hybrid mapping of the CNGA3 gene encoding the alpha-subunit of the cGMP gated cation channel in human cone photoreceptors resulted in a maximum lod score of 16.1 with marker D2S2311 combined with a calculated physical distance of 6.19cR10,000 . Screening of the CEPH YAC library and subsequent STS mapping indicated the physical order cen-D2S2222-D2S2175-(D2S2187/D2S2311)-qtel ofmarkers on 2q11 and showed that the CNGA3 gene maps most closely to D2S2187 and D2S2311 . These data indicate that the CNGA3 gene maps within the critical interval of the ACHM2 locus for rod monochromacy and thus is a candidate gene for this disease . J Mol Biol, 1998 Sep 4, 281(5), 949 - 68 Method for prediction of protein function from sequence using the sequence-to-structure-to-function paradigm with application to glutaredoxins/thioredoxins and T1 ribonucleases; Fetrow JS et al.; The practical exploitation of the vast numbers of sequences in the genome sequence databases is crucially dependent on the ability to identify the function of each sequence . Unfortunately, current methods, including global sequence alignment and local sequence motif identification, are limited by the extent of sequence similarity between sequences of unknown and known function; these methods increasingly fail as the sequence identity diverges into and beyond the twilight zone of sequence identity . To address this problem, a novel method for identification of protein function based directly on the sequence-to-structure-to-function paradigm is described . Descriptors of protein active sites, termed "fuzzy functional forms" or FFFs, are created based on the geometry and conformation of the active site . By way of illustration, the active sites responsible for the disulfide oxidoreductase activity of the glutaredoxin/thioredoxin family and the RNA hydrolytic activity of the T1 ribonuclease family are presented . First, the FFFs are shown to correctly identify their corresponding active sites in a library of exact protein models produced by crystallography or NMR spectroscopy, most of which lack the specified activity . Next, these FFFs are used to screen for active sites in low-to-moderate resolution models produced by ab initio folding or threading prediction algorithms . Again, the FFFs can specifically identify the functional sites of these proteins from their predicted structures . The results demonstrate that low-to-moderate resolution models as produced by state-of-the-art tertiary structure prediction algorithms are sufficient to identify protein active sites . Prediction of a novel function for the gamma subunit of a yeast glycosyl transferase and prediction of the function of two hypothetical yeast proteins whose models were produced via threading are presented . This work suggests a means for the large-scale functional screening of genomic sequence databases based on the prediction of structure from sequence, then on the identification of functional active sites in the predicted structure . J Mol Biol, 1998 Sep 4, 281(5), 871 - 84 Vibrational dynamics of transfer RNAs: comparison of the free and synthetase-bound forms; Bahar I et al.; The vibrational dynamics of transfer RNAs, both free, and complexed with the cognate synthetase, are analyzed using a model (Gaussian network model) which recently proved to satisfactorily describe the collective motions of folded proteins . The approach is similar to a normal mode analysis, with the major simplification that no residue specificity is taken into consideration, which permits us (i) to cast the problem into an analytical form applicable to biomolecular systems including about 10(3 )residues, and (ii) to acquire information on the essential dynamics of such large systems within computational times at least two orders of magnitude shorter than conventional simulations . On a local scale, the fluctuations calculated for yeast tRNAPhe and tRNAAsp in the free state, and for tRNAGln complexed with glutaminyl-tRNA synthetase (GlnRS) are in good agreement with the corresponding crystallographic B factors . On a global scale, a hinge-bending region comprising nucleotides U8 to C12 in the D arm, G20 to G22 in the D loop, and m7G46 to C48 in the variable loop (for tRNAPhe), is identified in the free tRNA, conforming with previous observations . The two regions subject to the largest amplitude anticorrelated fluctuations in the free form, i.e . the anticodon region and the acceptor arm are, at the same time, the regions that experience the most severe suppression in their flexibilities upon binding to synthetase, suggesting that their sampling of the conformational space facilitates their recognition by the synthetase . Likewise, examination of the global mode of motion of GlnRS in the complex indicates that residues 40 to 45, 260 to 270, 306 to 314, 320 to 327 and 478 to 485, all of which cluster near the ATP binding site, form a hinge-bending region controlling the cooperative motion, and thereby the catalytic function, of the enzyme . The distal beta-barrel and the tRNA acceptor binding domain, on the other hand, are distinguished by their high mobilities in the global modes of motion, a feature typical of recognition sites, also observed for other proteins . Most of the conserved bases and residues of tRNA and GlnRS are severely constrained in the global motions of the molecules, suggesting their having a role in stabilizing and modulating the global motion . Genes Chromosomes Cancer, 1998 Sep, 23(1), 74 - 7 Identification of a 100-kb region of common allelic loss on chromosome bands 10q25-q26 in human endometrial cancer; Yamakawa H et al.; In human endometrial cancer, we have previously identified a 790-kb region of common allelic loss in chromosome bands 10q25-q26, flanked by D10S587 and D10S1723 . We constructed a contig covering the entire deleted region using YACs, PACs, and BACs . Five overlapping cosmid clones derived from YAC clones completely covered the entire deleted region: its size was estimated to be no larger than 200 kb . We further performed two-color fluorescence in situ hybridization (FISH) analysis to confirm the deletion and narrowed down the deleted region to 100 kb or less; it was covered by three overlapping cosmid clones that were included in one BAC clone . Restriction endonuclease mapping identified a region in which NotI, SalI, SmaI, and Xhol were clustered, suggesting the possible existence of a CpG island. Life Sci, 1998, 63(8), 693 - 9 Sterically stabilized phospholipids attenuate human neutrophils chemotaxis in vitro; Hatipoglu U et al.; The purpose of this study was to determine whether sterically stabilized liposomes (SSL) and poly(ethylene glycol)-distearoylphosphatidylethanolamine (PEG-DSPE) attenuate polymorphonuclear neutrophils (PMNs) chemotaxis in vitro and, if so, whether incorporation of vasoactive intestinal peptide (VIP), a pleiotropic neuropeptide, on the surface of SSL amplifies SSL-induced responses . Using a modified blind-well chamber chemotaxis assay, we found that N-formyl-methionyl-leucyl-phenylalanine (FMLP; 0.1 microM) and zymosan opsonized with purified human complement (2 x 10(9) yeast wall particles/ml) elicit significant human PMNs chemotaxis (95+/-9 and 103+/-3 cells/high power field; p<0.05) . These effects are significantly attenuated by SSL and PEG-DSPE (p<0.05) . By contrast, aqueous VIP and VIP on SSL have no significant effects on FMLP- and zymosan-induced responses . We conclude that certain sterically stabilized liposomes and phospholipids attenuate human PMNs chemotaxis in vitro and that VIP does not modulate this response. Blood, 1998 Sep 1, 92(5), 1735 - 42 Consistent fusion of ZNF198 to the fibroblast growth factor receptor-1 in the t(8;13)(p11;q12) myeloproliferative syndrome; Reiter A et al.; The 8p11 myeloproliferative syndrome is a rare, aggressive condition associated with reciprocal translocations of chromosome band 8p11, most commonly the t(8;13)(p11;q12) . To identify the genes involved in this translocation, we used fluorescence in situ hybridization (FISH) analysis to show that the chromosome 8 breakpoints fell within YAC 899e2 and that the chromosome 13 breakpoints are clustered in a region flanked by YACs 929f11 and 911h8 . FISH using chromosome 13 PAC clones indicated that the t(8;13) is not simply a reciprocal translocation but also involves an inversion of 13q11-12 . Exon trapping of a PAC that spanned the chromosome 13 translocation breakpoints led to the identification of a gene, ZNF198, that detected rearranged bands when used as a probe against Southern blots of patient DNA . Conceptual translation of the full-length ZNF198 cDNA sequence predicts a protein of 1377 amino acids that shows significant homology to the DXS6673E/KIAA0385 and KIAA0425 proteins . Alignment of these three proteins revealed a novel, conserved Zn-finger-related motif (MYM domain) of the general form CX2C19-22CX3CX13-19CX2CX19-25FCX3CX3F/Y that is repeated five times in each protein . To identify the translocation partner gene on chromosome 8, 5' and 3' RACE polymerase chain reactions (PCRs) were performed on patient RNA with several combinations of ZNF198 primers . Clones were identified in which the ZNF198 was fused to exon 9 of the fibroblast growth factor receptor-1 (FGFR1), a gene known to map to 8p11 . An identical ZNF198-FGFR1 fusion was detected in the three patients with a t(8;13) for whom RNA was available; reciprocal FGFR1-ZNF198 transcripts were not detected . Rearrangements of both ZNF198 and FGFR1 were found in two further patients by Southern blotting . ZNF198-FGFR1 includes the five MYM domains of ZNF198 and the intracellular tyrosine kinase domain of FGFR1 . We hypothesize that this fusion leads to constitutive activation of the FGFR1 tyrosine kinase in a manner analogous to the activation of ABL by BCR in chronic myeloid leukemia . Eur J Pharmacol, 1998 Jul 10, 352(2-3), 299 - 305 Effects of non-steroidal anti-inflammatory drugs on the luminol and lucigenin amplified chemiluminescence of human neutrophils; Parij N et al.; A panel of non-steroidal anti-inflammatory drugs commonly used for therapeutic purposes was assessed for their effects on the respiratory burst of isolated human polymorphonuclear neutrophils . Cells were stimulated with opsonised yeast and the production of reactive oxygen species was measured by amplified chemiluminescence with luminol and lucigenin which are two luminogenic agents measuring different cellular events . A special attention was devoted to the establishment of dose-effect curves and calculation of ED50 . Some of the drugs tested (acemetacine, diclofenac, flufenamic acid and niflumic acid) were able to decrease both luminol and lucigenin chemiluminescence in a dose-dependent manner reflecting an inhibitory effect on the respiratory burst . The most potent derivative was flufenamic acid (ED50 8 and 78 microM, respectively, with luminol and lucigenin), followed by diclofenac (21 and 98 microM), niflumic acid (97 and 227 microM) and acemetacine (585 and 427 microM) . In contrast, several other drugs (flurbiprofen, ibuprofen, ketoprofen, piroxicam) stimulated both luminol and lucigenin chemiluminescence, suggesting a pro-oxidant activity . Acetylsalicylic acid (up to 1250 microM) was a modest inhibitor (maximum 25% inhibition) showing no dose-dependent effect and tolmetin (up to 125 microM) had no significant effect in both systems . The results were in agreement using both luminogenic agents, except for indomethacin, naproxen and tenoxicam which showed different kinds of effects . The unspecific and complex nature of the measurement systems used did not allow to give a complete mechanistic interpretation of the results, but the comparison with literature data gave some pertinent explanations for both anti- and pro-oxidant effects. Eur J Cell Biol, 1998 Jul, 76(3), 167 - 75 Isolation of nucleation-competent centrosomes from Dictyostelium discoideum; Graf R et al.; The centrosome of Dictyostelium discoideum is a box-shaped, layered core structure surrounded by a corona which is made up of dense nodules embedded in amorphous material . It is also known as nucleus-associated body . Because of its tight association with the nucleus the centrosome has resisted so far all attempts for isolation in sufficient purity and quantity for biochemical analysis . Here we report on the large-scale isolation of D . discoideum centrosomes after treatment of nucleus-centrosome complexes with a buffer containing sodium pyrophosphate . Following heparin treatment and a filtration step, centrosomes were further purified by density gradient centrifugation . Immunofluorescence analysis of the isolated centrosomes revealed the presence of the D . discoideum 350-kDa antigen, a centrosomal marker protein, gamma-tubulin, and the D . discoideum homologues of pericentrin, Spc110p, and Cdc31p . The structural integrity of the isolated centrosomes was demonstrated by confocal laser microscopy and electron microscopy . Microtubule nucleation assays with purified pig brain tubulin showed that the isolation procedure did not only preserve the structure but also the functionality of the isolated centrosomes . D . discoideum centrosomes should now become an attractive new model system in addition to, and for comparison with, centriolar centrosomes and yeast spindle pole bodies. Br J Cancer, 1998 Aug, 78(4), 495 - 503 Molecular characterization of a novel amplicon at 1q21-q22 frequently observed in human sarcomas; Forus A et al.; In a recent comparative genomic hybridization (CGH) study of a panel of sarcomas, we detected recurrent amplification of 1q21-q22 in soft tissue and bone tumours . Amplification of this region had not previously been associated with sarcoma development, but occasional amplification of CACY/S100A6 and MUC1 in 1q21 had been reported for melanoma and breast carcinoma respectively . Initial screening by Southern blot analysis showed amplification of S100A6, FLG and SPRR3 in several sarcomas and, in a first attempt to characterize the 1q21-q22 amplicon in more detail, we have now investigated the amplification status of these and 11 other markers in the region in 35 sarcoma samples . FLG was the most frequently amplified gene, and the markers located in the same 4.5-Mb region as FLG showed a higher incidence of amplification than the more distal ones . However, for most of the 14 markers, amplification levels were low, and only APOA2 and the anonymous marker D1S3620 showed high-level amplifications (> tenfold increases) in one sample each . We used fluorescence in situ hybridization (FISH) to determine the amplification patterns of two overlapping yeast artificial chromosomes (YACs) covering the region between D1S3620 and FLG (789f2 and 764a1), as well as two more distally located YACs in nine selected samples . Six samples had amplification of the YAC containing D1S3620 and, in three, 764a1 was also included . Five of these tumours showed normal copies of the more distal YACs; thus, it seems likely that an important gene may be located within 789f2, or very close . Two samples had high copy numbers of the most distal YACs . Taken together, FISH and molecular analyses indicate complex amplification patterns in 1q21-q22 with at least two amplicons: one located near D1S3620/789f2 and one more distal. Oncogene, 1998 Aug 13, 17(6), 727 - 31 Germline mutations in PTEN are an infrequent cause of genetic predisposition to breast cancer; FitzGerald MG et al.; Heterozygous germline mutations in PTEN are responsible for most cases of Cowden Syndrome, a rare familial trait characterized by hamartomas and by predisposition to cancer of the breast and thyroid . The variable and often subtle clinical findings that characterize Cowden Syndrome are frequently unrecognized, raising the possibility that germline PTEN mutations may confer susceptibility to breast cancer in women who have not been diagnosed with this syndrome . To determine whether such mutations contribute to genetic predisposition to breast cancer within the general population, we analysed a cohort of women with early-onset breast cancer (< age 40), a subset of the population at increased risk for genetic susceptibility . Lymphoblast cell lines were analysed using either direct nucleotide sequencing (28 cases), denaturing gradient gel electrophoresis (DGGE) (34 cases) or a yeast-based truncation assay (110 cases) . No definitive, truncating mutations were observed in 172 patients . Missense changes were noted in the germline of 2/60 patients analysed by direct nucleotide sequencing or DGGE, including a non-conservative amino acid substitution within the phosphatase domain, but neither showed loss of the wild-type allele in the corresponding breast tumor specimen . We conclude that germline mutations in PTEN are an uncommon cause of genetic predisposition to breast cancer within the general population. Gene, 1998 Jul 30, 215(2), 259 - 67 YAC/STS map of 15Mb of Xp21.3-p11.3, at 100kb resolution, with refined comparisons of genetic distances and DMD structure; Nagaraja R et al.; The 15<HSP SP = "0.25">Mb region between DXS997 and DXS8054 in Xp21.3-p11.3 has been mapped at seven-fold average coverage in yeast artificial chromosomes (YACs) and 100 kb inter-sequence tagged site (STS) distance . YACs from six different collections show self-consistent maps . The STSs include 18 (CA) repeat and one tetranucleotide repeat marker that detect polymorphism, as well as eight well-studied genes, a second site for MXS1 sequences, and three expressed sequence tags (ESTs) . One of the ESTs maps to intron 7 of Duchenne muscular dystrophy, and seems to be a processed intronic sequence with a poly(A) tail. Gene, 1998 Aug 17, 216(1), 139 - 47 Isolation and characterization of cDNAs encoding PDE5A, a human cGMP-binding, cGMP-specific 3',5'-cyclic nucleotide phosphodiesterase; Loughney K et al.; Human cGMP-binding, cGMP-specific 3',5'-cyclic nucleotide phosphodiesterase (PDE5A) cDNAs were isolated . A 3.1-kb composite DNA sequence assembled from overlapping cDNAs encodes an 875-amino-acid protein with a predicted molecular mass of 100012 Da (PDE5A1) . Extracts prepared from yeast expressing human PDE5A1 hydrolyzed cGMP . This activity was inhibited by the selective PDE5 inhibitors zaprinast and DMPPO . PDE5A mRNA is expressed in aortic smooth muscle cells, heart, placenta, skeletal muscle and pancreas and, to a much lesser extent, in brain, liver and lung . A 5'-splice variant, PDE5A2, encodes an 833-amino-acid protein with eight unique amino acids at the amino terminus . PDE5A maps to chromosome 4q 25-27. Biochim Biophys Acta, 1998 Aug 14, 1404(1-2), 9 - 31 SNAREs and membrane fusion in the Golgi apparatus; Nichols BJ et al.; Soluble factors, NSF and SNAPs, are required at many membrane fusion events within the cell . They interact with a class of type II integral membrane proteins termed SNAP receptors, or SNAREs . Interaction between cognate SNAREs on opposing membranes is a prerequisite for NSF dependent membrane fusion . NSF is an ATPase which will disrupt complexes composed of different SNAREs . However, there is increasingly abundant evidence that the SNARE complex recognised by NSF does not bridge the two fusing membranes, but rather is composed of SNAREs in the same membrane . The essential role of NSF may be to prime SNAREs for a direct role during fusion . The best characterised SNAREs in the Golgi are Sed5p in yeast and its mammalian homologue syntaxin 5, both of which are predominantly localised to the cis Golgi . The SNARE-SNARE interactions in which these two proteins are involved are strikingly similar . Sed5p and syntaxin 5 may mediate three distinct pathways for membrane flow into the cis Golgi, one from the ER, one from later Golgi cisternae, and possibly a third from endosomes . Syntaxin 5 is itself likely to cycle through the ER, and thus may be involved in homotypic fusion of ER derived transport vesicles . In all well characterised SNARE dependent membrane fusion events one of the interacting SNAREs is a syntaxin homologue . There are only eight members of the syntaxin family in yeast . Besides Sed5p two others, Tlg1p and Tlg2p, are found in the Golgi complex . They are present in a late Golgi compartment, but neither is required for transit of secreted proteins through the Golgi . We suggest that these observations are most compatible with a model for transit through the Golgi in which anterograde cargo is carried in cisternae, the enzymatic composition of which changes with time as Golgi resident enzymes are delivered in retrograde transport vesicles. Gene, 1998 Aug 17, 216(1), 1 - 11 A tale of two termini: a functional interaction between the termini of an mRNA is a prerequisite for efficient translation initiation; Gallie DR; A quarter of century following the prediction that mRNAs are translated in a circular form, recent biochemical and genetic evidence has accumulated to support the idea that communication between the termini of an mRNA is necessary to promote translation initiation . The poly(A)-binding protein (PABP) interacts with the cap-associated eukaryotic initiation factor (eIF) 4G (in yeast and plants) and eIF4B (in plants), a functional consequence of which is to increase the affinity of PABP for poly(A) and to increase the affinity of the cap-binding complex, eIF4F (of which eIF4G is a subunit) for the 5' cap structure . In mammals, PABP interacts with a novel PABP-interacting protein that also binds eIF4A . The interaction between PABP and those initiation factors associated with the 5' terminus of an mRNA may also explain the role of PABP during mRNA turnover, as it protects the 5' cap from attack by Dcp1p, the decapping enzyme . Several of those mRNAs that have evolved functional equivalents to a cap or a poly(A) tail nevertheless require a functional interaction between terminal regulatory elements similar to that observed between the 5' cap and poly(A) tail, suggesting that efficient translation is predicated on communication between largely-separated regulatory elements within an mRNA. Trends Cell Biol, 1998 Jul, 8(7), 282 - 8 The AP-3 complex: a coat of many colours; Odorizzi G et al.; A new adaptor protein complex, termed AP-3, has recently been identified in mammalian cells, and genetic studies in yeast have revealed a functional role for the AP-3 complex in cargo-selective transport via a new alternative trafficking pathway from the Golgi to the vacuole/lysosome . Here, the authors review what is currently known about the AP-3 complex and discuss recent insight into its function in multicellular organisms that has come from the finding that mutations in AP-3 subunits correspond to classical mutations in Drosophila and mice. Biochem Pharmacol, 1998 Jun 15, 55(12), 2007 - 15 Mechanisms of inhibition of aldehyde dehydrogenase by nitroxyl, the active metabolite of the alcohol deterrent agent cyanamide; DeMaster EG et al.; Nitroxyl, produced in the bioactivation of the alcohol deterrent agent cyanamide, is a potent inhibitor of aldehyde dehydrogenase (AIDH); however, the mechanism of inhibition of AlDH by nitroxyl has not been described previously . Nitroxyl is also generated from Angeli's salt (Na2N2O3) at physiological pH, and, indeed, Angeli's salt inhibited yeast AlDH in a time- and concentration-dependent manner, with IC50 values under anaerobic conditions with and without NAD+ of 1.3 and 1.8 microM, respectively . Benzaldehyde, a substrate for AlDH, competitively blocked the inhibition of this enzyme by nitroxyl in the presence of NAD+, but not in its absence, in accord with the ordered mechanism of this reaction . The sulfhydryl reagents dithiothreitol (5 mM) and reduced glutathione (10 mM) completely blocked the inhibition of AlDH by Angeli's salt . These thiols were also able to partially restore activity to the nitroxyl-inhibited enzyme, the extent of reactivation being dependent on the pH at which the inactivation occurred . This pH dependency indicates the formation of two inhibited forms of the enzyme, with an irreversible form predominant at pH 7.5 and below, and a reversible form predominant at pH 8.5 and above . The reversible form of the inhibited enzyme is postulated to be an intra-subunit disulfide, while the irreversible form is postulated to be a sulfinamide . Both forms of the inhibited enzyme are derived via a common N-hydroxysulfenamide intermediate produced by the addition of nitroxyl to active site cysteine thiol(s). J Gen Virol, 1998 Aug, 79 ( Pt 8), 2043 - 9 In vitro interactions between a potyvirus-encoded, genome-linked protein and RNA-dependent RNA polymerase; Fellers J et al.; Recent studies have shown that potyvirus VPg/ proteinases and RNA-dependent RNA polymerases are capable of protein-protein interactions in yeast cells . We have extended these studies in vitro . We found that tobacco vein mottling virus (TVMV) VPg is retained on glutathione-Sepharose matrices if co-incubated with a glutathione S-transferase (GST)-NIb fusion protein, but not with GST, which is suggestive of a direct physical interaction between these two proteins . However, a mutation in the VPg (Y1860S) that eliminates virus infectivity and the interaction in yeast cells had little effect on the in vitro interaction . We also found that the TVMV VPg and NIa proteins are capable of stimulating the polymerase activity of the NIb protein . Since this stimulatory activity is retained when the proteinase domain of the NIa is removed, we conclude that the VPg is the moiety responsible for the stimulation of polymerase activity . As with the interaction revealed by co-purification, the Y1860S mutation had little or no effect on the stimulation of polymerase activity . Moreover, the VPg was able to stimulate a mutant NIb with an altered 'GDD' motif . Our studies thus provide two lines of evidence indicative of in vitro interactions between the TVMV VPg and NIb proteins. J Vet Med Sci, 1998 Jul, 60(7), 863 - 5 Histoplasmosis in the skin and gingiva in a dog; Kagawa Y et al.; An 8-year-old, female mongrel dog had granulomatous lesions in the skull skin and gingiva of the left mandible . The lesions were macroscopically seen as grayish white papular granulomas, and microscopically consisted to numerous swollen macrophages and a few neutrophils without fibrocaseous necrosis . Macrophages contained many small oval or round-shaped yeast-like cells and a few rod-shaped organisms indicating a narrow based budding in their cytoplasm . The yeast-like cells were 2-5 microns (average 3.5 microns) in diameter, and appeared as a central, spherical, lightly basophilic body surrounded by a clear zone or "halo" . The cell wall and central body were stained by the periodic acid-Shiff, Grocott's methenamine silver impregnation, or Gridley fungus method . Immunohistochemically, yeast-like cells were positive to anti-histoplasma yeast antibody, and rod-shaped organisms were positive to anti-histoplasma mycelial antibody . The present paper describes the first case of canine histoplasmosis in Japan. J Am Vet Med Assoc, 1998 Aug 15, 213(4), 519 - 22 Comparison of diagnostic methods for detection of active infection with Tritrichomonas foetus in beef heifers; Kittel DR et al.; OBJECTIVE: To compare sensitivity of a generic trypticase-yeast extract-maltose (TYM) medium versus a commercial nutrient medium in the diagnosis of Tritrichomonas foetus infection in heifers and to assess sensitivity when incubation of samples inoculated into commercial medium pouches is delayed overnight . DESIGN: Prospective study . ANIMALS: 30 virgin beef heifers . PROCEDURES: 20 heifers vaccinated with a trichomonad antigen and 10 unvaccinated control heifers were exposed at synchronized estrus by intravaginal instillation of 10(6) T foetus organisms . Cervicovaginal mucus samples were collected every other week for 10 weeks from controls and once (10 weeks after exposure) from vaccinated heifers . Samples were inoculated into both media and immediately incubated at 37 C (98.6 F) . A duplicate inoculation from controls was made into commercial medium, and the pouch was shipped overnight to a diagnostic laboratory without prior incubation . RESULTS: For 40 of 50 samples from control heifers, there was agreement on diagnoses between media . There was agreement on a positive diagnosis for 3 of 20 samples from vaccinated heifers and on a negative diagnosis for 15 of these 20 samples . For samples shipped overnight before incubation, there were 10% fewer positive diagnoses, compared with samples incubated immediately in commercial medium and 10% more positive diagnoses, compared with samples immediately incubated in TYM . CLINICAL IMPLICATIONS: Use of the commercial medium is a more sensitive indicator of current infection in heifers than use of generic TYM medium . In herds where infection prevalence is high, this method is likely to identify more infected females, an important consideration when control programs include culling of infected cows. J Biol Chem, 1998 Aug 28, 273(35),