Yeasts

FEBS Lett, 1999 Feb 12, 444(2-3), 217 - 21
Hybridization of antisense oligonucleotides with the 3'part of tRNA(Phe); Petyuk VA et al.; The interaction of antisense oligodeoxyribonucleotides with yeast tRNA(Phe) was investigated . 14-15-mers complementary to the 3'-terminal sequence including the ACCA end bind to the tRNA under physiological conditions . At low oligonucleotide concentrations the binding occurs at the unique complementary site . At higher oligonucleotide concentrations, the second oligonucleotide molecule binds to the complex due to non-perfect duplex formation in the T-loop stabilized by stacking between the two bound oligonucleotides . In these complexes the acceptor stem is open and the 5'-terminal sequence of the tRNA is accessible for binding of a complementary oligonucleotide . The results prove that the efficient binding of oligonucleotides to the 3'-terminal sequence of the tRNA occurs through initial binding to the single-stranded sequence ACCA followed by invasion in the acceptor stem and strand displacement.

Plant Cell Physiol, 1998 Dec, 39(12), 1350 - 8
L-galactono-gamma-lactone dehydrogenase from sweet potato: purification and cDNA sequence analysis; Imai T et al.; L-Galactono-gamma-lactone dehydrogenase (EC 1.3.2.3, GLDHase) was partially purified from mitochondria of sweet potato tuberous roots over 600-fold on a specific activity basis, followed by purification of the enzyme protein of 56 kDa by a preparative SDS-PAGE . The absorption spectrum of the hydroxylapatite column-purified GLDH-ase showed peaks at 448 and 373 nm, suggesting the presence of flavin as a prosthetic group . The activity of GLDH-ase was inhibited by lycorine, an alkaloid which inhibits ascorbic acid biosynthesis in vivo . N-terminal partial sequences of four internal polypeptides generated by partial digestion of GLDHase with V8 protease were determined . The deduced nucleotide sequences were used to amplify a cDNA fragment of the GLDHase gene . The clone encoded a polypeptide of 581 amino acid residues with a molecular mass of 66 kDa . The deduced amino acid sequence showed 77% identity with that of cauliflower GLDHase, and significant homology to those of L-gulono-gamma-lactone oxidase (22% identity) from rat and L-galactono-gamma-lactone oxidase from yeast (17% identity), which are enzymes involved in L-ascorbic acid biosynthesis in these organisms . The absorption spectrum and cDNA sequence suggested that the flavin group bound noncovalently . We conclude that GLDHase, L-gulono-gamma-lactone oxidase and L-galactono-gamma-lactone oxidase are homologous in spite of the difference in substrates and electron acceptors . Genomic Southern analysis suggested that GLDHase gene exists as a single copy in the genome of sweet potato.

J Biochem (Tokyo), 1999 Mar, 125(3), 522 - 30
Structural characterization of the gene for human histidine-rich glycoprotein, reinvestigation of the 5'-terminal region of cDNA and a search for the liver specific promoter in the gene; Wakabayashi S et al.; Genomic DNA libraries were screened for the human histidine-rich glycoprotein (HRG) gene and a sequence of 15,499 nucleotides was determined . The gene is composed of 7 exons and 6 introns, and all the exon-intron boundaries match the consensus GT/AG sequence for donor and acceptor splice sites . Each of cystatin-like domains I and II of HRG is encoded by three exons, exons I to III and exons IV to VI, respectively, like those of other members of the cystatin superfamily . The entire C-terminal half of the molecule is encoded by the largest exon, VII . The first 103 nucleotides of the cDNA sequence reported for human HRG {Koide, T., Foster, D., Yoshitake, S . , and Davie, E.W . (1986) Biochemistry 25, 2220-2225} could not be found in the determined gene sequence . A homology search of this sequence against a database showed the complete matching to a part of the yeast mitochondrial DNA encoding 21S ribosomal RNA . Rapid amplification of cDNA 5' ends (5'-RACE) analysis revealed that the cDNA has multiple 5'-ends and that a possible starting point is nucleotide 104 of the reported cDNA sequence . These results suggest that the first 103 nucleotides of the cDNA sequence reported for human HRG originated from yeast mitochondrial DNA and were incidentally incorporated into the HRG cDNA in the process of the construction of a cDNA library . Various fragments obtained on restriction endonuclease digestion of the 5'-noncoding region of the HRG gene were ligated to the chloramphenicol acetyltransferase (CAT) gene and then transfected into HepG2 and 293 cells to analyze the promoter activity . The sequence between -262 and -21 from the putative translation initiation site supported the expression of CAT in HepG2 cells but not in 293 cells, suggesting that this segment promotes the liver-specific transcription of the human HRG gene.

Genetics, 1999 Mar, 151(3), 1065 - 79
Genetic analysis of viable Hsp90 alleles reveals a critical role in Drosophila spermatogenesis; Yue L et al.; The Hsp90 chaperone protein maintains the activities of a remarkable variety of signal transducers, but its most critical functions in the context of the whole organism are unknown . Point mutations of Hsp83 (the Drosophila Hsp90 gene) obtained in two different screens are lethal as homozygotes . We report that eight transheterozygous mutant combinations produce viable adults . All exhibit the same developmental defects: sterile males and sterile or weakly fertile females . We also report that scratch, a previously identified male-sterile mutation, is an allele of Hsp82 with a P-element insertion in the intron that reduces expression . Thus, it is a simple reduction in Hsp90 function, rather than possible altered functions in the point mutants, that leads to male sterility . As shown by light and electron microscopy, all stages of spermatogenesis involving microtubule function are affected, from early mitotic divisions to later stages of sperm maturation, individualization, and motility . Aberrant microtubules are prominent in yeast cells carrying mutations in HSP82 (the yeast Hsp90 gene), confirming that Hsp90 function is connected to microtubule dynamics and that this connection is highly conserved . A small fraction of Hsp90 copurifies with taxol-stabilized microtubule proteins in Drosophila embryo extracts, but Hsp90 does not remain associated with microtubules through repeated temperature-induced assembly and disassembly reactions . If the spermatogenesis phenotypes are due to defects in microtubule dynamics, we suggest these are indirect, reflecting a role for Hsp90 in maintaining critical signal transduction pathways and microtubule effectors, rather than a direct role in the assembly and disassembly of microtubules themselves.

Biochem Biophys Res Commun, 1999 Feb 24, 255(3), 631 - 8
Identification of ribosomal protein L34 as a novel Cdk5 inhibitor; Moorthamer M et al.; The cell cycle is regulated by sequential activation, inactivation of cyclin dependent kinases (Cdk-s) . Like all other Cdk-s, the catalytic subunit of Cdk5 is present in cycling cells . However, its highest concentration is found in differentiated neurons, and the only known protein that activates Cdk5 (i.e., p35) is expressed solely in the brain . Active Cdk5 is thought to be involved in the in vivo phosphorylation of the neurofilament proteins and tau which are hyperphosphorylated in neurodegenerative diseases . Recent reports suggest that Cdk5 may also contribute to cellular differentiation . Therefore, it would not be unusual to surmise that there exist specific proteins that regulate Cdk5 activity in cycling cells . In order to find if this was true, a cDNA library prepared from HeLa cells was screened using the yeast-two-hybrid system . The 60S ribosomal protein, L34, was identified as a Cdk5-interacting protein . Biochemical analyses reveal that L34 cannot activate Cdk5 but potently inhibits the p35-activated kinase . L34 also interacts with Cdk4 and, in parallel, inhibits the Cdk4/cyclin D1 activity . Interestingly, L34 does not interact with Cdk2 in the two-hybrid assay nor does it inhibit the Cdk2/cyclin A enzyme . The fact that a ribosomal protein inhibits Cdk5 and Cdk4 may suggest that these two kinases have a cellular role in translational regulation .

Biochem Biophys Res Commun, 1999 Feb 24, 255(3), 575 - 9
Molecular cloning and functional characterization of rat delta-6 fatty acid desaturase; Aki T et al.; Mammalian cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) sequence informations . These fragments were subsequently used to screen a rat liver cDNA library, yielding a 1573-bp clone . Expression of DNA fragment containing either of two possible open reading frames (nucleotide numbers 97-1431 and 148-1431) of the isolated clone in yeast led to the accumulation of gamma-linolenic acid in the presence of exogenous linoleic acid . In this system, the addition of alpha-linolenic acid also resulted in the accumulation of its Delta-6 desaturated product whereas dihomo-gamma-linolenic acid failed to be a substrate . These results indicate that the protein encoded by the rat cDNA is Delta-6 fatty acid desaturase, and the first 17 amino acids corresponding to the coding region 97-147 of the clone are not required to function in yeast .

Genomics, 1999 Feb 1, 55(3), 348 - 52
A physical map of the mouse shaker-2 region contains many of the genes commonly deleted in Smith-Magenis syndrome (del17p11.2p11.2); Probst FJ et al.; We report the construction of a physical map of the region of mouse chromosome 11 that encompasses shaker-2 (sh2), a model for the human nonsyndromic deafness DFNB3 . DFNB3 maps within the common deletion region of Smith-Magenis syndrome (SMS), del(17)(p11.2p11.2) . Eleven of the genes mapping within the SMS common deletion region have murine homologs on the sh2 physical map . The gene order in this region is not perfectly conserved between mouse and human, a finding to be considered as we engineer a mouse model of Smith-Magenis syndrome.

Genomics, 1999 Feb 1, 55(3), 335 - 40
Identification and characterization of a highly conserved protein absent in the Alport syndrome (A), mental retardation (M), midface hypoplasia (M), and elliptocytosis (E) contiguous gene deletion syndrome (AMME); Vitelli F et al.; We recently described a novel contiguous gene deletion syndrome (AMME) in Xq22.3 that includes Alport syndrome (A), mental retardation (M), midface hypoplasia (M), and elliptocytosis (E) . While the Alport syndrome is due to deletion of the COL4A5 gene, no other genes are known in the region with the exception of our recent finding of the FACL4 gene . In our effort to isolate additional genes from the deleted region, we have identified the gene named AMMECR1 (Alport syndrome, mental retardation, midface hypoplasia, and elliptocytosis chromosomal region gene 1) . RACE experiments and screening of cDNA libraries enabled us to obtain the entire ORF of the gene (1002 bp) followed by about 2 kb of 3'UTR . AMMECR1 is composed of six exons, shows a ubiquitous 6.5-kb transcript, and codes for a protein with a molecular mass of 35.5 kDa . Sequence analysis revealed that this gene is conserved in several species ranging from Caenorhabditis elegans and yeast to micro-organisms . Exon 2 of AMMECR1 encodes a domain consisting of six amino acids identically conserved throughout the course of evolution and whose function is as yet unknown . Analysis of the predicted protein product using ExPAsy tools raises the possibility that the gene may code for a regulatory factor potentially involved in the development of AMME contiguous gene deletion syndrome.

Genomics, 1999 Feb 1, 55(3), 268 - 74
Physical mapping of the rippling muscle disease locus; Stephan DA et al.; Rippling muscle disease (RMD) is an autosomal dominant disorder characterized by electrically silent, percussion-induced muscular contractions . We previously reported the localization of a gene for RMD to 1q41-q42 by genome-wide linkage analysis in a large family from Oregon . This RMD gene was initially found to be contained within a 12-cM interval with a maximum multipoint lod score of 3.56 . A YAC/BAC contig was assembled by STS content mapping and database searches spanning the nonrecombinant interval containing the RMD gene (RMD1) . The physical map, in conjunction with recent mapping information from various other sources, clarified the order of genetic markers in this region and necessitated redefinition of the RMD genetic interval by linkage analysis with the newly ordered markers . Polymorphisms that mapped to the YACs in this contig were genotyped in this family and used to provide statistical support for narrowing of the critical genetic interval to 3 cM, corresponding to a maximum possible physical distance of 4.0 Mb . In addition, recombination breakpoint mapping supported the evidence that RMD1 must reside within this interval between markers D1S446 and D1S2680 . ESTs (82) were mapped to the YACs spanning the region known to contain the RMD1 gene, and of these, 9 become strong positional candidates . The physical and refined genetic maps of this RMD locus set the stage for isolation of the responsible gene and elucidation of a novel patho-mechanism of calcium homeostasis in skeletal muscle.

Genomics, 1999 Feb 1, 55(3), 257 - 67
Cloning and characterization of two cytoplasmic dynein intermediate chain genes in mouse and human; Crackower MA et al.; Cytoplasmic dynein is a large multisubunit microtubule-based motor protein, which mediates movement of numerous intracellular organelles . We report here the identification of the human homologue of cytoplasmic dynein intermediate chain 1 gene (DNCI1) located on human chromosome 7q21.3-q22.1 . The mouse orthologue (Dnci1) was identified along with another highly related gene, Dnci2, and their RNA in situ expression patterns were examined during mouse embryogenesis . Dnci1 was found to have a highly restricted expression domain in the developing forebrain as well as the peripheral nervous system (PNS), while Dnci2 displayed a broad expression profile throughout the entire central nervous system and most of the PNS . A dynamic expression profile was also found for Dnci2 in the developing mouse limb bud . The data presented here provide a framework for the further analysis of the functional role of Dnci1 and Dnci2 in mouse and DNCI1 in human.

Protein Sci, 1999 Feb, 8(2), 435 - 8
Identification of a novel domain shared by putative components of the endocytic and cytoskeletal machinery; Kay BK et al.; We have identified a approximately 140 amino acid domain that is shared by a variety of proteins in budding and fission yeast, nematode, rat, mouse, frog, oat, and man . Typically, this domain is located within 20 residues of the N-terminus of the various proteins . The percent identity among the domains in the 12 proteins ranges from 42 to 93%, with 16 absolutely conserved residues: N-x(11-13)-V-x2-A-T-x(34-36)-R-x(7-8)-W-R-x3-K-x12-G-x-E-x15 -L-x11-12-D-x-G-R-x11-D-x7-R . Even though these proteins share little beyond their segment of homology, data are emerging that several of the proteins are involved in endocytosis and or regulation of cytoskeletal organization . We have named this protein segment the ENTH domain, for Epsin N-terminal Homology domain, and hypothesize that it is a candidate for binding specific ligands and/or enzymatic activity in the cell.

Toxicol Sci, 1998 Dec, 46(2), 282 - 93
Examination of the in vitro and in vivo estrogenic activities of eight commercial phthalate esters; Zacharewski TR et al.; The estrogenic activities of eight phthalate esters (i.e., di-2-ethylhexyl, di-n-butyl (DBP), butylbenzyl (BBP), di-hexyl (DHP), diiso-heptyl, di-n-octyl, diiso-nonyl, diiso-decyl) were investigated in vitro using estrogen receptor (ER) competitive ligand-binding and mammalian- and yeast-based gene expression assays . In vivo, their effects on uterine wet weight and vaginal cell cornification using ovariectomized Sprague-Dawley rats were assessed . DBP, BBP, and DHP weakly competed with 17 beta-estradiol (E2) for binding to the ER in competitive ligand-binding assays . In gene expression assays using MCF-7 cells transiently transfected with the Gal4-human estrogen receptor construct, Gal4-HEGO, and the Gal4-regulated luciferase reporter gene, 17m5-G-Luc, 10 microM DBP, BBP, or DHP exhibited 36, 42, and 20% activity, respectively, when compared to the 100% response observed with 10 nM E2 . Only BBP was found to induce luciferase activity (32%) in HeLa cells stably transfected with Gal4-HEGO and 17m5-G-Luc constructs and to impart minimal ER-mediated viability to the E2-dependent recombinant yeast strain, PL3, on selective medium . No significant responses were observed with the other phthalate esters in any of the in vitro assays . In vivo, none of the eight phthalate esters reproducibly induced significant increases in uterine wet weight in immature ovariectomized Sprague-Dawley rats treated with oral doses of 20, 200, or 2000 mg/kg of phthalate ester . In addition, treatment with phthalate esters at the same doses did not affect the degree of vaginal epithelial cell cornification in mature ovariectomized rats . These results indicate that only selected phthalate esters (i.e., DBP, BBP, and DHP) exhibit weak ER-mediated activity in some in vitro assays at high concentrations but none of the eight phthalate esters elicited in vivo estrogenic responses based upon results obtained from uterotrophic and vaginal cornification assays.

Curr Opin Cell Biol, 1999 Feb, 11(1), 142 - 51
Molecular genetic approaches to understanding the actin cytoskeleton; Sutherland JD et al.; New tools in molecular genetics, such as genetic interaction screens and conditional gene targeting, have advanced the study of actin dynamics in a number of model systems . Yeast, Dictyostelium, Caenorhabditis elegans, Drosophila, and mice have contributed much in recent years to a better understanding of both the numerous functions and modes of regulation of the actin cytoskeleton.

J Biol Chem, 1999 Mar 5, 274(10), 6035 - 8
Interaction of the small G protein RhoA with the C terminus of human phospholipase D1; Yamazaki M et al.; Mammalian phosphatidylcholine-specific phospholipase D1 (PLD1) is a signal transduction-activated enzyme thought to function in multiple cell biological settings including the regulation of membrane vesicular trafficking . PLD1 is activated by the small G proteins, ADP-ribosylation factor (ARF) and RhoA, and by protein kinase C-alpha (PKC-alpha) . This stimulation has been proposed to involve direct interaction and to take place at a distinct site in PLD1 for each activator . In the present study, we employed the yeast two-hybrid system to attempt to identify these sites . Successful interaction of ARF and PKC-alpha with PLD1 was not achieved, but a C-terminal fragment of human PLD1 (denoted "D4") interacted with the active mutant of RhoA, RhoAVal-14 . Deletion of the CAAX box from RhoAVal-14 decreased the strength of the interaction, suggesting that lipid modification of RhoA is important for efficient binding to PLD1 . The specificity of the interaction was validated by showing that the PLD1 D4 fragment interacts with glutathione S-transferase-RhoA in vitro in a GTP-dependent manner and that it associates with RhoAVal-14 in COS-7 cells, whereas the N-terminal two-thirds of PLD1 does not . Finally, we show that recombinant D4 peptide inhibits RhoA-stimulated PLD1 activation but not ARF- or PKC-alpha-stimulated PLD1 activation . These results conclusively demonstrate that the C-terminal region of PLD1 contains the RhoA-binding site and suggest that the ARF and PKC interactions occur elsewhere in the protein.

J Neurochem, 1999 Mar, 72(3), 999 - 1008
Presenilins interact with armadillo proteins including neural-specific plakophilin-related protein and beta-catenin; Levesque G et al.; Missense substitutions in the presenilin 1 (PS1) and presenilin 2 (PS2) proteins are associated with early-onset familial Alzheimer's disease . We have used yeast-two-hybrid and coimmunoprecipitation methods to show that the large cytoplasmic loop domains of PS1 and PS2 interact specifically with three members of the armadillo protein family, including beta-catenin, p0071, and a novel neuronal-specific armadillo protein--neural plakophilin-related armadillo protein (NPRAP) . The PS1:NPRAP interaction occurs between the arm repeats of NPRAP and residues 372-399 at the C-terminal end of the large cytoplasmic loop of PS1 . The latter residues contain a single arm-like domain and are highly conserved in the presenilins, suggesting that they form a functional armadillo protein binding site for the presenilins.

Environ Mol Mutagen, 1999, 33(1), 3 - 20
Human DNA repair systems: an overview; Yu Z et al.; DNA repair systems act to maintain genome integrity in the face of replication errors, environmental insults, and the cumulative effects of age . More than 70 human genes directly involved in the five major pathways of DNA repair have been described, including chromosomal location and cDNA sequence . However, a great deal of information as to the precise functions of these genes and their role in human health is still lacking . Hence, we summarize what is known about these genes and their contra part in bacterial, yeast, and rodent systems and discuss their involvement in human disease . While some associations are already well understood, it is clear that additional diseases will be found which are linked to DNA repair defects or deficiencies.

J Cell Sci, 1999 Mar, 112 ( Pt 6), 895 - 904
Homotypic and heterotypic interaction of the neurofibromatosis 2 tumor suppressor protein merlin and the ERM protein ezrin; Gronholm M et al.; Ezrin, radixin and moesin (ERM) are homologous proteins, which are linkers between plasma membrane components and the actin-containing cytoskeleton . The ERM protein family members associate with each other in a homotypic and heterotypic manner . The neurofibromatosis 2 (NF2) tumor suppressor protein merlin (schwannomin) is structurally related to ERM members . Merlin is involved in tumorigenesis of NF2-associated and sporadic schwannomas and meningiomas, but the tumor suppressor mechanism is poorly understood . We have studied the ability of merlin to self-associate and bind ezrin . Ezrin was coimmunoprecipitated with merlin from lysates of human U251 glioma cells and from COS-1 cells transfected with cDNA encoding for merlin isoform I . The interaction was further studied and the association domains were mapped with the yeast two-hybrid system and with blot overlay and affinity precipitation experiments . The heterotypic binding of merlin and ezrin and the homotypic association of merlin involves interaction between the amino- and carboxy-termini . The amino-terminal association domain of merlin involves residues 1-339 and has similar features with the amino-terminal association domain of ezrin . The carboxy-terminal association domain cannot be mapped as precisely as in ezrin, but it requires residues 585-595 and a more amino-terminal segment . Unlike ezrin, merlin does not require activation for self-association but native merlin molecules can interact with each other . Heterodimerization between merlin and ezrin, however, occurs only following conformational alterations in both proteins . These results biochemically connect merlin to the cortical cytoskeleton and indicate differential regulation of merlin from ERM proteins.

J Cell Sci, 1999 Mar, 112 ( Pt 6), 845 - 54
Syntaxin 11 is associated with SNAP-23 on late endosomes and the trans-Golgi network; Valdez AC et al.; SNARE proteins are known to play a role in regulating intracellular protein transport between donor and target membranes . This docking and fusion process involves the interaction of specific vesicle-SNAREs (e.g . VAMP) with specific cognate target-SNAREs (e.g . syntaxin and SNAP-23) . Using human SNAP-23 as the bait in a yeast two-hybrid screen of a human B-lymphocyte cDNA library, we have identified the 287-amino-acid SNARE protein syntaxin 11 . Like other syntaxin family members, syntaxin 11 binds to the SNARE proteins VAMP and SNAP-23 in vitro and also exists in a complex with SNAP-23 in transfected HeLa cells and in native human B lymphocytes . Unlike other syntaxin family members, no obvious transmembrane domain is present in syntaxin 11 . Nevertheless, syntaxin 11 is predominantly membrane-associated and colocalizes with the mannose 6-phosphate receptor on late endosomes and the trans-Golgi network . These data suggest that syntaxin 11 is a SNARE that acts to regulate protein transport between late endosomes and the trans-Golgi network in mammalian cells.

J Cell Sci, 1999 Mar, 112 ( Pt 6), 761 - 72
Structure and functions of nucleolin; Ginisty H et al.; Nucleolin is an abundant protein of the nucleolus . Nucleolar proteins structurally related to nucleolin are found in organisms ranging from yeast to plants and mammals . The association of several structural domains in nucleolin allows the interaction of nucleolin with different proteins and RNA sequences . Nucleolin has been implicated in chromatin structure, rDNA transcription, rRNA maturation, ribosome assembly and nucleo-cytoplasmic transport . Studies of nucleolin over the last 25 years have revealed a fascinating role for nucleolin in ribosome biogenesis . The involvement of nucleolin at multiple steps of this biosynthetic pathway suggests that it could play a key role in this highly integrated process.

Genomics, 1999 Feb 15, 56(1), 131 - 3
Cytogenetic and radiation hybrid mapping of human arachidonate 5-lipoxygenase-activating protein (ALOX5AP) to chromosome 13q12; Yandava CN et al.; Arachidonate 5-lipoxygenase-activating protein (ALOX5AP) is an arachidonic acid binding protein that has been shown to be critical in the biosynthesis of leukotrienes . We mapped the ALOX5AP gene to the chromosome 13q12 region by cytogenetic mapping, yeast artificial chromosome (YAC) pool screening, and radiation hybrid mapping . It was mapped to YAC contig WC13.2 by YAC pool screening with an unambiguous hit to WI-4874, which is at 78 cR on the radiation hybrid map, 3.36 cR, by radiation hybrid mapping, from WI-4874 .

Genomics, 1999 Feb 15, 56(1), 70 - 7
Revised genomic organization of FBN1 and significance for regulated gene expression; Biery NJ et al.; FBN1 encodes fibrillin-1, an extracellular matrix protein that is defective in Marfan syndrome . This gene is divided into 65 exons and was previously reported to be approximately 110 kb in length . The existence of 3 exons upstream of the exon containing the putative initiating methionine left open the possibility of alternative fibrillin-1 isoforms that vary at their N-termini . Detailed examination of YACs containing human FBN1 reveal that the gene is 200 kb, almost twice as large as previously thought . Characterization of the porcine FBN1 cDNA and 5' flanking sequence demonstrates extreme conservation between the pig and the human predicted proteins and argues against the possibility of alternative amino-terminal coding sequence . These data further our understanding of the regulatory requirements for gene expression and establish a framework for recombinant expression of fibrillin-1 .

Genomics, 1999 Feb 15, 56(1), 31 - 9
High-resolution human/goat comparative map of the goat polled/intersex syndrome (PIS): the human homologue is contained in a human YAC from HSA3q23; Vaiman D et al.; The genetic and cytogenetic map around the chromosome 1 region shown to be linked with polledness and intersexuality (PIS) in the domestic goat (Capra hircus) was refined . For this purpose, a goat BAC library was systematically screened with primers from human coding sequences, scraped chromosome 1 DNA, bovine microsatellites from the region, and BAC ends . All the BACs (n = 30) were mapped by fluorescence in situ hybridization (FISH) on goat chromosome 1q41-q45 . The genetic mapping of 30 new goat polymorphic markers, isolated from these BACs, made it possible to reduce the PIS interval to a region of less than 1 cM on goat chromosome 1q43 . The PIS locus is now located between the two genes ATP1B and COP, which both map to 3q23 in humans . Genetic, cytogenetic, and comparative data suggest that the PIS region is now probably circumscribed to an approximately 1-Mb DNA segment for which construction of a BAC contig is in progress . In addition, a human YAC contig encompassing the blepharophimosis-ptosis-epicanthus-inversus region was mapped by FISH to goat chromosome 1q43 . This human disease, mapped to HSA 3q23 and affecting the development and maintenance of ovarian function, could be a potential candidate for goat PIS .

Anal Biochem, 1999 Mar 1, 268(1), 94 - 101
An enzymatic cycling method using pyruvate orthophosphate dikinase and firefly luciferase for the simultaneous determination of ATP and AMP (RNA); Sakakibara T et al.; A novel bioluminescent enzymatic cycling assay for ATP and AMP with concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK) was developed . In this system, AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK . This resulted in constant luminescence once the stable phase had been reached . Background luminescence of the reagent was reduced with adenosine phosphate deaminase by degrading ATP and AMP in the reagent . The maximum recycling ratio calculated from the integrated luminescence value was 2.64 cycles/min . The measurable ranges for ATP and AMP were equal and were between 4 x 10(-13) and 4 x 10(-17) mol/assay . The amount of yeast RNA could be estimated in the range of 1 x 10(-8) to 1 x 10(-12) g/assay by estimating the amount of AMP resulting from the degradation of RNA with nuclease P1 . Various food samples were subjected to measurement of the amount of ATP + AMP + RNA to provide an index for hygiene monitoring . For beef extract, sensitivity was improved by more than 20 million compared to the previous methods relying only on the amount of ATP as an index .

Mol Biochem Parasitol, 1999 Jan 5, 98(1), 1 - 15
Molecular cloning and biochemical characterization of a VCP homolog in African trypanosomes; Roggy JL et al.; Through reverse transcription-polymerase chain reaction using degenerate oligonucleotide primers, a VCP homolog was identified in African trypanosomes . Sequence analysis shows a 72 and 64% deduced amino acid identity, respectively, with mouse VCP and yeast Cdc48p . Southern analysis indicates tbVCP to have a single locus with two alleles . Antibodies generated against recombinant protein recognize a 95 kDa protein in whole cell lysates of both procyclic and bloodstream trypanosomes . There is an approximately four-fold greater expression of TbVCP protein in the procyclic stage of the trypanosome life cycle . Subcellular fractionation and immunofluorescence with anti-TbVCP antibodies indicate the majority of TbVCP to be cytoplasmically localized with a small subset associated with membranes . Sucrose velocity sedimentation and gel filtration size analysis studies suggest that TbVCP is a homohexameric particle as has been demonstrated with other VCP homologs . Also like other VCP homologs, TbVCP contains an NEM-inhibitable ATPase activity . This is the first characterization of an AAA (ATPases Associated with a variety of cellular Activities) family member in African trypanosomes.

Drug Chem Toxicol, 1998, 21 Suppl 1, 101 - 21
Assessment of polystyrene extract for estrogenic activity in the rat uterotrophic model and an in vitro recombinant receptor reporter gene assay; Fail PA et al.; The purpose of the study was to determine whether polystyrene used in food-contact applications would elicit an estrogenic response when extracts simulating exaggerated conditions of use were subjected to in vivo and in vitro tests . A sample of polystyrene was subjected to extraction conditions that simulate, or exaggerate, the actual food-contact uses of polystyrene to maximize the amount of low molecular weight polystyrene extractables . The food-simulating solvent and the time and temperature conditions recommended by the Food and Drug Administration (FDA) were selected to maximize the level of extractable components from polystyrene . The extract was examined for its estrogenic response in vivo using the immature rat uterotrophic assay and in vitro using an estrogen receptor (ER)-mediated recombinant receptor reporter gene assay . In vivo, the uterine weights of juvenile female Sprague Dawley rats (10 rats/group) were determined after oral gavage exposure to the extract (two dosage levels: one represents the maximum potential daily human exposure to polystyrene extractables and the other represents one-tenth of the maximum exposure level), vehicle control (sesame oil), or positive control {diethylstilbestrol (DES), at 200 micrograms/kg body weight} . In addition, five treatment groups were dosed by subcutaneous injection of either estradiol (1, 50, and 500 micrograms/kg body weight) or DES (2 and 200 micrograms/kg body weight) . Dosing began on postnatal day (pnd) 21 and continued daily through pnd 23 . Body weights were collected at study initiation (pnd 21) and at necropsy (pnd 24) . Body weights were not different statistically between treatment groups at study initiation or at necropsy . Uterine wet weights and uterine weights relative to body weights were significantly increased (p < 0.05) for estradiol at 50 and 500 micrograms/kg, DES at 2 and 200 micrograms/kg, and DES at 200 micrograms/kg (oral) over vehicle control . The polystyrene extract had no effect on uterine wet weight or uterine weights relative to body weights at either level tested . An in vitro recombinant estrogen receptor/reporter gene assay that involved transiently transfecting MCF-7 human breast cancer cells with the chimeric human ER, Ga14-HEGO, consisting of the yeast Ga14 DNA binding domain linked to the ligand binding domain of the human ER and a Ga14 response element (17mer)-regulated reporter gene (17m5-G-Luc) was employed . Dose-dependent induction of the reporter gene, 17m5-G-Luc, was observed with the positive control, 17 beta-estradiol (E2) . Induction of greater than 100-fold was obtained following incubation of transfected MCF-7 cells with 10 nM E2 for 24 hours . No induction of reporter gene activity was observed with the polystyrene extracts dissolved in dimethylsulfoxide (0.01, 0.1 or 0.01 mg/ml) using the same assay conditions . These results indicate that polystyrene extract does not elicit ER-mediated activity using the Ga14-HEGO/17m5-G-Luc recombinant receptor/reporter gene assay . In conclusion, extracts from polystyrene produced no estrogenic response in either the rat uterotrophic assay or the MCF-7 cell assay for estrogen receptor-mediated activity.

Br J Haematol, 1999 Jan, 104(1), 37 - 43
Identification and characterization of two missense mutations causing factor XIIIA deficiency; Kangsadalampai S et al.; In this study, two amino acid substitutions, Arg260His and Val414Phe, have been identified in the factor XIIIA subunits of factor XIII deficient patients of Syrian and Indian descent, respectively . To confirm the deleterious effects of these substitutions, both variant sequences have been engineered into cDNA clones and the mutant enzymes expressed in yeast . Determination of the transglutaminase activity and immuno detection of the mutant enzymes together with mRNA hybridization revealed that the mutations dramatically reduce both the catalytic activity and the level of enzyme expressed in yeast . The mutations Arg260His and Val414Phe occur within the 'core' domain of the enzyme . Computer modelling of the mutant enzymes reveals that the substitution of the Arg260 by His results in the loss of a conserved electrostatic interaction whereas the effect of the Val414Phe substitution is a consequence of the large increase in side-chain volume . Although both mutations do not effect the active site directly, they are predicted to reduce the stability of the enzyme . The effects of these two amino acid substitutions on enzyme expression and three-dimensional structure strongly confirm that residues which are located outside of the active site can have a significant effect on protein stability and function.

Recent Results Cancer Res, 1998, 154, 156 - 73
The ataxia telangiectasia gene in familial and sporadic cancer; Yuille MA et al.; The ataxia telangiectasia (A-T) gene, ATM, predisposes affected homozygotes to a wide range of malignancies . It has been suggested that this is a consequence of the genomic instability associated with the syndrome . The elevated risk of malignancy is not, however, observed among A-T heterozygotes (except, apparently, regarding breast cancer) . In this report we describe results from the study of the rare sporadic disease, T cell prolymphocytic leukaemia (T-PLL) . In all individuals tested, we observed that at least one ATM allele was disrupted by rearrangement, that in many cases both alleles were disrupted and that there were additional mutations, predominantly missense, that clustered toward the 3' end of the gene corresponding to the protein's phosphatidylinositol 3-kinase (PIK)-related domain . We conclude that the ATM gene can act as a tumour suppressor in the development of sporadic T-PLL . Our finding of a surfeit of mutations within ATM may reflect the involvement of the gene at more than one step in tumorigenesis . In particular, we suggest that the clustering of missense mutations may pertain to the late-onset character of both sporadic and A-T-related T-PLL, since the closest homologue of Atm protein is the yeast TEL1 protein that maintains telomere length . ATM inactivation may not be the initiating event in T-PLL tumorigenesis: prior mutation of another gene--perhaps TCL1 activation--may be obligate . This would explain the recessive character of T-PLL risk in A-T.

Neurology, 1999 Feb, 52(3), 510 - 5
Confirmation of linkage of type 1 hereditary sensory neuropathy to human chromosome 9q22; Bejaoui K et al.; OBJECTIVES: 1) To confirm linkage of hereditary sensory neuropathy type 1 (HSN-I) to human chromosome 9q22 in a large American family of German origin . 2) To construct a yeast artificial chromosome (YAC) contig spanning the HSN-I candidate interval . 3) To investigate the HSN-I contig for potential candidate genes . BACKGROUND: HSN-I is a rare peripheral neuropathy characterized by loss of temperature sensation, ulceration and osteomyelitis of the digits, and subtle distal weakness . A gene for HSN-I has previously been mapped to human chromosome 9q22.1-q22.3 between markers D9S318 and D9S176 in an 8-cM interval in four Australian families . METHODS: In a large German-American family with HSN-I, genome-wide linkage analysis was performed on 68 family members extending over five generations and including 17 affected members . Genotyping was performed with PCR, and the resulting genotypes were analyzed with two-point linkage analysis with Fastlink . A YAC contig was constructed based on the Whitehead Institute YAC contig WC9.3 . RESULTS: Two-point linkage analysis resulted in a maximum lod score of 8.2 at theta = 0 for marker D9S1815 . Haplotype analysis locates the HSN-I gene between markers D9S1797 and D9S197 . Using YAC clones from the Centre d'Etude du Polymorphism Humain YAC Library, we constructed a YAC contig spanning these markers . Based on the radiation hybrid map of the human genome, we estimate that the size of this interval is less than 2,500 kb . CONCLUSIONS: Our study confirms linkage of a putative HSN-I gene to chromosome 9q22, considerably narrows the HSN-I locus, and provides a basis for identification of the HSN-I gene.

J Biol Chem, 1999 Feb 26, 274(9), 5746 - 54
Calmodulin mediates calcium-dependent activation of the intermediate conductance KCa channel, IKCa1; Fanger CM et al.; Small and intermediate conductance Ca2+-activated K+ channels play a crucial role in hyperpolarizing the membrane potential of excitable and nonexcitable cells . These channels are exquisitely sensitive to cytoplasmic Ca2+, yet their protein-coding regions do not contain consensus Ca2+-binding motifs . We investigated the involvement of an accessory protein in the Ca2+-dependent gating of hIKCa1, a human intermediate conductance channel expressed in peripheral tissues . Cal- modulin was found to interact strongly with the cytoplasmic carboxyl (C)-tail of hIKCa1 in a yeast two-hybrid system . Deletion analyses defined a requirement for the first 62 amino acids of the C-tail, and the binding of calmodulin to this region did not require Ca2+ . The C-tail of hSKCa3, a human neuronal small conductance channel, also bound calmodulin, whereas that of a voltage-gated K+ channel, mKv1.3, did not . Calmodulin co-precipitated with the channel in cell lines transfected with hIKCa1, but not with mKv1 . 3-transfected lines . A mutant calmodulin, defective in Ca2+ sensing but retaining binding to the channel, dramatically reduced current amplitudes when co-expressed with hIKCa1 in mammalian cells . Co-expression with varying amounts of wild-type and mutant calmodulin resulted in a dominant-negative suppression of current, consistent with four calmodulin molecules being associated with the channel . Taken together, our results suggest that Ca2+-calmodulin-induced conformational changes in all four subunits are necessary for the channel to open.

J Biol Chem, 1999 Feb 26, 274(9), 5738 - 45
A plant 126-kDa phosphatidylinositol 4-kinase with a novel repeat structure . Cloning and functional expression in baculovirus-infected insect cells; Xue HW et al.; Phosphatidylinositol metabolism plays a central role in signaling pathways in animals and is also believed to be of importance in signal transduction in higher plants . We report here the molecular cloning of a cDNA encoding a previously unidentified 126-kDa phosphatidylinositol (PI) 4-kinase (AtPI4Kbeta) from the higher plant Arabidopsis thaliana . The novel protein possesses the conserved domains present in animal and yeast PI 4-kinases, namely a lipid kinase unique domain and a catalytic domain . An additional domain, approximately 300 amino acids long, containing a high percentage (46%) of charged amino acids is specific to this plant enzyme . Recombinant AtPI4Kbeta expressed in baculovirus-infected insect (Spodoptera frugiperda) cells phosphorylated phosphatidylinositol exclusively at the D4 position of the inositol ring . Recombinant protein was maximally activated by 0.6% Triton X-100 but was inhibited by adenosine with an IC50 of approximately 200 microM . Wortmannin at a concentration of 10 microM inhibited AtPI4Kbeta activity by approximately 90% . AtPI4Kbeta transcript levels were similar in all tissues analyzed . Light or treatment with hormones or salts did not change AtPI4Kbeta transcript levels to a great extent, indicating constitutive expression of the AtPI4Kbeta gene.

J Biol Chem, 1999 Feb 26, 274(9), 5723 - 30
Characterization of a mutant pancreatic eIF-2alpha kinase, PEK, and co-localization with somatostatin in islet delta cells; Shi Y et al.; Phosphorylation of eukaryotic translation initiation factor-2alpha (eIF-2alpha) is one of the key steps where protein synthesis is regulated in response to changes in environmental conditions . The phosphorylation is carried out in part by three distinct eIF-2alpha kinases including mammalian double-stranded RNA-dependent eIF-2alpha kinase (PKR) and heme-regulated inhibitor kinase (HRI), and yeast GCN2 . We report the identification and characterization of a related kinase, PEK, which shares common features with other eIF-2alpha kinases including phosphorylation of eIF-2alpha in vitro . We show that human PEK is regulated by different mechanisms than PKR or HRI . In contrast to PKR or HRI, which are dependent on autophosphorylation for their kinase activity, a point mutation that replaced the conserved Lys-614 with an alanine completely abolished the eIF-2alpha kinase activity, whereas the mutant PEK was still autophosphorylated when expressed in Sf-9 cells . Northern blot analysis indicates that PEK mRNA was predominantly expressed in pancreas, though low expression was also present in several tissues . Consistent with the high levels of mRNA in pancreas, the PEK protein was only detected in human pancreatic islets, and the kinase co-localized with somatostatin, a pancreatic delta cell-specific hormone . Thus PEK is believed to play an important role in regulating protein synthesis in the pancreatic islet, especially in islet delta cells.

J Biol Chem, 1999 Feb 26, 274(9), 5522 - 31
Identification and cloning of xp95, a putative signal transduction protein in Xenopus oocytes; Che S et al.; A 95-kDa protein in Xenopus oocytes, Xp95, was shown to be phosphorylated from the first through the second meiotic divisions during progesterone-induced oocyte maturation . Xp95 was purified and cloned . The Xp95 protein sequence exhibited homology to mouse Rhophilin, budding yeast Bro1, and Aspergillus PalA, all of which are implicated in signal transduction . It also contained three conserved features including seven conserved tyrosines, a phosphorylation consensus sequence for the Src family of tyrosine kinases, and a proline-rich domain near the C terminus that contains multiple SH3 domain-binding motifs . We showed the following: 1) that both Xp95 isolated from Xenopus oocytes and a synthetic peptide containing the Src phosphorylation consensus sequence of Xp95 were phosphorylated in vitro by Src kinase and to a lesser extent by Fyn kinase; 2) Xp95 from Xenopus oocytes or eggs was recognized by an anti-phosphotyrosine antibody, and the relative abundance of tyrosine-phosphorylated Xp95 increased during oocyte maturation; and 3) microinjection of deregulated Src mRNA into Xenopus oocytes increased the abundance of tyrosine-phosphorylated Xp95 . These results suggest that Xp95 is an element in a tyrosine kinase signaling pathway that may be involved in progesterone-induced Xenopus oocyte maturation.

DNA Cell Biol, 1999 Jan, 18(1), 27 - 37
Differential regulation of human T-plastin gene in leukocytes and non-leukocytes: identification of the promoter, enhancer, and CpG island; Lin CS et al.; Plastins (fimbrins) are a family of actin-bundling proteins conserved from yeast to humans . In humans, three tissue-specific plastin isoforms have been identified . The T isoform (T-plastin) is unique in that it is expressed in all tissues except leukocytes . To investigate how the T-plastin gene is differentially regulated in leukocytes and non-leukocytes, we isolated a genomic clone that included 9 kb of the upstream flanking region, 0.1 kb of the first exon, and 5.9 kb of the first intron . From this clone, we obtained a continuous sequence of 5535 bp, including 3138 bp of the upstream flanking region, the first exon, and 2286 bp of the first intron . A cluster of four transcription initiation sites was located by S1 mapping . A region spanning these sites and extending 1.4 kb into the first intron had the characteristics of a CpG island . Three CG-containing restriction sites within this island were analyzed and found all or variably methylated in four T-plastin-negative leukemia cell lines . In contrast, the same sites were not methylated in three T-plastin-expressing cell lines or in a sample of normal blood lymphocytes . A basal promoter was located 250 bp upstream from the transciption initiation sites . It comprised a CCAAT box, an Sp1 motif, and four AP2 motifs . No TATA or Inr sequence was found . The basal promoter exhibited weak activity when assayed in fibrosarcoma cells . Stronger promoter activities were found in the presence of the SV40 enhancer or a T-plastin enhancer located some 500 bp from the basal promoter . In T-plastin-negative leukemia cells, the T-plastin basal promoter could be activated by the SV40 enhancer but not by the T-plastin enhancer . DNA footprinting identified the T-plastin enhancer as two inverted symmetric octamers (AGATAACCTC and GAGGTCAGCT) separated by 17 nucleotides.

Semin Cell Dev Biol, 1997 Jun, 8(3), 305 - 310
Phospholipase D; Wakelam MJ et al.; Phospholipase D catalyses the hydrolysis of phosphatidylcholine to generate phosphatidate . The regulation of PLD activity is complex involving a number of small GTP binding proteins, but in particular Arf and Rho, phosphatidylinositol 4,5-bisphosphate and protein kinase C . The cDNA for PLD1 has recently been cloned and shows homology to the yeast and plant genes but only within four domains . Domains I and IV each contain a putative catalytic triad . PLD activity has been detected in plasma membranes, Golgi membranes and in nuclear membranes; it is unclear if different isoenzymes are responsible for this variation, or if the PLDs are differently regulated . The product of PLD activity, PA, appears to be a messenger molecule regulating the actin cytoskeleton and maybe playing a role in the control of membrane traffic and secretion.

RNA, 1999 Feb, 5(2), 318 - 29
Photocrosslinking of 4-thio uracil-containing RNAs supports a side-by-side arrangement of domains 5 and 6 of a group II intron; Podar M et al.; Previous studies suggested that domains 5 and 6 (D5 and D6) of group II introns act together in splicing and that the two helical structures probably do not interact by helix stacking . Here, we characterized the major Mg2+ ion- and salt-dependent, long-wave UV light-induced, intramolecular crosslinks formed in 4-thiouridine-containing D56 RNA from intron 5gamma (aI5gamma) of the COXI gene of yeast mtDNA . Four major crosslinks were mapped and found to result from covalent bonds between nucleotides separating D5 from D6 {called J(56)} and residues of D6 near and including the branch nucleotide . These findings are extended by results of similar experiments using 4-thioU containing D56 RNAs from a mutant allele of aI5gamma and from the group IIA intron, aI1 . Trans-splicing experiments show that the crosslinked wild-type aI5gamma D56 RNAs are active for both splicing reactions, including some first-step branching . An RNA containing the 3-nt J(56) sequence and D6 of aI5gamma yields one main crosslink that is identical to the most minor of the crosslinks obtained with D56 RNA, but in this case in a cation-independent fashion . We conclude that the interaction between J(56) and D6 is influenced by charge repulsion between the D5 and D6 helix backbones and that high concentrations of cations allow the helices to approach closely under self-splicing conditions . The interaction between J(56) and D6 appears to be a significant factor establishing a side-by-side (i.e., not stacked) orientation of the helices of the two domains.

RNA, 1999 Feb, 5(2), 167 - 79
The C-terminal region of hPrp8 interacts with the conserved GU dinucleotide at the 5' splice site; Reyes JL et al.; A U5 snRNP protein, hPrp8, forms a UV-induced crosslink with the 5' splice site (5'SS) RNA within splicing complex B assembled in trans- as well as in cis-splicing reactions . Both yeast and human Prp8 interact with the 5'SS, branch site, polypyrimidine tract, and 3'SS during splicing . To begin to define functional domains in Prp8 we have mapped the site of the 5'SS crosslink within the hPrp8 protein . Immunoprecipitation analysis limited the site of crosslink to the C-terminal 5060-kDa segment of hPrp8 . In addition, size comparison of the crosslink-containing peptides generated with different proteolytic reagents with the pattern of fragments predicted from the hPrp8 sequence allowed for mapping of the crosslink to a stretch of five amino acids in the C-terminal portion of hPrp8 (positions 1894-1898) . The site of the 5'SS:hPrp8 crosslink falls within a segment spanning the previously defined polypyrimidine tract recognition domain in yPrp8, suggesting that an overlapping region of Prp8 may be involved both in the 5'SS and polypyrimidine tract recognition events . In the context of other known interactions of Prp8, these results suggest that this protein may participate in formation of the catalytic center of the spliceosome.

RNA, 1999 Feb, 5(2), 153 - 7
Rpp14 and Rpp29, two protein subunits of human ribonuclease P; Jarrous N et al.; In HeLa cells, the tRNA processing enzyme ribonuclease P (RNase P) consists of an RNA molecule associated with at least eight protein subunits, hPop1, Rpp14, Rpp20, Rpp25, Rpp29, Rpp30, Rpp38, and Rpp40 . Five of these proteins (hPop1p, Rpp20, Rpp30, Rpp38, and Rpp40) have been partially characterized . Here we report on the cDNA cloning and immunobiochemical analysis of Rpp14 and Rpp29 . Polyclonal rabbit antibodies raised against recombinant Rpp14 and Rpp29 recognize their corresponding antigens in HeLa cells and precipitate catalytically active RNase P . Rpp29 shows 23% identity with Pop4p, a subunit of yeast nuclear RNase P and the ribosomal RNA processing enzyme RNase MRP . Rpp14, by contrast, exhibits no significant homology to any known yeast gene . Thus, human RNase P differs in the details of its protein composition, and perhaps in the functions of some of these proteins, from the yeast enzyme.

Oncogene, 1999 Jan 28, 18(4), 995 - 1005
Physical interaction of the bHLH LYL1 protein and NF-kappaB1 p105; Ferrier R et al.; The LYL1 gene was first identified upon the molecular characterization of the t(7;9)(q35;p13) translocation associated with some human T-cell acute leukemias (T-ALLs) . In adult tissues, LYL1 expression is restricted to hematopoietic cells with the notable exclusion of the T cell lineage . LYL1 encodes a basic helix-loop-helix (bHLH) protein highly related to TAL-1, whose activation is also associated with a high proportion of human T-ALLs . A yeast two-hybrid system was used to identify proteins that specifically interact with LYL1 and might mediate its activities . We found that p105, the precursor of NF-kappaB1 p50, was the major LYL1-interacting protein in this system . The association between LYL1 and p105 was confirmed both in vitro and in vivo in mammalian cells . Biochemical studies indicated that the interaction was mediated by the bHLH motif of LYL1 and the ankyrin-like motifs of p105 . Ectopic expression of LYL1 in a human T cell line caused a significant decrease in NF-kappaB-dependent transcription, associated with a reduced level of NF-kappaB1 proteins.

Oncogene, 1999 Jan 28, 18(4), 849 - 54
The human F box protein beta-Trcp associates with the Cul1/Skp1 complex and regulates the stability of beta-catenin; Latres E et al.; Ubiquitin-conjugation targets numerous cellular regulators for proteasome-mediated degradation . Thus, the identification of ubiquitin ligases and their physiological substrates is crucially important, especially for those cases in which aberrant levels of regulatory proteins (e.g., beta-catenin, p27) result from a deregulated ubiquitination pathway . In yeast, the proteolysis of several G1 regulators is controlled by ubiquitin ligases (or SCFs) formed by three subunits: Skp1, Cul A (Cdc53), and one of many F-box proteins . Specific F-box proteins (Fbps) recruit different substrates to the SCF . Although many Fbps have been identified in mammals, their specific substrates and the existence of multiple SCFs have not yet been reported . We have found that one human Fbp, beta-Trcp (beta-Transducin repeat containing protein), does indeed form a novel SCF with human Skp1 and Cul1 . Consistent with recent reports indicating that Xenopus and Drosophila beta-Trcp homologs act as negative regulators of the Wnt/beta-catenin signaling pathway, we report here that human beta-Trcp interacts with beta-catenin in vivo . Furthermore, beta-catenin is specifically stabilized in vivo by the expression of a dominant negative beta-Trcp . These results indicate that the Cul1/Skp1/beta-Trcp complex forms a ubiquitin ligase that mediates the degradation of beta-catenin.

Genome Res, 1999 Feb, 9(2), 182 - 8
Isolation of human transcripts expressed in hamster cells from YACs by cDNA representational difference analysis; Gu J et al.; Gene isolation methods used during positional cloning rely on physical contigs consisting of bacterial artificial chromosomes, P1, or cosmid clones . However, in most instances, the initial framework for physical mapping consists of contigs of yeast artificial chromosome (YACs), large vectors that are suboptimal substrates for gene isolation . Here we report a strategy to identify gene sequences contained within a YAC by using cDNA representational difference analysis (RDA) to directly isolate transcripts expressed from the YAC in mammalian cells . The RDA tester cDNAs were generated from a previously reported hamster cell line derived by stable transfer of a 590-kb YAC (911D5) that expressed NPC1, the human gene responsible for Niemann-Pick type C (NP-C) . The driver cDNAs were generated from a control hamster cell line that did not contain the YAC that expressed NPC1 . Among the gene fragments obtained by RDA, NPC1 was the most abundant product . In addition, two non-NPC1 fragments were isolated that were mapped to and expressed from 911D5 . One of these RDA gene fragments (7-R) spans more than one exon and has 98% sequence identity with a human cDNA clone reported previously as an expressed sequence tag (EST), but not mapped to a chromosomal region . The other fragment (2-R) that had no significant sequence similarities with known mammalian genes or ESTs, was further localized to the region of overlap between YACs 911D5 and 844E3 . The latter YAC is part of a contig across the NP-C candidate region, but does not contain NPC1 . This two-part approach in which stable YAC transfer is followed by cDNA RDA should be a useful adjunct strategy to expedite the cloning of human genes when a YAC contig is available across a candidate interval.

Genome Res, 1999 Feb, 9(2), 130 - 6
Comparative sequence analysis of human minisatellites showing meiotic repeat instability; Murray J et al.; The highly variable human minisatellites MS32 (D1S8), MS31A (D7S21), and CEB1 (D2S90) all show recombination-based repeat instability restricted to the germline . Mutation usually results in polar interallelic conversion or occasionally in crossovers, which, at MS32 at least, extend into DNA flanking the repeat array, defining a localized recombination hotspot and suggesting that cis-acting elements in flanking DNA can influence repeat instability . Therefore, comparative sequence analysis was performed to search for common flanking elements associated with these unstable loci . All three minisatellites are located in GC-rich DNA abundant in dispersed and tandem repetitive elements . There were no significant sequence similarities between different loci upstream of the unstable end of the repeat array . Only one of the three loci showed clear evidence for putative coding sequences near the minisatellite . No consistent patterns of thermal stability or DNA secondary structure were shared by DNA flanking these loci . This work extends previous data on the genomic environment of minisatellites . In addition, this work suggests that recombinational activity is not controlled by primary or secondary characteristics of the DNA sequence flanking the repeat array and is not obviously associated with gene promoters as seen in yeast.

Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics, 1995 Sep, 52(3), 2939 - 50
Systematic analysis of coding and noncoding DNA sequences using methods of statistical linguistics; Mantegna RN et al.; We compare the statistical properties of coding and noncoding regions in eukaryotic and viral DNA sequences by adapting two tests developed for the analysis of natural languages and symbolic sequences . The data set comprises all 30 sequences of length above 50 000 base pairs in GenBank Release No . 81.0, as well as the recently published sequences of C . elegans chromosome III (2.2 Mbp) and yeast chromosome XI (661 Kbp) . We find that for the three chromosomes we studied the statistical properties of noncoding regions appear to be closer to those observed in natural languages than those of coding regions . In particular, (i) a n-tuple Zipf analysis of noncoding regions reveals a regime close to power-law behavior while the coding regions show logarithmic behavior over a wide interval, while (ii) an n-gram entropy measurement shows that the noncoding regions have a lower n-gram entropy (and hence a larger "n-gram redundancy") than the coding regions . In contrast to the three chromosomes, we find that for vertebrates such as primates and rodents and for viral DNA, the difference between the statistical properties of coding and noncoding regions is not pronounced and therefore the results of the analyses of the investigated sequences are less conclusive . After noting the intrinsic limitations of the n-gram redundancy analysis, we also briefly discuss the failure of the zeroth- and first-order Markovian models or simple nucleotide repeats to account fully for these "linguistic" features of DNA . Finally, we emphasize that our results by no means prove the existence of a "language" in noncoding DNA.

Mol Cell Biol, 1999 Mar, 19(3), 2425 - 34
The LIM-only protein PINCH directly interacts with integrin-linked kinase and is recruited to integrin-rich sites in spreading cells; Tu Y et al.; PINCH is a widely expressed and evolutionarily conserved protein comprising primarily five LIM domains, which are cysteine-rich consensus sequences implicated in mediating protein-protein interactions . We report here that PINCH is a binding protein for integrin-linked kinase (ILK), an intracellular serine/threonine protein kinase that plays important roles in the cell adhesion, growth factor, and Wnt signaling pathways . The interaction between ILK and PINCH has been consistently observed under a variety of experimental conditions . They have interacted in yeast two-hybrid assays, in solution, and in solid-phase-based binding assays . Furthermore, ILK, but not vinculin or focal adhesion kinase, has been coisolated with PINCH from mammalian cells by immunoaffinity chromatography, indicating that PINCH and ILK associate with each other in vivo . The PINCH-ILK interaction is mediated by the N-terminal-most LIM domain (LIM1, residues 1 to 70) of PINCH and multiple ankyrin (ANK) repeats located within the N-terminal domain (residues 1 to 163) of ILK . Additionally, biochemical studies indicate that ILK, through the interaction with PINCH, is capable of forming a ternary complex with Nck-2, an SH2/SH3-containing adapter protein implicated in growth factor receptor kinase and small GTPase signaling pathways . Finally, we have found that PINCH is concentrated in peripheral ruffles of cells spreading on fibronectin and have detected clusters of PINCH that are colocalized with the alpha5beta1 integrins . These results demonstrate a specific protein recognition mechanism utilizing a specific LIM domain and multiple ANK repeats and suggest that PINCH functions as an adapter protein connecting ILK and the integrins with components of growth factor receptor kinase and small GTPase signaling pathways.

EMBO J, 1999 Feb 15, 18(4), 949 - 58
Deletion of a region that is a candidate for the difference between the deletion forms of hereditary persistence of fetal hemoglobin and deltabeta-thalassemia affects beta- but not gamma-globin gene expression; Calzolari R et al.; The analysis of a number of cases of beta-globin thalassemia and hereditary persistence of fetal hemoglobin (HPFH) due to large deletions in the beta-globin locus has led to the identification of several DNA elements that have been implicated in the switch from human fetal gamma- to adult beta-globin gene expression . We have tested this hypothesis for an element that covers the minimal distance between the thalassemia and HPFH deletions and is thought to be responsible for the difference between a deletion HPFH and deltabeta-thalassemia, located 5' of the delta-globin gene . This element has been deleted from a yeast artificial chromosome (YAC) containing the complete human beta-globin locus . Analysis of this modified YAC in transgenic mice shows that early embryonic expression is unaffected, but in the fetal liver it is subject to position effects . In addition, the efficiency of transcription of the beta-globin gene is decreased, but the developmental silencing of the gamma-globin genes is unaffected by the deletion . These results show that the deleted element is involved in the activation of the beta-globin gene perhaps through the loss of a structural function required for gene activation by long-range interactions.

Mol Cell Endocrinol, 1998 Nov 25, 146(1-2), 69 - 76
Receptor-interacting protein 140 interacts with and inhibits transactivation by, peroxisome proliferator-activated receptor alpha and liver-X-receptor alpha; Miyata KS et al.; Receptor interacting protein 140 (RIP140), a previously identified putative ligand-dependent coactivator of nuclear hormone receptors, was isolated by yeast two-hybrid cloning as a factor that interacts with peroxisome proliferator-activated receptor alpha (PPARalpha) . This interaction in yeast required the integrity of the carboxyl-terminal, ligand-dependent activation domain of PPARalpha . However, protein binding studies carried out in vitro showed that full-length RIP140 bound efficiently to PPARalpha in the absence of exogenously added ligand . RIP140 also bound strongly to the liver-X-receptor (LXRalpha) in the absence of an activator for this receptor . In contrast, a strong interaction of RIP140 with the PPARalpha and LXRalpha heterodimerization partner retinoid-X-receptor alpha (RXRalpha) required the presence of its cognate ligand, 9-cis retinoic acid . Transfection analysis in mammalian cells demonstrated that RIP140 antagonized PPARalpha/RXRalpha- and LXRalpha/RXRalpha-mediated signaling . Our findings identify RIP140 as a novel modulator of transcriptional activation mediated by PPARalpha and LXRalpha and indicate that RIP140 can also bind to nuclear hormone receptors in a ligand-independent manner and repress their activity.

Pharmacogenetics, 1998 Apr, 8(2), 101 - 8
Use of heterologously expressed human cytochrome P450 1A2 to predict tacrine-fluvoxamine drug interaction in man; Becquemont L et al.; The aim of the present study was to evaluate the use of recombinant human cytochrome P-450 1A2 (rH-CYP1A2) in studies performed in vitro in order to predict metabolic drug-drug interactions occurring in man . In vitro metabolism of tacrine (a CYP1A2 probe) in the presence and absence of fluvoxamine, a CYP1A2 inhibitor, was investigated in human liver mircrosomes and with different rH-CYP . Vmax, Km and Ki determined with human liver microsomes were compared with those observed using rH-CYP1A2, assuming that 1 mg of liver microsomes contains, on average, 69 pmol of CYP1A2 . The extent of tacrine metabolism inhibition procured by fluvoxamine with rH-CYP1A2, was compared with previous results observed in man . The Vax and Km for 1-hydroxytacrine formation rates obtained with rH-CYP1A2 were in good agreement with those observed in human liver microsomes (175+/-9 versus 140+/-60 pmol/min/mg for Vmax and 14+/-2 versus 16+/-2 microM for Km, respectively . The Ki of fluvoxamine on 1-hydroxytacrine formation rate observed with rH-CYP1A2 was similar to that observed with human liver microsome (0.35+/-0.05 versus 0.20+/-0.20 microM, respectively) . Using the Km, Vmax and Ki determined with rH-CYP1A2, we calculated that fluvoxamine produced an inhibition of 1-, 2- and 4-hydroxytacrine formation rate of 91, 87 and 88%, respectively, in the range of tacrine and fluvoxamine concentrations observed in man . These percentages of inhibition calculated in vitro were in agreement with the percentage of fluvoxamine-dependent decrease in tacrine apparent oral clearance previously observed in man (83+/-13%) . We conclude that human CYP1A2 expressed in yeast is a powerful tool to predict and to quantify drug-drug interactions in man.

Toxicol Lett, 1998 Dec 28, 102-103, 41 - 5
Telomerase enzyme activation and human cell immortalization; Meyerson M; Activity of telomerase, the enzyme that synthesizes the telomere ends of linear eukaryotic chromosomes, is repressed in most normal human somatic cells but induced in most human cancers . Normal human cells that lack telomerase activity progressively lose telomere sequences . In contrast, most immortalized cell lines and malignant human tumors appear to maintain constant telomere length via telomerase activity . Telomerase is composed of at least two subunits, an RNA subunit that templates telomere synthesis, and a catalytic protein subunit . The gene encoding the catalytic protein subunit of telomerase has recently been identified, first in yeast and ciliates and then in humans . This catalytic subunit belongs to the reverse transcriptase family . Studies of telomerase subunits further define a role for telomerase in the control of mammalian cell lifespan . The expression of the human telomerase catalytic subunit gene, hTERT, is induced in immortalized cells and primary tumors . When hTERT is ectopically expressed in hitherto telomerase-negative cells, telomerase enzyme activity appears, and an extended lifespan has been observed in some cells . In contrast, disruption of the mouse telomerase RNA subunit gene, mTERC, results in a delayed failure of cell proliferation . Telomerase activity therefore appears to be necessary for the prolonged survival of mammalian cells.

Oncogene, 1999 Feb 4, 18(5), 1209 - 17
TIF1gamma, a novel member of the transcriptional intermediary factor 1 family; Venturini L et al.; We report the cloning and characterization of a novel member of the Transcriptional Intermediary Factor 1 (TIF1) gene family, human TIF1gamma . Similar to TIF1alpha and TIF1beta, the structure of TIF1beta is characterized by multiple domains: RING finger, B boxes, Coiled coil, PHD/TTC, and bromodomain . Although structurally related to TIF1alpha and TIF1beta, TIF1gamma presents several functional differences . In contrast to TIF1alpha, but like TIF1beta, TIF1 does not interact with nuclear receptors in yeast two-hybrid or GST pull-down assays and does not interfere with retinoic acid response in transfected mammalian cells . Whereas TIF1alpha and TIF1beta were previously found to interact with the KRAB silencing domain of KOX1 and with the HP1alpha, MODI (HP1beta) and MOD2 (HP1gamma) heterochromatinic proteins, suggesting that they may participate in a complex involved in heterochromatin-induced gene repression, TIF1gamma does not interact with either the KRAB domain of KOX1 or the HP1 proteins . Nevertheless, TIF1gamma, like TIF1alpha and TIF1beta, exhibits a strong silencing activity when tethered to a promoter . Since deletion of a novel motif unique to the three TIF1 proteins, called TIF1 signature sequence (TSS), abrogates transcriptional repression by TIF1gamma, this motif likely participates in TIF1 dependent repression.

Oncogene, 1999 Feb 4, 18(5), 1157 - 64
11q23.1 and 11q25-qter YACs suppress tumour growth in vivo; Koreth J et al.; Frequent allelic deletion at chromosome 11q22-q23.1 has been described in breast cancer and a number of other malignancies, suggesting putative tumour suppressor gene(s) within the approximately 8 Mb deleted region . In addition, we recently described another locus, at the 11q25-qter region, frequently deleted in breast cancer, suggesting additional tumour suppressor gene(s) in this approximately 2 Mb deleted region . An 11q YAC contig was accessed and three YACs, one containing the candidate gene ATM at 11q23.1, and two contiguous YACs (overlapping for approximately 400-600 kb) overlying most of the 11q25 deleted region, were retrofitted with a G418 resistance marker and transfected into murine A9 fibrosarcoma cells . Selected A9 transfectant clones (and control untransfected and 'irrelevant' alphoid YAC transfectant A9 clones) were assayed for in vivo tumorigenicity in athymic female Balb c-nu/nu mice . All the 11q YAC transfectant clones demonstrated significant tumour suppression compared to the control untransfected and 'irrelevant' YAC transfected A9 cells . These results define two discrete tumour suppressor loci on chromosome 11q by functional complementation, one to a approximately 1.2 Mb region on 11q23.1 (containing the ATM locus) and another to a approximately 400-600 kb subterminal region on 11q25-qter.

Curr Biol, 1999 Jan 28, 9(2), R66 - 9
Meiosis: MeiRNA hits the spot; Ohno M et al.; The protein Mei2 performs at least two functions required in fission yeast for the switch from mitotic to meiotic cell cycles . One of these functions also requires meiRNA . It appears that meiRNA targets Mei2 to the nucleus, where it can promote the first meiotic division.

Proc Natl Acad Sci U S A, 1999 Feb 16, 96(4), 1761 - 6
The tomato DWARF enzyme catalyses C-6 oxidation in brassinosteroid biosynthesis
Bishop GJ, Nomura T, Yokota T, Harrison K, Noguchi T, Fujioka S, Takatsuto S, Jones JD, Kamiya Y.
Brassinosteroids (BRs) are steroidal plant hormones essential for normal plant growth and development . Mutants in the biosynthesis or perception of BRs are usually dwarf . The tomato Dwarf gene (D), which was predicted to encode a cytochrome P450 enzyme (P450) with homology to other P450s involved in BR biosynthesis, was cloned previously . Here, we show that DWARF catalyses the C-6 oxidation of 6-deoxocastasterone (6-deoxoCS) to castasterone (CS), the immediate precursor of brassinolide . To do this, we first confirmed that the D cDNA complemented the mutant light- and dark-grown phenotypes of the extreme dwarf (dx) allele of tomato . To identify a substrate for the DWARF enzyme, exogenous application of BR intermediates to dx plants was carried out . C-6 oxoBR intermediates enhanced hypocotyl elongation whereas the C-6 deoxoBR, 6-deoxoCS, had little effect . Quantitative analysis of endogenous BR levels in tomato showed mainly the presence of 6-deoxoBRs . Furthermore, dx plants were found to lack CS and had a high level of 6-deoxoCS in comparison to D plants that had CS and a lower level of 6-deoxoCS . Confirmation that DWARF catalyzed the C-6 oxidation of 6-deoxoCS to CS was obtained by functional expression of DWARF in yeast . In these experiments, the intermediate 6alpha-hydroxycastasterone was identified, indicating that DWARF catalyzes two steps in BR biosynthesis . These data show that DWARF is involved in the C-6 oxidation in BR biosynthesis.

Genes Dev, 1999 Feb 1, 13(3), 270 - 83
The SCFbeta-TRCP-ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IkappaBalpha and beta-catenin and stimulates IkappaBalpha ubiquitination in vitro; Winston JT et al.; Ubiquitin-mediated proteolysis has a central role in controlling the intracellular levels of several important regulatory molecules such as cyclins, CKIs, p53, and IkappaBalpha . Many diverse proinflammatory signals lead to the specific phosphorylation and subsequent ubiquitin-mediated destruction of the NF-kappaB inhibitor protein IkappaBalpha . Substrate specificity in ubiquitination reactions is, in large part, mediated by the specific association of the E3-ubiquitin ligases with their substrates . One class of E3 ligases is defined by the recently described SCF complexes, the archetype of which was first described in budding yeast and contains Skp1, Cdc53, and the F-box protein Cdc4 . These complexes recognize their substrates through modular F-box proteins in a phosphorylation-dependent manner . Here we describe a biochemical dissection of a novel mammalian SCF complex, SCFbeta-TRCP, that specifically recognizes a 19-amino-acid destruction motif in IkappaBalpha (residues 21-41) in a phosphorylation-dependent manner . This SCF complex also recognizes a conserved destruction motif in beta-catenin, a protein with levels also regulated by phosphorylation-dependent ubiquitination . Endogenous IkappaBalpha-ubiquitin ligase activity cofractionates with SCFbeta-TRCP . Furthermore, recombinant SCFbeta-TRCP assembled in mammalian cells contains phospho-IkappaBalpha-specific ubiquitin ligase activity . Our results suggest that an SCFbeta-TRCP complex functions in multiple transcriptional programs by activating the NF-kappaB pathway and inhibiting the beta-catenin pathway.

Acta Cytol, 1997 Jul-Aug, 41(4 Suppl), 1317 - 9
Cutaneous blastomycosis . Report of a case with diagnosis by fine needle aspiration cytology; Desai AP et al.; BACKGROUND: Blastomycosis is rare in India . Clinically, cutaneous blastomycosis may be mistaken for keratoacanthoma, squamous cell carcinoma, tuberculosis, tertiary syphilis, leprosy or pyoderma . CASE: A 19-year-old male presented with a soft, fluctuant swelling at the medial canthus of the eye . Fine needle aspiration biopsy (FNAB) showed many single, spherical structures with thick walls . They had basophilic protoplasm and six nuclei in the center . A few of them showed budding with a broad base . CONCLUSION: The cytomorphology of Blastomyces dermatitidis is characteristic, and the organism can be differentiated from other yeast forms . The disease is sensitive to antifungal imidazole derivatives and can be cured.

Rev Esp Quimioter, 1998 Dec, 11(4), 295 - 315
A reevaluation of nystatin in prophylaxis and treatment of oropharyngeal candidiasis; Alvarez Alvarez ME et al.; The incidence of oropharyngeal candidiasis is growing . The species of the genus Candida are extremely frequent among human colonizers . The changes in the yeast-human interaction by aging, debilitating, and immunosuppressive diseases, and the more aggressive medical interventions can explain this phenomenon . Antifungals are used both in prophylaxis and therapy, but the number of available agents remains scarce . Acquired resistance to the more commonly used antifungal agents, the azole compounds, is also an increasing threat, Policies for antifungal use should be established in order to maintain the therapeutic possibilities of the current compounds, The widespread use of systemic azoles, agents useful in deep mycosis, may increasingly exert a selective power for resistant variants . Superficial infections, such as oropharyngeal candidiasis, can be successfully controlled by nystatin, a classic polyene, which is very well tolerated and has very low rates of resistance . This review on the importance of oropharyngeal candidiasis emphasizes this therapeutic possibility, and is complemented by in vitro studies documenting the excellent activity of nystatin on both azole-susceptible and resistant strains.

J Appl Toxicol, 1999 Jan-Feb, 19(1), 39 - 45
Partial and weak oestrogenicity of the red wine constituent resveratrol: consideration of its superagonist activity in MCF-7 cells and its suggested cardiovascular protective effects; Ashby J et al.; It was recently reported that the red wine phytoestrogen resveratrol (RES) acts as a superagonist to oestrogen-responsive MCF-7 cells . This activity of RES was speculated to be relevant to the 'French paradox' in which moderate red wine consumption is reported to yield cardiovascular health benefits to humans . We report here that RES binds to oestrogen receptors (ER) isolated from rat uterus with an affinity approximately 5 orders of magnitude lower than does either the reference synthetic oestrogen diethylstilboestrol (DES) or oestradiol (E2) . In comparison with E2 or DES, RES is only a weak and partial agonist in a yeast hER-alpha transcription assay and in cos-1 cell assays employing transient transfections of ER-alpha or ER-beta associated with two different ER-response elements . Resveratrol was also concluded to be inactive in immature rat uterotrophic assays conducted using three daily administrations of 0.03-120 mgkg(-1)/day(-1) RES (administered by either oral gavage or subcutaneous injection) . These data weaken the suggestion that the oestrogenicity of RES may account for the reported cardiovascular protective effects of red wine consumption, and they raise questions regarding the extent to which oestrogenicity data derived for a chemical using MCF-7 cells (or any other single in vitro assay) can be used to predict the hormonal effects likely to occur in animals or humans.

Nature, 1999 Feb 4, 397(6718), 446 - 50
The transcriptional cofactor complex CRSP is required for activity of the enhancer-binding protein Sp1; Ryu S et al.; Activation of gene transcription in metazoans is a multistep process that is triggered by factors that recognize transcriptional enhancer sites in DNA . These factors work with co-activators to direct transcriptional initiation by the RNA polymerase II apparatus . One class of co-activator, the TAF(II) subunits of transcription factor TFIID, can serve as targets of activators and as proteins that recognize core promoter sequences necessary for transcription initiation . Transcriptional activation by enhancer-binding factors such as Sp1 requires TFIID, but the identity of other necessary cofactors has remained unknown . Here we describe a new human factor, CRSP, that is required together with the TAF(II)s for transcriptional activation by Sp1 . Purification of CRSP identifies a complex of approximate relative molecular mass 700,000 (M(r) approximately 700K) that contains nine subunits with M(r) values ranging from 33K to 200K . Cloning of genes encoding CRSP subunits reveals that CRSP33 is a homologue of the yeast mediator subunit Med7, whereas CRSP150 contains a domain conserved in yeast mediator subunit Rgr1 . CRSP p200 is identical to the nuclear hormone-receptor co-activator subunit TRIP2/PBP . CRSPs 34, 77 and 130 are new proteins, but the amino terminus of CRSP70 is homologous to elongation factor TFIIS . Immunodepletion studies confirm that these subunits have an essential cofactor function . The presence of common subunits in distinct cofactor complexes suggests a combinatorial mechanism of co-activator assembly during transcriptional activation.

Oncogene, 1999 Jan 21, 18(3), 797 - 806
Mechanism of activation of Pak1 kinase by membrane localization; Lu W et al.; Pak kinases are a family of serine/threonine protein kinases homologous to Ste20p of yeast . Paks can be activated in vivo and in vitro by binding to GTP-bound Cdc42 and Rac1, members of the Rho family of small GTPases implicated in regulating the organization of the actin cytoskeleton . We have previously reported that the SH2/SH3-containing adaptor protein Nck binds Pak kinase through its second SH3 domain . Pak1 can be targeted to the membrane by Nck in response to tyrosine phosphorylation, and membrane association of Pak1 is sufficient to increase its specific activity . The mechanism whereby Pak is activated by membrane localization, however, is unknown . We show here that expression of three proteins that inhibit Rho-family GTPases by different mechanisms (RhoGDI, Bcr and D57Y Cdc42) all block the activation of Pak by a membrane-targeted Nck SH3 domain, demonstrating that the in vivo activation of Pak1 induced by membrane localization is dependent on Rho-family GTPases . This implies that Pak activity can be regulated in cells both by the level of GTP loading of various Rho-family GTPases and the local concentration of Pak relative to these GTPases . Our data also suggest the existence of Rho-family GTPases in addition to Cdc42 and Rac1 that can activate Pak on membranes.

Oncogene, 1999 Jan 21, 18(3), 689 - 701
The product of the cph oncogene is a truncated, nucleotide-binding protein that enhances cellular survival to stress; Velasco JA et al.; Cph was isolated from neoplastic Syrian hamster embryo fibroblasts initiated by 3-methylcholanthrene (MCA), and was shown to be a single copy gene in the hamster genome, conserved from yeast to human cells, expressed in fetal cells and most adult tissues, and acting synergistically with H-ras in the transformation of murine NIH3T3 fibroblasts . We have now isolated Syrian hamster full-length cDNAs for the cph oncogene and proto-oncogene . Nucleotide sequence analysis revealed that cph was activated in MCA-treated cells by a point-mutational deletion at codon 214, which caused a shift in the normal open reading frame (ORF) and brought a translation termination codon 33 amino acids downstream . While proto-cph encodes a protein (pcph) of 469 amino acids, cph encodes a truncated protein (cph) of 246 amino acids with a new, hydrophobic C-terminus . Similar mechanisms activated cph in other MCA-treated Syrian hamster cells . The cph and proto-cph proteins have partial sequence homology with two protein families: GDP/GTP exchange factors and nucleotide phosphohydrolases . In vitro translated, gel-purified cph proteins did not catalyze nucleotide exchange for H-ras, but were able to bind nucleotide phosphates, in particular ribonucleotide diphosphates such as UDP and GDP . Steady-state levels of cph mRNA increased 6.7-fold in hamster neoplastic cells, relative to a 2.2-fold increase in normal cells, when they were subjected to a nutritional stress such as serum deprivation . Moreover, cph-transformed NIH3T3 cells showed increased survival to various forms of stress (serum starvation, hyperthermia, ionizing radiation), strongly suggesting that cph participates in cellular mechanisms of response to stress.

Cell, 1999 Jan 8, 96(1), 143 - 52
Menin interacts with the AP1 transcription factor JunD and represses JunD-activated transcription; Agarwal SK et al.; MEN1 is a tumor suppressor gene that encodes a 610 amino acid nuclear protein (menin) of previously unknown function . Using a yeast two-hybrid screen with menin as the bait, we have identified the transcription factor JunD as a direct menin-interacting partner . Menin did not interact directly with other Jun and Fos family members . The menin-JunD interaction was confirmed in vitro and in vivo . Menin repressed transcriptional activation mediated by JunD fused to the Gal4 DNA-binding domain from a Gal4 responsive reporter, or by JunD from an AP1-responsive reporter . Several naturally occurring and clustered MEN1 missense mutations disrupted menin interaction with JunD . These observations suggest that menin's tumor suppressor function involves direct binding to JunD and inhibition of JunD activated transcription.

Cell, 1999 Jan 8, 96(1), 121 - 30
Functional association of Nmi with Stat5 and Stat1 in IL-2- and IFNgamma-mediated signaling; Zhu M et al.; Using the coiled-coil region of Stat5b as the bait in a yeast two-hybrid screen, we identified the association of Nmi, a protein of unknown function previously reported as an N-Myc interactor . We further show that Nmi interacts with all STATs except Stat2 . We evaluated two cytokine systems, IL-2 and IFNgamma, and demonstrate that Nmi augments STAT-mediated transcription in response to these cytokines . Interestingly, Nmi lacks an intrinsic transcriptional activation domain; instead, Nmi enhances the association of CBP/p300 coactivator proteins with Stat1 and Stat5, and together with CBP/p300 can augment IL-2- and IFNgamma-dependent transcription . Therefore, our data not only reveal that Nmi can potentiate STAT-dependent transcription, but also suggest that it can augment coactivator protein recruitment to at least some members of a group of sequence-specific transcription factors.

Nat Genet, 1999 Feb, 21(2), 216 - 9
Receptors for polytropic and xenotropic mouse leukaemia viruses encoded by a single gene at Rmc1; Yang YL et al.; The onset of leukaemia caused by type C retroviruses (MLV) in mice is accelerated by the emergence of recombinant polytropic or mink cell focus-forming (MCF) viruses . Susceptibility to infection by polytropic/MCF and also by closely related xenotropic MLV has been mapped to Rmc1 on mouse chromosome 1 (refs 5-7) . To identify this gene, we introduced an expression cDNA library prepared from mouse NIH3T3 fibroblasts into nonpermissive hamster cells and screened these cells for acquired susceptibility to MCF viruses encoding beta-galactosidase and G418 resistance . From hamster cell clones identified in the screen, we recovered a mouse cDNA that maps to Rmc1 and confers MCF MLV infection when expressed in nonpermissive cell lines . It encodes a membrane protein related to Syg1p (suppressor of yeast G alpha deletion; ref . 8) . The receptor-binding domain of the MCF MLV envelope protein binds specifically to Xenopus laevis oocytes that express mouse Syg1, suggesting it functions as a receptor that mediates virus entry . We also obtained the cDNA encoding human SYG1 . When expressed in hamster cells, it establishes infectivity by MCF MLV as well as xenotropic MLV, which do not infect laboratory mice.

J Biol Chem, 1999 Feb 19, 274(8), 4839 - 47
Identification of inhibitor specificity determinants in a mammalian phosphodiesterase; Atienza JM et al.; Mammalian phosphodiesterase types 3 and 4 (PDE3 and PDE4) hydrolyze cAMP and are essential for the regulation of this intracellular second messenger in many cell types . Whereas these enzymes share structural and biochemical similarities, each can be distinguished by its sensitivity to isozyme-specific inhibitors . By using a series of chimeric enzymes, we have localized the region of PDE4 that confers sensitivity to selective inhibitors . This inhibitor specificity domain lies within a short sequence at the carboxyl terminus of the catalytic domain of the protein, consistent with the competitive nature of inhibition by these compounds . Surprisingly, the identified region also includes some of the most highly conserved residues among PDE isoforms . A yeast-based expression system was used for the isolation and characterization of mutations within this area that confer resistance to the PDE4-specific inhibitor rolipram . Analysis of these mutants indicated that both conserved and unique residues are required for isoform-specific inhibitor sensitivity . In some cases, combined point mutations contribute synergistically to the reduction of sensitivity (suppression of IC50) . We also report that several mutations display differential sensitivity changes with respect to distinct structural classes of inhibitors.

J Biol Chem, 1999 Feb 19, 274(8), 4577 - 85
Identification of a cDNA encoding a retinoid X receptor homologue from Schistosoma mansoni . Evidence for a role in female-specific gene expression; Freebern WJ et al.; Schistosoma mansoni, a multicelluar eukaryotic blood fluke, is a major cause of morbidity worldwide in humans . The study of female parasite growth, development, and gene regulation is important because the eggs produced are responsible for the pathogenesis observed in schistosomiasis . p14, an eggshell precursor gene expressed only in sexually mature females in response to a male stimulus, is a model for female-specific gene regulation . The upstream region of the p14 gene shares sequences present in insect genes known to be regulated in a sex-, temporal-, and tissue-specific manner by members of the steroid receptor superfamily . Herein, we report the identification and characterization of a cDNA that encodes the S . mansoni (Sm) RXR homologue . Sequence analysis predicts and Western blot analysis confirms the synthesis of a 74-kDa protein, the largest member of the RXR family reported to date . We show by electrophoretic mobility shift assay analysis that SmRXR binds to cis-elements of the p14 gene including a direct repeat that follows the "3-4-5" rule of binding elements recognized by members of the steroid receptor superfamily . Furthermore, we demonstrate that SmRXR can act as a transcription activator in the yeast one-hybrid system . Through quantitative reverse transcriptase-polymerase chain reaction, we show that the SmRXR gene is constitutively expressed and thus must play multiple roles throughout the schistosome life cycle.

Med Mycol, 1998, 36 Suppl 1, 68 - 78
Standardization of antifungal susceptibility testing and clinical relevance; Espinel-Ingroff A et al.; Standardization of antifungal susceptibility testing became essential to overcome the problem of interlaboratory variability and to determine the clinical relevance of in vitro data . This evolving process began for the yeasts and consequently broth macrodilution and microdilution methods (NCCLS M27 document) have been developed . These tests may not be useful for testing all organism-drug combinations or be the most convenient techniques for routine use in the clinical laboratory . Different alternatives to the NCCLS methods are currently under investigation . The identification of standard guidelines for antifungal susceptibility testing has reduced interlaboratory variability and further progress has been achieved with the determination of tentative interpretive breakpoints for certain drug-yeast combinations . However, these breakpoints are not adequate for interpretations of MICs for fungi-drug combinations beyond the setting for which they were determined . The NCCLS Subcommittee has also generated data for proposed testing guidelines for the moulds.

Eur J Pharmacol, 1999 Jan 22, 365(2-3), 259 - 66
Anti-inflammatory and analgesic effects of a novel pyrazole derivative, FR140423; Ochi T et al.; The pharmacological profile of a novel and newly discovered non-steroidal anti-inflammatory and analgesic compound, 3-(difluoromethyl)-1-(4-methoxyphenyl)-5-{4-(methylsulfinyl)phenyl}pyraz ole (FR140423), was investigated . In recombinant human cyclooxygenase enzyme assays, the inhibition of prostaglandin E2 formation by FR140423 was 150 times more selective for cyclooxygenase-2 than cyclooxygenase-1 . Oral administration of FR140423 dose dependently reduced carrageenin-induced paw edema and adjuvant arthritis . These effects were two- to three-fold more potent than those of indomethacin . Unlike indomethacin, FR140423 did not induce mucosal lesions in the stomach . FR140423 showed dose-dependent anti-hyperalgesic effects in the yeast-induced hyperalgesic model . This effect was five-fold more potent than that of indomethacin . Furthermore, FR140423 increased the pain threshold in non-inflamed paws and, unlike indomethacin, it produced an analgesic effect in the tail-flick test . These analgesic effects were blocked by the mu-opioid antagonist naloxone . These results suggest that FR140423, a selective cyclooxygenase-2 inhibitor, is a potent non-steroidal anti-inflammatory drug (NSAID) without gastrointestinal side effects and is a unique compound having morphine-like analgesic effects.

Science, 1999 Feb 12, 283(5404), 985 - 7
Conserved structures of mediator and RNA polymerase II holoenzyme; Asturias FJ et al.; Single particles of the mediator of transcriptional regulation (Mediator) and of RNA polymerase II holoenzyme were revealed by electron microscopy and image processing . Mediator alone appeared compact, but at high pH or in the presence of RNA polymerase II it displayed an extended conformation . Holoenzyme contained Mediator in a fully extended state, partially enveloping the globular polymerase, with points of apparent contact in the vicinity of the polymerase carboxyl-terminal domain and the DNA-binding channel . A similarity in appearance and conformational behavior of yeast and murine complexes indicates a conservation of Mediator structure among eukaryotes.

Fungal Genet Biol, 1998 Nov, 25(2), 119 - 33
Lovastatin triggers an apoptosis-like cell death process in the fungus Mucor racemosus; Roze LV et al.; The filamentous dimorphic fungus Mucor racemosus possesses three ras genes, Mras1, 2, and 3, whose expression is correlated to morphogenesis of the fungus . Lovastatin, an indirect inhibitor of protein prenylation, altered the processing of MRas1 protein, blocked the accumulation of MRas3 protein, and caused the MRas1/p20 protein complex to disappear in M . racemosus . Concurrently it arrested sporangiospore germination, decreased growth rate, caused a loss of cell viability accompanied by cell shrinkage, increased cell density and cytoplasm condensation, and triggered DNA fragmentation, resulting in nucleosomes and nucleosome multimers . The specific morphological and biochemical events seen in Mucor cell death, particularly DNA fragmentation, resemble the best known characteristics of classical apoptosis in mammalian cells and prompted us to classify lovastatin-induced cell death as an apoptosis-like process . Lovastatin did not cause cell death in a leucine auxotroph of Mucor grown in YNB minimal medium, conditions which support only spherical growth during spore germination . Exogenous dibutyryl-cAMP initiated morphogenesis from hyphal (polar) growth to yeast-like (spherical) growth during spore germination and strongly prevented cell death which resulted from lovastatin treatment . Wortmannin added together with dibutyryl-cAMP showed a synergistic effect in the prevention of fungal cell death . These data suggest that the regulation of lovastatin-induced cell death in Mucor requires a signal transduction pathway(s) involving cAMP whose function is specific to a particular developmental stage.

Am J Hum Genet, 1999 Feb, 64(2), 462 - 70
Identification and characterization of a mutation, in the human UDP-galactose-4-epimerase gene, associated with generalized epimerase-deficiency galactosemia; Wohlers TM et al.; Epimerase-deficiency galactosemia results from impairment of the human enzyme UDP-galactose-4-epimerase (hGALE) . We and others have identified substitution mutations in the hGALE alleles of patients with the clinically mild, peripheral form of epimerase deficiency . We report here the first identification of an hGALE mutation in a patient with the clinically severe, generalized form of epimerase deficiency . The mutation, V94M, was found on both GALE alleles of this patient . This same mutation also was found in the homozygous state in two additional patients with generalized epimerase deficiency . The specific activity of the V94M-hGALE protein expressed in yeast was severely reduced with regard to UDP-galactose and partially reduced with regard to UDP-N-acetylgalactosamine . In contrast, two GALE-variant proteins associated with peripheral epimerase deficiency, L313M-hGALE and D103G-hGALE, demonstrated near-normal levels of activity with regard to both substrates, but a third allele, G90E-hGALE, demonstrated little, if any, detectable activity, despite near-normal abundance . G90E originally was identified in a heterozygous patient whose other allele remains uncharacterized . Thermal lability and protease-sensitivity studies demonstrated compromised stability in all of the partially active mutant enzymes.

J Immunol, 1999 Feb 15, 162(4), 2281 - 90
The beta-glucan-binding lectin site of mouse CR3 (CD11b/CD18) and its function in generating a primed state of the receptor that mediates cytotoxic activation in response to iC3b-opsonized target cells; Xia Y et al.; Mouse leukocyte CR3 (Mac-1, alphaMbeta2 integrin) was shown to function as a receptor for beta-glucans in the same way as human CR3 . Soluble zymosan polysaccharide (SZP) or pure beta-glucans labeled with FITC or 125I bound in a saturable and reversible manner to neutrophils, macrophages, and NK cells . This lectin activity was blocked by anti-CD11b mAb M1/70 or 5C6 and did not occur with leukocytes from CR3-/- (CD11b-deficient) mice . SZP preparations containing primarily mannose or glucose bound to CR3, and the binding of 125I-labeled beta-glucan to CR3 was competitively inhibited by beta-glucans from barley or seaweed, but not by yeast alpha-mannan . Also, as with human CR3, the lectin site of mouse CR3 was inhibited by alpha- or beta-methylglucoside (but not D-glucose), alpha- or beta-methylmannoside, and N-acetyl-D-glucosamine . Phagocytosis of zymosan and serum-opsonized zymosan was partially inhibited by anti-CR3 and was reduced to <40% of normal with leukocytes from CR3-/- mice . As with neutrophils from patients with CD18 deficiency, neutrophils from CR3-/- mice exhibited no phagocytosis of particulate beta-glucan . SZP or beta-glucans primed CR3 of neutrophils, macrophages, and NK cells for cytotoxicity of iC3b-opsonized tumor cells that otherwise did not trigger killing . beta-Glucan priming for cytotoxicity was inhibited by anti-CR3 and did not occur with leukocytes from CR3-/- mice . The primed state of macrophage and NK cell CR3 remained detectable for 18 to 24 h after pulsing with beta-glucans . The similarity of mouse and human CR3 in response to beta-glucans highlights the utility of mouse tumor models for development of therapeutic beta-glucans.

Cancer Res, 1999 Feb 1, 59(3), 689 - 95
N-(2-chloroethyl)-N-nitrosourea tethered to lexitropsin induces minor groove lesions at the p53 cDNA that are more cytotoxic than mutagenic; Inga A et al.; Many different N-chloroethyl-N-nitrosourea (CENU) derivatives have been synthesized in an attempt to minimize carcinogenic activity while favoring antineoplastic activity . CENU derivatives linked to the dipeptide lexitropsin (lex) showed significant changes in groove- and sequence-selective DNA alkylation inducing thermolabile N3-alkyladenines (N3-Alkyl-As) at lex equilibrium binding sites . CENU-lex sequence specificity for DNA alkylation was determined using 32P-end-labeled restriction fragments of the p53 cDNA . The adducted sites were converted into single-strand breaks by sequential heating at neutral pH and exposure to piperidine . To establish the mutagenic and lethal properties of CENU-lex-specific lesions, a yeast expression vector harboring a human wild-type p53 cDNA was treated in vitro with CENU-lex and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter . p53 mutants were isolated from independent ade- transformants . The results revealed that: (a) CENU-lex preferentially induces N3-Alkyl-A at specific lex equilibrium binding sites, the formations of which are strongly inhibited by distamycin; (b) reactivity toward Gs is still present, albeit to a lesser extent when compared to N-(2-chloroethyl)-N-cyclohexyl-N-nitrosourea and to CENU; (c) 91% of the 49 CENU-lex p53 mutations (45 of 49) were bp substitutions, 29 of which were GC-->AT transitions, mainly at 5' purine G sites; (d) all AT-targeted mutations but one were AT-->TA transversions; (e) the distribution of the CENU-lex mutations along the p53 cDNA was not random, with position 273 (codon 91), where only GC-->AT transitions were observed, being a real (n = 3, P < 0.0002) CENU-lex mutation hot spot; and (f) a shift in DNA alkylation sites between lesion spectra induced by CENU-lex and N-(2-chloroethyl-N-cyclohexyl-N-nitrosourea was associated with an increased lethality and a decreased mutagenicity, whereas no dramatic change in mutational specificity was observed . Hence, it is tempting to conclude that, in this experimental system, N3-Alkyl-A is more lethal than mutagenic, whereas O6-alkylguanine is a common premutational lesion formed at non-lex binding sites . These results suggest that CENU derivatives with virtually absolute specificity for A residues would make targeting of lethal, nonmutagenic lesions at A+T-rich regions possible, and this may represent a new strategy for the development of new chemotherapeutic agents with a higher therapeutic index.

Comp Biochem Physiol C Pharmacol Toxicol Endocrinol, 1998 Nov, 121(1-3), 107 - 37
Rainbow trout cytochrome P450s: purification, molecular aspects, metabolic activity, induction and role in environmental monitoring; Buhler DR et al.; Cytochromes P450 (P450s or CYPs) constitute a superfamily of heme-thiolate proteins that play important roles in oxidative metabolism of endogenous and exogenous compounds . This review provides some limited history but addresses mainly the research progress on the cytochrome P450s in rainbow trout (Oncorhynchus mykiss), their purification, structures at the primary level, role in metabolism, responses to chemicals and environmental pollutants, application to biomonitoring and the effect of various factors on their expression or activities . Information obtained to date suggests that the rainbow trout P450 systems are as complex as those seen in mammals . Fourteen P450s have been purified from liver or trunk kidney to relatively high specific content . cDNAs belonging to seven different P450 families have been documented from trout liver, kidney and ovary . Two CYP1A genes, nine cDNAs containing open reading frames, and a cDNA fragment were entered into GenBank . Among them, CYP2K1, CYP2K3, CYP2K4, CYP2M1, CYP3A27 and CYP4T1 are the most recently described forms . CYP2K1, CYP2M1 and CYP4T1 represent newly identified P450 subfamilies first described in the rainbow trout . In many cases, the cloned rainbow trout P450s have subsequently been expressed in heterologous expressions systems such as COS-7 cells, yeast and baculovirus infected insect cells . Some of the overexpressed P450 isoforms have been partially characterized . Potential future research directions are discussed.

Diagn Cytopathol, 1999 Feb, 20(2), 74 - 7
Fine-needle aspiration cytologic diagnosis of lymphocutaneous sporotrichosis: a case report; Zaharopoulos P; A case of lymphocutaneous sporotrichosis initially diagnosed by fine-needle aspiration (FNA) cytology and confirmed by tissue biopsy and culture study is presented . Asteroid bodies and yeast cells with budding, highly suggestive of the disease, were seen in the cytologic and histologic preparations . The pathology of this unusual fungal disease and the role of cytology in the diagnosis are discussed . This is the first case of lymphocutaneous sporotrichosis reported in the cytologic literature as diagnosed by FNA cytology.

J Neurosci, 1999 Feb 15, 19(4), 1324 - 34
The sec6/8 complex is located at neurite outgrowth and axonal synapse-assembly domains; Hazuka CD et al.; The molecules that specify domains on the neuronal plasma membrane for the delivery and accumulation of vesicles during neurite outgrowth and synapse formation are unknown . We investigated the role of the sec6/8 complex, a set of proteins that specifies vesicle targeting sites in yeast and epithelial cells, in neuronal membrane trafficking . This complex was found in layers of developing rat brain undergoing synaptogenesis . In cultured hippocampal neurons, the sec6/8 complex was present in regions of ongoing membrane addition: the tips of growing neurites, filopodia, and growth cones . In young axons, the sec6/8 complex was also confined to periodic domains of the plasma membrane . The distribution of synaptotagmin, synapsin1, sec6, and FM1-43 labeling in cultured neurons suggested that the plasma membrane localization of the sec6/8 complex preceded the arrival of synaptic markers and was downregulated in mature synapses . We propose that the sec6/8 complex specifies sites for targeting vesicles at domains of neurite outgrowth and potential active zones during synaptogenesis.

Arch Latinoam Nutr, 1998 Sep, 48(3), 247 - 9
{Co-crystallization of cucumber concentrate (Cucumis sativa L.)}; Vazquez A et al.; A sucrose syrup of 70 degrees Brix was concentrated until a concentration greater than 95 degrees Brix was attained . It was studied the effect of concentration (20, 25 and 35 degrees Brix) on the physical properties of the cucumber (Cucumis sativa L.) granules . Moisture content, solubility, and density were determined . The best results were found for the concentrated at 30 degrees Brix . Lemon juice was added to the concentrated to decrease pH from 5.5 to 4.0 to improve flavor and to avoid growth of molds and yeast . No significant differences in the higroscopicity were found between both pH (s) . Sensory evaluation shows that 30 judges of 45 preferred the sample made with the co-crystallizate containing lemon juice.

Hum Mol Genet, 1999 Mar, 8(3), 425 - 30
The Friedreich's ataxia mutation confers cellular sensitivity to oxidant stress which is rescued by chelators of iron and calcium and inhibitors of apoptosis; Wong A et al.; Expansions of an intronic GAA repeat reduce the expression of frataxin and cause Friedreich's ataxia (FRDA), an autosomal recessive neurodegenerative disease . Frataxin is a mitochondrial protein, and disruption of a frataxin homolog in yeast results in increased sensitivity to oxidant stress, increased mitochondrial iron and respiration deficiency . These previous data support the hypothesis that FRDA is a disease of mitochondrial oxidative stress, a hypothesis we have tested in cultured cells from FRDA patients . FRDA fibroblasts were hypersensitive to iron stress and significantly more sensitive to hydrogen peroxide than controls . The iron chelator deferoxamine rescued FRDA fibroblasts more than controls from oxidant-induced death, consistent with a role for iron in the differential kinetics of death; however, mean mitochondrial iron content in FRDA fibroblasts was increased by only 40% . Treatment of cells with the intracellular Ca2+chelator BAPTA-AM rescued both FRDA fibroblasts and controls from oxidant-induced death . Treatment with apoptosis inhibitors rescued FRDA but not control fibroblasts from oxidant stress, and staurosporine-induced caspase 3 activity was higher in FRDA fibroblasts, consistent with the possibility that an apoptotic step upstream of caspase 3 is activated in FRDA fibroblasts . These results demonstrate that FRDA fibroblasts are sensitive to oxidant stress, and may be a useful model in which to elucidate the FRDA mechanism and therapeutic strategies.

Parasitol Res, 1999 Feb, 85(2), 158 - 61
In vitro development of histotropic larvae of Oesophagostomum dentatum under various conditions of cultivation; Daugschies A et al.; We performed a total of 14 trials to evaluate the culture conditions suited best for the in vitro production of fourth-stage larvae (L4) of Oesophagostomum dentatum . Chicken embryo extract was shown to be redundant, whereas pig blood serum, trypticase, liver extract, and yeast extract proved to be important medium ingredients . Development to L4 could be moderately accelerated by increases of temperature (40 degrees C) and of CO2 concentration (20% in air), but the total yield of L4 could not be improved . Thus, conditions of 38.5 degrees C and 10% CO2 are recommended for routine application.

J Gen Virol, 1999 Jan, 80 ( Pt 1), 261 - 8
Novel endogenous retroviral sequences in the chicken genome closely related to HPRS-103 (subgroup J) avian leukosis virus; Smith LM et al.; HPRS-103, the prototype of avian leukosis virus (ALV) subgroup J, is a recently identified retrovirus associated with myeloid leukosis in meat-type chickens . Although this virus shows high sequence identity to other ALV subgroups within the gag and pol genes, its env gene is highly diverged (with only about 40% sequence identity) from other ALV subgroups . On the other hand, the sequence of the env gene of HPRS-103 was 75% identical to that of E51, a member of the EAV family of endogenous avian retroviruses . It is reported here that the chicken genome also contains another EAV-related element, EAV-HP, showing much greater sequence identity (over 97%) to the HPRS-103 env gene . Southern blotting analysis showed that EAV-HP-related sequences were distinct from EAV-O and were present in all lines of chicken examined and in grey jungle fowl, but were absent from several other avian species . The potential role of these endogenous sequences in the evolution of ALV subgroup J viruses is discussed.

J Biol Chem, 1999 Feb 12, 274(7), 4160 - 5
Ser-534 in the hinge 1 region of Arabidopsis nitrate reductase is conditionally required for binding of 14-3-3 proteins and in vitro inhibition; Kanamaru K et al.; 14-3-3 proteins bind to the hinge 1 region of nitrate reductase (NR) and inhibit its activity . To determine which residues of NR are required for 14-3-3-inhibitory interactions, wild-type and mutant forms of Arabidopsis NR were examined in the yeast two-hybrid system and in vitro inhibition assays . NR fragments with or without hinge 1 were introduced into yeast with one of seven Arabidopsis 14-3-3 isoforms (called GF14s) . NR fragments (residues 1-562 or 487-562) containing hinge 1 interacted with all GF-14s tested; an NR fragment (residues 1-487) lacking hinge 1 did not . GF14 binding to NR fragments was dependent on Ser-534, since Asp or Ala substitutions at this site blocked the interaction . Revertants with second site substitutions restoring interaction between GF14omega and the Ala- or Asp-substituted NR fragments were identified . One isolate had a Lys to Glu substitution at position 531, which is in hinge 1, and six isolates had Ile to Leu or Phe substitutions at 561 in the heme binding region . Double mutant forms of holo-NR (S534D plus K531E, I561F, or I561L) were constructed and found to be partially inhibited by protein extracts from Arabidopsis containing 14-3-3 proteins . Wild-type NR is phosphorylated and inhibited by these extracts, but S534D single mutant forms are not . These results show that inhibitory NR/14-3-3 interactions are dependent on Ser-534 but only in the context of the wild-type sequence, since substitutions at second sites render 14-3-3 binding and in vitro NR inhibition independent of Ser-534.

J Biol Chem, 1999 Feb 12, 274(7), 3937 - 40
The human homologue of Drosophila TRF-proximal protein is associated with an RNA polymerase II-SRB complex; Xiao H et al.; Mammalian RNA polymerase II holoenzymes are large complexes that have been reported to contain, in addition to RNA polymerase II, homologues of several yeast SRBs, various general transcription factors, and other polypeptides . On the basis of its copurification with an SRB-containing RNA polymerase II complex by conventional chromatography procedures, we have identified a human homologue of Drosophila TRF-proximal protein, designated hTRFP, and isolated its cognate cDNA . Antibody specific for SRB7 can immunoprecipitate hTRFP and RNA polymerase II and, reciprocally, antibody specific for hTRFP can immunoprecipitate RNA polymerase II and SRB7 . These data indicate that hTRFP is an integral component of an RNA polymerase II-SRB complex . Whereas the precise function of hTRFP remains to be determined, the hTRFP-containing RNA polymerase II-SRB complex supports basal level transcription and, relative to RNA polymerase II alone, enhances transcriptional activation by Gal4-VP16 in the presence of cofactor PC4 . Thus, hTRFP may regulate transcription of class II genes through association with the RNA polymerase II-SRB complex.

Genomics, 1999 Jan 15, 55(2), 185 - 93
Physical delineation of a 700-kb region overlapping the Looptail mutation on mouse chromosome 1; Underhill DA et al.; The mouse looptail (Lp) mutation is an established model for neural tube defects with homozygous Lp embryos showing an open neural tube from the caudal midbrain to the tip of the tail . Heterozygous Lp mice are characterized by a "looped-tail" and wobbly head movements . The Lp gene has been mapped to a 0.6-cM interval on mouse chromosome 1 delineated by two clusters of markers, Fcer1gamma/Usf1/D1Mit113/D1Wsu1 on the proximal side and Fcer1alpha/Spna1/D1Mit149 distally . In the present study, we have created a high-resolution physical map of the Lp genetic interval that is based on long-range restriction mapping by PFGE, fluorescence in situ hybridization analysis of interphase nuclei and extended chromatid fibers, and the assembly of a cloned contig . This contig consists of 25 independent and overlapping BAC clones and 3 YAC clones . The combined analysis indicates that the 0.6-cM genetic interval for Lp corresponds to a minimal physical interval of 700 kb that is delineated by D1Mit113 proximally (two crossovers) and Fcer1alpha distally (one crossover) . The overall gene order and intergene distances for the region were determined to be D1Mit113-<150 kb-Nhlh1-250 kb-Atp1alpha2-280 kb-Fcer1alpha . Partial sequencing of BAC clones from the contig yielded 42 new STS markers for this region of mouse chromosome 1 . Sequence analysis of the BAC clones and assignment of ESTs from the human transcript map to the cloned contig allowed the placement of four new transcription units within this region: Pc326, Kiaa0253, and Pea15 were positioned in the Nhlh1/Atp1alpha2 nonrecombinant interval, while Girk3 was located distal to Atp1alpha2 .

Genomics, 1999 Jan 15, 55(2), 157 - 63
TTC4, a novel human gene containing the tetratricopeptide repeat and mapping to the region of chromosome 1p31 that is frequently deleted in sporadic breast cancer; Su G et al.; The 1p31 region shows loss of heterozygosity in up to 50% of human breast cancers, indicating the presence of a tumor suppressor gene in this location . We have mapped six novel ESTs to a 15-Mb contig of yeast artificial chromosomes spanning the critical region of 1p31 . One of these ESTs was localized within the contig to the region most commonly undergoing loss of heterozygosity in breast cancer . The corresponding gene sequence for this EST was established by cDNA cloning and RACE procedures . This gene is 2 kb long and contains a tetratricopeptide repeat motif and a coiled-coil domain . This family of genes has been implicated in a wide variety of functions, including tumorigenesis . This is the fourth member of the human gene family, and so we have named this gene TTC4 . Northern blot analysis demonstrates a ubiquitous pattern of gene expression that includes breast tissue . A preliminary screen of human breast cancer cell lines shows that TTC4 is expressed in all cases, but SSCP analysis of the coding region of this gene following RT-PCR failed to reveal any mutations . Clearly, because of its map location, a more extensive analysis is warranted to establish whether subtle mutations are present in breast cancers .

Prog Nucleic Acid Res Mol Biol, 1999, 62, 1 - 17
Structural and functional characteristics of Dyrk, a novel subfamily of protein kinases with dual specificity; Becker W et al.; Dyrk-related kinases represent a novel subfamily of protein kinases with unique structural and enzymatic features . Its members have been identified in distantly related organisms . The yeast kinase, Yak1, has been characterized as a negative regulator of growth . Mnb from Drosophila is encoded by the minibrain gene, whose mutation results in specific defects in neurogenesis . Its mammalian homolog, Dyrk1A, is activated by tyrosine phosphorylation in the activation loop between subdomains VII and VIII of the catalytic domain . The human gene for Dyrk1A is located in the "Down syndrome critical region" of chromosome 21 and is therefore a candidate gene for mental retardation in Down syndrome . More recently, six additional mammalian Dyrk-related kinases have been identified (Dyrk1B, Dyrk1C, Dyrk2, Dyrk3, Dyrk4A, and Dyrk4B) . All members of the Dyrk family contain in the activation loop the tyrosines that are essential for the full activity of Dyrk1A . Outside their catalytic domains, Dyrk kinases exhibit little sequence similarity except for a small segment immediately preceding the catalytic domain (DH-box, Dyrk homology box) . An unusual enzymatic property of Dyrk-related kinases is their ability to catalyze tyrosine-directed autophosphorylation as well as phosphorylation of serine/threonine residues in exogenous substrates . The exact cellular function of the Dyrk kinases is yet unknown . However, it appears reasonable to assume that they are involved in the regulation of cellular growth and/or development.

Gene, 1999 Jan 21, 226(2), 155 - 63
Characterization of cDNAs encoding cytoplasmic ribosomal protein L15 and L27a in petunia (Petunia hybrida): primary structures and coordinate expression; Lee HS et al.; We have isolated two cDNA clones, PhRL15 and PhRL27a, encoding cytoplasmic ribosomal proteins L15 and L27a, from a cDNA library prepared from petunia petal protoplast cultures . An expressed sequence tag (EST) strategy was employed . PhRL15 and PhRL27a contained open reading frames corresponding to proteins of 204 and 150 amino acids and molecular weights of 24,100 and 17,000Da, respectively . The deduced amino acid sequences of these clones are about 60% to 70%, identical with those of yeast and rat . Southern blot analysis indicates that each gene may be encoded by a small multigene . The transcription levels of both clones were high in young vegetative organs, at the early event of floral development, and in dividing petal protoplast, and relatively low in highly differentiated reproductive organs . Our results demonstrate that the expression levels of both clones are correlated with the rate of growth and coordinatively controlled in petunia plant.

Gene, 1998 Dec 28, 225(1-2), 125 - 30
Eimeria tenella: cloning and characterization of cDNA encoding a S3a ribosomal protein; Ouarzane M et al.; A lambda Zap II cDNA library was constructed from Eimeria tenella first- generation schizonts mRNA and screened with a mouse serum raised against this parasitic stage . This serum identified a clone encoding a S3a ribosomal protein (EtS3a) . The 858-bp cDNA fragment, containing the entire parasitic gene encoded a highly basic protein of 264 amino acids (aa) with a molecular weight of 29.780kDa . Based upon amino acid sequence comparison, EtS3a is highly homologous to v-fos transformation effector (encoded by the fte-1 gene) and cyc-07 (a plant homologue of fte-1) and similar to the yeast MFT1 (encoded by the mitochondrial fusion targeting gene) . The expressions of mammalian fte-1, plant cyc-07 and yeast MFT1 have all been shown to be cell-cycle-regulated and involved in protein synthesis at the level of the ribosome . Since EtS3a expression is also developmentally regulated, we suggest that this gene product is a functional homologue of fte-1, cyc-07 and MFT1 and an important molecule regulating the development of Eimeria tenella.

Gene, 1999 Feb 4, 227(1), 111 - 6
Physical map covering a 2 Mb region in human xp11.3 distal to DX6849; Stoddart KL et al.; A 2Mb contig was constructed of yeast artificial chromosomes (YACs) and P1 artificial chromosomes (PACs), extending from DXS6849 to a new marker EC7034R, 1Mb distal to UBE1, within the p11.3 region of the human X chromosome . This contig, which has on average four-fold cloned coverage, was assembled using 37 markers, including 13 new sequence tagged sites (STSs) developed from YAC and PAC end-fragments, for an average inter-marker distance of 55kb . The inferred marker order predicted from SEGMAP analysis, STS content and cell hybrid data is Xpter-EC7034R-EC8058R-FB20E11-DXS7804-D XS8308-(DXS1264, DXS1055)-DXS1003-UBE1-(UHX), PCTK1)-DXS1364-DXS1266-DXS337-SYN1-DXS6 849-cen . One (TC)n dinucleotide sequence from an end-clone was identified and found to be polymorphic (48% heterozygosity) . The contig is merged with published physical maps both in the distal and in the centromeric direction of Xp, and provides reagents to aid in the DNA sequencing and the finding of genes in this region of the human genome.

Gene, 1998 Dec 11, 224(1-2), 53 - 8
Identification of Ce-AF-6, a novel Caenorhabditis elegans protein, as a putative Ras effector; Watari Y et al.; Mammalian Ras proteins associate with multiple effectors, including Raf, Ral guanine nucleotide dissociation stimulator, phosphoinositide 3-kinase and AF-6 . In the nematode Caenorhabditis elegans, LIN-45/Raf has been identified genetically as an effector of LET-60/Ras . To search for other effectors in C . elegans, we carried out a yeast two-hybrid screening for LET-60-associating proteins . The screening identified a novel protein, designated Ce-AF-6, which exhibited a strong structural homology with human AF-6, rat Afadin and Drosophila melanogaster Canoe and possessed both the Ras-associating (RA) domain and the PSD-95/DlgA/ZO-1 (PDZ) domain . Ce-AF-6 associated with human Ha-Ras in a GTP-dependent manner, with an efficiency comparable to that of human Raf-1 Ras-binding domain . When the effects of mutations of the Ras effector region residues were examined for associations with various effectors, Ce-AF-6 was found to possess a distinct and the most rigorous requirement for the effector region residues . These results strongly suggest that Ce-AF-6 is a putative effector of Ras that possesses a distinct recognition mechanism for association with Ras.

J Neurochem, 1999 Feb, 72(2), 605 - 13
Effects of cyclin-dependent kinase inhibitors on transcription and ocular circadian rhythm of Aplysia; Sankrithi N et al.; Cyclin-dependent kinases (CDKs) mediate cell-cycle phase transitions . Recently, CDKs have been associated with non-cell-cycle roles such as DNA repair, transcription, and phosphate metabolism in yeast . The cyclical processes, circadian rhythms and the eukaryotic cell cycle, are similar in many respects . It is possible that a kinase like CDK is involved in the control of circadian rhythms . In this study, the effects of CDK inhibitors (olomoucine, roscovitine, and butyrolactone I) on the Aplysia ocular circadian rhythm were investigated . Continuous treatments with olomoucine (10 microM) lengthened the free-running period of the rhythm, and pulse treatments of olomoucine (6 h, 100 microM) delayed the rhythm . The effects of olomoucine on the rhythm were qualitatively similar to those of a reversible inhibitor of transcription, 5,6-dichloro-beta-1-ribobenzimidazole . Subsequently, olomoucine was found to inhibit RNA synthesis in the eye of Aplysia and Bulla . All of the other CDK inhibitors used in this study also inhibited transcription in the eye of Aplysia, and their effects on transcription correlated with their effects on the circadian rhythm . This study adds substantial evidence to that previously obtained by using 5,6-dichloro-beta-1-ribobenzimidazole for a role of transcription in the mechanism responsible for circadian rhythmicity in the eye of Aplysia . Also, these results indicate that caution is warranted in interpreting results obtained by using CDK inhibitors, because these drugs appear to inhibit transcription as well as CDKs.

Nippon Ishinkin Gakkai Zasshi, 1999, 40(1), 21 - 5
A clinical isolate of Candida palmioleophila formerly identified as Torulopsis candida; Sugita T et al.; A strain of yeast labeled Torulopsis candida, which was isolated from a clinical specimen in Canada and reported as a new opportunistic pathogen causing intravenous catheter-associated fungemia, was found to be a strain of Candida palmioleophila in a DNA-DNA reassociation experiment.

Cancer Lett, 1998 Nov 13, 133(1), 89 - 94
p53 inactivating mutations in Chinese nasopharyngeal carcinomas; Lung ML et al.; Previously a low frequency of p53 mutations was detected in nasopharyngeal carcinoma (NPC) using molecular techniques to screen for mutations, yet immunohistochemical staining revealed a high frequency of p53 aberrant proteins . These findings might be attributed to the occurrence of p53 mutations outside the common hot spots and/or the inactivation of the protein through interactions with cellular or viral proteins . Using a previously established simple and sensitive p53 yeast functional assay, we blindly screened 25 nasopharyngeal biopsies for p53 mutations from exons 4 to 11 . p53 was mutated in 27.3% of NPC specimens and in 0% of the nasopharyngeal biopsies from patients with non-malignant diseases . Two p53 mutations were detected in exon 7 and two were detected in exon 8 . Interestingly, the exon 8 mutations observed in NPC lie in codons which appear to be hot spots for mutations in other head and neck cancers.

Development, 1999 Feb, 126(5), 1065 - 75
Expression of the cell cycle in sperm of Arabidopsis: implications for understanding patterns of gametogenesis and fertilization in plants and other eukaryotes; Friedman WE; The relationship between developmental events and the cell cycle was examined in sperm of Arabidopsis thaliana . Sperm of Arabidopsis rapidly enter the S (synthesis) phase of the cell cycle after inception from mitosis of the generative cell . Sperm in pollen grains within anthers continue to synthesize DNA, and at the time of pollination, contain approximately 1.5C DNA . Following pollination, sperm continue through the S phase of the cell cycle during pollen tube growth . By the time pollen tubes reach the ovary, sperm nuclei contain approximately 1.75C DNA . Just prior to double fertilization, sperm nuclei within embryo sacs contain the 2C quantity of DNA . These data indicate that molecular programs associated with the G1-S transition and the S phase of the cell cycle are expressed in sperm cells of developing pollen grains and pollen tubes in Arabidopsis . This pattern of prefertilization S phase activity in the sperm of a flowering plant stands in marked contrast to all other non-plant eukaryotes (from ciliates to yeast to sea urchins to mammals) where sperm remain in G1 during development, prior to the initiation of gametic fusion . In addition, when patterns of cell cycle activity in sperm of Arabidopsis and other flowering plants are compared, developmental analysis reveals that heterochronic alterations (changes in the relative timing of ontogenetic events) in cell cycle activity are a central cause of the diversification of patterns of gametogenesis in higher plants . Finally, comparative analysis of the patterns of cell cycle activity in Arabidopsis and other angiosperms may be used to predict which flowering plants will be amenable to development of successful in vitro fertilization techniques.

Genome Res, 1999 Jan, 9(1), 44 - 52
Localization of Jacobsen syndrome breakpoints on a 40-Mb physical map of distal chromosome 11q; Tunnacliffe A et al.; Jacobsen syndrome is a haploinsufficiency disorder caused, most frequently by terminal deletion of part of the long arm of chromosome 11, with breakpoints in 11q23.3-11q24.2 . Inheritance of an expanded p(CCG)n trinucleotide repeat at the folate-sensitive fragile site FRA11B has been implicated in the generation of the chromosome breakpoint in several Jacobsen syndrome patients . The majority of such breakpoints, however, map distal to this fragile site and are not linked with its expression . To characterize these distal breakpoints and ultimately to further investigate the mechanisms of chromosome breakage, a 40-Mb YAC contig covering the distal long arm of chromosome 11 was assembled . The utility of the YAC contig was demonstrated in three ways: (1) by rapidly mapping the breakpoints from two new Jacobsen syndrome patients using FISH; (2) by demonstrating conversion to high resolution PAC contigs after direct screening of PAC library filters with a YAC clone containing a Jacobsen syndrome breakpoint; and (3) by placing 23 Jacobsen syndrome breakpoints on the physical map . This analysis has suggested the existence of at least two new Jacobsen syndrome breakpoint cluster regions in distal chromosome 11.

Oncogene, 1999 Jan 14, 18(2), 315 - 26
Cell cycle regulation of the double stranded RNA activated protein kinase, PKR; Zamanian-Daryoush M et al.; The interferon (IFN)-induced, double stranded RNA (dsRNA)-activated serine/threonine kinase, PKR, is a potent negative regulator of cell growth when overexpressed in yeast or mammalian cells . To determine whether endogenous PKR plays a role in cell growth control, we have investigated the regulation of PKR levels and activity during the cell cycle in human glioblastoma T98G cells . The steady-state level of PKR mRNA in T98G cells was highest in growth arrested cells, dropped sharply within 3 h of serum stimulation then gradually increased as cells progressed through G1, reaching a plateau in early S phase . PKR protein level increased following serum stimulation reaching a peak at the G2+M boundary and declining thereafter . In contrast, PKR kinase activity exhibited two peaks, in early G1 and at the G1/S boundary, declining sharply in early S phase . Thus, the activity profile did not follow the protein profile indicating a tight regulation of PKR at the level of activity . In T98G cells expressing the catalytically inactive PKRK296R the dsRNA-induced activation of NF-kappaB and IRF-1 was suppressed and the mutant cells exhibited resistance to stress induced apoptosis . Cell cycle distribution analysis showed that the mutant expressing cells exhibited longer G1 phase and fewer cells engaged in S phase . Furthermore, early passage mouse embryo fibroblasts derived from PKR knockout mice grew more slowly compared with the control cells . Taken together these results suggest that PKR may play a role in cell cycle progression.

Oncogene, 1999 Jan 7, 18(1), 269 - 75
Functional association of TGF-beta receptor II with cyclin B; Liu JH et al.; Utilizing the cytoplasmic tail of Transforming Growth Factor Receptor Type II (TGFbeta RII) as bait in a yeast two hybrid system, we have identified human cyclin B2 as a direct physical partner of TGFbeta RII . Analysis of deletion mutants of glutathione-S-transferase (GST)-cyclin B2 mapped its binding domain for TGFbeta RII to the C-terminal and revealed a negative regulatory region immediately upstream of the cyclin box . Using recombinant proteins, Cdc2 was demonstrated to indirectly interact with TGFbeta RII via cyclin B2 . This interaction was reproduced in THP-1 monocytic cells, where TGFbeta treatment markedly enhanced the ability of cyclin B2 and, correspondingly, Cdc2 from TGFbeta-treated THP-1 cells, to bind the GST-TGFbeta RII fusion protein . More importantly, TGFbeta RII co-precipitated with cyclin B2 in TGFbeta-treated THP-1 cells . TGFbeta treatment also caused threonine phosphorylation of Cdc2 in the TGFbeta RII-cyclin B2-Cdc2 complex in THP1 cells, in parallel with down regulation of Cdc2 function as measured by histone H1 kinase activity . Cyclin B1 had the same capacity to bind TGFbeta RII and mediate indirect Cdc2 binding . These results suggest an alternative mechanism that cell cycle arrest in the G1/S phase caused by TGFbeta may, in part, be due to inactivation of cyclin B/Cdc2 kinase, which is needed for entry into the G2/M phase.

Oncogene, 1999 Jan 7, 18(1), 249 - 56
Chk1 complements the G2/M checkpoint defect and radiosensitivity of ataxia-telangiectasia cells; Chen P et al.; Cells from patients with the human genetic disorder ataxia-telangiectasia (A-T) are defective in the activation of cell cycle checkpoints in response to ionizing radiation damage . In order to understand the role of ATM in checkpoint control we investigated whether Schizosaccaromyces pombe chk1, a protein kinase implicated in controlling the G2 DNA damage checkpoint, might alter the radiosensitive phenotype in A-T cells . The fission yeast chkl gene was cloned into an EBV-based vector under the control of a metallothionein promoter and transfected into A-T lymphoblastoid cells . Induction of chk1 enhanced the survival of an A-T cell line in response to radiation exposure as determined by cell viability and reduction of radiation-induced chromosome aberrations . This can be accounted for at least in part by the restoration of the G2 checkpoint to chk1 expressing cells . There was no evidence that chk1 expression corrected either the G1/S checkpoint or radioresistant DNA synthesis in S phase in these cells . These results suggest that chk1 when overexpressed acts downstream from ATM to restore the G2 checkpoint in these cells and correct the radiosensitive phenotype . These data allow us to dissociate individual checkpoint events and relate them to the radiosensitive phenotype in A-T cells.

Genes Dev, 1999 Jan 15, 13(2), 202 - 12
Transcriptional repression by the Caenorhabditis elegans germ-line protein PIE-1; Batchelder C et al.; In the early Caenorhabditis elegans embryo, maternally expressed PIE-1 protein is required in germ-line blastomeres to inhibit somatic differentiation, maintain an absence of mRNA transcription, and block phosphorylation of the RNA polymerase II large subunit (Pol II) carboxy-terminal domain (CTD) . We have determined that PIE-1 can function as a transcriptional repressor in cell culture assays . By fusing PIE-1 sequences to the yeast GAL4 DNA-binding domain, we have identified a PIE-1 repression domain that appears to inhibit the transcriptional machinery directly . A sequence element that is required for this repressor activity is similar to the Pol II CTD heptapeptide repeat, suggesting that the PIE-1 repression domain might target a protein complex that can bind the CTD . An alteration of this sequence element that blocks repression also impairs the ability of a transgene to rescue a pie-1 mutation, suggesting that this repressor activity may be important for PIE-1 function in vivo.

Antimicrob Agents Chemother, 1999 Feb, 43(2), 322 - 8
Comparison of a new triazole antifungal agent, Schering 56592, with itraconazole and amphotericin B for treatment of histoplasmosis in immunocompetent mice; Connolly P et al.; A murine model of intratracheally induced histoplasmosis was used to evaluate a new triazole antifungal agent, Schering (SCH) 56592, for treatment of histoplasmosis . MICs were determined for SCH 56592, amphotericin B, and itraconazole by testing yeast-phase isolates from 20 patients by a macrobroth dilution method . The MICs at which 90% of the isolates are inhibited were for 0.019 microgram/ml for SCH 56592, 0.5 microgram/ml for amphotericin B, and < or = 0.019 microgram/ml for itraconazole . Survival studies were done on groups of 10 B6C3F1 mice with a lethal inoculum of 10(5) . All mice receiving 5, 1, or 0.25 mg of SCH 56592 per kg of body weight per day, 2.5 mg of amphotericin B per kg every other day (qod), or 75 mg of itraconazole per kg per day survived to day 29 . Only 44% of mice receiving 5 mg of itraconazole/kg/day survived to day 29 . Fungal burden studies done in similar groups of mice with a sublethal inoculum of 10(4) showed a reduction in CFUs and Histoplasma antigen levels in lung and spleen tissue in animals treated with 2 mg of amphotericin B/kg qod, 1 mg of SCH 56592/kg/day, and 75 mg of itraconazole/kg/day, but not in those treated with lower doses of the study drugs (0.2 mg of amphotericin B/kg qod, 0.1 mg of SCH 56592/kg/day, or 10 mg of itraconazole/kg/day) . Serum drug concentrations were measured 3 and 24 h after the last dose in mice (groups of five to seven mice), each treated for 7 days with SCH 56592 (10 and 1 mg/kg/day) and itraconazole (75 and 10 mg/kg/day) . Mean levels measured by bioassay were as follows: SCH 56592, 10 mg/kg/day (2.15 micrograms/ml at 3 h and 0.35 microgram/ml at 24 h); SCH 56592, 1 mg/kg/day (0.54 microgram/ml at 3 h and none detected at 24 h); itraconazole, 75 mg/kg/day (22.53 micrograms/ml at 3 h and none detected at 24 h); itraconazole, 10 mg/kg/day (1.33 micrograms/ml at 3 h and none detected at 24 h) . Confirmatory results were obtained by high-pressure liquid chromatography assay . These studies show SCH 56592 to be a promising candidate for studies of treatment of histoplasmosis in humans.

Int J Biochem Cell Biol, 1998 Dec, 30(12), 1297 - 318
Properties and functions of the thiamin diphosphate dependent enzyme transketolase; Schenk G et al.; This review highlights recent research on the properties and functions of the enzyme transketolase, which requires thiamin diphosphate and a divalent metal ion for its activity . The transketolase-catalysed reaction is part of the pentose phosphate pathway, where transketolase appears to control the non-oxidative branch of this pathway, although the overall flux of labelled substrates remains controversial . Yeast transketolase is one of several thiamin diphosphate dependent enzymes whose three-dimensional structures have been determined . Together with mutational analysis these structural data have led to detailed understanding of thiamin diphosphate catalysed reactions . In the homodimer transketolase the two catalytic sites, where dihydroxyethyl groups are transferred from ketose donors to aldose acceptors, are formed at the interface between the two subunits, where the thiazole and pyrimidine rings of thiamin diphosphate are bound . Transketolase is ubiquitous and more than 30 full-length sequences are known . The encoded protein sequences contain two motifs of high homology; one common to all thiamin diphosphate-dependent enzymes and the other a unique transketolase motif . All characterised transketolases have similar kinetic and physical properties, but the mammalian enzymes are more selective in substrate utilisation than the nonmammalian representatives . Since products of the transketolase-catalysed reaction serve as precursors for a number of synthetic compounds this enzyme has been exploited for industrial applications . Putative mutant forms of transketolase, once believed to predispose to disease, have not stood up to scrutiny . However, a modification of transketolase is a marker for Alzheimer's disease, and transketolase activity in erythrocytes is a measure of thiamin nutrition . The cornea contains a particularly high transketolase concentration, consistent with the proposal that pentose phosphate pathway activity has a role in the removal of light-generated radicals.

Genome, 1998 Dec, 41(6), 806 - 17
Arabidopsis YAC restriction mapping; Morroll SM et al.; The approach of partial restriction mapping and vector hybridisation has been used to restriction map and align six yeast artificial chromosomes (YACs) corresponding to the top arm (approximately 27.9 centiMorgans, cM) of Arabidopsis chromosome 5 and confirm the chimeric nature of a further four clones which map to this region . The restriction endonucleases Sma1 and Sfi1 which recognise rare-medium frequency sites in the Arabidopsis genome were used . This work has restriction mapped a 315 kb region that includes a number of genes implicated in floral development, namely PISTILLATA and TOUSLED, and a number of uncharacterized genes involved in male gametogenesis (e.g., Ms1 and Ms37) . The information generated can be used to transcriptionally map genes to this contig and will provide data for the isolation of several uncharacterized floral development genes which lie in this region . This approach has demonstrated how large tracts of YAC DNA can be mapped and aligned to show the presence/absence of chimeric YAC clones and provide detailed restriction knowledge for a large genomic region to help facilitate the positional cloning of genes.

Nature, 1999 Jan 14, 397(6715), 172 - 5
Nuclear localization of Cdc25 is regulated by DNA damage and a 14-3-3 protein; Lopez-Girona A et al.; DNA damage activates a cell-cycle checkpoint that prevents mitosis while DNA repair is under way . The protein Chk1 enforces this checkpoint by phosphorylating the mitotic inducer Cdc25 . Phosphorylation of Cdc25 by Chk1 creates a binding site in Cdc25 for 14-3-3 proteins, but it is not known how 14-3-3 proteins regulate Cdc25 . Rad24 is a 14-3-3 protein that is important in the DNA-damage checkpoint in fission yeast . Here we show that Rad24 controls the intracellular distribution of Cdc25 . Elimination of Rad24 causes nuclear accumulation of Cdc25 . Activation of the DNA-damage checkpoint causes the net nuclear export of Cdc25 by a process that requires Chk1, Rad24 and nuclear-export machinery . Mutation of a putative nuclear-export signal in Rad24 impairs the nuclear exclusion of Rad24, the damage-induced nuclear export of Cdc25 and the damage checkpoint . Thus, Rad24 appears to function as an attachable nuclear-export signal that enhances the nuclear export of Cdc25 in response to DNA damage.

Biochem Cell Biol, 1998, 76(2-3), 223 - 34
NMR spectroscopic studies of I = 1/2 metal ions in biological systems; Oz G et al.; This article reviews the use of nuclear magnetic resonance methods of spin 1/2 metal nuclei to probe the metal binding site(s) in a variety of metalloproteins . The majority of the studies have involved native Zn(II) and Ca(II) metalloproteins where there has been isostructural substitution of these metal ions with the I = 1/2 (111/113)Cd(II) ion . Also included are recent studies that have utilized the 109Ag(I) ion to probe Cu(I) sites in yeast metallothionein and 199Hg(II) as a probe of the metal binding sites in mercury resistance proteins . Pertinent aspects for the optimal execution of these experiments along with the procedures for the metal substitution reactions are discussed together with the presentation of a 113Cd chemical shift correlation map with ligand type and coordination number . Specific examples of protein systems studied using the (111/113)Cd and 109Ag nuclei include the metallothionein superfamily of Zn(II)- and Cu(I)-binding proteins from mammalian, invertebrate, and yeast systems . In addition to the structural features revealed by these metal ion nuclear magnetic resonance studies, important new information is frequently provided about the dynamics at the active-site metal ion . In an effort for completeness, other less frequently used spin 1/2 metal nuclei are mentioned.

Mol Cell Endocrinol, 1998 Oct 25, 145(1-2), 21 - 5
Intracellular mechanisms of ovarian cell apoptosis; Hsu SY et al.; Apoptosis is an active cell 'suicide' essential for the elimination of superfluous cells during diverse physiological processes in essentially all animal species . Although regulation of apoptosis by extracellular mediators is cell type-specific, new insights based on characterization of conserved intracellular effectors have suggested that intracellular pathways leading to apoptosis in diverse organisms is regulated by a group of evolutionarily conserved genes including ced-9/Bcl-2, ced-4/Apaf-1 and ced3/caspases gene families . To study whether the Bcl-2 family proteins are important in the regulation of ovarian cell apoptosis, we have used transgenic mice and yeast 2-hybrid protein protein interaction assay to characterize the roles of Bcl-2 family proteins in ovarian atresia . The use of 2-hybrid analysis resulted in the isolation of a novel pro-apoptotic Bcl-2 protein, Bcl-2-related ovarian killer (Bok) and the identification of upstream mediators for ovarian cell apoptosis.

Hum Genet, 1998 Dec, 103(6), 674 - 80
A transcript map of an 800-kb region on human chromosome 11q13, part of the candidate region for SCA5 and BBS1; Zhu S et al.; The exon-amplification method was used to identify putative transcribed sequences from an 800-kb region that includes the genes for phospholipase Cbeta3 and PYGM on human chromosome 11q13 . The clone contig consisted of ten cosmids, three bacterial artificial chromosomes, and one P1 artificial chromosome . A total of 83 exons were generated of which 23 were derived from known genes and expressed sequence tags (ESTs) . Five different EST cDNA clones were identified and mapped on the contig . One is a homolog of the human p70S6 kinase (p70s6 k) gene whose function involves the translational regulation of ribosomal protein synthesis and thereby impacts on ribosomal biogenesis . The gene for p70s6 k is expressed universally, including within adipose cells and retina, and it could play a role in Bardet-Biedl syndrome type 1, which has been mapped to 11q13.

Braz J Med Biol Res, 1998 Nov, 31(11), 1425 - 8
Thioglycollate-elicited murine macrophages are cytotoxic to Mycoplasma arginini-infected YAC-1 tumor cells; Ribeiro-Dias F et al.; Macrophages are important components of natural immunity involved in inhibition of tumor growth and destruction of tumor cells . It is known that these cells can be activated for tumoricidal activity by lymphokines and bacterial products . We investigated whether YAC-1 tumor cells infected with Mycoplasma arginini stimulate nitric oxide (NO) release and macrophage cytotoxic activity . Thioglycollate-elicited macrophages from male BALB/c mice were co-cultured for 20 h with YAC-1 tumor cells infected or not with Mycoplasma arginini . The cytotoxic activity was evaluated by MTT assay and nitrite levels were determined with the Griess reagent . Thioglycollate-elicited macrophages co-cultured with noninfected YAC-1 cells showed low cytotoxic activity (34.7 +/- 8.6%) and low production of NO (4.7 +/- 3.1 microM NO2-) . These macrophages co-cultured with mycoplasma-infected YAC-1 cells showed significantly higher cytotoxic activity (61.4 +/- 9.1%; P < 0.05) and higher NO production (48.5 +/- 13 microM NO2-; P < 0.05) . Addition of L-NAME (10 mM), an inhibitor of NO synthesis, to these co-cultures reduced the cytotoxic activity to 37.4 +/- 2% (P < 0.05) and NO production to 3 +/- 4 microM NO2- (P < 0.05) . The present data show that Mycoplasma arginini is able to induce macrophage cytotoxic activity and that this activity is partially mediated by NO.

J Biol Chem, 1999 Feb 5, 274(6), 3446 - 52
Kinetic and thermodynamic analysis of mutant type II DNA topoisomerases that cannot covalently cleave DNA; Morris SK et al.; DNA topoisomerase II catalyzes two different chemical reactions as part of its DNA transport cycle: ATP hydrolysis and DNA breakage/religation . The coordination between these reactions was studied using mutants of yeast topoisomerase II that are unable to covalently cleave DNA . In the absence of DNA, the ATPase activities of these mutant enzymes are identical to the wild type activity . DNA binding stimulates the ATPase activity of the mutant enzymes, but with steady-state parameters different from those of the wild type enzyme . These differences were examined through DNA binding experiments and pre-steady-state ATPase assays . One mutant protein, Y782F, binds DNA with the same affinity as wild type protein . This mutant topologically traps one DNA circle in the presence of a nonhydrolyzable ATP analog under the same conditions that the wild type protein catenates two circles . Rapid chemical quench and pulse-chase ATPase experiments reveal that the mutant proteins bound to DNA have the same sequential hydrolysis reaction cycle as the wild type enzyme . Binding of ATP to the mutants is not notably impaired, but hydrolysis of the first ATP is slower than for the wild type enzyme . Models to explain these results in the context of the entire DNA topoisomerase II reaction cycle are discussed.

J Biol Chem, 1999 Feb 5, 274(6), 3439 - 45
Identification of a domain of Axin that binds to the serine/threonine protein phosphatase 2A and a self-binding domain; Hsu W et al.; Axin is a negative regulator of embryonic axis formation in vertebrates, which acts through a Wnt signal transduction pathway involving the serine/threonine kinase GSK-3 and beta-catenin . Axin has been shown to have distinct binding sites for GSK-3 and beta-catenin and to promote the phosphorylation of beta-catenin and its consequent degradation . This provides an explanation for the ability of Axin to inhibit signaling through beta-catenin . In addition, a more N-terminal region of Axin binds to adenomatous polyposis coli (APC), a tumor suppressor protein that also regulates levels of beta-catenin . Here, we report the results of a yeast two-hybrid screen for proteins that interact with the C-terminal third of Axin, a region in which no binding sites for other proteins have previously been identified . We found that Axin can bind to the catalytic subunit of the serine/threonine protein phosphatase 2A through a domain between amino acids 632 and 836 . This interaction was confirmed by in vitro binding studies as well as by co-immunoprecipitation of epitope-tagged proteins expressed in cultured cells . Our results suggest that protein phosphatase 2A might interact with the Axin.APC.GSK-3.beta-catenin complex, where it could modulate the effect of GSK-3 on beta-catenin or other proteins in the complex . We also identified a region of Axin that may allow it to form dimers or multimers . Through two-hybrid and co-immunoprecipitation studies, we demonstrated that the C-terminal 100 amino acids of Axin could bind to the same region as other Axin molecules.

J Biol Chem, 1999 Feb 5, 274(6), 3355 - 62
Characterization of genes encoding known and novel human mast cell tryptases on chromosome 16p13.3; Pallaoro M et al.; Tryptases are serine proteases implicated in asthma and are very highly expressed in human mast cells . They fall into two groups, alpha and beta . Although several related tryptase mRNAs are known, it is unclear which if any are transcripts of separate haploid genes . The studies described here investigated the nature and number of human tryptases and sought possibly novel members of the family . To this end, two human bacterial artificial chromosome (BAC) clones containing tryptase genes were identified and mapped to chromosome 16p13.3, of which approximately 2.2 megabases are syntenic with the part of mouse chromosome 17 containing tryptase genes mouse mast cell protease (mMCP)-6 and -7 . Sequencing and restriction mapping suggest that the BACs may partially overlap . Sequenced BAC genes correspond to three known beta-tryptases (betaI, betaII, and betaIII), an alpha-like gene, and a pair of novel hybrid genes related partly to alpha/beta-tryptases and partly to orthologs of mMCP-7 . betaII and betaIII, betaI and alphaII, as well as the two mMCP-7-like genes, may be alleles at single loci; in total, there are at least three nonallelic tryptase genes in the isolated BAC clones . DNA blotting and restriction analysis suggest that the BACs include most members of the immediate tryptase family . Thus, chromosome 16p13.3 harbors a cluster of known and previously undescribed members of the tryptase gene family.

Blood, 1999 Feb 1, 93(3), 1025 - 31
Fusion of ETV6 to the caudal-related homeobox gene CDX2 in acute myeloid leukemia with the t(12;13)(p13;q12); Chase A et al.; The t(12;13)(p13;q12) is a rare, recurrent translocation reported in a range of hematological malignancies . We have analyzed the molecular basis of this lesion in three patients with acute myeloid leukemia (AML), two of whom were known to have chromosome 12 breakpoints within the ETV6 gene . Fluorescence in situ hybridization (FISH) with ETV6 cosmids indicated that this gene was also disrupted in the third patient, while the normal ETV6 allele was retained . 3' rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR) from bone marrow mRNA of this individual identified a novel sequence fused to ETV6 that was homologous to a region just upstream of the mouse CDX2 homeobox gene, the human homologue of which has previously been mapped to chromosome 13q12 . PCR primers designed to amplify an ETV6-CDX2 fusion identified two major transcripts from this patient . First, a direct in-frame fusion between exon 2 of ETV6 and exon 2 of CDX2, and second, a transcript that had an additional sequence of unknown origin spliced between these same exons . Surprisingly, apparently normal CDX2 transcripts, usually expressed only in intestinal epithelium, were also detectable in cDNA from this patient . Neither normal nor fusion CDX2 mRNA was detectable in the two other patients with a t(12;13), indicating that this translocation is heterogeneous at the molecular level . Reverse transcription-PCR analysis showed that CDX2 mRNA, but not ETV6-CDX2 mRNA, was strongly expressed in 1 of 10 patients with chronic myeloid leukemia in transformation, suggesting that deregulation of this gene may be more widespread in leukemia . CDX2 is known to regulate class I homeobox genes and its expression in hematopoietic cells may critically alter the balance between differentiation and proliferation.

Biochem Biophys Res Commun, 1999 Jan 8, 254(1), 203 - 10
TFAR19, a novel apoptosis-related gene cloned from human leukemia cell line TF-1, could enhance apoptosis of some tumor cells induced by growth factor withdrawal; Liu H et al.; Using the cDNA-representative differences analysis (cDNA-RDA) approach, we identified a novel gene, TFAR19 (TF-1 cell apoptosis related gene-19), from TF-1 cells undergoing apoptosis . The human TFAR19 encodes a protein which shares significant homology to the corresponding proteins of species ranging from yeast to mice . TFAR19 exhibits a ubiquitous expression pattern and its expression is upregulated in the tumor cells undergoing apoptosis . Overexpression of TFAR19 in tumor cells enhances apoptosis triggered by growth factor or serum deprivation . We propose that TFAR19 may play a general role in the apoptotic process .

Biochem Biophys Res Commun, 1999 Jan 8, 254(1), 1 - 4
Senescence marker protein-30 (SMP30): structure and biological function; Fujita T; Senescence marker protein-30 (SMP30), which we previously identified, is notable for its androgen-independent decrease in the livers of aging rats . Hepatocytes and renal tubular epithelia express large amounts of SMP30 in their cytosol throughout the tissue-maturing process and adulthood, but its level decreases thereafter . Upon cloning cDNAs that encode SMP30 in rats, mice, and humans, we found that the amino acid sequence of SMP30 is well conserved with remarkable homology among these species . However, this gene, which is so strongly conserved in these higher animals, does not appear in yeast . We also determined the genome organization and 5' flanking region of SMP30 in mouse genome . In the meantime, SMP30 turned out be identical to a Ca2+-binding protein called regucalcin (RC) . To learn how this protein functions, we transfected Hep G2 cells with human SMP30 cDNA so that these cells stably express large amounts of SMP30 . The results suggest that SMP30 regulates Ca2+ homeostasis by enhancing Ca2+-pumping activity in the plasma membranes . Thus, SMP30 seems to play a critical role in the highly differentiated functions of the liver and kidney and to exert a major impact on Ca2+ homeostasis . If so, down-regulation of SMP30 with aging would attribute greatly to the related deterioration of these organs, as indicated in this brief overview of the structure, expression, and function of SMP30 .

Biochim Biophys Acta, 1998 Dec 10, 1448(2), 254 - 63
Clustered organization of S100 genes in human and mouse; Ridinger K et al.; S100 Ca2+-binding proteins became of major interest because of their differential expression in tissues and their association with human diseases . Earlier studies showed that 13 S100 genes are located as a cluster on human chromosome 1q21 . Since a number of mouse S 100 genes, such as S100A4 and S100A6, have been localized to a syntenic region on mouse chromosome 3, we investigated if the S100 gene cluster exists in mouse and is structurally conserved during evolution . First we identified the cDNA sequences of mouse S100A1, S100A3 and S100A5 . Then we isolated a 490 kb mouse YAC clone which gives a specific signal by FISH most likely on chromosome 3 . Hybridization studies with different mouse S100 cDNAs revealed that eight mouse S100 genes are arranged in a clustered organization similar to that in human . The linkage relationships between the genes S100A8-S100A9 and S100A3-S100A4-S100A5-S100A6 were conserved during divergence of human and mouse about 70 million years ago . However, the separation of the mouse S100 genes S100A1 and S100A13 in comparison to the human linkage group suggests rearrangement processes between human and mouse . Our data demonstrate that the S100 gene cluster is structurally conserved during evolution . Further studies on the genomic organization of the S100 genes including various species could generate new insights into gene regulatory processes and phylogenetic relationships.

J Parasitol, 1998 Dec, 84(6), 1261 - 3
Maintenance of Acanthamoeba culbertsoni by cryopreservation; Alejandre-Aguilar R et al.; In order to determine the viability of trophozoites of Acanthamoeba culbertsoni under cryopreservation conditions, cultures in serum-casein-glucose-yeast extract medium were subject to 5%, 7.5%, and 10% concentrations of dimethylsulfoxide (DMSO) . With the methodology followed, the percentages of recovery varied between 75.6% and 86.6% with DMSO at 10%, between 54.5% and 73.5% with DMSO at 7.5%, and between 43.6% and 68.5% with DMSO at 5% . The amebae were kept in liquid nitrogen for 30-210 days . The highest viability of trophozoites was founded when DMSO was used at a final concentration of 10% and an equilibrium temperature of 4 C . Gross cultural or morphological changes were not noted in trophozoites thawed from frozen suspensions.

Epidemiol Mikrobiol Imunol, 1998 Dec, 47(4), 137 - 40
{Candida krusei (Castellani) Berkhout--a potential pathogen with growing importance}; Otcenasek M et al.; The authors collect information on the biological properties, ecology and pathogenicity of Candida krusei . They give a list of forms of diseases caused by this yeast and a profile of its sensitivity to antimycotics . They point out the difference between C . krusei and species of C . albicans and analyze the factors enabling its propagation . In their opinion, the resistance of this yeast to fluconazole is the main cause of the increased number of colonizations and infections produced by C . krusei . The population of C . krusei resistant to fluconazole is selected from other, sensitive species of the genus Candida primarily in hospitalized patients . Though there is no consensus of opinion at present on the influence of fluconazole on the yeast spectrum, the authors suppose this systemic antimycotic plays a distinct role in the growing importance of C . krusei.

Somat Cell Mol Genet, 1998 Mar, 24(2), 135 - 40
Toward expression mapping of albinism-deafness syndrome (ADFN) locus on chromosome Xq26; Jacob AN et al.; We have employed a direct cDNA selection methodology to isolate transcribed sequences encoded in the human chromosomal interval Xq26 that contains the gene for X-chromosome linked albinism deafness syndrome (ADFN) . ADFN had been previously mapped to an 8 centi Morgan region on chromosome Xq26 . We have constructed six cDNA libraries specific to six YACs mapping to a 1.5 mb span at the distal boundary of the ADFN locus . The YAC specific libraries were characterized for the presence of unique cDNAs . We have identified 15 transcribed sequences from the selected cDNA libraries . These cDNAs matched to three well characterized sequences corresponding to steroid 5-alpha reductase, ribosomal protein L28, and a short transcript that has been shown to be expressed in human brain cortex . Seven of the cDNAs matched to expressed sequence tags or other sequences of unknown function, and five cDNAs shared no homology with sequences in the public data bases . Each one of these sequences was represented as 3-10 clones in the set that was subjected to sequencing . Further characterization of these transcribed sequences may indicate potential candidates responsible for ADFN . We have discussed the utility of cDNA selection methodology in assembling transcript maps and identifying potential candidates for genetic deafness.

Biochem Biophys Res Commun, 1999 Jan 19, 254(2), 417 - 23
Identification and characterization of nuclear pore subcomplexes in mitotic extract of human somatic cells; Matsuoka Y et al.; In higher eukaryotic cells, it is generally thought that nuclear pore complex disassembles at the beginning of mitosis and reassembles at the end . Using a monoclonal antibody that recognizes nuclear pore antigens, we found that, at mitosis of mammalian somatic Hela cells, the nuclear pore complex disassembles into at least three subcomplexes, termed subcomplexes A, B and C (molecular mass; 2 MDa<, approximately 660 kDa, and approximately 440 kDa, respectively) . The direct partial amino acid sequencing of the components of these subcomplexes indicates that the A subcomplex contains CAN/Nup214/p250 and p62 and the B subcomplex also contains p62, indicating that p62 is contained in two different subcomplexes . Subcomplex C was shown to consist of Nup98 and human RAE1, a human homolog of yeast Rae1p/Gle2p . Since Nup98 and Rae1p/Gle2p have been reported to be involved in mRNA export, this suggests that some of the mitotic subcomplex may be formed by functionally related proteins .

Biochem Biophys Res Commun, 1998 Dec 30, 253(3), 703 - 6
Oxylipin formation in fungi: biotransformation of arachidonic acid to 3-hydroxy-5,8-tetradecadienoic acid by Mucor genevensis; Pohl CH et al.; The soil fungus Mucor genevensis was shown to convert exogenous arachidonic acid to the oxylipin 3-hydroxy-5Z,8Z-tetradecadienoic acid (3-HTDE) as determined by gas chromatography/mass spectrometry . This metabolite was only found in the aqueous supernatant together with free linoleic acid, but not in the final fungal biomass . In contrast, the corresponding primary arachidonic acid metabolite (3R)-hydroxy-(5Z,8Z,11Z,14Z)-eicosatetraenoic acid (3-HETE), which has been earlier shown to be produced by the yeast Dipodascopsis uninucleata, could not be detected . These observations may be plausibly explained by a retroconversion by M . genevensis of arachidonic acid to linoleic acid before the latter is metabolised to 3-HTDE.

Anal Biochem, 1999 Feb 1, 267(1), 227 - 33
A high-yield, enzymatic synthesis of GDP-D-{3H}arabinose and GDP-L-{3H}fucose; Mengeling BJ et al.; For assays involving glycosyltransferases or transporters, several GDP-sugars are either commercially unavailable or expensive . We describe an enzymatic synthesis of GDP-d-{3H}arabinosep and GDP-l-{3H}fucose that yields 66-95% nucleotide-sugar from the appropriate radiolabeled sugar in less than 30 min . The coupled reaction requires Mg2+, ATP, and GTP along with the appropriate radioactive monosaccharide, sugar-1-kinase, and pyrophosphorylase . The latter two activities are present in a cytosolic fraction of Crithidia fasciculata, which is easily grown at room temperature in simple culture medium without serum or added CO2 . Addition of commercial yeast inorganic pyrophosphatase shifts the equilibrium of the pyrophosphorylase reaction toward nucleotide-sugar formation . To verify that these nucleotide-sugars are biologically active, we tested their ability to serve as substrates for glycosyltransferases . GDP-l-{3H}fucose functions as the donor substrate for recombinant human fucosyltransferase V, and GDP-d-{3H}arabinosep serves as the donor substrate for the arabinosyltransferase activities present in Leishmania major microsomes .

Acta Biochim Pol, 1998, 45(3), 721 - 33
Annexins and ATP in membrane traffic: a comparison with membrane fusion machinery; Bandorowicz-Pikula J et al.; Annexins, calcium- and membrane-binding multifunctional proteins, have been implicated in N-ethylmaleimide (NEM)-independent fusion of vesicular structures involved in membrane traffic . This view is based on intracellular localization of annexins, which are frequently associated with endosomes, chromaffin granules, caveolae, clathrin-coated pits, and other membrane compartments, engaged in endo- and exocytosis . Moreover, annexins were found to modulate budding and aggregation of vesicle membranes, to interact with cytoskeletal proteins, and, upon binding to membranes, to change the structure of lipid bilayer, leading to membrane fusion . In addition, some annexins are substrates for various protein kinases and, in membrane-bound form, reveal calcium channel activity . Recently, annexins were observed to interact in vitro and in vivo with nucleotides, ATP, GTP or cAMP, which are potent mediators of membrane traffic processes . In addition, annexin VII showed hydrolytic activity towards GTP, and similarities in the mechanism of action to that of small GTP-binding proteins were found . The aim of the present review is to summarize the observations implying annexins as possible effectors in endo- and exocytosis and to compare them with well known complexes of cytosolic and membrane proteins forming the true membrane fusion machinery within a cell, conserved from yeast to the neurons of humans.

Acta Biochim Pol, 1998, 45(3), 669 - 76
Cloning and sequencing of cDNA encoding the rice methionyl-tRNA synthetase; Deniziak M et al.; Three overlapping clones of cDNA, Mos43, Mos28 and Mos60, coding for methionyl-tRNA synthetase were obtained by screening the Oryza sativa lambda gt11 library . Their nucleotide sequence of 2850 bp was determined . The deduced amino-acid sequence of the isolated clones contains a HLGN and KFSKS motifs, which are conserved for this family of enzymes and have been proposed to be the signature sequences for class I aminoacyl-tRNA synthetases . A comparison of the rice MetRS primary structure with those deposited in EMBL/GenBank points to its high homology to yeast, human and Caenorhabditis elegans MetRSs . Interestingly, a great similarity of its C terminus to endothelial-monocyte-activating polypeptide II (EMAPII) and yeast protein G4p1 was observed.

Oncogene, 1998 Dec 24, 17(25), 3277 - 85
Stress signals for apoptosis: ceramide and c-Jun kinase; Basu S et al.; Mammalian systems respond to environmental stress by either adapting or undergoing programmed cell death . While there is general agreement that the caspase family of proteases serve as the effectors of the apoptotic death response, the signaling apparatus involved in the decision to activate the caspase system is less clear . In the past few years, the sphingomyelin and c-Jun Kinase (JNK)/Stress-activated Protein Kinase (SAPK) pathways have been linked to the death response in many cellular systems . These signaling systems are found throughout the animal kingdom, and ceramide signaling is conserved through yeast . Since yeast do not undergo apoptosis, the sphingomyelin pathway appears evolutionarily older than the caspase-mediated death programs . While recent reviews by several groups have broadly surveyed ceramide signaling in apoptosis, this paper examines the role of sphingomyelinases and the JNK/SAPK pathway in coordinate signaling of apoptosis.

J Biol Chem, 1999 Jan 29, 274(5), 3159 - 64
A common binding site on the microsomal triglyceride transfer protein for apolipoprotein B and protein disulfide isomerase; Bradbury P et al.; The assembly of triglyceride-rich lipoproteins requires the formation in the endoplasmic reticulum of a complex between apolipoprotein B (apoB), a microsomal triglyceride transfer protein (MTP), and protein disulfide isomerase (PDI) . In the MTP complex, the amino-terminal region of MTP (residues 22-303) interacts with the amino-terminal region of apoB (residues 1-264) . Here, we report the identification and characterization of a site on apoB between residues 512 and 721, which interacts with residues 517-603 of MTP . PDI binds in close proximity to this apoB binding site on MTP . The proximity of these binding sites on MTP for PDI and amino acids 512-721 of apoB was evident from studies carried out in a yeast two-hybrid system and by co-immunoprecipitation . The expression of PDI with MTP and apoB16 (residues 1-721) in the baculovirus expression system reduced the amount of MTP co-immunoprecipitated with apoB by 73% . The interaction of residues 512-721 of apoB with MTP facilitates lipoprotein production . Mutations of apoB that markedly reduced this interaction also reduced the level of apoB-containing lipoprotein secretion.

J Biol Chem, 1999 Jan 29, 274(5), 3151 - 8
Pituitary tumor-transforming gene protein associates with ribosomal protein S10 and a novel human homologue of DnaJ in testicular cells; Pei L; Pituitary tumor-transforming gene (PTTG) is a recently characterized proto-oncogene that is expressed specifically in adult testis . In this study, we have used in situ hybridization and developmental Northern blot assays to demonstrate that PTTG mRNA is expressed stage-specifically in spermatocytes and spermatids during rat spermatogenic cycle . We have used the yeast two-hybrid system to identify proteins that interact with PTTG in testicular cells . Two positive clones were characterized . One of the clones is the ribosomal protein S10, the other encodes a novel human DnaJ homologue designated HSJ2 . Northern blot analysis showed that testis contains higher levels of HSJ2 mRNA than other tissues examined, and the expression pattern of HSJ2 mRNA in postnatal rat testis is similar to PTTG . S10 mRNA levels do not vary remarkably among different tissues and remains unchanged during testicular germ cell differentiation . In vitro binding assays demonstrated that both S10 and HSJ2 bind to PTTG specifically and that PTTG can be co-immunoprecipitated with S10 and HSJ2 from transfected cells . Moreover, the binding sites for both proteins were located within the C-terminal 75 amino acids of the PTTG protein . These results suggest that PTTG may play a role in spermatogenesis.

J Biol Chem, 1999 Jan 29, 274(5), 3094 - 102
Differential role of insulin receptor substrate (IRS)-1 and IRS-2 in L6 skeletal muscle cells expressing the Arg1152 --> Gln insulin receptor; Miele C et al.; In L6 muscle cells expressing the Arg1152 --> Gln insulin receptor (Mut), basal tyrosine phosphorylation of insulin receptor substrate (IRS)-1 was increased by 35% compared with wild-type cells (WT) . Upon exposure to insulin, IRS-1 phosphorylation increased by 12-fold in both the Mut and WT cells . IRS-2 was constitutively phosphorylated in Mut cells and not further phosphorylated by insulin . The maximal phosphorylation of IRS-2 in basal Mut cells was paralleled by a 4-fold increased binding of the kinase regulatory loop binding domain of IRS-2 to the Arg1152 --> Gln receptor . Grb2 and phosphatidylinositol 3-kinase association to IRS-1 and IRS-2 reflected the phosphorylation levels of the two IRSs . Mitogen-activated protein kinase activation and {3H}thymidine incorporation closely correlated with IRS-1 phosphorylation in Mut and WT cells, while glycogen synthesis and synthase activity correlated with IRS-2 phosphorylation . The Arg1152 --> Gln mutant did not signal Shc phosphorylation or Shc-Grb2 association in intact L6 cells, while binding Shc in a yeast two-hybrid system and phosphorylating Shc in vitro . Thus, IRS-2 appears to mediate insulin regulation of glucose storage in Mut cells, while insulin-stimulated mitogenesis correlates with the activation of the IRS-1/mitogen-activated protein kinase pathway in these cells . IRS-1 and Shc-mediated mitogenesis may be redundant in muscle cells.

J Biol Chem, 1999 Jan 29, 274(5), 2953 - 62
Identification and characterization of golgin-84, a novel Golgi integral membrane protein with a cytoplasmic coiled-coil domain; Bascom RA et al.; The cytoplasmic face of the Golgi contains a variety of proteins with coiled-coil domains . We identified one such protein in a yeast two-hybrid screen, using as bait the peripheral Golgi phosphatidylinositol(4,5)P2 5-phosphatase OCRL1 that is implicated in a human disease, the oculocerebrorenal syndrome . The approximately 2.8-kilobase mRNA is ubiquitously expressed and abundant in testis; it encodes a 731-amino acid protein with a predicted mass of 83 kDa . Antibodies against the sequence detect a novel approximately 84-kDa Golgi protein we termed golgin-84 . Golgin-84 is an integral membrane protein with a single transmembrane domain close to its C terminus . In vitro, the protein inserts post-translationally into microsomal membranes with an N-cytoplasmic and C-lumen orientation . Cross-linking indicates that golgin-84 forms dimers, consistent with the prediction of an approximately 400-residue dimerizing coiled-coil domain in its N terminus . The dimerization potential is supported by a data base search that showed that the N-terminal 497 residues of golgin-84 contain a coiled-coil domain that when fused to the RET tyrosine kinase domain had the ability to activate it, forming the RET-II oncogene . Data base searching also indicates golgin-84 is similar in structure and sequence to giantin, a membrane protein that tethers coatamer complex I vesicles to the Golgi.

J Biol Chem, 1999 Jan 29, 274(5), 2938 - 52
Cloning and characterization of PRAX-1 . A new protein that specifically interacts with the peripheral benzodiazepine receptor; Galiegue S et al.; Using a cytoplasmic domain of the peripheral benzodiazepine receptor (PBR) as a bait in the yeast two-hybrid system, we have isolated a cDNA encoding a new protein that specifically interacts with PBR . We named it PRAX-1, for peripheral benzodiazepine receptor-associated protein 1 . PRAX-1 is a 1857-amino acid protein, the sequence of which was structurally unrelated to any known proteins . The gene encoding PRAX-1 is located in the q22-q23 region of the long arm of the human chromosome 17 . The PRAX-1 mRNA is 7.5 kilobase pairs, predominantly expressed in the central nervous system, pituitary gland, and thymus . At the protein level, we found the PRAX-1 as a single 220-250-kDa protein in the brain and in many different human cell lines tested using specific antibody raised against PRAX-1 . Parallel analysis of the PRAX-1 mRNA and protein expression performed in mouse and rat gave similar results . Immunocytochemistry analysis carried out to define the distribution of the PRAX-1 protein in the rat brain showed that PRAX-1 was prevalent in the mesolimbic system, specially abundant in the CA1 subfield of the hippocampus . Exhibiting several domains involved in protein-protein interaction (three proline-rich domains, three leucine-zipper motifs, and an Src homology region 3-like domain), the PRAX-1 may be looked upon as a new adaptator protein . We show that both the Src homology region 3-like domain and a proline-rich domain in PRAX-1 are required for the interaction with PBR . PRAX-1 is a cytoplasmic protein that also partially colocalizes with PBR in the mitochondria, as determined by confocal microscopy and Western blotting . Altogether our observations support a model of interaction implicating PBR and this newly described protein, PRAX-1 . As being the first cytoplasmic protein associated with PBR, PRAX-1 is a new tool that opens new fields for exploring PBR biological roles.

J Biol Chem, 1999 Jan 29, 274(5), 2780 - 5
Intermolecular and interdomain interactions of a dynamin-related GTP-binding protein, Dnm1p/Vps1p-like protein; Shin HW et al.; Dnm1p/Vps1p-like protein (DVLP) is a mammalian member of the dynamin GTPase family, which is classified into subfamilies on the basis of the structural similarity . Mammalian dynamins constitute the dynamin subfamily . DVLP belongs to the Vps1 subfamily, which also includes yeast Vps1p and Dnm1p . Typical structural features that discriminate between members of the Vps1 and dynamin subfamilies are that the former lacks the pleckstrin homology and Pro-rich domains . Dynamin exists as tetramers under physiological salt conditions, whereas under low salt conditions, it can polymerize into spirals that resemble the collar structures seen at the necks of constricted coated pits . In this study, we found that DVLP is also oligomeric, probably tetrameric, under physiological salt conditions and forms sedimentable large aggregates under low salt conditions . The data indicate that neither the pleckstrin homology nor Pro-rich domain is required for the self-assembly . Analyses using the two-hybrid system and co-immunoprecipitation show that the N-terminal region containing the GTPase domain and a domain (DVH1) conserved across members of the dynamin and Vps1 subfamilies, can interact with the C-terminal region containing another conserved domain (DVH2) . The data on the interdomain interaction of DVLP is compatible with the previous reports on the interdomain interaction of dynamin . Thus, the self-assembly mechanism of DVLP appears to resemble that of dynamin, suggesting that DVLP may also be involved in the formation of transport vesicles.

Chromosoma, 1998 Dec, 107(6-7), 424 - 9
GFP tagging reveals human Polo-like kinase 1 at the kinetochore/centromere region of mitotic chromosomes; Arnaud L et al.; Polo-like kinases (Plks) have been implicated in various aspects of M-phase progression in organisms ranging from yeast to man . In vertebrates, Plks participate in centrosome maturation and spindle assembly, as well as the activation of the Cdk1/cyclin B complex . Moreover, Plks are required for the destruction of mitotic cyclins, indicating that they play an important role in the regulation of the ubiquitin-dependent proteolytic degradation machinery that controls exit from M-phase . Here, we have fused Green Fluorescent Protein (GFP) to the N-terminus of human Plk1, and expressed this chimeric construct in human cells . We found that GFP-Plk1 associates with centrosomes, the equatorial spindle midzone and the postmitotic bridge of dividing cells, confirming and extending previous results obtained with conventional immunofluorescence microscopy . In addition, however, we observed fluorescence emanating from the midbody between dividing cells, and from discrete dots associated with mitotic chromosomes . This latter staining pattern being reminiscent of centromeres, we performed double-labeling experiments with antibodies against the centromeric marker CENP-B, and reexamined the subcellular localization of endogenous Plk1 using different fixation procedures . Our data clearly show that both GFP-tagged Plk1 and endogenous Plk1 associate with the kinetochore/centromere region of human mitotic chromosomes . This novel localization of Plk1 suggests that substrates and/or regulators of Plks may be found among kinetochore-associated proteins with important functions in chromosome segregation and/or spindle checkpoint mechanisms.

Chromosoma, 1998 Dec, 107(6-7), 406 - 16
Assay of centromere function using a human artificial chromosome; Masumoto H et al.; In order to define a functional human centromere sequence, an artificial chromosome was constructed as a reproducible DNA molecule . Mammalian telomere repeats and a selectable marker were introduced into yeast artificial chromosomes (YACs) containing alphoid DNA from the centromere region of human chromosome 21 in a recombination-deficient yeast host . When these modified YACs were introduced into cultured human cells, a YAC with the alphoid DNA from the alpha21-I locus, containing CENP-B boxes at a high frequency and a regular repeat array, efficiently formed minichromosomes that were maintained stably in the absence of selection and bound CENP-A, CENP-B, CENP-C and CENP-E . The minichromosomes, 1-5 Mb in size and composed of multimers of the introduced YAC DNA, aligned at metaphase plates and segregated to opposite poles correctly in anaphase . Extensive cytological analyses strongly suggested that the minichromosomes had not acquired host sequences and were formed in all cases by a de novo mechanism . In contrast, minichromosomes were never produced with a modified YAC containing alphoid DNA from the alpha21-II locus, which contains no CENP-B boxes and has a less regular sequence arrangement . We conclude that alpha21-I alphoid DNA can induce de novo assembly of active centromere/kinetochore structures on minichromosomes.

Immunogenetics, 1999 Mar, 49(3), 183 - 95
Mhc class I and non-class I gene organization in the proximal H2-M region of the mouse; Jones EP et al.; A bacterial artificial chromosome (BAC) contig was constructed across the proximal part of the H2-M region from the major histocompatibility complex (Mhc) of mouse strain 129 (H2bc) . The contig is composed of 28 clones that span approximately 1 megabasepair (Mb), from H2-T1 to Mog, and contains three H2-T genes and 18 H2-M genes . We report the fine mapping of the H2-M class I gene cluster, which includes the previously reported M4-M6, the M1 family, the M10 family, and four additional class I genes . All but two of the H2-M class I genes are conserved among haplotypes H2k, H2b, and H2bc, and only two genes are found in polymorphic HindIII fragments . Six evolutionarily conserved non-class I genes were mapped to a 180 kilobase interval in the distal part of the class I region in mouse, and their order Znf173-Rfb30-Tctex5-Tctex6- Tctex4-Mog was found conserved between human and mouse . In this Znf173-Mog interval, three mouse class I genes, M6, M4, and M5, which are conserved among haplotypes, occupy the same map position as the human HLA-A class I cluster, which varies among haplotypes and is diverged in sequence from the mouse genes . These results further support the view that class I gene diverge and evolve independently between species.

Curr Opin Cell Biol, 1998 Dec, 10(6), 776 - 83
Polo-like kinases: positive regulators of cell division from start to finish; Nigg EA; Present in organisms ranging from yeast to man, homologues of the Drosophila Polo kinase control multiple stages of cell division . At the onset of mitosis, Polo-like kinases (Plks) function in centrosome maturation and bipolar spindle formation, and they contribute to the activation of cyclin-dependent kinase (Cdk)1-cyclin B . Subsequently, they are required for the inactivation of Cdk1 and exit from mitosis . In the absence of Plk function, mitotic cyclins fail to be destroyed, indicating that Plks are important regulators of the anaphase-promoting complex/cyclosome (APC/C), a key component of the ubiquitin-dependent proteolytic degradation pathway . Finally, recent evidence implicates Plks in the temporal and spatial coordination of cytokinesis.

Development, 1999 Feb, 126(4), 733 - 42
The trithorax group gene osa encodes an ARID-domain protein that genetically interacts with the brahma chromatin-remodeling factor to regulate transcription; Vazquez M et al.; The trithorax group gene brahma (brm) encodes the ATPase subunit of a chromatin-remodeling complex involved in homeotic gene regulation . We report here that brm interacts with another trithorax group gene, osa, to regulate the expression of the Antennapedia P2 promoter . Regulation of Antennapedia by BRM and OSA proteins requires sequences 5' to the P2 promoter . Loss of maternal osa function causes severe segmentation defects, indicating that the function of osa is not limited to homeotic gene regulation . The OSA protein contains an ARID domain, a DNA-binding domain also present in the yeast SWI1 and Drosophila DRI proteins . We propose that the OSA protein may target the BRM complex to Antennapedia and other regulated genes.

Biol Chem, 1998 Dec, 379(12), 1397 - 405
Recruitment of the RNA polymerase II holoenzyme and its implications in gene regulation; Barberis A et al.; In yeast cells, interaction between a DNA-bound protein and a single component of the RNA polymerase II (poIII) holoenzyme is sufficient to recruit the latter to a promoter and thereby activate gene transcription . Here we review results which have suggested such a simple mechanism for how genes can be turned on . The series of experiments which eventually led to this model was originally instigated by studying gene expression in a yeast strain which carries a point mutation in Gal11, a component of the poIII holoenzyme . In cells containing this mutant protein termed Gall11P, a derivative of the transcriptional activator Gal4 devoid of any classical activating region is turned into a strong activator . This activating function acquired by an otherwise silent DNA-binding protein is solely due to a novel and fortuitous interaction between Gal11P and a fragment of the Gal4 dimerization region generated by the P mutation . The simplest explanation for these results is that tethering Gal11 to DNA recruits the poIII holoenzyme and, consequently, activates gene transcription . Transcription factors that are believed not to be integral part of the poIII holoenzyme but are nevertheless required for this instance of gene activation, e.g . the TATA-binding TFIID complex, may bind DNA cooperatively with the holoenzyme when recruited to a promoter, thus forming a complete poIII preinitiation complex . One prediction of this model is that recruitment of the entire poIII transcription complex and consequent gene activation can be achieved by tethering different components to DNA . Indeed, fusion of a DNA-binding domain to a variety of poIII holoenzyme components and TFIID subunits leads to activation of genes bearing the recognition site for the DNA-binding protein . These results imply that accessory factors, which are required to remove or modify nucleosomes do not need to be directly contacted by activators, but can rather be engaged in the activation process when the poIII complex is recruited to DNA . In fact, recruitment of the poIII holoenzyme suffices to remodel nucleosomes at the PHO5 promoter and presumably at many other promoters . Other events in the process of gene expression following recruitment of the transcription complex, e.g . initiation, promoter clearance, elongation and termination, could unravel as a consequence of the recruitment step and the formation of an active preinitiation complex on DNA . This view does not exclude the possibility that classical activators also act directly on chromatin remodeling and post-recruitment steps to regulate gene expression.

Biochemistry, 1999 Jan 19, 38(3), 881 - 9
Electrostatic channeling of oxaloacetate in a fusion protein of porcine citrate synthase and porcine mitochondrial malate dehydrogenase; Shatalin K et al.; Mitochondrial malate dehydrogenase and citrate synthase are sequential enzymes in the Krebs tricarboxylic acid cycle . We have shown {Lindbladh, C., Rault, M., Hagglund, C., Small, W . C., Mosbach, K., Bulow, L., Evans, C., and Srere, P.A (1994) Biochemistry 33, 11692-11698} that a fusion protein of yeast mitochondrial citrate synthase and yeast mitochondrial malate dehydrogenase channels oxaloacetate between the active sites . A Brownian dynamics simulation model of porcine mitochondrial enzymes of citrate synthase and malate dehydrogenase was used {Elcock, A . H., and McCammon, A . M . (1996) Biochemistry 35, 12652-12658}, showing that a positive electrostatic surface potential between the active sites of the fusion protein could account for the channeling of oxaloacetate we observed with the yeast fusion protein . Since the data were established with a yeast fusion protein and the model was with porcine fusion protein, we have now prepared and studied the porcine fusion protein . The channeling of the oxaloacetate intermediate was the same for the porcine fusion protein as it was for the yeast fusion protein . This channeling behavior is eliminated at high ionic strength . A fusion protein of porcine citrate synthase and porcine cytosolic malate dehydrogenase does not exhibit any channeling of oxaloacetate . A model of the fusion protein with the cytosolic malate dehydrogenase shows no clear positive electrostatic potential surface between the two active sites, thus distinguishing it from the fusion protein with the mitochondrial malate dehydrogenase . These results establish the electrostatic nature of channeling in mitochondrial fusion proteins.

Proc Natl Acad Sci U S A, 1999 Jan 19, 96(2), 349 - 54
Tissue-specific and developmental stage-specific DNA binding by a mammalian SWI/SNF complex associated with human fetal-to-adult globin gene switching; O'Neill D et al.; SWI/SNF complexes in yeast and higher eukaryotes are thought to facilitate gene activation and transcription factor binding by disrupting repressive chromatin structures . Little is known, however, about how these complexes target specific genes for activation . We now have purified a specialized SWI/SNF-related complex (PYR complex) from murine erythroleukemia (MEL) cell nuclear extract that binds pyrimidine-rich elements at the human and murine beta-globin loci . PYR complex DNA-binding activity is restricted to definitive hematopoietic cells and is both DNA sequence- and length-dependent . Mass spectrometric identification of purified peptides and antibody supershift assays indicate that PYR complex contains at least four known mammalian SWI/SNF subunits: BAF57, INI1, BAF60a, and BAF170 . PYR complex broadly footprints a 250-bp pyrimidine-rich element between the human fetal and adult beta-globin genes . A short intergenic deletion that removes this element from a human globin locus cosmid construct results in delayed human fetal-to-adult globin gene switching in transgenic mice . Taken together, the data suggest that PYR complex may act through this intergenic element to facilitate human fetal-to-adult globin gene switching, presumably by opening the locus in the region of the adult genes to permit the binding of beta-globin transcriptional activators.

Genes Chromosomes Cancer, 1999 Jan, 24(1), 78 - 82
Fluorescence in situ hybridization mapping of breakpoints in pleomorphic adenomas with 8q12-13 abnormalities identifies a subgroup of tumors without PLAG1 involvement; Roijer E et al.; Recently, we identified the PLAG1 gene as the target gene in pleomorphic adenomas with chromosome abnormalities involving 8q12 . The majority of breakpoints were shown to reside within the 5' noncoding region of the gene . We now report three pleomorphic adenomas with breakpoints located distal to PLAG1 in band 8q13 . These tumors had the following chromosome 8 abnormalities: ins(8;12)(q12-13;q14q15), t(8;12)(q13;q15), and t(6;8)(p21.3-22;q13) . Fluorescence in situ hybridization mapping of the chromosome 8 breakpoints revealed a yeast artificial chromosome clone spanning the breakpoints in two tumors . In none of the cases was PLAG1 activated and/or disrupted . Three candidate genes, N8, HMGIC, and HMGIY, were analyzed for rearrangements and/or abnormal expression by using reverse transcriptase-polymerase chain reaction, rapid amplification of 3' cDNA ends, and Northern blot analyses.

Biol Trace Elem Res, 1998 Dec, 65(3), 221 - 36
Effect of selenium and vitamin E supplementation on lipid abnormalities in plasma, aorta, and adipose tissue of Zucker rats; Douillet C et al.; Twenty-nine obese female Zucker rats (fa/fa) were fed with a laboratory chow supplemented or not with a selenium-rich yeast (Selenion), or Selenion + vitamin E, or vitamin E alone . Twelve lean female Zucker rats (Fa/Fa) of the same littermates fed with the same diet were used as control . After 32 wk of diet, obesity induced a large increase in plasma insulin and lipid levels . A significant decrease in the plasma vitamin E/triglycerides ratio (p<0.005) and an increase in plasma thiobarbituric reactive substances (TBARS) (p<0.005) were also observed . Plasma selenium and vitamin E increased in all supplemented rats . The plasma insulin level was decreased by selenion supplementation and the vitamin E/triglycerides ratio was completely corrected by double supplementation with Selenion + vitamin E . TBARS were also efficiently decreased in two obese groups receiving vitamin E . In plasma, adipose tissue and aorta, obesity induced an increase in palmitic acid (C16:0), a very large increase in monounsaturated fatty acids (palmitoleic acid C16:1, stearic acid C18:1) associated with a decrease in polyunsaturated n-6 fatty acids (linoleic acid C18:2 n-6, arachidonic C20:4 n-6) . These alterations in fatty acid distribution were only partly modulated by Se and vitamin E supplements . However, in the aorta, antioxidant treatment in obese rats significantly reduced the increase in C16:0 and C16:1 (p<0.05 and p<0.01, respectively) and the decrease in arachidonic acid (p<0.05) . These changes could be beneficial in the reduction of insulin resistance and help to protect the vascular endothelium.

Mol Endocrinol, 1999 Jan, 13(1), 167 - 75
P450c17 mutations R347H and R358Q selectively disrupt 17,20-lyase activity by disrupting interactions with P450 oxidoreductase and cytochrome b5; Geller DH et al.; Cytochrome P450c17 catalyzes steroid 17alpha-hydroxylase and 17,20-lyase activities and hence is a key enzyme in the production of human glucocorticoids and sex steroids . These two activities are catalyzed in a single substrate-binding site but are regulated independently in human physiology . We have recently shown that cytochrome b5 facilitates 17,20-lyase activity by allosterically promoting the interaction of P450c17 with P450 oxidoreductase (OR) and that the human P450c17 mutations, R347H and R358Q, selectively destroy 17,20-lyase activity while sparing 17alpha-hydroxylase activity . We transfected COS-1 cells with vectors for these P450c17 mutants and found that an excess of OR and b5 restored a small amount of 17,20-lyase activity, suggesting the mutations interfere with electron donation . To determine whether these mutations selectively interfere with the interaction of P450c17 and its electron-donating system, we expressed each P450cl7 mutant in yeast with or without OR, b5, or both, and measured enzyme kinetics in yeast microsomes using pregnenolone and 17alpha-hydroxypregnenolone as substrates . The apparent Michaelis-Menten (Km) values for the R347H mutant with and without coexpressed OR were 0.2 and 0.6 microM, respectively, and for the R358Q mutant with and without OR they were 0.3 and 0.4 microM, respectively; these values did not differ significantly from the wild-type values of 0.4 and 0.8 microM with and without OR, respectively . Furthermore, coincubation with 17alpha-hydroxypregnenolone showed a competitive mechanism for interference of catalysis . The similar kinetics and the competitive inhibition prove that the mutations did not affect the active site . Coexpression of the mutants with OR yielded insignificant 17,20-lyase activity, but addition of a 30:1 molar excess cytochrome b5 to these microsomes restored partial 17,20-lyase activity, with the R358Q mutant achieving twice the activity of the R347H mutant . These data indicate that both mutations selectively interfere with 17,20-lyase activity by altering the interaction of P450c17 with OR, thus proving that the lyase activity was disrupted by interfering with electron transfer . Furthermore, the data offer the first evidence that R347 is a crucial component of the site at which b5 interacts with the P450c17 x OR complex to promote electron transfer.

Biochem Mol Biol Int, 1998 Dec, 46(6), 1161 - 74
Characterization of mouse ubiquitin-like SMT3A and SMT3B cDNAs and gene/pseudogenes; Chen A et al.; Mouse SMT3A and SMT3B cDNAs encoding ubiquitin-like proteins of 110 and 95 amino acids, respectively, were isolated and sequenced . The sequence of the first 92 amino acids (ending with the conserved Gly-Gly) of mouse SMT3A exhibited two differences at amino acid no . 38 and 76 in comparison with that of human SMT3A . The C-terminal 18 amino acid sequence of mouse SMT3A was completely different from the C-terminal 11 amino acid sequence of human SMT3A . Mouse and human SMT3B were identical for a sequence of 95 amino acids . Mouse SMT3A genomic DNAs were amplified by polymerase-chain-reaction and sequenced . The nucleotide sequence of a PCR-amplified SMT3A genomic DNA fragment was found to be identical to that of SMT3A cDNA, indicating the absence of intron(s) in its protein coding region . Another genomic DNA fragment of 1,531 nucleotides, containing 7% differences from that of cDNA, is unable to encode a functional protein, and thus, it is a SMT3A processed pseudogene . Three mouse SMT3B processed pseudogenes were cloned and sequenced . The genuine mouse SMT3B gene has not yet been isolated . Mouse SMT3A transcript of 1.8 kb was predominantly expressed in most tissues, while SMT3B transcript of 1.0 kb was abundantly present in all tissues analyzed . A family of ubiquitin-like proteins was recently discovered . One distinguishing feature of ubiquitin and ubiquitin-like proteins is the capacity to conjugate with other proteins post-translationally . The ubiquitin-like proteins are cleaved endoproteolytically after a diglycine sequence, corresponding to the C-terminal Gly75-Gly76 of ubiquitin . The cleavage activates the molecule for conjugation . The yeast SMT3 gene was originally identified as a suppressor of mutations in MIF2 gene, which encodes an essential protein binding to the A+T-rich CDEII region of centromere DNA (1) . Studies using temperature-sensitive mutants showed that the loss of yeast Mif2 protein function results in chromosome missegregation, mitotic delay, and aberrant microtubule morphologies (2) . The yeast Mif2 protein shares at least two regions of similarity with mammalian centromere protein CENP-C, an integral component of active kinetochores (3, 4) . Human SMT3A cDNA was identified from the genome sequencing project of chromosome 21 (5) . We have cloned human SMT3B (formerly designated as HSMT3) cDNA (6) . Human SMT3C protein was independently isolated by several groups and denoted as SUMO-1 (7), GMP1 (8), PICI (9), UBL1 (10), sentrin (11) . SUMO-1/GMP1 was found to be covalently linked to the Ran GTPase-activating protein RanGAP1, and attachment of SUMO-1 targets the otherwise cytosolic RanGAP1 to the nuclear pore complex . The modified form of RanGAP1 also appeared to associate with the mitotic spindle apparatus during mitosis (7, 8) . PIC1 was shown to interact with the PML component of nuclear multiprotein complex that is disrupted in acute promyelocytic leukemia (9) . UBL1 was found to associate with human RAD51/RAD52 proteins involved in DNA recombination and DNA double-strand break repair (10) . Sentrin was shown to interact with Fas/APO-1 or the TNF receptor 1 death domain, and the overexpression of sentrin provided protection against both anti-Fas/APO-1 and TNF-induced cell death (11) . Here we report the characterization of mouse SMT3A and SMT3B cDNAs, gene/pseudogenes, and mRNA expression.

Mol Cell Biol, 1999 Feb, 19(2), 1558 - 68
Localization of distant urogenital system-, central nervous system-, and endocardium-specific transcriptional regulatory elements in the GATA-3 locus; Lakshmanan G et al.; We found previously that neither a 6-kbp promoter fragment nor even a 120-kbp yeast artificial chromosome (YAC) containing the whole GATA-3 gene was sufficient to recapitulate its full transcription pattern during embryonic development in transgenic mice . In an attempt to further identify tissue-specific regulatory elements modulating the dynamic embryonic pattern of the GATA-3 gene, we have examined the expression of two much larger (540- and 625-kbp) GATA-3 YACs in transgenic animals . A lacZ reporter gene was first inserted into both large GATA-3 YACs . The transgenic YAC patterns were then compared to those of embryos bearing the identical lacZ insertion in the chromosomal GATA-3 locus (creating GATA-3/lacZ "knock-ins") . We found that most of the YAC expression sites and tissues are directly reflective of the endogenous pattern, and detailed examination of the integrated YAC transgenes allowed the general localization of a number of very distant transcriptional regulatory elements (putative central nervous system-, endocardium-, and urogenital system-specific enhancers) . Remarkably, even the 625-kbp GATA-3 YAC, containing approximately 450 kbp and 150 kbp of 5' and 3' flanking sequences, respectively, does not contain the full transcriptional regulatory potential of the endogenous locus and is clearly missing regulatory elements that confer tissue-specific expression to GATA-3 in a subset of neural crest-derived cell lineages.

J Biol Chem, 1999 Jan 22, 274(4), 2440 - 5
Identification of a new family of higher eukaryotic histone deacetylases . Coordinate expression of differentiation-dependent chromatin modifiers; Verdel A et al.; The histone deacetylase domain of almost all members of higher eukaryotic histone deacetylases already identified (HDAC family) is highly homologous to that of yeast RPD3 . In this paper we report the cloning of two cDNAs encoding members of a new family of histone deacetylase in mouse that show a better homology to yeast HDA1 histone deacetylase . These cDNAs encode relatively large proteins, presenting an in vitro trichostatin A-sensitive histone deacetylase activity . Interestingly, one, mHDA2, encodes a protein with two putative deacetylase domains, and the other, mHDA1, contains only one deacetylase homology domain, located at the C-terminal half of the protein . Our data showed that these newly identified genes could belong to a network of genes coordinately regulated and involved in the remodeling of chromatin during cell differentiation . Indeed, the expression of mHDA1 and mHDA2 is tightly linked to the state of cell differentiation, behaving therefore like the histone H1 degrees-encoding gene . Moreover, like histone H1(0) gene, mHDA1 and mHDA2 gene expression is induced upon deacetylase inhibitor treatment . We postulate the existence of a regulatory mechanism, commanding a coordinate expression of a group of genes involved in the remodeling of chromatin not only during cell differentiation but also after abnormal histone acetylation.

J Biol Chem, 1999 Jan 22, 274(4), 2118 - 25
A novel human STE20-related protein kinase, HGK, that specifically activates the c-Jun N-terminal kinase signaling pathway; Yao Z et al.; The yeast serine/threonine kinase STE20 activates a signaling cascade that includes STE11 (mitogen-activated protein kinase kinase kinase), STE7 (mitogen-activated protein kinase kinase), and FUS3/KSS1 (mitogen-activated protein kinase) in response to signals from both Cdc42 and the heterotrimeric G proteins associated with transmembrane pheromone receptors . Using degenerate polymerase chain reaction, we have isolated a human cDNA encoding a protein kinase homologous to STE20 . This protein kinase, designated HPK/GCK-like kinase (HGK), has nucleotide sequences that encode an open reading frame of 1165 amino acids with 11 kinase subdomains . HGK was a serine/threonine protein kinase that specifically activated the c-Jun N-terminal kinase (JNK) signaling pathway when transfected into 293T cells, but it did not stimulate either the extracellular signal-regulated kinase or p38 kinase pathway . HGK also increased AP-1-mediated transcriptional activity in vivo . HGK-induced JNK activation was inhibited by the dominant-negative MKK4 and MKK7 mutants . The dominant-negative mutant of TAK1, but not MEKK1 or MAPK upstream kinase (MUK), strongly inhibited HGK-induced JNK activation . TNF-alpha activated HGK in 293T cells, as well as the dominant-negative HGK mutants, inhibited TNF-alpha-induced JNK activation . These results indicate that HGK, a novel activator of the JNK pathway, may function through TAK1, and that the HGK --> TAK1 --> MKK4, MKK7 --> JNK kinase cascade may mediate the TNF-alpha signaling pathway.

Nucleic Acids Res, 1999 Feb 1, 27(3), 743 - 9
Covalent crosslinks introduced via a triple helix-forming oligonucleotide coupled to psoralen are inefficiently repaired; Barre FX et al.; Triple helix-forming oligonucleotides (TFOs) represent potentially powerful tools to artificially modulate gene activity . In particular, they can be used to specifically introduce a lesion into a selected target sequence: interstrand crosslinks and monoadducts can be introduced via TFOs coupled to psoralen . The efficiency of these strategies depends on the cell ability to repair these lesions, an issue which is still controversial . Here we show, using psoralen-coupled TFOs and the yeast as a convenient cellular test system, that interstrand crosslinks are quantitatively poorly repaired, resulting in an efficient modification of target gene activity . In addition, these lesions result in the introduction of mutations in a high proportion of cells . We show that these mutations are generated by the Error-Prone Repair pathway, alone or in combination with Nucleotide Excision Repair . Taken together, these results suggest that TFOs coupled to psoralen could be used to inactivate a gene with significant efficiency.

Curr Biol, 1999 Jan 14, 9(1), 1 - 10
A human homologue of the checkpoint kinase Cds1 directly inhibits Cdc25 phosphatase; Blasina A et al.; BACKGROUND: In human cells, the mitosis-inducing kinase Cdc2 is inhibited by phosphorylation on Thr14 and Tyr15 . Disruption of these phosphorylation sites abrogates checkpoint-mediated regulation of Cdc2 and renders cells highly sensitive to agents that damage DNA . Phosphorylation of these sites is controlled by the opposing activities of the Wee1/Myt1 kinases and the Cdc25 phosphatase . The regulation of these enzymes is therefore likely to be crucial for the operation of the G2-M DNA-damage checkpoint . RESULTS: Here, we show that the activity of Cdc25 decreased following exposure to ionizing radiation . The irradiation-induced decrease in Cdc25 activity was suppressed by wortmannin, an inhibitor of phosphatidylinositol (PI) 3-kinases, and was dependent on the function of the gene that is mutated in ataxia telangiectasia . We also identified two human kinases that phosphorylate and inactivate Cdc25 in vitro . One is the previously characterized Chk1 kinase . The second is novel and is homologous to the Cds1/Rad53 family of checkpoint kinases in yeast . Human Cds1 was found to be activated in response to DNA damage . CONCLUSIONS: These results suggest that, in human cells, the DNA-damage checkpoint involves direct inactivation of Cdc25 catalyzed by Cds1 and/or Chk1.

Genomics, 1999 Jan 1, 55(1), 113 - 7
The mouse mitotic checkpoint gene bub1b, a novel bub1 family member, is expressed in a cell cycle-dependent manner; Davenport JW et al.; A search for genes differentially expressed in normal and leukemic mouse thymocytes yielded a homolog of the yeast mitotic checkpoint protein Bub1 . This novel protein ("mBub1b") has 40% sequence similarity to the mouse Bub1 ("mBub1a") previously described by Taylor and McKeon (1997, Cell 89, 727-735) over four extended domains . Differences between the Bub1 sequences suggest that the two proteins may have different substrate specificities and that Bub1b alone has a putative "destruction" box that can target proteins for degradation by proteosomes during mitosis . Northern blots of normal tissues show that mouse Bub1a and Bub1b genes are expressed in thymus and spleen, but not in nondividing tissues . In synchronized cells, expression of both Bub1 genes is undetectable in G1; Bub1 gene expression peaks in G2/M with Bub1b delayed by 6 h relative to Bub1a . This cell cycle-dependent expression explains the tissue distribution and the abundance of Bub1 mRNAs in rapidly dividing cell lines . The human equivalent of mBub1b was isolated and mapped to chromosome 15q15 . The existence in mammals of two separate Bub1 genes encoding distinct proteins, coupled with the different timing of peak expression, suggests that Bub1a and Bub1b have distinct roles in the mitotic checkpoint .

Genomics, 1999 Jan 1, 55(1), 78 - 87
Integrated mapping package--a physical mapping software tool kit; Zhang P et al.; We have developed an integrated physical mapping computer software package (IMP), originally designed to support the physical mapping of human chromosome 13 and expanded to support several gene-identification projects based on the positional candidate approach . IMP displays map data in a form that provides useful guidelines to the end users . An integrated map with high resolution and confidence is constructed from different types of mapping data, including hybridization experiments, STS-based PCR assays, genetic linkage mapping, cDNA localization, and FISH data . The map is also designed to provide suggestions for specific experiments that are required to obtain maps with even higher resolution and confidence . To this end, the optimization employs multiple constraints that take into account already established STS "scaffold" maps . This software thus serves as an important general tool kit for physical mapping, sequencing, and gene-hunting projects .

Biochemistry, 1999 Jan 12, 38(2), 596 - 604
The PUMILIO-RNA interaction: a single RNA-binding domain monomer recognizes a bipartite target sequence; Zamore PD et al.; Translational repression of hunchback (hb) mRNA in the posterior of the Drosophila embryo requires two copies of a bipartite sequence, the Nanos Response Element (NRE), located in the 3' untranslated region of the mRNA . The PUMILIO (PUM) protein is thought to bind the NREs and thereby repress hb translation . The RNA-binding domain of PUM defines an evolutionarily conserved family of RNA-binding proteins, the PUM-Homology Domain (PUM-HD) proteins, which have been identified in yeast, plants, and animals . The PUM RNA-binding domain, the Drosophila PUM-HD (DmPUM-HD), has been shown previously to recognize nucleotides in both the 5' and 3' halves of the NRE, suggesting that a dimer of PUM might recognize one NRE . Here, we analyze the RNA-binding affinity and stoichiometry of the DmPUM-HD and find that one DmPUM-HD monomer binds independently and with equal affinity to each NRE (KD approximately 0.5 nM) . We detect no cooperative interactions between DmPUM-HD monomers bound at adjacent sites . Our results imply that a single DmPUM-HD protein recognizes nucleotides in both the 5' and 3' NRE half-sites . Based on our estimate of the intraembryonic concentration of PUM (>40 nM), we propose that in vivo nearly all NREs are occupied by a PUM monomer.

Endocr Res, 1998 Aug-Nov, 24(3-4), 817 - 25
The molecular basis of isolated 17,20 lyase deficiency; Miller WL et al.; Human P450c17 catalyzes the 17alpha-hydroxylation of pregnenolone to 17OH pregnenolone and of progesterone to 17alpha-OH progesterone; the same P450c17 enzyme also catalyzes 17,20 lyase activity on the same active site, converting 17OH-pregnenolone to DHEA . Rodent and porcine P450c17 also catalyze 17,20 lyase activity with delta4 substrates, converting 17OH-progesterone to delta4 androstenedione, but human P450c17 catalyzes this reaction very inefficiently, so that virtually all human C19 sex steroids are made via 17OH pregnenolone and DHEA . P450c17 is encoded by a single gene and a single species of mRNA . Many mutations of this gene have been described, but until recently all of these either entirely eliminated both 17alpha-hydroxylase and 17,20 lyase activity, or affected each activity equivalently . We have identified and characterized the first patients with P450c17 mutations that selectively ablate 17,20 lyase activity while retaining 17alpha-hydroxylase activity . Through a combination of enyzmologic experiments in transfected mammalian cells and in genetically manipulated yeast, plus a computer model of human P450c17, we have proven that the responsible mutations, R347H and R358Q lie in the redox-partner binding site of P450c17 . This site, through which P450c17 interacts with P450 oxidoreductase to receive the electrons needed for catalysis, can be allosterically influenced by cytochrome b5 . These two mutations have contributed substantially to our understanding of the mechanisms by which 17alpha-hydroxylase and 17,20 lyase activities are regulated independently, and thus have contributed to the study of regulated 17,20 lyase activity in adrenarche, aging, and the polycystic ovary syndrome.

Immunogenetics, 1999 Feb, 49(2), 99 - 105
Linkage of the NKG2 and CD94 receptor genes to D12S77 in the human natural killer gene complex; Sobanov Y et al.; The human natural killer (NK) gene complex is located on the short arm of chromosome 12 and contains a number of genes encoding C-type lectin receptors important for natural killer cell function . Among these are CD94 and the five NKG2 genes . The CD94 protein associates with different NKG2 isoforms to heterodimeric receptors which function to inhibit or trigger cytotoxicity of NK cells depending on the NKG2 isoform . We selected two yeast artificial chromosome clones comprising approximately 1.5 Mb of the NK gene complex and established a contig of underlying P1-derived artificial chromosome clones containing all NKG2 and the CD94 genes . A detailed analysis shows that all six genes are found within a region of 100 to 200 kilobases proximal of the marker D12S77 . The gene order established is D12S77 - CD94 - NKG2D - NKG2F - NKG2E - NKG2C - NKG2A . The NKG2 genes are of identical transcriptional orientation, whereas the CD94 gene is placed in opposite orientation . The tight genomic linkage of these genes and the identical orientation of the NKG2 genes suggest coordinate regulation of expression during the differentiation of natural killer cells.

Virology, 1999 Jan 5, 253(1), 17 - 34
Sequence and analysis of the genome of a baculovirus pathogenic for Lymantria dispar; Kuzio J et al.; The genome of the Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV) was sequenced and analyzed . It is composed of 161,046 bases with a G + C content of 57.5% and contains 163 putative open reading frames (ORFs) of >/=150 nucleotides . Homologs were found to 95 of the 155 genes predicted for the Autographa californica MNPV (AcMNPV) genome . More than 9% of the LdMNPV genome was occupied by 16 repeated genes related to AcMNPV ORF2 . Readily identifiable homologs of several genes that have been reported to play important roles in the AcMNPV life cycle are not present; these include ie-2, a transcriptional transactivator, and gp64, a major envelope glycoprotein of the nonoccluded form of the virus . A number of genes lacking in AcMNPV but present in other baculoviruses were identified; these include two viral enhancing factor homologs, a second copy of a conotoxin-like gene, and a dutpase homolog . Although a single gene predicted to encode a large subunit of ribonucleotide reductase was found, two different copies of the small subunit gene were present . In addition, homologs of genes not previously reported for baculoviruses were identified, including a predicted protein with homology to DNA ligases and another that has motifs most closely related to a yeast mitochondrial helicase . Thirteen homologous regions (hrs) containing 54 repeated sequences that include 30-bp imperfect palindromes were identified . The imperfect palindromes are related to those from other baculoviruses .

Genes Chromosomes Cancer, 1999 Feb, 24(2), 95 - 104
Molecular mapping of chromosome 2 deletions in murine radiation-induced AML localizes a putative tumor suppressor gene to a 1.0 cM region homologous to human chromosome segment 11p11-12; Silver A et al.; Radiation-induced acute myeloid leukemias (AMLs) in the mouse are characterized by chromosome 2 deletions . Previous studies showed that a minimal deleted region (mdr) of approximately 6.5 cM is lost from one homologue in chromosome 2-deleted AMLs . An AML tumor suppressor gene is proposed to map within this mdr . In this study, we refine the mdr to a I cM interval between markers D2Mit126 and D2Mit185 by microsatellite analysis of 21 primary radiation-induced F I AMLs . The construction of a partial yeast artificial chromosome (YAC) contig spanning the mdr and the location of six known genes indicated that the 1 cM mdr is homologous to human 11p11-12, a region implicated in some human AMLs . Screening of five cell lines derived from primary radiation-induced AMLs for homozygous loss of microsatellites and genes mapping within the mdr revealed loss of both copies of the hemopoietic tissue-specific transcription factor Sfpi1(PU.1/Spi1) in one cell line . Studies of primary and F1 AMLs failed to implicate Sfpi1 as the AML tumor suppressor gene . YAC contig construction, together with data suggesting that the critical gene flanks Sfpi1, represents significant progress toward identifying an AML tumor suppressor gene.

Genes Dev, 1999 Jan 1, 13(1), 49 - 63
Intermediates in formation and activity of the RNA polymerase II preinitiation complex: holoenzyme recruitment and a postrecruitment role for the TATA box and TFIIB; Ranish JA et al.; Assembly and activity of yeast RNA polymerase II (Pol II) preinitiation complexes (PIC) was investigated with an immobilized promoter assay and extracts made from wild-type cells and from cells containing conditional mutations in components of the Pol II machinery . We describe the following findings: (1) In one step, TFIID and TFIIA assemble at the promoter independently of holoenzyme . In another step, holoenzyme is recruited to the promoter . Mutations in the CTD of Pol II, Srb2, Srb4, and Srb5, and two mutations in TFIIB disrupt recruitment of all holoenzyme components tested without affecting TFIID and TFIIA recruitment . These results indicate that the stepwise assembly pathway is blocked after TFIID/TFIIA binding . (2) Both the Gal4-AH and Gal4-VP16 activators stimulate formation of active PICs by increasing the extent of PIC formation . The Gal4-AH activator stimulated PIC formation by enhancing the binding of TFIID and TFIIA, whereas Gal4-VP16 could enhance the recruitment of TFIID, TFIIA, and holoenzyme . (3) Extracts deficient in TFIIA activity showed reduced assembly of all PIC components . These and other results suggest that TFIIA acts at an early step by enhancing the stable recruitment of TFIID . (4) An extract containing the TFIIB mutant E62G, had no defect in PIC formation, but had a severe defect in transcription . Similarly, mutation of the TATA box reduced PIC formation only two- to fourfold, but severely compromised transcription . These results demonstate an involvement of TFIIB and the TATA box in one or more steps after recruitment of factors to the promoter.

J Immunol, 1999 Jan 1, 162(1), 387 - 91
Opsonic complement component C3 in the solitary ascidian, Halocynthia roretzi; Nonaka M et al.; The recent identification of two mannose-binding lectin-associated serine protease clones from Halocynthia roretzi, an ascidian, suggested the presence of a complement system in urochordates . To elucidate the structure and function of this possibly primitive complement system, we have isolated cDNA clones for ascidian C3 (AsC3) and purified AsC3 protein from body fluid . The deduced primary structure of AsC3 shows overall similarity to mammalian C3, including a typical thioester site with the His residue required for nucleophilic activation of the thioester . AsC3 has a two-subunit chain structure, and the alpha-chain is cleaved at a specific site near to the N terminus upon activation . Ascidian body fluid contains an opsonic activity which enhances phagocytosis of yeast by ascidian blood cells, and Ab against AsC3 inhibits this opsonic activity . These results indicate that the complement system played a pivotal role in innate immunity by enhancing phagocytosis before the emergence of the vertebrates and well ahead of the establishment of adaptive immunity, which is believed to have occurred at about the time of the appearance of cartilaginous fish.

Mol Cell, 1998 Dec, 2(6), 877 - 85
PRC1: a human mitotic spindle-associated CDK substrate protein required for cytokinesis; Jiang W et al.; We have identified a novel human protein, PRC1, that is involved in cytokinesis . PRC1 is a good substrate for several CDKs in vitro and is phosphorylated in vivo at sites that are phosphorylated by CDK in vitro, strongly suggesting that PRC1 is an in vivo CDK substrate . PRC1 has sequence homology to the budding yeast anaphase spindle elongation factor Ase1p . Like Ase1p, PRC1 protein levels are high during S and G2/M and drop dramatically after cells exit mitosis and enter G1 . PRC1 is a nuclear protein in interphase, becomes associated with mitotic spindles in a highly dynamic manner during mitosis, and localizes to the cell mid-body during cytokinesis . Microinjection of anti-PRC1 antibodies into HeLa cells blocked cellular cleavage, but not nuclear division, indicating a functional role for PRC1 in the process of cytokinesis.

J Cell Sci, 1999 Feb, 112 ( Pt 3), 381 - 93
SUMO-1 modification of the acute promyelocytic leukaemia protein PML: implications for nuclear localisation; Duprez E et al.; PML is a nuclear phosphoprotein that was first identified as part of a translocated chromosomal fusion product associated with acute promyelocytic leukaemia (APL) . PML localises to distinct nuclear multi-protein complexes termed ND10, Kr bodies, PML nuclear bodies and PML oncogenic domains (PODs), which are disrupted in APL and are the targets for immediate early viral proteins, although little is known about their function . In a yeast two-hybrid screen, we first identified a ubiquitin-like protein named PIC1 (now known as SUMO-1), which interacts and co-localises with PML in vivo . More recent studies have now shown that SUMO-1 covalently modifies a number of target proteins including PML, RanGAP1 and IkappaBalpha and is proposed to play a role in either targeting modified proteins and/or inhibiting their degradation . The precise molecular role for the SUMO-1 modification of PML is unclear, and the specific lysine residues within PML that are targeted for modification and the PML sub-domains necessary for mediating the modification in vivo are unknown . Here we show that SUMO-1 covalently modifies PML both in vivo and in vitro and that the modification is mediated either directly or indirectly by the interaction of UBC9 with PML through the RING finger domain . Using site-specific mutagenesis, we have identified the primary PML-SUMO-1 modification site as being part of the nuclear localisation signal (Lys487 or Lys490) . However SUMO-1 modification is not essential for PML nuclear localisation as only nuclear PML is modified . The sequence of the modification site fits into a consensus sequence for SUMO-1 modification and we have identified several other nuclear proteins which could also be targets for SUMO-1 . We show that SUMO-1 modification appears to be dependant on the correct subcellular compartmentalisation of target proteins . We also find that the APL-associated fusion protein PML-RARA is efficiently modified in vitro, resulting in a specific and SUMO-1-dependent degradation of PML-RARA . Our results provide significant insights into the role of SUMO-1 modification of PML in both normal cells and the APL disease state.

J Cell Sci, 1999 Feb, 112 ( Pt 3), 317 - 27
Clathrin, adaptors and eps15 in endosomes containing activated epidermal growth factor receptors; Sorkina T et al.; Activation of the epidermal growth factor receptor (EGFR) by EGF results in binding of clathrin adaptor protein complex AP-2 to the receptor cytoplasmic tail . The transient interaction with AP-2 is thought to be responsible for the selective recruitment of the EGFR into coated pits during endocytosis . In this study we found that EGF-induced EGFR/AP-2 association, measured by co-immunoprecipitation, persists after receptor internalization . Double-label immunofluorescence of EGF-treated A-431 and COS-1 cells revealed the presence of AP-2, clathrin and eps15, another component of the plasma membrane coated pits, in the large perinuclear endosomes loaded with EGFRs . By optical sectioning and image deconvolution, the immunoreactivities were seen to be distributed within vesicular and tubular elements of these endosomes . In addition, these compartments contained the transferrin receptors and a EEA.1 protein, markers of early endosomes . Furthermore, Golgi clathrin adaptor complex AP-1 was found in EGFR-containing endosomes and EGFR immunoprecipitates in A-431 cells . The direct interaction of the EGFR with micro1 as well as micro2 subunits of AP-1 and AP-2, correspondingly, was shown using the yeast two-hybrid assay . Brefeldin A, a drug that releases AP-1 from the trans-Golgi membranes, had no effect on AP-1 association with endosomes and its co-precipitation with EGFR . Taken together, the data suggest that endosomal EGFR-AP complexes make up a significant portion of the total amount of these complexes detectable by co-immunoprecipitation . It can be proposed that APs are capable of binding to the endosomal membrane via a mechanism that requires AP interaction with the intracellular tails of multimeric receptors like activated EGFR, which in turn allows recruitment of clathrin and eps15 . The hypothesis that the competition between adaptor complexes for binding to the receptor tails in endosomes may regulate of the sorting of receptors is discussed.

Am J Ind Med, 1999 Jan, 35(1), 68 - 75
Respiratory and immunological findings in brewery workers; Godnic-Cvar J et al.; BACKGROUND: Occupational exposure of brewery workers to organic dusts such as hops, barley, and brewery yeast has the potential to change respiratory function and immunological status . METHODS: Ninety-seven male workers employed in a brewery plant were studied . The mean age of the workers in this plant was 40 years, the mean duration of their employment was 16 years . In addition, a group of 76 unexposed workers was studied as a control . Respiratory symptoms were recorded . Lung function was measured by recording maximum expiratory flow-volume (MEFV) curves . Immunological testing was performed on all brewery workers and some control volunteers using skin prick testing with hops, barley, and yeast antigens as well as other nonoccupational allergens, and by determining total serum IgE levels . RESULTS: There was a significantly higher prevalence of most of the chronic respiratory symptoms in brewery workers compared to controls (P < 0.01) . Occupational asthma, however, was recorded in only 2 (2.1%) of the brewery workers . Logistic regression analysis showed that smoking was the major studied factor responsible for the high prevalence of chronic respiratory symptoms in workers . A large number of brewery workers complained of acute symptoms that developed during the work shift . Lung function tests were decreased compared to predicted . Multivariate analysis of these respiratory function parameters suggested the importance of workplace exposure in explaining lung function abnormalities . Significantly higher prevalences of positive skin prick tests were recorded in 37 brewery workers for molds, hops, and barley than in controls . Increased serum levels of total IgE were documented in 34/97 (45.1%) brewery workers and in 1/76 (2.7%) of the control workers (P < 0.01) . However, workers with positive skin prick tests had prevalences of chronic respiratory symptoms and lung function changes similar to those of workers with negative skin prick tests . CONCLUSION: Our data suggest that both smoking and dust exposure in the brewery industry may be responsible for the development of respiratory impairment and immunological reactions.

J Cell Biol, 1999 Jan 11, 144(1), 113 - 24
Alf1p, a CLIP-170 domain-containing protein, is functionally and physically associated with alpha-tubulin; Feierbach B et al.; Tubulin is a heterodimer of alpha- and beta-tubulin polypeptides . Assembly of the tubulin heterodimer in vitro requires the CCT chaperonin complex, and a set of five proteins referred to as the tubulin cofactors (Tian, F., Y . Huang, H . Rommelaere, J . Vandekerckhove, C . Ampe, and N.J . Cowan . 1996 . Cell . 86:287-296; Tian, G., S.A . Lewis, B . Feierbach, T . Stearns, H . Rommelaere, C . Ampe, and N.J . Cowan . 1997 . J . Cell Biol . 138:821-832) . We report the characterization of Alf1p, the yeast ortholog of mammalian cofactor B . Alf1p interacts with alpha-tubulin in both two-hybrid and immunoprecipitation assays . Alf1p and cofactor B contain a single CLIP-170 domain, which is found in several microtubule-associated proteins . Mutation of the CLIP-170 domain in Alf1p disrupts the interaction with alpha-tubulin . Mutations in alpha-tubulin that disrupt the interaction with Alf1p map to a domain on the cytoplasmic face of alpha-tubulin; this domain is distinct from the region of interaction between alpha-tubulin and beta-tubulin . Alf1p-green fluorescent protein (GFP) is able to associate with microtubules in vivo, and this localization is abolished either by mutation of the CLIP-170 domain in Alf1p, or by mutation of the Alf1p-binding domain in alpha-tubulin . Analysis of double mutants constructed between null alleles of ALF1 and PAC2, which encodes the other yeast alpha-tubulin cofactor, suggests that Alf1p and Pac2p act in the same pathway leading to functional alpha-tubulin . The phenotype of overexpression of ALF1 suggests that Alf1p can act to sequester alpha-tubulin from interaction with beta-tubulin, raising the possibility that it plays a regulatory role in the formation of the tubulin heterodimer.

J Cell Biol, 1999 Jan 11, 144(1), 59 - 69
Vinexin: a novel vinculin-binding protein with multiple SH3 domains enhances actin cytoskeletal organization; Kioka N et al.; Using the yeast two-hybrid system and an in vitro binding assay, we have identified a novel protein termed vinexin as a vinculin-binding protein . By Northern blotting, we identified two types of vinexin mRNA that were 3 and 2 kb in length . Screening for full-length cDNA clones and sequencing indicated that the two mRNA encode 82- and 37-kD polypeptides termed vinexin alpha and beta, respectively . Both forms of vinexin share a common carboxyl-terminal sequence containing three SH3 domains . The larger vinexin alpha contains an additional amino-terminal sequence . The interaction between vinexin and vinculin was mediated by two SH3 domains of vinexin and the proline-rich region of vinculin . When expressed, vinexin alpha and beta localized to focal adhesions in NIH 3T3 fibroblasts, and to cell-cell junctions in epithelial LLC-PK1 cells . Furthermore, expression of vinexin increased focal adhesion size . Vinexin alpha also promoted upregulation of actin stress fiber formation . In addition, cell lines stably expressing vinexin beta showed enhanced cell spreading on fibronectin . These data identify vinexin as a novel focal adhesion and cell- cell adhesion protein that binds via SH3 domains to the hinge region of vinculin, which can enhance actin cytoskeletal organization and cell spreading.

Blood, 1999 Jan 15, 93(2), 703 - 12
Correct function of the locus control region may require passage through a nonerythroid cellular environment; Vassilopoulos G et al.; The function of the beta-globin locus control region (LCR) has been studied both in cell lines and in transgenic mice . We have previously shown that when a 248-kb beta-locus YAC was first microinjected into L-cells and then transferred into MEL cells by fusion, the YAC loci of the LxMEL hybrids displayed normal expression and developmental regulation.To test whether direct transfer of a beta-globin locus (beta-YAC) into MEL cells could be used for studies of the function of the LCR, a 155-kb beta-YAC that encompasses the entire beta-globin locus was used . This YAC was retrofitted with a PGK-neo selectable marker and with two I-PpoI sites at the vector arm-cloned insert junctions, allowing detection of the intact globin loci on a single I-PpoI fragment by pulsed field gel electrophoresis (PFGE) . The Ppo-155 beta-YAC was used to directly lipofect MEL 585 cells . In 7 beta-YAC MEL clones with at least one intact copy of the YAC, the levels of total human globin mRNA (ie, epsilon + gamma + beta) per copy of integrated beta-YAC varied more than 97-fold between clones . These results indicated that globin gene expression was strongly influenced by the position of integration of the beta-YAC into the MEL cell genome and suggested that the LCR cannot function properly when the locus is directly transferred into an erythroid cell environment as naked beta-YAC DNA . To test whether passage of the beta-YAC through L-cells before transfer into MEL cells was the reason for the previously observed correct developmental regulation of human globin genes in the LxMEL hybrid cells, we transfected the YAC into L-cells by lipofection . Three clones carried the intact 144-kb I-PpoI fragment and transcribed the human globin genes with a fetal-like pattern . Subsequent transfer of the YAC of these L(beta-YAC) clones into MEL cells by fusion resulted in LxMEL hybrids that synthesized human globin mRNA . The variation in human beta-globin mRNA (ie, epsilon + gamma + beta) levels between hybrids was 2.5-fold, indicating that globin gene expression was independent of position of integration of the transgene, as expected for normal LCR function . The correct function of the LCR when the YAC is first transferred into the L-cell environment raises the possibility that normal activation of the LCR requires interaction with the transcriptional environment of an uncommitted, nonerythroid cell . We propose that the activation of the LCR may represent a multistep process initiated by the binding of ubiquitous transcription factors early during the differentiation of hematopoietic stem cells and completed with the binding of erythroid type of factors in the committed erythroid progenitors.

Diagn Microbiol Infect Dis, 1998 Nov, 32(3), 211 - 6
Azole resistance among oral Candida species isolates from AIDS patients under ketoconazole exposure; Milan EP et al.; This is a prospective study designed to investigate species distribution and azole susceptibility profile among Candida spp . isolated from the oral cavities of AIDS patients . One hundred thirty-two AIDS patients sequentially admitted at a teaching tertiary care hospital were enrolled in this study . Samples were obtained by swabbing the oral cavities of the patients . Yeast isolates were identified by classical methods and the antifungal susceptibility profile was further determined according to the NCCLS microbroth assay . Among all patients with prescriptions of systemic antifungal drugs, ketoconazole had been elected to treat 56% of patients . We found 82% of oral yeast carriage, 22% of them harboring non-albicans species . Overall rates of susceptibility dose dependent/resistance to azoles was 16% for itraconazole, 13% for ketoconazole, and 10% for fluconazole with a high agreement rate among the susceptibility profiles of all isolates tested against the triazoles.

Dev Genet, 1998, 23(4), 264 - 74
Clonal analysis of cmp44E, which encodes a conserved putative transmembrane protein, indicates a requirement for cell viability in Drosophila; Faulkner DL et al.; We have identified the cmp44E gene which encodes a putative multi-pass transmembrane protein that is conserved from yeast to humans . The expression of cmp44E during embryogenesis is ubiquitous with notably higher levels in the CNS and brain . It is also expressed in the germline during the germarial stages as well as several later stages of oogenesis . Utilizing a P-element insertion at the 5' end of cmp44E we have isolated several deletions, created by imprecise excision events which eliminate most or all of its coding region . Analysis of these deficiencies has revealed that cmp44E is an essential gene required for embryogenesis . Results obtained from germline clone analysis indicate that cmp44E is not only required in the germline slem cells early in oogenesis, but is also required in other tissues probably due to it being required for cell viability . Finally, using germline transformation, we have identified a minimal genomic fragment capable of fully rescuing a null allele of cmp44E.

Anim Genet, 1998 Dec, 29(6), 446 - 52
Targeting of marker loci to chicken chromosome 16 by representational difference analysis; Wain HM et al.; Representational difference analysis (RDA) was initially used to identify differences between two inbred lines of chickens, line N and line 15I, on which the Compton mapping reference population is based . RDA was subsequently used to identify marker loci targeted specifically to chicken chromosome 16 . Chromosome 16 contains the major histocompatibility complex (MHC), nucleolar organiser region (NOR) and Rfp-Y complex . To generate markers specific for this chromosome a bird was selected from the Compton mapping reference population which had inherited N line alleles for the MHC, NOR and Rfp-Y regions on this chromosome . DNA from this bird was compared with pooled DNA from 16 of its siblings, all of which had inherited line 15I alleles for the MHC, NOR and Rfp-Y regions . Initially amplicons were derived from BamHI digested samples, RDA products were cloned after the first round of hybridisation and 113 clones were investigated: 45 of these identified BamHI polymorphisms in this population . Of the 45 polymorphic clones, 17 have been mapped in the reference population so far, and these have identified seven new loci on chromosome 16 . Interestingly a group of 16 other loci were linked on chromosome 4 . The same birds were also compared by RDA following digestion with TaqI . Again large numbers of clones were generated of which 65 were investigated . Of these 17 clones were polymorphic and of five clones mapped so far three lie on chromosome 16 . Two of the loci mapped to chromosome 16 have been used to identify yeast artificial chromosome (YAC) clones.

Biochem J, 1999 Jan 15, 337 ( Pt 2), 319 - 27
Changes in the protein pattern of H1 histones associated with apoptotic DNA fragmentation; Kratzmeier M et al.; Characteristic steps in the course of cellular apoptosis are the induction of chromatin condensation and cleavage of the DNA, leading to the formation of oligomers of nucleosomes . Since the H1 histones represent functional elements that are essential for the generation of highly condensed chromatin structures, we analysed the total cellular H1 histones of five leukaemic and three solid human tumour cell lines, comparing the H1 pattern of exponentially growing cells with that of apoptotic cells . For the induction of apoptosis, cell lines were treated with the water-soluble camptothecin derivative, topotecan (a topoisomerase I inhibitor), or with an apoptosis-inducing monoclonal anti-CD95 (Fas/APO-1) antibody . Total histone H1 proteins were isolated by extraction with 5% perchloric acid and were analysed by means of capillary zone electrophoresis (CZE) separation . The identities of the peaks representing different histone H1 subtypes on CZE electropherograms were confirmed by analysis of preparations (recombinant proteins purified from transformed yeast used as internal standards) mixed with each of the subtypes respectively . The progress of topotecan- or anti-CD95-induced cell death was monitored by flow cytometry analysis and also by agarose electrophoresis of fragmented DNA . During early apoptosis of three of these cell lines, we observed the induction of internucleosomal DNA cleavage and, simultaneously, a typical change in the histone H1 protein pattern, leading to an increase in the relative amounts of histone subtypes H1.4 and H1.5 . Upon apoptosis induction, these changes were only observed in correlation with the occurrence of DNA fragmentation, thus possibly reflecting a prerequisite for DNA accessibility and/or endonuclease activity.

J Virol, 1999 Feb, 73(2), 1468 - 78
Polo-like kinase 1 as a target for human cytomegalovirus pp65 lower matrix protein; Gallina A et al.; Human cytomegalovirus (HCMV) pp65 protein is the major constituent of viral dense bodies but is dispensable for viral growth in vitro . pp65 copurifies with a S/T kinase activity and has been implicated in phosphorylation of HCMV IE1 immediate-early protein and its escape from major histocompatibility complex 1 presentation . Furthermore, the presence of pp65 correlates with a virion-associated kinase activity . To clarify the role of pp65, yeast two-hybrid system (THS) screening was performed to identify pp65 cellular partners . A total of 18 out of 48 yeast clones harboring cDNAs for putative pp65 binding proteins encoded the Polo-like kinase 1 (Plk1) C-terminal domain . Plk1 behaved as a bona fide pp65 partner in THS control crosses, and the interaction was confirmed by in vitro binding experiments . Endogenous Plk1 was coimmunoprecipitated with pp65 from transiently transfected COS7 cells . In infected fibroblasts, Plk1 was coimmunoprecipitated with pp65 at late infection stages . Furthermore, Plk1 was detected within wild-type HCMV particles but not within the particles of a pp65-negative mutant (RVAd65) . The hydrophilic region of pp65 was phosphorylated in vitro by Plk1 . These results suggest that one function of pp65 may be to capture a cell kinase, perhaps in order to alter its activity, nucleotide preference, substrate specificity, or subcellular localization to the advantage of HCMV.

J Virol, 1999 Feb, 73(2), 1350 - 61
Interactions of the cytoplasmic domains of human and simian retroviral transmembrane proteins with components of the clathrin adaptor complexes modulate intracellular and cell surface expression of envelope glycoproteins; Berlioz-Torrent C et al.; The cytoplasmic domains of the transmembrane (TM) envelope proteins (TM-CDs) of most retroviruses have a Tyr-based motif, YXXO, in their membrane-proximal regions . This signal is involved in the trafficking and endocytosis of membrane receptors via clathrin-associated AP-1 and AP-2 adaptor complexes . We have used CD8-TM-CD chimeras to investigate the role of the Tyr-based motif of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and human T-leukemia virus type 1 (HTLV-1) TM-CDs in the cell surface expression of the envelope glycoprotein . Flow cytometry and confocal microscopy studies showed that this motif is a major determinant of the cell surface expression of the CD8-HTLV chimera . The YXXO motif also plays a key role in subcellular distribution of the envelope of lentiviruses HIV-1 and SIV . However, these viruses, which encode TM proteins with a long cytoplasmic domain, have additional determinants distal to the YXXO motif that participate in regulating cell surface expression . We have also used the yeast two-hybrid system and in vitro binding assays to demonstrate that all three retroviral YXXO motifs interact with the micro1 and micro2 subunits of AP complexes and that the C-terminal regions of HIV-1 and SIV TM proteins interact with the beta2 adaptin subunit . The TM-CDs of HTLV-1, HIV-1, and SIV also interact with the whole AP complexes . These results clearly demonstrate that the cell surface expression of retroviral envelope glycoproteins is governed by interactions with adaptor complexes . The YXXO-based signal is the major determinant of this interaction for the HTLV-1 TM, which contains a short cytoplasmic domain, whereas the lentiviruses HIV-1 and SIV have additional determinants distal to this signal that are also involved.

DNA Cell Biol, 1998 Dec, 17(12), 1047 - 55
Identification of novel mRNA transcripts of the nm23-M1 gene that are modulated during mouse embryo development and are differently expressed in adult murine tissues; Gervasi F et al.; The nm23-M1, a putative metastasis-suppressor gene, and its homologs are involved in development and differentiation . We have shown previously that in vitro neuronal cell proliferation and differentiation can be modulated by nm23-M1 expression levels . In the present study, by the yeast two-hybrid system, we have shown that, at the onset of mouse tissue differentiation, the Nm23-M1 protein forms either homodimers, or heterodimers with Nm23-M2 . Furthermore, we have isolated two cDNA variants of the nm23-M1 gene in the 3'-untranslated region (UTR) . The two variants related to novel mRNA transcripts that are modulated in mouse embryo and are differently expressed in adult murine tissues.

Mamm Genome, 1998 Dec, 9(12), 1042 - 8
Structural and evolutionary characterization of the human sorbitol dehydrogenase gene duplication; Carr IM et al.; We have established that two very closely homologous human sorbitol dehydrogenase sequences lie within 0.5 Mb on Chromosome 15 . We have defined the relative orientation of SORD1 and SORD2 genes with respect to both the centromere and each other and established their exact chromosome location . In addition, we have identified polymorphic variants in the locus, which may be useful, in association studies to predict predisposition to clinical problems resulting from decreased conversion of cellular sorbitol to fructose . To define the evolutionary relationship of these human genes, SORD from the marmoset was also sequenced for comparison . Marmoset SORD, which appears to be a single gene in this species, shows significantly less homology with either SORD1 or SORD2 than they do with each other, suggesting that the human homologs represent a recent gene duplication event . A hypothesis is presented to explain the retention of the redundant SORD2 sequence in the human genome.

J Biol Chem, 1999 Jan 15, 274(3), 1635 - 45
HEED, the product of the human homolog of the murine eed gene, binds to the matrix protein of HIV-1; Peytavi R et al.; heed, the human homolog of mouse eed and Drosophila esc, two members of the trithorax (trx) and Polycomb group (Pc-G) of genes, was isolated by screening an activated lymphocyte cDNA library versus the immunodeficiency virus type 1 (HIV-1) MA protein used as a bait in a two-hybrid system in yeast . The human EED protein (HEED) had 99 . 5% identity with the mouse EED protein and contained seven WD repeats . Two heed gene transcripts were identified, with a putative 407-nucleotide-long intron, giving rise to two HEED protein isoforms of 535 and 494 residues in length, respectively . The shorter HEED isoform, originated from the unspliced message, lacked the seventh WD repeat . HEED was found to bind to MA protein in vitro, as efficiently as in vivo in yeast cells . Site-directed mutagenesis and phage biopanning suggested that the interaction between HEED and MA involved the N-terminal region of the MA protein, including the first polybasic signal, in a MA conformation-dependent manner . In the HEED protein, however, two discrete linear MA-binding motifs were identified within residues 388-403, overlapping the origin of the fifth WD repeat . Deletion of the C-terminal 41 residues of HEED, spanning the seventh WD repeat, as in the 494-residue HEED protein, was detrimental to HEED-MA interaction in vivo, suggesting the existence of another C-terminal binding site and/or a conformational role of the HEED C-terminal domain in the MA-HEED interaction . MA and HEED proteins co-localized within the nucleus of co-transfected human cells and of recombinant baculovirus co-infected insect cells . This and the failure of HEED to bind to uncleaved GAG precursor suggested a role of HEED at the early stages of virus infection, rather than late in the virus life cycle.

J Biol Chem, 1999 Jan 15, 274(3), 1621 - 7
Molecular cloning and characterization of a mitogen-activated protein kinase-associated intracellular chloride channel; Qian Z et al.; ERK7, a member of the mitogen-activated protein kinase family, has a carboxyl-terminal tail that is required for ERK7 activation, cellular localization, and its ability to inhibit DNA synthesis . To identify proteins that interact with ERK7, we utilized a yeast two-hybrid screen with the COOH-terminal tail of ERK7 as bait and isolated the cDNA for a novel protein termed CLIC3 . The interaction between CLIC3 and ERK7 in mammalian cells was confirmed by co-immunoprecipitation . CLIC3 has significant homology to human intracellular chloride channels 1 (NCC27/CLIC1) and 2 and bovine kidney chloride channel p64 . Like NCC27/CLIC1, CLIC3 is predominantly localized in the nucleus and stimulates chloride conductance when expressed in cells . Taken together, these results suggest that CLIC3 is a new member of the human CLIC family . The observed interaction between CLIC3 and ERK7 is the first demonstration of a stable complex between a protein that activates chloride ion transport and a member of the mitogen-activated protein kinase family of signal transducers . The specific association of CLIC3 with the COOH-terminal tail of ERK7 suggests that CLIC3 may play a role in the regulation of cell growth.

J Biol Chem, 1999 Jan 15, 274(3), 1388 - 93
The hematopoietic transcription factor SCL binds the p44 subunit of TFIIH; Zhao XF et al.; SCL is a basic domain helix-loop-helix (bHLH) oncoprotein that is involved in T-cell acute lymphoblastic leukemia as well as in normal hematopoiesis . Although it is believed that SCL functions as a tissue-specific transcription factor, no molecular mechanism has thus far been identified for this putative function . In this report, we show that SCL interacts with p44, a subunit of the basal transcription factor TFIIH . The minimal region of SCL that interacts with p44 was mapped to a 101-amino acid sequence that includes, but is not limited to, the bHLH region; the SCL-binding site of p44 is located in the carboxyl-terminal half of p44 . This interaction was confirmed by glutathione S-transferase fusion protein pull-down assays and a co-immunoprecipitation assay . As analyzed with a yeast two-hybrid system, p44 interacts specifically with SCL, but not with the other class A or B bHLH proteins tested . E2A did not compete with p44 for SCL binding, as demonstrated by an in vitro binding assay . These findings document a previously unsuspected interaction between SCL and a subunit of the basal transcription factor TFIIH, suggesting a potential means by which SCL might modulate transcription.

Plant Cell, 1999 Jan, 11(1), 31 - 42
Polymorphisms and genomic organization of repetitive DNA from centromeric regions of Arabidopsis chromosomes; Heslop-Harrison JS et al.; A highly abundant repetitive DNA sequence family of Arabidopsis, AtCon, is composed of 178-bp tandemly repeated units and is located at the centromeres of all five chromosome pairs . Analysis of multiple copies of AtCon showed 95% conservation of nucleotides, with some alternative bases, and revealed two boxes, 30 and 24 bp long, that are 99% conserved . Sequences at the 3' end of these boxes showed similarity to yeast CDEI and human CENP-B DNA-protein binding motifs . When oligonucleotides from less conserved regions of AtCon were hybridized in situ and visualized by using primer extension, they were detected on specific chromosomes . When used for polymerase chain reaction with genomic DNA, single primers or primer pairs oriented in the same direction showed negligible amplification, indicating a head-to-tail repeat unit organization . Most primer pairs facing in opposite directions gave several strong bands corresponding to their positions within AtCon . However, consistent with the primer extension results, some primer pairs showed no amplification, indicating that there are chromosome-specific variants of AtCon . The results are significant because they elucidate the organization, mode of amplification, dispersion, and evolution of one of the major repeated sequence families of Arabidopsis . The evidence presented here suggests that AtCon, like human alpha satellites, plays a role in Arabidopsis centromere organization and function.

Plant Physiol, 1999 Jan, 119(1), 65 - 72
Temporal and spatial patterns of accumulation of the transcript of Myo-inositol-1-phosphate synthase and phytin-containing particles during seed development in rice; Yoshida KT et al.; Myo-inositol-1-phosphate (I{1}P) synthase (EC 5.5.1.4) catalyzes the reaction from glucose 6-phosphate to I(1)P, the first step of myo-inositol biosynthesis . Among the metabolites of I(1)P is inositol hexakisphosphate, which forms a mixed salt called phytin or phytate, a storage form of phosphate and cations in seeds . We have isolated a rice (Oryza sativa L.) cDNA clone, pRINO1, that is highly homologous to the I(1)P synthase from yeast and plants . Northern analysis of total RNA showed that the transcript accumulated to high levels in embryos but was undetectable in shoots, roots, and flowers . In situ hybridization of developing seeds showed that the transcript first appeared in the apical region of globular-stage embryos 2 d after anthesis (DAA) . Strong signals were detected in the scutellum and aleurone layer after 4 DAA . The level of the transcript in these cells increased until 7 DAA, after which time it gradually decreased . Phytin-containing particles called globoids appeared 4 DAA in the scutellum and aleurone layer, coinciding with the localization of the RINO1 transcript . The temporal and spatial patterns of accumulation of the RINO1 transcript and globoids suggest that I(1)P synthase directs phytin biosynthesis in rice seeds.

J Med Microbiol, 1998 Feb, 47(2), 103 - 10
Interactions between Candida species and platelets; Willcox MD et al.; Candida spp . are able to cause disseminated disease in immunocompromised patients . This study examined the interactions of Candida spp . with platelets, complement and polymorphonuclear leucocytes (PMNLs) . With the exception of C . albicans, all other Candida spp., including a C . albicans strain previously classified as C . stellatoidia, aggregated human platelets at a ratio of yeast cells: platelets of 1:80 . Usually, those species and strains that aggregated platelets were either killed or prevented from growing in platelet-rich plasma indicating that the aggregation released microbicidal or microbistatic substances that were active against Candida spp . All Candida spp . were resistant to attack by complement in 50% serum . However, all species activated complement as determined by the presence of C3 fragments on their surface, in particular a 195-kDa fragment corresponding to C3c, two fragments at 67 and 40 kDa corresponding to iC3b, and a 33-kDa fragment corresponding to C3d . When strains were tested for their ability to stimulate the release of pro-inflammatory substances from platelets and PMNLs, it was found that most strains stimulated PMNLs to release interleukin(IL)-8 but not IL-1beta or leucotriene B4 . The ability of C . albicans to evade complement-mediated killing and not to aggregate platelets may contribute to the survival of this species in the blood during vascular infections.

Mol Biochem Parasitol, 1998 Nov 30, 97(1-2), 81 - 95
The human malaria parasite Plasmodium falciparum exports the ATP-binding cassette protein PFGCN20 to membrane structures in the host red blood cell; Bozdech Z et al.; PFGCN20 is a member of the ATP-binding cassette family of proteins that is closely related to the yeast translational regulator Gcn20p . We have generated a polyclonal antibody against the N-terminal region of PFGCN20 and studied the cellular localization of PFGCN20 throughout the erythrocytic life cycle of Plasmodium falciparum . PFGCN20 was found to be present at all stages and a pronounced export of PFGCN20 into the erythrocyte was observed in the trophozoite and schizont stages . In the indirect immunofluorescence assay, PFGCN20 was found to display significant colocalization with antigens detected by the monoclonal antibody 41E11 . In contrast, there was only a minimal overlap of PFGCN20 localization with EMP2 and HRP2 . Immunoelectron microscopy demonstrated a pronounced accumulation of PFGCN20 in the lumen of the parasitophorous vacuole and deconvolution fluorescence microscopy showed membrane association with selective regions of a tubovesicular network in the red cell . We also observed a concentration of PFGCN20 in electron-dense plaques just underneath the parasite's plasma membrane and an association of PFGCN20 with cytoplasmic vesicular structures within the parasite . The observed export of PFGCN20 and its association with the tubovesicular network in host red cells, may be indicative of the fact that PFGCN20 functions as ATP-binding subunit of an unknown multimeric ABC-transporter . The cytoplasmic localization of PFGCN20 in the parasite, however, suggests that the involvement of PFGCN20 in translational regulation or other cytoplasmic biological functions cannot be ruled out.

Biochim Biophys Acta, 1999 Jan 6, 1426(2), 239 - 57
The dolichol pathway of N-linked glycosylation; Burda P et al.; The oligosaccharide substrate for the N-linked protein glycosylation is assembled at the membrane of the endoplasmic reticulum . Dolichyl pyrophosphate serves as a carrier in this biosynthetic pathway . In this review, we discuss the function of the lipid carrier dolichol in oligosaccharide assembly and give an overview of the biosynthesis of the different sugar donors required for the building of the oligosaccharide . Yeast genetic techniques have made it possible to identify many different loci encoding specific glycosyltransferases required for the precise and ordered assembly of the dolichyl pyrophosphate-linked oligosaccharide . Based on the knowledge obtained from studying this pathway in yeast, we compare it to the process of N-linked protein glycosylation in archaea . We suggest that N-linked glycosylation in eukaryotes and in archaea share a common evolutionary origin.

Mutat Res, 1999 Jan, 436(1), 1 - 9
Cyclin-dependent kinases at the G1-S transition of the mammalian cell cycle; Hengstschlager M et al.; In the mammalian cell cycle, the transition from the G1 phase to S phase, in which DNA replication occurs, is dependent on tight cell size control and has been shown to be regulated by the cyclin-dependent kinases (Cdks) 2, 3, 4 and 6 . Activities of Cdks are controlled by association with cyclins and reversible phosphorylation reactions . An additional level of regulation is provided by inhibitors of Cdks . G1-S and S phase substrates of these enzymes include proteins implicated in replication and transcription . Whereas the regulation and role of Cdk2, 4 and 6 has intensively been studied, less is known about Cdk3 . Recent data provide first insights into the regulation of Cdk3-associate kinase activity and suggest a model how Cdk3 participates in the regulation of the G1-S transition . Although it has been shown that these G1-Cdks are absolutely essential for a proper transition into S phase, their physiological activation is not sufficient to directly initiate replication independently of cell size . Evidence obtained from yeast and Xenopus indicate the initiation of DNA replication to be a two-step process: the origin recognition complex, Cdc6 and Mcm proteins are required for establishing the prereplicative complex and the activities of Cdks and of Cdc7 kinase then trigger the G1-S transition . Recent findings provide evidence that the overall mechanism of initiation of replication is conserved in mammalian cells .

Biochem Biophys Res Commun, 1998 Dec 18, 253(2), 532 - 43
Isolation of ATMEKK1 (a MAP kinase kinase kinase)-interacting proteins and analysis of a MAP kinase cascade in Arabidopsis; Ichimura K et al.; In plants, a number of MAP kinase (MAPK), MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK) homologues have been reported . However, there have been no reports of protein-protein interactions between these kinases or molecular analysis of MAPK cascades in higher plants . To analyze a possible MAPK cascade in Arabidopsis thaliana, we took two molecular approaches . One is the two-hybrid screening of ATMEKK1 (a MAPKKK)-interacting proteins; the other is an analysis of physical and functional interactions among isolated MAPK, MAPKK, and MAPKKK homologues from Arabidopsis . In two-hybrid screening using ATMEKK1 as bait, we isolated a novel MAPKK homologue, ATMKK2, a MAPK homologue, ATMPK4, and an unknown protein . ATMKK2 has high sequence similarity with MEK1 (a MAPKK) in Arabidopsis . Based on yeast two-hybrid analysis, we detected protein-protein interactions between ATMEKK1 and ATMKK2/MEK1 (MAPKKs), between ATMKK2/MEK1 and ATMPK4 (a MAPK), and between ATMPK4 and ATMEKK1 . ATMPK4 and ATMKK2/MEK1 interacted with two distinct regions of ATMEKK1, the N-terminal regulatory domain and the C-terminal kinase domain, respectively . Coexpression of ATMEKK1 increased the ability of two closely related MAPKKs, ATMKK2 and MEK1, to complement a growth defect of the yeast pbs2 mutant . Coexpression of ATMPK4 and MEK1 complemented a growth defect of the yeast mpk1 and bck1 mutants . By contrast, other combinations of MAPKs and MAPKKs did not suppress these yeast mutations . These results suggest that ATMEKK1, ATMKK2/MEK1, and ATMPK4 may constitute a MAP kinase cascade .

Biochem Biophys Res Commun, 1998 Dec 18, 253(2), 443 - 7
Molecular cloning and expression of human grap-2, a novel leukocyte-specific SH2- and SH3-containing adaptor-like protein that binds to gab-1; Qiu M et al.; The SH2- and SH3-containing adaptor molecules serve to recruit cytosolic signal-transducing molecules to the activated receptor tyrosine kinases . In this study, we report the molecular cloning of a novel adaptor-like protein, Grap-2 (Grb-2 related adaptor protein 2), using the multisubstrate docking protein Gab-1 as bait in the yeast two-hybrid system . Sequence analysis revealed that Grap-2 contains a SH3-SH2-SH3 structure that has a high degree of sequence homology to those of the Grb-2 and Grap adaptor molecules . However, unlike in Grap and Grb-2, the SH2 and the C-terminal SH3 domains of Grap-2 are separated by a 120-amino-acid glutamine-rich sequence that shows no apparent homology to any known molecule or structural motif . The C-terminal SH3 domain of Grap-2 alone is sufficient to bind to Gab-1 . Furthermore, Northern blot analysis demonstrated that Grap-2 has two major transcripts of 1.4 and 4.0 kb that can only be detected in tissues rich in leukocytes and in two leukemia cell lines . This highly restricted pattern of expression suggests that Grap-2 may participate in leukocyte-specific protein tyrosine kinase signaling .

J Magn Reson, 1998 Dec, 135(2), 288 - 97
Automated peak picking and peak integration in macromolecular NMR spectra using AUTOPSY; Koradi R et al.; A new approach for automated peak picking of multidimensional protein NMR spectra with strong overlap is introduced, which makes use of the program AUTOPSY (automated peak picking for NMR spectroscopy) . The main elements of this program are a novel function for local noise level calculation, the use of symmetry considerations, and the use of lineshapes extracted from well-separated peaks for resolving groups of strongly overlapping peaks . The algorithm generates peak lists with precise chemical shift and integral intensities, and a reliability measure for the recognition of each peak . The results of automated peak picking of NOESY spectra with AUTOPSY were tested in combination with the combined automated NOESY cross peak assignment and structure calculation routine NOAH implemented in the program DYANA . The quality of the resulting structures was found to be comparable with those from corresponding data obtained with manual peak picking .

J Mol Biol, 1999 Jan 15, 285(2), 581 - 93
Determination of the angle between the acceptor and anticodon stems of a truncated mitochondrial tRNA; Frazer-Abel AA et al.; Significant departures from the canonical (cloverleaf) secondary structure of transfer (t)RNAs can be found among the mitochondrial (m)tRNAs of higher metazoans; these mtRNAs thus pose a challenge to the concept of an invariant, L-shaped tertiary conformation for all tRNAs . For bovine mtRNASer(AGY), which lacks the entire "dihydrouridine" (dhU) arm, two distinct tertiary models have been proposed: the first model preserves the L-shaped conformation at the expense of overall size; the second model preserves the absolute distance between the 3' terminus and the anticodon loop, while allowing the acceptor-anticodon interstem angle to vary . We have tested the central predictions of these two models by performing a series of transient electric birefringence measurements on bovine mtRNASer(AGY) constructs in which the aminoacyl-acceptor and anticodon stems were each extended by approximately 70 bp . This mtRNA species is particularly amenable to analysis, since the native bovine (heart) mtRNA is completely unmodified outside of the anticodon loop . For magnesium ion concentrations above 1 mM, the interstem angle for the extended mtRNA, 120(+/-5) degrees, is approximately 50% larger than the corresponding angle for yeast tRNAPhe (70-80 degrees) under the same ionic conditions . Furthermore, the interstem angles of the two tRNAs exhibit strikingly different responses to the addition of magnesium ions: the interstem angle for yeast tRNAPhe is reduced by nearly 50 % upon addition of 2 mM magnesium ions, whereas the angle for mtRNASer(AGY) increases by about 10% . Our data thus support a central prediction of the second model; namely, that truncated mtRNAs will possess more open interstem angles . In addition, we demonstrate that birefringence amplitude data can be used to provide model-independent estimates for the interstem angles .

J Mol Biol, 1999 Jan 8, 285(1), 149 - 61
Novel organization and sequences of five genes encoding all six enzymes for de novo pyrimidine biosynthesis in Trypanosoma cruzi; Gao G et al.; A 25 kb segment of genomic DNA from Trypanosoma cruzi, the causative agent of Chagas' disease, was sequenced . It contains five genes, pyr1, pyr2, pyr3, pyr4, and pyr6-5, encoding all six enzymes involved in de novo pyrimidine biosynthesis, glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydroorotase, dihydroorotate dehydrogenase, and orotidine-5'-phosphate decarboxylase linked with orotate phosphoribosyltransferase, respectively . The pyr genes constitute a polycistronic transcription unit on an 800 kb chromosomal DNA in the order of pyr1, pyr3, pyr6-5, pyr2, and pyr4 from the 5' terminus, with intervening sequences of 2.2, 0.4, 8.1, and 0.8 kb . The amino acid sequences deduced from the trypanosomatid pyr genes, except for pyr6, showed closer similarities to mammalian and yeast sequences, and less similarity to archaeal and bacterial sequences . The last two enzymes encoded by a single gene, pyr6-5, are covalently linked in the order opposite to mammalian pyr5-6, and possess a putative glycosomal targeting signal tripeptide, serine-lysine-leucine, at the C terminus . The calculated isoelectric points of 9.3 and 9.9 are also diagnostic of the glycosomal localization of these enzymes . We conclude that the T . cruzi pyr gene organization represents an early progenitor in de novo pyrimidine biosynthesis in eukaryotic lineage, and that the independent pyr genes may have evolved before the gene fusion events that resulted in the three mammalian-type genes, pyr1-3-2, pyr4, and pyr5-6, for UMP synthesis . Peculiarities in the trypanosomatid pyr6-5 gene product are discussed .

J Mol Biol, 1999 Jan 8, 285(1), 85 - 95
Characterization of two subunits of Arabidopsis 19S proteasome regulatory complex and its possible interaction with the COP9 complex; Kwok SF et al.; The nuclear localized, multi-subunit COP9 complex (or COP9 signalosome) is a key developmental modulator involved in repression of photomorphogenesis . In an effort to further define the molecular actions of the COP9 complex, a yeast two hybrid interactive screen was undertaken to identify proteins that could directly interact with one subunit of this complex, namely FUS6/COP11 . This screen identified one specific interactive protein, AtS9, that is likely the Arabidopsis non-ATPase S9 (subunit 9) of the 19S regulatory complex from the 26S proteasome . AtS9 specifically interacts with FUS6/COP11 via the C-terminal domain of FUS6/COP11, which is distinct from the N-terminal domain necessary for FUS6/COP11 to interact with itself . As anticipated, AtS9 is not a member of the COP9 complex, but tightly associates with an ATPase subunit of the Arabidopsis 19S proteasome regulatory complex, AtS6A . Since all three proteins, FUS6/COP11, AtS9, and AtS6A, are present as complexed forms in vivo, the observed interaction implies that the COP9 complex may directly interact with the 19S regulatory complex of the 26S proteasome or other potential AtS9-containing complex . This notion is consistent with the parallel tissue-specific expression patterns and the similar, predominantly nuclear localization of both the COP9 complex and the AtS9 protein .

J Mol Biol, 1998 Dec 18, 284(5), 1289 - 99
Symmetry and chirality in topoisomerase II-DNA crossover recognition; Timsit Y et al.; Several experimental data support the notion that the recognition of DNA crossovers play an important role in the multiple functions of topoisomerase II . Here, a theoretical analysis of the possible modes of assembly of yeast topoisomerase II with right and left-handed tight DNA crossovers is performed, using the crystal coordinates of the docking partners . The DNA crossovers are assumed to be clamped into the central hole of the enzyme . Taking into account the rules for building symmetric ternary complexes and the structural constraints imposed by DNA-DNA and protein-DNA interactions, this analysis shows that two geometric solutions could exist, depending on the chirality of the DNA crossovers . In the first one, the two DNA segments are symmetrically recognized by the enzyme while each single double helix binds asymmetrically the protein dimer . In the second one, each double helix is symmetrically recognized by the protein around its dyad axis, while the two DNA segments have their own binding modes . The finding of potential DNA-binding domains which could interact with the crossovers provides structural supports for each model . The structural similarity of a loop containing a cluster of conserved basic residues pointing into the central hole of topoisomerase II and the second DNA-binding site of histone H5 which binds DNA crossover is of particular interest . Each solution, which is consistent with different sets of experimental data found in the literature, could either correspond to different functions of the enzyme or different steps of the reaction . This work provides structural insights for better understanding the role of chirality and symmetry in topoisomerase II-DNA crossover recognition, suggests testable experiments to further elucidate the structure of ternary complexes, and raises new questions about the relationships between the mechanism of strand-passage and strand-exchange catalyzed by the enzyme .

Genomics, 1998 Dec 15, 54(3), 556 - 9
Patchy fur, a mouse coat mutation associated with X-Y nondisjunction, maps to the pseudoautosomal boundary region; Korobova O et al.; Patchy fur is a semidominant X-linked mutation in the mouse, resulting in a sparse coat . The Paf mutation also alters the normal segregation of the X and the Y chromosomes during male meiosis by causing nondisjunction at anaphase I . Analysis of 1139 female meioses from an intersubspecific backcross using 15 PCR-based markers localizes Paf to an approximately 0.2-cM interval that includes the pseudoautosomal boundary . The meiotic nondisjunction phenotype may result from a chromosomal rearrangement that includes pseudoautosomal sequences and affects XY pairing .

Genomics, 1998 Dec 15, 54(3), 529 - 41
Analysis of the human RAD51L1 promoter region and its activation by UV light; Peng L et al.; The human REC2/RAD51B gene (HGMW-approved symbol RAD51L1) encodes a 350-amino-acid protein with regional homologies to members of the RAD52 epistasis group . It is induced by DNA-damaging agents, and the overexpression of this gene product causes G1/S cell cycle arrest . In this report, the promoter region, containing the UV-responsive element, is revealed . Deletion analyses of a 1699-base fragment at the 5' end of the human REC2/RAD51B cDNA identified a 116-base sequence that appears to be responsible for radiation induction . This fragment contains many DNA sequences that have been identified in the promoter regions of other radiation-inducible genes in yeast and humans . Within this region are "consensus" binding sites for both the AP2 and the p53 proteins that may act to regulate the expression of the human REC2/RAD51B gene . Five putative transcripts have been identified from regions 5' of the promoter element that splice near the ATG translation start site . None of the transcripts contain the UV-inducible element nor the consensus transcription factor binding sites .

Genomics, 1998 Dec 15, 54(3), 505 - 10
Characterization of the mouse Src homology 3 domain gene Sh3d2c on Chr 7 demonstrates coexpression with huntingtin in the brain and identifies the processed pseudogene Sh3d2c-ps1 on Chr 2; Zechner U et al.; Formation of intracellular protein complexes is often mediated by Src homology 3 domain-containing proteins interacting with proline-rich target sequences on other proteins . The Sh3d2c gene or its rat/human orthologs have been implicated in synaptic vesicle recycling due to interaction with dynamin I and synaptojanin in nerve terminals . In a yeast two-hybrid system, association with a huntingtin fragment containing an elongated stretch of polyglutamines was observed recently . By genetic mapping and fluorescence in situ hybridization we demonstrate the localization of Sh3d2c on mouse chromosome 7 . A processed pseudogene of Sh3d2c, Sh3d2c-ps1, was identified and mapped to mouse chromosome 2 . Using RNA in situ hybridization, we show that Sh3d2c is transcribed in various regions of the brain . The striatum, hippocampus, cortex, basal hypothalamus, brain stem, and cerebellum are the most prominent sites of expression . Because huntingtin and Sh3d2c are coexpressed in most regions of the brain, it can be speculated that there is a link between the association of huntingtin/Sh3d2c and the pathogenesis of Huntington disease .

Genomics, 1998 Dec 15, 54(3), 469 - 76
The human lamin B receptor/sterol reductase multigene family; Holmer L et al.; LBR (lamin B receptor) is an integral protein of the inner nuclear membrane encoded by a gene on human chromosome 1q42.1 . LBR has a nucleoplasmic, amino-terminal domain of approximately 200 amino acids followed by a carboxyl-terminal domain similar in sequence to yeast and plant sterol reductases . We have determined the primary structures of two human proteins with strong sequence similarity to the carboxyl-terminal domain of LBR and sterol reductases . Their genes have recently been assigned the symbols TM7SF2 and DHCR7 . TM7SF2 mRNA is most predominantly expressed in heart and DHCR7 mRNA mostly in liver and brain . Whereas LBR is localized to the inner nuclear membrane, these two related proteins are in the endoplasmic reticulum . The TM7SF2 gene contains 10 coding exons, and its intron positions are exactly conserved in the part of the LBR gene encoding its carboxyl-terminal domain . Intron positions in the DHCR7 gene are also similar . Both of these new LBR-like genes are on chromosome 11q13 . These results describe a human gene family encoding proteins of the inner nuclear membrane and endoplasmic reticulum that function in nuclear organization and/or sterol metabolism .

Genomics, 1998 Dec 15, 54(3), 437 - 42
A cluster of keratin-associated proteins on mouse chromosome 10 in the region of conserved linkage with human chromosome 21; Cole SE et al.; A gene cluster of three to five high-cysteine keratin-associated proteins (KAPs) has been identified on mouse Chromosome 10 (MMU10) in the region of conserved linkage with human chromosome 21 (HSA21) . One of these genes, Krtap12-1, has been sequenced in its entirety and shown to be an intronless gene encoding a predicted 130-amino-acid protein . Krtap12-1 is most closely related to two previously identified KAP4 genes, but variation in sequence and cysteine content suggests that it represents a new KAP family . Krtap12-1 is expressed in the skin of a 3-day-old mouse . The corresponding region of HSA21, between ITGB2 (integrin beta2) and PFKL (the liver isoform of phosphofructokinase), has proven refractory to cloning, and thus mapping of this region at high resolution has been problematic . Based on the KAP gene cluster position in mouse, evidence has been found for an orthologous human KAP cluster on HSA21q22.3, reinforcing the observation that comparative genomics can play an essential and practical role in determining mammalian genome organization .

Genomics, 1998 Dec 15, 54(3), 415 - 23
A STS content physical and transcription map across the ky, kyphoscoliosis, nonrecombinant region; Blanco G et al.; The ky mouse mutant exhibits a degenerative muscle disease resulting in chronic deformation of the spinal column . Following a previous report describing the mapping of the ky locus to a small region of mouse chromosome 9 (Skynner et al., 1995, Genomics 25, 207-213), we have now undertaken a positional cloning approach to identify candidate genes for ky . A YAC/BAC contig encompassing the ky locus was constructed comprising 48 YAC clones and 48 newly generated STSs . The results from the combined physical and genetic analyses showed that only two overlapping BAC clones, which together do not exceed 260 kb, span the ky nonrecombinant region . A combination of gene hunting methods on the critical BACs has led to the identification of seven coding fragments, which have been tested for expression . The expression analysis and the position of the coding fragments on the contig suggest their grouping in at least four transcription units . One of these transcription units is expressed exclusively in skeletal muscle, making it a suitable candidate for this muscle defect in the ky mouse .

Cell Biol Int, 1998, 22(2), 145 - 60
Proteins involved in membrane transport between the ER and the Golgi apparatus: 21 putative plant homologues revealed by dbEST searching; Andreeva AV et al.; Numerous proteins have been identified in yeast and mammalian cells which are involved in trafficking between the endoplasmic reticulum and the Golgi apparatus . A great number of partial cDNA sequences now available from the two major plant model species, Arabidopsis thaliana and Oryza sativa, makes it possible to identify putative plant homologues of known genes/proteins from non-plant species . The authors used this approach to screen the database of Expressed Sequence Tags (dbEST) in order to detect plant homologues of proteins involved in membrane transport between ER and Golgi . Availability of these partial sequences will facilitate the screening of cDNA and genomic libraries otherwise performed using heterologous probes derived from animal and yeast genes . As the plant Golgi complex differs in many respects from its mammalian and yeast counterparts, the dbEST clones found can be directly used for various functional assays (immunoprecipitation, two-hybrid analysis, transgenic plants etc.) to test the exact roles of the encoded proteins and identify their functional partners, some of which may be specific for plants .

Toxicol Appl Pharmacol, 1998 Nov, 153(1), 12 - 9
Some alkyl hydroxy benzoate preservatives (parabens) are estrogenic; Routledge EJ et al.; The inadvertent estrogenicity of certain synthetic chemicals, and their subsequent effects on the endocrine system of humans and wildlife, is of concern . In this paper we report findings from in vitro and in vivo (uterotrophic) studies which confirm that a range of alkyl hydroxy benzoate preservatives (parabens) are weakly estrogenic . In a receptor-binding assay, butylparaben was able to compete with 3H-estradiol for binding to the rat estrogen receptor with an affinity approximately 5 orders of magnitude lower than that of diethylstilboestrol, and between 1 and 2 orders of magnitude less than nonylphenol . In an in vitro yeast-based estrogen assay, the four most widely used parabens (namely methyl-, ethyl-, propyl-, and butylparaben) were all found to be weakly estrogenic with the most potent (butylparaben) being 10,000-fold less potent than 17 beta-estradiol . The estrogenic activity of parabens was inhibited by 4-hydroxy tamoxifen in vitro, illustrating the requirement of these chemicals to interact with the estrogen receptor in order to activate the yeast . When administered orally to immature rats, the parabens were inactive . However, subcutaneous administration of butylparaben produced a positive uterotrophic response in vivo, although it was approximately 100,000 times less potent than 17 beta-estradiol . Given their use in a wide range of commercially available topical preparations, it is suggested that the safety in use of these chemicals should be reassessed, with particular attention being paid to estimation of the actual levels of systemic exposure of humans exposed to these chemicals . The acquisition of such data is a prerequisite to the derivation of reliable estimates of the possible human risk of exposure to parabens.

Biochem Biophys Res Commun, 1998 Dec 9, 253(1), 33 - 7
MAPKKK6, a novel mitogen-activated protein kinase kinase kinase, that associates with MAPKKK5; Wang XS et al.; MAPKKK5/ASK1 activates c-Jun N-terminal kinase (JNK) and p38 kinase signaling pathways and induces apoptosis when expressed in stably transfected cells . Using MAPKKK5 as bait in yeast two-hybrid screening, a novel protein that interacts with MAPKKK5 was identified and cloned . This novel protein is predicted to contain all 11 kinase subdomains and shares 45% amino acid identity with MAPKKK5 and thus is designated MAPKKK6 . The interaction of MAPKKK6 with MAPKKK5 in vivo was confirmed by coexpression of MAPKKK5 and MAPKKK6 in 293 cells followed by immunoprecipitation . In contrast to MAPKKK5, which activated both JNK and p38 kinase pathways, MAPKKK6 only weakly activated JNK but not ERK or p38 kinase pathways.

Biochem Biophys Res Commun, 1998 Dec 9, 253(1), 16 - 20
Polar amino acid-rich sequences bind to polyglutamine tracts; Imafuku I et al.; Polyglutamine tracts are found in different proteins including transcription factors and cofactors as well as in triplet repeat disease gene products . To characterize the protein motif that binds to the polyglutamine tract, we screened a human embryonic brain cDNA library with the polyglutamine tract of Brn-2 as bait using the yeast two-hybrid method . All six isolated clones encoding polyglutamine tract binding proteins were rich in polar amino acids . Three of these clones could form polar helical structures . These observations suggest that polar amino acid-rich sequences are essential for binding to the polyglutamine tract.

Proc Natl Acad Sci U S A, 1999 Jan 5, 96(1), 29 - 34
A change in actin conformation associated with filament instability after Pi release; Belmont LD et al.; The ability of actin to both polymerize into filaments and to depolymerize permits the rapid rearrangements of actin structures that are essential for actin's function in most cellular processes . Filament polarity and dynamic properties are conferred by the hydrolysis of ATP on actin filaments . Release of inorganic phosphate (Pi) from filaments after ATP hydrolysis promotes depolymerization . We identify a yeast actin mutation, Val-159 to Asn, which uncouples Pi release from the conformational change that results in filament destabilization . Three-dimensional reconstructions of electron micrographs reveal a conformational difference between ADP-Pi filaments and ADP filaments and show that ADP V159N filaments resemble ADP-Pi wild-type filaments . Crystal structures of mammalian beta-actin in which the nucleotide binding cleft is in the "open" and "closed" states can be used to model actin filaments in the ADP and ADP-Pi conformations, respectively . We propose that these two conformations of G-actin may be related to two functional states of F-actin.

Pathobiology, 1999, 67(1), 39 - 44
Cellular immune response to retinal S-antigen and interphotoreceptor retinoid-binding protein fragments in Eales' disease patients; Saxena S et al.; The role of retinal antigens in Eales' disease was studied in 24 patients and an equal number of healthy controls . Lymphocyte proliferative responses were tested in vitro against native S-antigen, its uveitopathogenic peptides (peptide M and peptide G), yeast histone H3 peptide and uveitopathogenic fragment of interphotoreceptor retinoid-binding protein (IRBP; R16) to establish their role in the pathogenesis of Eales' disease . Out of 24 Eales' disease patients, 6 showed significant proliferative response against S-antigen, its uveitogenic fragments or IRBP . None among the controls showed any response to any retinal antigen used in this study . There was no statistically significant difference in the response to purified protein derivative between patients and controls . These results suggest that retinal antigens may play a role in the etiopathogenesis of Eales' disease . An extraneous agent that could result in exposure of normally sequestered uveitopathogenic antigens of the immune system, leading to an exuberant immune response in the eye may initiate the disease.

Bioorg Med Chem Lett, 1998 Sep 22, 8(18), 2603 - 8
Synthesis and binding of stable bisubstrate ligands for phosphoglycerate kinase; Williams DM et al.; Stable bisubstrate ligands of phosphoglycerate kinase (PGK) have been synthesized with AMP or ADP conjugated to hydrolytically-stable, symmetrical analogues of 1,3-bisphosphoglycerate and their binding to yeast PGK evaluated . Their Kds decrease with net negative charge, with a penta-anionic analogue 7 showing highest affinity-in accordance with its approximation to the transition state for the reaction catalysed by PGK.

J Biol Chem, 1999 Jan 8, 274(2), 1124 - 30
Extracellular signal-regulated kinase 1/2-mediated phosphorylation of JunD and FosB is required for okadaic acid-induced activator protein 1 activation; Rosenberger SF et al.; Previously, we reported that in papilloma-producing 308 mouse keratinocytes, the tumor promoter okadaic acid, a serine-threonine phosphatase inhibitor, increased binding of activator protein 1 (AP-1) to a consensus 12-O-tetradecanoylphorbol-13-acetate-responsive element (Rosenberger, S . F., and Bowden, G . T . (1996) Oncogene 12, 2301-2308) . In this study, we investigated the correlation between AP-1 DNA binding and transactivation and examined molecular mechanisms involved in this process . Using a luciferase reporter driven by region -74 to +63 of the human collagenase gene, we demonstrated induction of AP-1-mediated transcription following okadaic acid treatment . By performing in vitro kinase assays, we found elevated activities of extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase . The ERK-1/2-specific inhibitor PD 98059 completely abrogated okadaic acid-induced AP-1 transactivation without altering AP-1 expression, DNA binding, or complex composition . Phosphorylation analyses indicated that inhibition of ERK-1/2 decreased okadaic acid-elevated phosphorylation of JunD and FosB . To further examine the role of JunD and FosB in okadaic acid-induced AP-1 transactivation, we generated fusion proteins of the DNA-binding domain of the yeast transcription factor Gal4 and the transactivation domain of either JunD or FosB . Cotransfection experiments of these constructs with a Gal4-luciferase reporter demonstrated that both JunD and FosB are required for okadaic acid-induced transcription . Treatment with PD 98059 reduced JunD/FosB-dependent transactivation, suggesting that ERK-1/2-mediated phosphorylation is a critical component in this process.

J Biol Chem, 1999 Jan 8, 274(2), 1116 - 23
Ceruloplasmin ferroxidase activity stimulates cellular iron uptake by a trivalent cation-specific transport mechanism; Attieh ZK et al.; The balance required to maintain appropriate cellular and tissue iron levels has led to the evolution of multiple mechanisms to precisely regulate iron uptake from transferrin and low molecular weight iron chelates . A role for ceruloplasmin (Cp) in vertebrate iron metabolism is suggested by its potent ferroxidase activity catalyzing conversion of Fe2+ to Fe3+, by identification of yeast copper oxidases homologous to Cp that facilitate high affinity iron uptake, and by studies of "aceruloplasminemic" patients who have extensive iron deposits in multiple tissues . We have recently shown that Cp increases iron uptake by cultured HepG2 cells . In this report, we investigated the mechanism by which Cp stimulates cellular iron uptake . Cp stimulated the rate of non-transferrin 55Fe uptake by iron-deficient K562 cells by 2-3-fold, using a transferrin receptor-independent pathway . Induction of Cp-stimulated iron uptake by iron deficiency was blocked by actinomycin D and cycloheximide, consistent with a transcriptionally induced or regulated transporter . Cp-stimulated iron uptake was completely blocked by unlabeled Fe3+ and by other trivalent cations including Al3+, Ga3+, and Cr3+, but not by divalent cations . These results indicate that Cp utilizes a trivalent cation-specific transporter . Cp ferroxidase activity was required for iron uptake as shown by the ineffectiveness of two ferroxidase-deficient Cp preparations, copper-deficient Cp and thiomolybdate-treated Cp . We propose a model in which iron reduction and subsequent re-oxidation by Cp are essential for an iron uptake pathway with high ion specificity.

Genetics, 1999 Jan, 151(1), 211 - 20
The dominant temperature-sensitive lethal DTS7 of Drosophila melanogaster encodes an altered 20S proteasome beta-type subunit; Smyth KA et al.; Proteasomes are multicatalytic complexes that function as the major proteolytic machinery in regulated protein degradation . The eukaryotic 20S proteasome proteolytic core structure comprises 14 different subunits: 7 alpha-type and 7 beta-type . DTS7 is a dominant temperature-sensitive (DTS) lethal mutation at 29 degrees that also acts as a recessive lethal at ambient temperatures . DTS7 maps to cytological position 71AB . Molecular characterization of DTS7 reveals that this is caused by a missense mutation in a beta-type subunit gene, beta2 . A previously characterized DTS mutant, l(3)73Ai1, results from a missense mutation in another beta-type subunit gene, beta6 . These two mutants share a very similar phenotype, show a strong allele-specific genetic interaction, and are rescued by the same extragenic suppressor, Su(DTS)-1 . We propose that these mutants might act as "poison subunits," disrupting proteasome function in a dosage-dependent manner, and suggest how they may interact on the basis of the structure of the yeast 20S proteasome.

Plant Cell Physiol, 1998 Oct, 39(10), 1073 - 9
Changes in protein interactions of cell cycle-related genes during the dormancy-to-growth transition in pea axillary buds; Shimizu S et al.; Apical dominance is a phenomenon in which a terminal bud grows predominantly and the growth of the axillary buds is suppressed . Here, we investigated the molecular mechanisms associated with cell cycle control that occur in pea axillary buds as a result of decapitation . Proliferating cell nuclear antigen (PCNA) protein was detected in both dormant and growing buds, while PCNA mRNA was absent in dormant buds . Pissa; CycB1;2 and Cdc2 proteins were undetectable during dormancy . To analyze an interaction between PCNA and Pissa;CycD3;1, we performed anti-PCNA immunoaffinity column chromatography . Pissa;CycD3;1 protein was detected in the eluate prepared from the dormant buds, but not in the eluate prepared from the growing buds . Furthermore, we performed anti-Pissa;CycD3;1 immunoaffinity column chromatography . PCNA protein was detected in the eluate prepared from the dormant buds, but not in the eluate prepared from the growing buds . These results indicated that PCNA associated with Pissa;CycD3;1 only during dormancy . In addition, the interaction between PCNA and Pissa;CycD3;1 was confirmed by a yeast two-hybrid system.

Plant Mol Biol, 1998 Dec, 38(6), 1043 - 52
Physical mapping of unique nucleotide sequences on identified rice chromosomes; Ohmido N et al.; A physical mapping method for unique nucleotide sequences on specific chromosomal regions was developed combining objective chromosome identification and highly sensitive fluorescence in situ hybridisation (FISH) . Four unique nucleotide sequences cloned from rice genomic DNAs, varying in size from 1.3 to 400 kb, were mapped on a rice chromosome map . A yeast artificial chromosome (YAC) clone with a 399 kb insert of rice genomic DNA was localised at the distal end of the long arm of rice chromosome (1q2.1) and a bacterial artificial chromosome (BAC) clone (180 kb) containing the rice leaf blast-resistant gene (Pi-b) was shown to occur at the distal end of the long arm of chromosome 2 (2q2.1) . A cosmid (35 kb) with the resistance gene (Xa-21) against bacterial leaf blight was mapped on the interstitial region of the long arm on chromosome 11 (11q1.3) . Furthermore a single RFLP marker, 1.29 kb in size, was mapped successfully to the distal region of the long arm of rice chromosome 4 (4q2.1) . For precise localisation of the nucleotide sequences within the chromosome region, image analyses were effective . The BAC clone was localised to the specific region, 2q2.1:96.16, by image analysis . The result was compared with the known location of the BAC clone on the genetic map and the consistency was confirmed . The effectiveness and reliability in physically mapping nucleotide sequences on small plant chromosomes achieved by the FISH method using a variety of probes was unequivocally demonstrated.

Jpn J Pharmacol, 1998 Nov, 78(3), 365 - 71
Profile of JTE-522 as a human cyclooxygenase-2 inhibitor; Wakitani K et al.; Inhibitory activity and the mechanism of action of JTE-522 (4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzenesulfonamid e), a novel selective cyclooxygenase (COX)-2 inhibitor, on human COX-1 and COX-2 were investigated and compared with those of reference compounds . In an enzyme assay, JTE-522 inhibited yeast-expressed human recombinant COX-2 with an IC50 value of 0.085 microM . In contrast, JTE-522 did not inhibit human COX-1 prepared from human platelets at concentrations up to 100 microM . In a cell-based assay, JTE-522 diminished lipopolysaccharide-induced prostaglandin E2 production in human peripheral blood mononuclear cells (COX-2) (IC50 value = 15.1 nM) . On the other hand, JTE-522 was less potent at inhibiting calcium ionophore-induced thromboxane B2 production in washed human platelets (COX-1) (IC50 value = 6210 nM) . JTE-522 showed highly selective inhibition of human COX-2, and its activity was more selective than that of other COX-2 inhibitors (NS-398 and SC-58635) . Human recombinant COX-2 activity was attenuated by JTE-522 in a dose-dependent and time-dependent manner . In contrast, the inhibitory activity of JTE-522 on human COX-1 was not affected by preincubation time . COX-2 inhibition by JTE-522 could not be recovered by gel filtration . These results indicate that JTE-522 is a highly selective, time-dependent and irreversible inhibitor of human COX-2.

Arch Pharm Res, 1998 Dec, 21(6), 707 - 11
Possible implication for an indirect interaction between basic fibroblast growth factor and (Na,K)ATPase; Oh J et al.; The (Na,K)ATPase is responsible for generating the ionic gradients and membrane potentials by the exchange of intracellular Na+ for K+ . It has been recently shown that (Na,K)ATPase is involved in the exocytic pathway of basic fibroblast growth factor (bFGF), although it is not known that bFGF is secreted to the outside of cell through direct interaction with (Na,K)ATPase . To understand the role for (Na,K)ATPase in the secretory pathway of bFGF, we have sought to identify the cytoplasmic domains of the alpha 1 isoform of (Na,K)ATPase interacting with bFGF by yeast two-hybrid system . We have also investigated the interaction between the alpha 2 isoform of (Na,K)ATPase and bFGF to find out whether the interaction is isoform-specific . We found that none of the cytoplasmic domains of (Na,K)ATPase isoforms interacted with bFGF . The result suggests that the interaction between bFGF and (Na,K)ATPase might be indirect, thus requiring other proteins which are involved in the formation of protein complexes for the interaction, although we cannot exclude the possibility that the interaction requires the element of the whole alpha subunit structure that was not present in the isolated alpha subunit cytoplasmic domains.

Dev Growth Differ, 1998 Dec, 40(6), 583 - 90
Histone deacetylase mRNA temporally and spatially regulated in its expression in sea urchin embryos; Nemer M; SpHDAC1, a cDNA homolog of the yeast Rpd3 and higher eukaryotic histone deacetylases (HDAC), was cloned from the sea urchin Strongylocentrotus purpuratus . Its predicted polypeptide and the Rpd3 homologs were highly identical in two-thirds of their lengths, but diverged in their carboxyl-terminal regions in both length and sequence . SpHDAC1 transcripts, which reached maximal concentration at the blastula stages, and diminished thereafter, were neither ubiquitously expressed nor restricted to particular cell lineages, but appeared successively in distinct embryonic regions . In the blastula, transcripts were concentrated in a ring within the vegetal plate, comprising primordial endoderm, and, at the outset of gastrulation, in primordial hindgut endoderm . However, in early to mid-gastrula transcripts, they also appeared in oral ectoderm . In the late-stage gastrula, expression developed in the foregut . These shifts in spatial expression, together with an observed developmental blockage prior to sea urchin gastrulation by the histone deacetylase inhibitor trichostatin A, suggest a stepwise involvement of SpHDAC1 gene expression or SpHDAC1 functionality in the events of normal gastrulation.

Cancer Res, 1998 Dec 15, 58(24), 5859 - 65
Cytogenetic and fluorescence in situ hybridization characterization of chromosome 1 rearrangements in head and neck carcinomas delineate a target region for deletions within 1p11-1p13; Jin Y et al.; Cytogenetic analyses have revealed structural rearrangements of chromosome 1 in a large fraction of head and neck carcinomas (HNCA) . These aberrations frequently affect chromosomal band 1p13 and the centromeric region, the latter often in the form of isochromosome i(1q) and whole-arm translocations . To delineate the critical region involved in rearrangements of proximal 1p, we have undertaken a more precise breakpoint mapping in 13 HNCAs, using metaphase fluorescence in situ hybridization with 11 yeast artificial chromosome (YAC) clones spanning 1p . All of the tumors had chromosome 1 changes at G-banding analyses . Fluorescence in situ hybridization showed that in almost all of the cases, at least one copy of chromosome 1 was affected by centromeric rearrangement . By the use of YAC clones mapped to juxtacentromeric regions and a centromere-specific alpha-satellite probe, we detected variable breakpoints in the whole-arm translocations . At the cytogenetic level, 1p13 rearrangements were frequent . However, molecular breakpoints within this band varied among the HNCAs tested . The lack of consistently rearranged chromosome segments indicates that the pathogenetically important consequence of 1p rearrangements in HNCAs is loss and/or gain of genes outside the breakpoint regions . In an assessment of the genomic imbalances, partial or complete overrepresentation of 1q was seen in eight cases . Loss of 1p material was also identified in eight cases; and in four of them, the deleted segments were too small to be discovered by G-banding analysis . The minimal overlapping deleted region was in the interval between YAC 959C4 (band p11-p12) and the centromere (p10) . Our findings indicate that a target region potentially harboring tumor suppressor gene(s) crucial for HNCA is located within chromosomal bands 1p11-p13.

Cancer Res, 1998 Dec 15, 58(24), 5695 - 700
Modulation of aromatase expression in the breast tissue by ERR alpha-1 orphan receptor; Yang C et al.; We have previously identified a silencer element (S1) that is situated between promoters I.3 and II of the human aromatase gene and that down-regulates the action of these promoters . We recently applied the yeast one-hybrid approach to screen a human breast tissue hybrid cDNA expression library for genes encoding the proteins binding to the silencer region . Most proteins identified from this approach belong to the nuclear receptor superfamily . Fifty % of the positive clones encode for ERR alpha-1, and other positive clones include EAR-2, EAR-3 (COUP-TF1), RAR gamma, and p120E4F . Because ERR alpha-1 was found to be the major protein interacting with S1, we decided to examine the regulatory action of ERR alpha-1 on promoter I.3 of the human aromatase gene . Using a reporter plasmid that includes the aromatase genomic fragment containing promoter I.3 and S1, ERR alpha-1 was found to have a positive regulatory function in breast cancer SK-BR-3 cells . Gel mobility shift assays have confirmed that ERR alpha-1 binds to S1 in a dose-dependent manner, and DNase I footprinting analysis has revealed that ERR alpha-1 binds to a region, 5'-AAGGTCAGAAAT-3', which is within S1 and between 96 and 107 bp relative to the transcriptional start site of promoter I.3 . In addition, despite the fact that the nuclear receptor SF1 was shown previously to bind to the same site and to mediate a cAMP response in ovary, our yeast one-hybrid screening did not find any SF-1 clones . Gel mobility shift assays further revealed that SF-1 can bind to the silencer element with an affinity comparable with ERR alpha-1 . Because our reverse transcription-PCR analysis was not able to detect SF1 mRNA in breast cancer tissue or in SK-BR-3 cells, it is thought that SF1 protein is not expressed in breast cancer tissue . Two ERR alpha-1 RNA variants with differences at the 5'-end have been reported . Our reverse transcription-PCR analysis identified the shorter variant in 28 of 32 breast tumor specimens and the longer variant in only 1 specimen . In addition, the shorter variant was detected in breast cancer SK-BR-3 cells as well as in a breast tumor fibroblast line WS3TF . The results suggest that ERR alpha-1 is one of the nuclear proteins interacting with S1 in breast cancer tissue . It is thought that the silencer element in the human aromatase gene may function differently in different tissues because of distinct expression patterns of transcription factors.

Infect Immun, 1999 Jan, 67(1), 342 - 9
Partial protection against Plasmodium vivax blood-stage infection in Saimiri monkeys by immunization with a recombinant C-terminal fragment of merozoite surface protein 1 in block copolymer adjuvant; Yang C et al.; Merozoite surface protein 1 is a candidate for blood-stage vaccines against malaria parasites . We report here an immunization study of Saimiri monkeys with a yeast-expressed recombinant protein containing the C terminus of Plasmodium vivax merozoite surface protein 1 and two T-helper epitopes of tetanus toxin (yP2P30Pv20019), formulated in aluminum hydroxide (alum) and block copolymer P1005 . Monkeys immunized three times with yP2P30Pv20019 in block copolymer P1005 had significantly higher prechallenge titers of immunoglobulin G (IgG) antibodies against the immunogen and asexual blood-stage parasites than those immunized with yP2P30Pv20019 in alum, antigen alone, or phosphate-buffered saline (PBS) (P < 0.05) . Their peripheral blood mononuclear cell proliferative responses to immunogen stimulation 4 weeks after the second immunization were also significantly higher than those from the PBS control group (P < 0.05) . Upon challenge with 100,000 asexual blood-stage parasites 5 weeks after the last immunization, monkeys immunized with yP2P30Pv20019 in block copolymer P1005 had prepatent periods longer than those for the control alone group (P > 0.05) . Three of the five animals in this group also had low parasitemia (peak parasitemia, </=20 parasites/microliter of blood) . Partially protected monkeys had significantly higher levels of prechallenge antibodies against the immunogen than those unprotected (P < 0.05) . There was also a positive correlation between the prepatent period and titers of IgG antibodies against the immunogen and asexual blood-stage parasites and a negative correlation between accumulated parasitemia and titers of IgG antibodies against the immunogen (P < 0.05) . These results indicate that when combined with block copolymer and potent T-helper epitopes, the yeast-expressed P2P30Pv20019 recombinant protein may offer some protection against malaria.

Infect Immun, 1999 Jan, 67(1), 94 - 101
Effect of melanin and carotenoids of Exophiala (Wangiella) dermatitidis on phagocytosis, oxidative burst, and killing by human neutrophils; Schnitzler N et al.; The black yeast Exophiala (Wangiella) dermatitidis is an increasingly recognized pathogen and a leading cause of severe pheohyphomycosis . Melanin is thought to contribute to the virulence of E . dermatitidis . Whereas the synthesis and the redox properties of melanin have been studied intensively, the influence of melanin and carotenoids on the phagocytosis, the oxidative burst, and the killing of E . dermatitidis by human neutrophils has not been studied . To study their effects on these phenomena, we applied a combination of flow cytometry and a colony-count-dependent method . Using E . dermatitidis wild-type strain 8565 and several melanin-deficient mutants that have been described previously, we demonstrate that melanin prevents this pathogen from being killed in the phagolysosome of the neutrophils . Melanin did not influence the phagocytosis or the oxidative burst of the neutrophils involved . The carotenoids torulene and torularhodine were not found to contribute to the prevention of killing . The ability of E . dermatitidis to block the effects of the neutrophil oxidative burst may critically impair the potential of the host to sufficiently eliminate this fungal pathogen and thus may play an important role in the pathogenesis of phaeohyphomycosis.

Zhonghua Yu Fang Yi Xue Za Zhi, 1997 Nov, 31(6), 325 - 9
{Effects of green tea on growth inhibition and immune regulation of Lewis lung cancer in mice}; Zhu M et al.; C57/BL6J mice were inoculated with Lewis lung cancer cells as an experimental model to study the effects of green tea on cancer prevention, inhibition of tumor growth and immune regulation in mice with tumor . Results showed that weight of thymus in C57/BL6J mice and its index declined, proportion of positive CD4 subgroup of T lymphocyte and ratio of CD4+, to CD8+ reduced, baseline chemilumi-nescence decreased in peripheral white blood cells, yeast zymosan stimulated chemiluminescence increased, and number of immunoglobulin M formation cells decreased . It indicated that green tea had obvious inhibition in Lewis lung cancer and protective effects, to various extent, on adverse changes of above indices.

Nucleic Acids Res, 1999 Jan 15, 27(2), 587 - 95
U1 snRNA is cleaved by RNase III and processed through an Sm site-dependent pathway; Seipelt RL et al.; Core snRNP proteins bind snRNA through the conserved Sm site, PuA(U)n>/=3GPu . While yeast U1 snRNA has three matches to the Sm consensus, the U1 3'-terminal Sm site was found to be both necessary and sufficient for U1 function . Mutation of this site inhibited pre-mRNA splicing, blocked cell division and resulted in the accumulation of two 3'-extended forms of the U1 snRNA . Cells which harbor the Sm site mutation lack mature U1 RNA (U1alpha) but have a minor polyadenylated species, U1gamma, and a prominent, non-polyadenylated species, U1beta . Metabolic depletion of the essential Sm core protein, Smd1p, also resulted in the increased accumulation of U1beta and U1gamma . In vitro, synthetic U1 precursors were cleaved by Rnt1p (RNase III) very near the U1beta 3'-end observed in vivo . We propose that U1beta is an Rnt1p-cleaved intermediate and that U1 maturation to the U1alpha form occurs through an Sm-sensitive step . Interestingly, both U1alpha and a second, much longer RNA, U1straightepsilon, were produced in an rnt1 mutant strain . These results suggest that yeast U1 snRNA processing may progress through Rnt1p-dependent and Rnt1p-independent pathways, both of which require a fun-ctional Sm site for final snRNA maturation.

Nucleic Acids Res, 1999 Jan 15, 27(2), 439 - 45
Alternative function of a protein kinase homology domain in 2', 5'-oligoadenylate dependent RNase L; Dong B et al.; RNase L is the 2',5'-oligoadenylate (2-5A)-dependent endoribonuclease that functions in interferon action and apoptosis . One of the intriguing, albeit unexplained, features of RNase L is its significant homology to protein kinases . Despite the homology, however, no protein kinase activity was detected during activation and RNA cleavage reactions with human RNase L . Similarly, the kinase plus ribonuclease domains of RNase L produced no detectable protein kinase activity in contrast to the phosphorylation obtained with homologous domains of the related kinase and endoribonuclease, yeast IRE1p . In addition, neither ATP nor pA(2'p5'A)3was hydrolyzed by RNase L . To further investigate the function of the kinase homology in RNase L, the conserved lysine at residue 392 in protein kinase-like domain II was replaced with an arginine residue . The resulting mutant, RNase LK392R, showed >100-fold decreases in 2-5A-dependent ribonuclease activity without reducing 2-5A- or RNA-binding activities . The greatly reduced activity of RNase LK392Rwas correlated to a defect in the ability of RNase L to dimerize . These results demonstrate a critical role for lysine 392 in the activation and dimerization of RNase L, thus suggesting that these two activities are intimately linked.

Eur J Immunol, 1998 Dec, 28(12), 4345 - 55
Protection from Plasmodium berghei infection by priming and boosting T cells to a single class I-restricted epitope with recombinant carriers suitable for human use; Plebanski M et al.; The desirability of inducing cytotoxic T cell responses to defined epitopes in humans has led to the development of a variety of recombinant delivery systems . Recombinant protein particles derived from a yeast retrotransposon (Ty) and the modified Ankara vaccinia (MVA) virus can deliver large epitope strings or even whole proteins . Both have previously been administered safely in humans . Immunization with recombinant Ty and MVA containing a single Plasmodium berghei class I-binding epitope provided 95% sterile protection against malaria in mice . The sequence of immunization, Ty followed by MVA, was critical to elicit high levels of IFN-gamma-producing cells and protection . The reciprocal sequence (MVA/TY) or homologous boosting was not protective . Both constructs (Ty and MVA) contain the H-2Kd-restricted pb9 CTL epitope from the circumsporozoite protein of P . berghei among a string of 8-15 human P . falciparum-derived CTL epitopes restricted through 7 common HLA alleles as well as widely recognized CD4 T cell epitopes . Thus, the novel recombinant Ty/MVA prime/boost combination with these constructs provides a safe alternative for evaluation for human vaccination against P . falciparum malaria.

Plant Mol Biol, 1998 Nov, 38(5), 699 - 712
Prediction of functional regions of the maize streak virus replication-associated proteins by protein-protein interaction analysis; Horvath GV et al.; The replication of the geminiviruses depends on the viral encoded early (complementary-sense) proteins and on host genome encoded factors . Additionally, some of the early proteins (the AL2 protein of subgroup III, and the RepA (formerly known as C1) or Rep (C1:C2) proteins of subgroup I geminiviruses) can function as transcriptional activators of virion- (V-)sense gene expression . The yeast two-hybrid system has allowed us to predict some of the functionally important regions of the maize streak virus (MSV) early proteins RepA and Rep . Defined domains of these proteins were shown to act as transactivators in yeast cells . We detected the association of the RepA and Rep proteins, and their subfragments, with the maize retinoblastoma protein (ZmRb1) which is likely to be one of the interacting host proteins . We showed the self-association capability of the MSV proteins and suggest that homo- or hetero-oligomerization may play an important role in virus replication . These results provide new insights into the role of different regions of the MSV proteins in relation to transcriptional activation and regulation of viral DNA replication.

Mycoses, 1998 Sep-Oct, 41(7-8), 273 - 5
Anti-Malassezia furfur antibodies in the population; Faggi E et al.; The authors carried out research into anti-Malassezia precipitating antibodies in the population, specifying the distribution by age and sex . A total of 868 serum specimens from subjects of both sexes, aged between 0 and 80 years, were studied . An immunoelectroprecipitation reaction was used using a M . furfur culture filtrate as antigen . No antibodies were found in children under 11 years, whereas they were present after that age and reached maximum frequency in subjects between 31 and 40 years of age . In subjects up to 50 years of age they were more frequent in women, but in subjects over the age of 50 years they were more frequent in men . Globally, antibodies were found in 31% (270/868) of the subjects examined . The presence of antibodies correlates with data from the literature regarding the isolation of this yeast in relation to age.

Proc Natl Acad Sci U S A, 1998 Dec 22, 95(26), 15781 - 6
Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs; Fasshauer D et al.; SNARE {soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor} proteins are essential for membrane fusion and are conserved from yeast to humans . Sequence alignments of the most conserved regions were mapped onto the recently solved crystal structure of the heterotrimeric synaptic fusion complex . The association of the four alpha-helices in the synaptic fusion complex structure produces highly conserved layers of interacting amino acid side chains in the center of the four-helix bundle . Mutations in these layers reduce complex stability and cause defects in membrane traffic even in distantly related SNAREs . When syntaxin-4 is modeled into the synaptic fusion complex as a replacement of syntaxin-1A, no major steric clashes arise and the most variable amino acids localize to the outer surface of the complex . We conclude that the main structural features of the neuronal complex are highly conserved during evolution . On the basis of these features we have reclassified SNARE proteins into Q-SNAREs and R-SNAREs, and we propose that fusion-competent SNARE complexes generally consist of four-helix bundles composed of three Q-SNAREs and one R-SNARE.

Biochemistry, 1998 Dec 8, 37(49), 17105 - 11
Divalent cations stabilize unstacked conformations of DNA and RNA by interacting with base pi systems; McFail-Isom L et al.; Nucleic acid structure, stability, and reactivity are governed substantially by cations . We propose that magnesium and other biological inorganic ions unstack bases of DNA and RNA . This unstacking function of cations opposes their previously accepted role in stabilizing DNA and RNA duplexes and higher assemblies . We show that cations interact favorably with pi-systems of nucleic acid bases . These cation-pi interactions require access of cations or their first hydration shells to faces of nucleic acid bases . We observe that hydrated magnesium ions located in the major groove of B-DNA pull cytosine bases partially out from the helical stack, exposing pi-systems to positive charge . A series of critical cation-pi interactions contribute to the stability of the anticodon arm of yeast-tRNAphe, and to the magnesium core of the Tetrahymena group I intron P4-P6 domain . The structural consequences of divalent cation-pi interactions are clearly distinct from, and some cases in opposition to, cation-electron lone pair interactions . These observations of cation-pi interactions suggest a number of new mechanistic roles for cations in DNA bending, DNA-protein recognition, base-flipping, RNA folding, and catalysis.

Cytogenet Cell Genet, 1998, 82(3-4), 273 - 7
A serpin gene cluster on human chromosome 6p25 contains PI6, PI9 and ELANH2 which have a common structure almost identical to the 18q21 ovalbumin serpin genes; Sun J et al.; The human genes encoding the "ovalbumin" subgroup of closely related serine proteinase inhibitors (serpins) are located at 18q21.3 and 6p25 . Those at 6p25 include proteinase inhibitor 6 (PI-6; gene symbol PI6), proteinase inhibitor 9 (PI-9; gene symbol PI9) and monocyte neutrophil elastase inhibitor (M/NEI; gene symbol ELANH2) . Here we describe the fine mapping of these genes to a 200-kb region of chromosome 6 that includes the markers WI-8835 and D6S1338, and the establishment of the gene order: tel-PI6-PI9-ELANH2-cen . PI6 and ELANH2 are transcribed towards the telomere, and structural analysis shows that PI6 and PI9 are organized identically, having seven exons and six introns . PI6 and PI9 are almost identical in structure to the ovalbumin serpin genes at 18q21.3 . The 18q21.3 genes have an extra exon and intron, otherwise all the other exon/intron boundaries are conserved between the two groups . These results represent the first detailed map of the chromosome 6 serpin gene cluster, and demonstrate that although they are very closely related, the 6p25 and 18q21-->q23 ovalbumin serpin genes form two structurally distinct groups . These findings do not support a previously proposed model for evolution of the clusters which invoked an inter-chromosomal duplication of the entire 6p25 group to 18q21.3.

Cytogenet Cell Genet, 1998, 82(3-4), 238 - 46
Identification of the WBSCR9 gene, encoding a novel transcriptional regulator, in the Williams-Beuren syndrome deletion at 7q11.23; Peoples RJ et al.; We have identified a novel gene (WBSCR9) within the common Williams-Beuren syndrome (WBS) deletion by interspecies sequence conservation . The WBSCR9 gene encodes a roughly 7-kb transcript with an open reading frame of 1483 amino acids and a predicted protein product size of 170.8 kDa . WBSCR9 is comprised of at least 20 exons extending over 60 kb . The transcript is expressed ubiquitously throughout development and is subject to alternative splicing . Functional motifs identified by sequence homology searches include a bromodomain; a PHD, or C4HC3, finger; several putative nuclear localization signals; four nuclear receptor binding motifs; a polyglutamate stretch and two PEST sequences . Bromodomains, PHD motifs and nuclear receptor binding motifs are cardinal features of proteins that are involved in chromatin remodeling and modulation of transcription . Haploinsufficiency for WBSCR9 gene products may contribute to the complex phenotype of WBS by interacting with tissue-specific regulatory factors during development.

Cytogenet Cell Genet, 1998, 82(3-4), 189 - 91
Molecular cytogenetic mapping of 24 CEPH YACs and 24 gene-specific large insert probes to chromosome 17; Barlund M et al.; Defining boundaries of chromosomal rearrangements at the molecular level would benefit from landmarks that link the cytogenetic map to physical, genetic, and transcript maps, as well as from large-insert FISH probes for such loci to detect numerical and structural rearrangements in metaphase or interphase cells . Here, we determined the locations of 24 genetically mapped CEPH-Mega YACs along the FLpter scale (fractional length from p-telomere) by quantitative fluorescence in situ hybridization analysis . This generated a set of cytogenetically mapped probes for chromosome 17 with an average spacing of about 5 cM . We then developed large-insert YAC, BAC, PAC, or P1 clones to the following 24 known genes, and determined refined map locations along the same FLpter scale: pter-TP53-TOP3-cen-TNFAIP1-ERBB2-TOP2A- BRCA1-TCF11-NME1-HLF-ZNF147/CL N80-BCL5/MPO/SFRS1-TBX2-PECAM1-DDX5/ PRKCA-ICAM2-GH1/PRKAR1A-GRB2-CDK3 /FKHL13-qter . Taken together, these 48 cytogenetically mapped large-insert probes provide tools for the molecular analysis of chromosome 17 rearrangements, such as mapping amplification, deletion, and translocation breakpoints in this chromosome, in cancer and other diseases.

Eur J Cell Biol, 1998 Nov, 77(3), 161 - 5
Mint 3: a ubiquitous mint isoform that does not bind to munc18-1 or -2; Okamoto M et al.; Mint 1 and 2 are proteins that bind to munc18-1, an essential component of the synaptic vesicle fusion machinery, and are detectably expressed only in neurons {Okamoto and Sudhof, J . Biol . Chem . 272, 31459-31464 (1997)} . Mint 1 and 2 are composed of a variable N-terminal region that includes a conserved munc18-1-binding site, and a constant C-terminal region that contains one PTB and two PDZ domains . We have now identified a third mint isoform, mint 3 . Similar to mint 1 and 2, the C-terminal half of mint 3 is composed of one PTB domain and two PDZ domains . However, in contrast to mint 1 and 2, mint 3 lacks an N-terminal munc18-binding domain and does not interact with munc18-1 in yeast two-hybrid assays . Mint 3 is ubiquitously expressed in all tissues, with lowest levels in brain and testis whereas mint 1 and 2 appear to be brain-specific . Our data suggest that mints form a diverse family of proteins with specialized neuronal and ubiquitous isoforms.

Mol Cell Biol, 1999 Jan, 19(1), 807 - 16
Ribosomal pausing and scanning arrest as mechanisms of translational regulation from cap-distal iron-responsive elements; Paraskeva E et al.; Iron regulatory protein 1 (IRP-1) binding to an iron-responsive element (IRE) located close to the cap structure of mRNAs represses translation by precluding the recruitment of the small ribosomal subunit to these mRNAs . This mechanism is position dependent; reporter mRNAs bearing IREs located further downstream exhibit diminished translational control in transfected mammalian cells . To investigate the underlying mechanism, we have recapitulated this position effect in a rabbit reticulocyte cell-free translation system . We show that the recruitment of the 43S preinitiation complex to the mRNA is unaffected when IRP-1 is bound to a cap-distal IRE . Following 43S complex recruitment, the translation initiation apparatus appears to stall, before linearly progressing to the initiation codon . The slow passive dissociation rate of IRP-1 from the cap-distal IRE suggests that the mammalian translation apparatus plays an active role in overcoming the cap-distal IRE-IRP-1 complex . In contrast, cap-distal IRE-IRP-1 complexes efficiently repress translation in wheat germ and yeast translation extracts . Since inhibition occurs subsequent to 43S complex recruitment, an efficient arrest of productive scanning may represent a second mechanism by which RNA-protein interactions within the 5' untranslated region of an mRNA can regulate translation . In contrast to initiating ribosomes, elongating ribosomes from mammal, plant, and yeast cells are unaffected by IRE-IRP-1 complexes positioned within the open reading frame . These data shed light on a characteristic aspect of the IRE-IRP regulatory system and uncover properties of the initiation and elongation translation apparatus of eukaryotic cells.

Mol Cell Biol, 1999 Jan, 19(1), 733 - 44
The E6 oncoproteins of high-risk papillomaviruses bind to a novel putative GAP protein, E6TP1, and target it for degradation; Gao Q et al.; The high-risk human papillomaviruses (HPVs) are associated with carcinomas of the cervix and other genital tumors . Previous studies have identified two viral oncoproteins, E6 and E7, which are expressed in the majority of HPV-associated carcinomas . The ability of high-risk HPV E6 protein to immortalize human mammary epithelial cells (MECs) has provided a single-gene model to study the mechanisms of E6-induced oncogenic transformation . In this system, the E6 protein targets the p53 tumor suppressor protein for degradation, and mutational analyses have shown that E6-induced degradation of p53 protein is required for MEC immortalization . However, the inability of most dominant-negative p53 mutants to induce efficient immortalization of MECs suggests the existence of additional targets of the HPV E6 oncoprotein . Using the yeast two-hybrid system, we have isolated a novel E6-binding protein . This polypeptide, designated E6TP1 (E6-targeted protein 1), exhibits high homology to GTPase-activating proteins for Rap, including SPA-1, tuberin, and Rap1GAP . The mRNA for E6TP1 is widely expressed in tissues and in vitro-cultured cell lines . The gene for E6TP1 localizes to chromosome 14q23.2-14q24.3 within a locus that has been shown to undergo loss of heterozygosity in malignant meningiomas . Importantly, E6TP1 is targeted for degradation by the high-risk but not the low-risk HPV E6 proteins both in vitro and in vivo . Furthermore, the immortalization-competent but not the immortalization-incompetent HPV16 E6 mutants target the E6TP1 protein for degradation . Our results identify a novel target for the E6 oncoprotein and provide a potential link between HPV E6 oncogenesis and alteration of a small G protein signaling pathway.

Mol Cell Biol, 1999 Jan, 19(1), 646 - 56
Cell cycle-dependent regulation of human DNA polymerase alpha-primase activity by phosphorylation; Voitenleitner C et al.; DNA polymerase alpha-primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180 and p68 subunits . Here we show that phosphorylation of purified human DNA polymerase alpha-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity . Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 and poorly by cyclin E/cdk2 . The p180 phosphopeptides were identical with both kinases . By mass spectrometry, the p68 peptide family was identified as residues 141 to 160 . Cyclin A/cdk2- and cyclin A/cdc2-modified p68 also displayed a phosphorylation-dependent shift to slower electrophoretic mobility . Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity . Phosphopeptide maps of the p68 subunit of DNA polymerase alpha-primase from human cells, synchronized and labeled in G1/S and in G2, revealed a cyclin E/cdk2-like pattern in G1/S and a cyclin A/cdk2-like pattern in G2 . The slower-electrophoretic-mobility form of p68 was absent in human cells in G1/S and appeared as the cells entered G2/M . Consistent with this, the ability of DNA polymerase alpha-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G1/S, decreased as the cells completed S phase, and reached a minimum in G2/M . These results suggest that the replication activity of DNA polymerase alpha-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.

Mol Cell Biol, 1999 Jan, 19(1), 216 - 28
FLP recombinase-mediated induction of Cu/Zn-superoxide dismutase transgene expression can extend the life span of adult Drosophila melanogaster flies; Sun J et al.; Yeast FLP recombinase was used in a binary transgenic system ("FLP-OUT") to allow induced overexpression of catalase and/or Cu/Zn-superoxide dismutase (Cu/ZnSOD) in adult Drosophila melanogaster . Expression of FLP recombinase was driven by the heat-inducible hsp70 promoter . Once expressed, FLP catalyzed the rearrangement and activation of a target construct in which expression of catalase or Cu/ZnSOD cDNAs was driven by the constitutive actin5C promoter . In this way a brief heat pulse (120 or 180 min, total) of young adult flies activated transgene expression for the rest of the life span . FLP-OUT allows the effects of induced transgene expression to be analyzed in control (no heat pulse) and experimental (heat pulse) populations with identical genetic backgrounds . Under the conditions used, the heat pulse itself always had neutral or slightly negative effects on the life span . Catalase overexpression significantly increased resistance to hydrogen peroxide but had neutral or slightly negative effects on the mean life span . Cu/ZnSOD overexpression extended the mean life span up to 48% . Simultaneous overexpression of catalase with Cu/ZnSOD had no added benefit, presumably due to a preexisting excess of catalase . The data suggest that oxidative damage is one rate-limiting factor for the life span of adult Drosophila . Finally, experimental manipulation of the genetic background demonstrated that the life span is affected by epistatic interactions between the transgene and allele(s) at other loci.

EMBO J, 1998 Dec 15, 17(24), 7480 - 9
A newly identified N-terminal amino acid sequence of human eIF4G binds poly(A)-binding protein and functions in poly(A)-dependent translation; Imataka H et al.; Most eukaryotic mRNAs possess a 5' cap and a 3' poly(A) tail, both of which are required for efficient translation . In yeast and plants, binding of eIF4G to poly(A)-binding protein (PABP) was implicated in poly(A)-dependent translation . In mammals, however, there has been no evidence that eIF4G binds PABP . Using 5' rapid amplification of cDNA, we have extended the known human eIF4GI open reading frame from the N-terminus by 156 amino acids . Co-immunoprecipitation experiments showed that the extended eIF4GI binds PABP, while the N-terminally truncated original eIF4GI cannot . Deletion analysis identified a 29 amino acid sequence in the new N-terminal region as the PABP-binding site . The 29 amino acid stretch is almost identical in eIF4GI and eIF4GII, and the full-length eIF4GII also binds PABP . As previously shown for yeast, human eIF4G binds to a fragment composed of RRM1 and RRM2 of PABP . In an in vitro translation system, an N-terminal fragment which includes the PABP-binding site inhibits poly(A)-dependent translation, but has no effect on translation of a deadenylated mRNA . These results indicate that, in addition to a recently identified mammalian PABP-binding protein, PAIP-1, eIF4G binds PABP and probably functions in poly(A)-dependent translation in mammalian cells.

EMBO J, 1998 Dec 15, 17(24), 7395 - 403
Evidence that P-TEFb alleviates the negative effect of DSIF on RNA polymerase II-dependent transcription in vitro; Wada T et al.; Recently, a positive and a negative elongation factor, implicated in 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibition of transcription elongation, has been identified . P-TEFb is a positive transcription elongation factor and the DRB-sensitive kinase that phosphorylates the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) . PITALRE, a member of the Cdc2 family of protein kinases, is the catalytic subunit of P-TEFb . DSIF is a human homolog of the yeast Spt4-Spt5 complex and renders elongation of transcription sensitive to DRB . DRB sensitivity-inducing factor (DSIF) binds to RNA Pol II and may directly regulate elongation . Here we show a functional interaction between P-TEFb and DSIF . The reduction of P-TEFb activity induced by either DRB, antibody against PITALRE, or immunodepletion resulted in a negative effect of DSIF on transcription . DSIF acts at an early phase of elongation, and the prior action of P-TEFb makes transcription resistant to DSIF . The state of phosphorylation of CTD determines the DSIF-RNA Pol II interaction, and may provide a direct link between P-TEFb and DSIF . Taken together, this study reveals a molecular basis for DRB action and suggests that P-TEFb stimulates elongation by alleviating the negative action of DSIF.

EMBO J, 1998 Dec 15, 17(24), 7273 - 81
Mona, a novel hematopoietic-specific adaptor interacting with the macrophage colony-stimulating factor receptor, is implicated in monocyte/macrophage development; Bourette RP et al.; The production, survival and function of monocytes and macrophages are regulated by the macrophage colony-stimulating factor (M-CSF or CSF-1) through its tyrosine kinase receptor Fms . Binding of M-CSF results in Fms autophosphorylation on specific tyrosines that act as docking sites for intracellular signaling molecules containing SH2 domains . Using a yeast two-hybrid screen, we cloned a novel adaptor protein which we called 'Mona' for monocytic adaptor . Mona contains one SH2 domain and two SH3 domains related to the Grb2 adaptor . Accordingly, Mona interacts with activated Fms on phosphorylated Tyr697, which is also the Grb2-binding site . Furthermore, Mona contains a unique proline-rich region located between the SH2 domain and the C-terminal SH3 domain, and is apparently devoid of any catalytic domain . Mona expression is restricted to two hematopoietic tissues: the spleen and the peripheral blood mononuclear cells, and is induced rapidly during monocytic differentiation of the myeloid NFS-60 cell line in response to M-CSF . Strikingly, overexpression of Mona in bone marrow cells results in strong reduction of M-CSF-dependent macrophage production in vitro . Taken together, our results suggest an important role for Mona in the regulation of monocyte/macrophage development as controlled by M-CSF.

J Biol Chem, 1998 Dec 25, 273(52), 35273 - 81
Cbl-mediated negative regulation of the Syk tyrosine kinase . A critical role for Cbl phosphotyrosine-binding domain binding to Syk phosphotyrosine 323; Lupher ML Jr et al.; The proto-oncogene product Cbl has emerged as a potential negative regulator of the Syk tyrosine kinase; however, the nature of physical interactions between Cbl and Syk that are critical for this negative regulation remains unclear . Here we show that the phosphotyrosine-binding (PTB) domain within the N-terminal transforming region of Cbl (Cbl-N) binds to phosphorylated Tyr323 in the linker region between the Src homology 2 and kinase domains of Syk, confirming recent results by another laboratory using the yeast two-hybrid approach (Deckert, M., Elly, C., Altman, A., and Liu, Y . C . (1998) J . Biol . Chem . 273, 8867-8874) . A PTB domain-inactivating point mutation (G306E), corresponding to a loss-of-function mutation in the Caenorhabditis elegans Cbl homologue SLI-1, severely compromised Cbl-N/Syk binding in vitro and Cbl/Syk association in transfected COS-7 cells . Using heterologous expression in COS-7 cells, we investigated the role of Cbl PTB domain binding to Syk Tyr323 in the negative regulation of Syk . Co-expression of Cbl with Syk in COS-7 cells led to a dose-dependent decrease in the autophosphorylated pool of Syk and in phosphorylation of an in vivo substrate, CD8-zeta . Unexpectedly, these effects were largely due to the loss of Syk protein . Both the decrease in Syk and CD8-zeta phosphorylation and reduction in Syk protein levels were blocked by either G306E mutation in Cbl or by Y323F mutation in Syk . These results demonstrate a critical role for the Cbl PTB domain in the recruitment of Cbl to Syk and in Cbl-mediated negative regulation of Syk.

J Invest Dermatol, 1998 Dec, 111(6), 1015 - 22
Interaction of BP180 (type XVII collagen) and alpha6 integrin is necessary for stabilization of hemidesmosome structure; Hopkinson SB et al.; The hemidesmosome is a multimolecular complex that integrates the extracellular matrix with the keratin cytoskeleton and that stabilizes epithelial attachment to connective tissue . A 180 kDa protein (BP180, type XVII collagen), first identified by its reactivity with autoantibodies in the serum of patients with a blistering skin disease called bullous pemphigoid (BP), is a transmembrane component of the hemidesmosome with a collagen-like extracellular domain . Here, using recombinantly expressed molecules and the yeast two-hybrid assay, we have identified alpha6 integrin as a BP180-binding partner . The association between specific domains of the BP180 and alpha6 integrin molecules is inhibited by a 14 mer peptide, whose sequence is identical to amino acid residues 506-519 in the noncollagenous region of the ectodomain of the BP180 molecule, as well as by antibodies raised against this peptide . The 14 mer peptide sequence is part of an epitope recognized by autoantibodies that are pathogenic in BP . In vivo, when 804G cells are plated into medium containing the same peptide, they fail to assemble hemidesmosomes . Furthermore, although BP180 and certain cytoplasmic components of the hemidesmosome colocalize in the peptide-treated cells, they are aberrantly distributed and fail to show extensive association with (alpha6beta4 integrin . Taken together, our results indicate that BP180 is a novel transmembrane ligand of the alpha6beta4 integrin heterodimer . In addition, our data provide support for the possibility that BP180 and alpha6 integrin interaction is not only mediated by the BP epitope but is necessary for hemidesmosome formation.

J Biol Chem, 1998 Dec 25, 273(52), 35039 - 47
Identification of an interaction between the m-band protein skelemin and beta-integrin subunits . Colocalization of a skelemin-like protein with beta1- and beta3-integrins in non-muscle cells; Reddy KB et al.; Signaling across integrins is regulated by interaction of these receptors with cytoskeletal proteins and signaling molecules . To identify molecules interacting with the cytoplasmic domain of the beta3-integrin subunit (glycoprotein IIIa), a placental cDNA library was screened in the yeast two-hybrid system . Two identical clones coding for a 96-amino acid sequence were identified . This sequence was 100% identical to a sequence in skelemin, a protein identified previously in skeletal muscle . Skelemin is a member of a superfamily of cytoskeletal proteins that contain fibronectin-type III-like motifs and immunoglobulin C2-like motifs and that regulate the organization of myosin filaments in muscle . The amino acid residues in the isolated clones encompassed C2 motifs 4 and 5 of skelemin . A recombinant skelemin protein consisting of C2 motifs 3-7 interacted with beta1- and beta3-integrin cytoplasmic domains expressed as glutathione S-transferase (GST) fusion proteins, but not with GST-beta2-integrin cytoplasmic tail or GST alone . The skelemin-binding region was in the membrane proximal cytoplasmic domains of the integrins . Full-length skelemin interacted with integrin in intact cells as demonstrated by the colocalization of hemagglutinin-tagged skelemin in Chinese hamster ovary (CHO) cells containing alphaIIbbeta3-integrin and by the finding that microinjection of C2 motif 4 of skelemin into C2C12 mouse myoblast cells caused spread cells to round up . A skelemin-like protein was detected in CHO cells, endothelial cells, and platelets, and this protein colocalized with beta1- and beta3-integrins in CHO cells . This study suggests the presence of a skelemin-like protein in non-muscle cells and provides evidence that it may be involved in linking integrins to the cytoskeleton.

J Biol Chem, 1998 Dec 25, 273(52), 34737 - 44
Raf-1 is involved in the regulation of the interaction between guanine nucleotide exchange factor and Ha-ras . Evidences for a function of Raf-1 and phosphatidylinositol 3-kinase upstream to Ras; Giglione C et al.; The observation that activated c-Ha-Ras p21 interacts with diverse protein ligands suggests the existence of mechanisms that regulate multiple interactions with Ras . This work studies the influence of the Ras effector c-Raf-1 on the action of guanine nucleotide exchange factors (GEFs) on Ha-Ras in vitro . Purified GEFs (the catalytic domain of yeast Sdc25p and the full-length and catalytic domain of mouse CDC25Mm) and the Ras binding domains (RBDs) of Raf-1 (Raf (1-149) and Raf (51-131)) were used . Our results show that not only the intrinsic GTP/GTP exchange on Ha-Ras but also the GEF-stimulated exchange is inhibited in a concentration-dependent manner by the RBDs of Raf . Conversely, the scintillation proximity assay, which monitors the effect of GEF on the Ras.Raf complex, showed that the binding of Raf and GEF to Ha-Ras.GTP is mutually exclusive . The various GEFs used yielded comparable results . It is noteworthy that under more physiological conditions mimicking the cellular GDP/GTP ratio, Raf enhances the GEF-stimulated GDP/GTP exchange on Ha-Ras, in agreement with the sequestration of Ras.GTP by Raf . Consistent with our results, the GEF-stimulated exchange of Ha-Ras.GTP was also inhibited by another effector of Ras, the RBD (amino acid residues 133-314) of phosphatidylinositol 3-kinase p110alpha . Our data show that Raf-1 and phosphatidylinositol 3-kinase can influence the upstream activation of Ha-Ras . The interference between Ras effectors and GEF could be a regulatory mechanism to promote the activity of Ha-Ras in the cell.

J Biol Chem, 1998 Dec 25, 273(52), 34671 - 4
Identification of STRAP, a novel WD domain protein in transforming growth factor-beta signaling; Datta PK et al.; Transforming growth factor-beta1 (TGF-beta1) is the prototype of a large family of proteins that regulate a variety of biological processes . The pleiotropic responses to TGF-beta are mediated via ligand-induced heteromeric complex formation by type I (TbetaR-I) and type II (TbetaR-II) serine-threonine kinase receptors . Several studies have shown that TbetaR-II acts as a primary receptor, binding TGF-beta and phosphorylating TbetaR-I, whose kinase activity then propagates the signals . Therefore, intracellular proteins that interact with type I receptors are likely to play important roles in TGF-beta signaling . We have identified a novel WD domain-containing protein, designated STRAP (serine-threonine kinase receptor-associated protein), which interacts with TbetaR-I in a yeast two-hybrid system . STRAP associates with both functional TbetaR-I and TbetaR-II in vivo . Overexpression of STRAP leads to inhibition of TGF-beta-mediated transcriptional activation . It also shows synergistic inhibition of TGF-beta signaling in concert with Smad7, but not with Smad6, as measured by TGF-beta-dependent transcriptional reporters . The existence of the STRAP gene from yeast to mammals indicates an evolutionarily conserved function in eukaryotes . The data suggest a potential role for STRAP in TGF-beta signal transduction.

J Med Microbiol, 1998 Dec, 47(12), 1047 - 57
The relative pathogenicity of Candida krusei and C . albicans in the rat oral mucosa; Samaranayake YH et al.; The relative pathogenicity of Candida krusei and C . albicans was investigated by assessing their colonisation and infectivity of the Sprague-Dawley rat oral mucosa . During an initial 21-week period with intermittent oral inoculation, both Candida spp . demonstrated variable surface colonisation of the oral mucosa . After 3 days of oral inoculation, both yeast species were recovered from all animals . During the 21-week period the mean oral load of C . albicans in the control group of rats varied between (26-274) x 10(1) cfu/ml whereas the two test groups of rats carrying C . krusei CK9 and CK13 had a mean load of (2-10) x 10(1) cfu/ml . Although oral colonisation by C . albicans was greater than that of C . krusei, neither species induced candidal infection during this period . Subsequent immunosuppression of the rats by intramuscular cyclophosphamide (40 mg/kg body weight) initiated C . albicans infection of the dorsal tongue (around the conical papillae area) after 4 weeks, in all of three animals while similar lesions due to C . krusei were seen--albeit after 5-7 weeks--in three of eight animals . Characteristic histological changes of mucosal candidosis were discernible on the lingual mucosa of rats infected with both Candida spp . including parakeratosis, absence of a stratum granulosum, thickened rete ridges, micro-abscess formation and polymorph infiltration of the lingual epithelium . Although both species produced fungal hyphae that penetrated the epithelium up to the prickle cell layer, C . albicans hyphae tended to be relatively more profuse . Taken together these results substantiate, for the first time in an animal model, the clinical evidence that C . krusei, once considered an innocuous commensal, is capable of transforming into an invasive pathogen under conditions of immunosuppression.

Neuron, 1998 Nov, 21(5), 1115 - 22
Molecular analysis of mammalian timeless; Zylka MJ et al.; We cloned the mouse cDNA of a mammalian homolog of the Drosophila timeless (tim) gene and designated it mTim . The mTim protein shows five homologous regions with Drosophila TIM . mTim is weakly expressed in the suprachiasmatic nuclei (SCN) but exhibits robust expression in the hypophyseal pars tuberalis (PT) . mTim RNA levels do not oscillate in the SCN nor are they acutely altered by light exposure during subjective night . mTim RNA is expressed at low levels in several peripheral tissues, including eyes, and is heavily expressed in spleen and testis . Yeast two-hybrid assays revealed an array of interactions between the various mPER proteins but no mPER-mTIM interactions . The data suggest that PER-PER interactions have replaced the function of PER-TIM dimers in the molecular workings of the mammalian circadian clock.

Mol Pharmacol, 1998 Dec, 54(6), 954 - 61
An evolutionarily conserved cysteine protease, human bleomycin hydrolase, binds to the human homologue of ubiquitin-conjugating enzyme 9; Koldamova RP et al.; Bleomycin hydrolase (BH) is a highly conserved cysteine proteinase that deamidates and inactivates the anticancer drug bleomycin . Yeast BH self-assembles to form a homohexameric structure, which resembles a 20 S proteasome and may interact with other proteins . Therefore, we searched for potential human BH (hBH) partners using the yeast two-hybrid system with a HeLa cDNA library and identified the full-length human homologue of yeast ubiquitin-conjugating enzyme 9 (UBC9) . Cotransformation assays using hBH deletion mutants revealed that the carboxyl terminus of hBH (amino acids 356-455), which contains two of the three essential catalytic amino acids, was not critical for protein binding in the yeast two-hybrid environment . In vitro translated human UBC9 was precipitated by glutathione S-transferase-hBH fusion protein but not by glutathione S-transferase . Efficient in vitro binding occurred in the absence of the first 24 amino acids of UBC9 and the catalytic Cys93 of UBC9 . We confirmed that hBH and UBC9 interacted in vivo by affinity copurification of proteins overexpressed in mammalian cells . Using immunocytochemical analysis, hBH was colocalized with UBC9 . Coexpression of hBH and UBC9 in mammalian cells did not markedly alter the bleomycin-hydrolyzing activity of hBH or apparent small ubiquitin-related modifier 1 addition . This is the first reported heteromeric interaction with hBH, and it suggests a role for hBH in intracellular protein processing and degradation.

Nat Biotechnol, 1998 Dec, 16(13), 1338 - 42
Selection system for genes encoding nuclear-targeted proteins; Ueki N et al.; Nuclear proteins have essential roles in cell proliferation and differentiation . We have developed a yeast selection system-the nuclear transportation trap (NTT)-to identify genes encoding nuclear transport signals . Both unknown and previously identified nuclear localization signals were identified from a human fetal brain cDNA library . The majority (75%) of the unknown proteins examined were exclusively localized to the nucleus in COS-7 cells . We propose that NTT is an efficient method for isolating cDNAs that encode nuclear targeted proteins that can be applied to the retrieval of novel nuclear proteins and to annotate gene function.

J Pept Sci, 1998 Nov, 4(7), 426 - 35
Solution structure of peptides from HIV-1 Vpr protein that cause membrane permeabilization and growth arrest; Yao S et al.; Vpr, one of the accessory gene products encoded by HIV-1, is a 96-residue protein with a number of functions, including targeting of the viral pre-integration complex to the nucleus and inducing growth arrest of dividing cells . We have characterized by 2D NMR the solution conformations of bioactive synthetic peptide fragments of Vpr encompassing a pair of H(F/S)RIG sequence motifs (residues 71-75 and 78-82 of HIV-1 Vpr) that cause cell membrane permeabilization and death in yeast and mammalian cells . Due to limited solubility of the peptides in water, their structures were studied in aqueous trifluoroethanol . Peptide Vpr59-86 (residues 59-86 of Vpr) formed an alpha-helix encompassing residues 60-77, with a kink in the vicinity of residue 62 . The first of the repeated sequence motifs (HFRIG) participated in the well-defined alpha-helical domain whereas the second (HSRIG) lay outside the helical domain and formed a reverse turn followed by a less ordered region . On the other hand, peptides Vpr71-82 and Vpr71-96, in which the sequence motifs were located at the N-terminus, were largely unstructured under similar conditions, as judged by their C(alpha)H chemical shifts . Thus, the HFRIG and HSRIG motifs adopt alpha-helical and turn structures, respectively, when preceded by a helical structure, but are largely unstructured in isolation . The implications of these findings for interpretation of the structure-function relationships of synthetic peptides containing these motifs are discussed.

J Microsc, 1998 Nov, 192 ( Pt 2), 186 - 93
Towards automatic cell identification in DIC microscopy; Young D et al.; A general method is proposed for constructing templates of cells in differential interference contrast (DIC) microscopy . This takes account of the optics which generate DIC images, and is applicable to both transparent and semi-transparent cells of simple and complex shapes . Then, a template matching methodology is presented, which uses fast Fourier transforms to fit templates of a range of sizes and orientations to images . For illustration, this is used to automatically identify and measure individual Candida yeast cells in clusters.

Int J Mol Med, 1998 Apr, 1(4), 665 - 70
Identification of a novel Rac3-interacting protein C1D; Haataja L et al.; Rac3 is a small GTPase of the Rho family, members of which have been implicated in tumorigenesis, cell growth/death and organization of the actin cytoskeleton . Many Rac-interacting proteins or effectors identified to date have a role in cytoskeletal organization . Using the yeast two-hybrid system, we have isolated a novel Rac3-interacting protein from a human placenta cDNA library which has no homology to other previously identified Rac-interacting proteins . Sequence analysis revealed that this protein is C1D, the human homolog of the murine SUN-CoR protein which acts as a corepressor for the thyroid hormone receptor . In yeast cells, C1D binds to constitutively activated but not to GDP-bound Rac3 . When coexpressed with Rac3 in COS cells, C1D complexed with constitutively active Rac3 but not with wild-type Rac3, demonstrating that C1D-Rac3 interactions take place in vivo in mammalian cells and that C1D appears to be an effector of Rac3 . The C1D gene was mapped to human chromosome 2, which frequently shows deletions in human follicular thyroid carcinomas.

Int J Mol Med, 1998 Jan, 1(1), 71 - 6
Characterization of the nuclear localization signal in the DNA helicase involved in Werner's syndrome; Matsumoto T et al.; The nuclear localization signal (NLS) of the DNA helicase involved in Werner's syndrome (WS) was studied . Previously, we noted that the C-terminal region of WS helicase contains the NLS . In this study, we generated in HeLa cells various chimeric proteins consisting of the N-terminal tagged with an enhanced green fluorescent protein and the C-terminal fragments of the WS helicase that were truncated either from N- or C-termini, and we examined the ability of fragments to transfer the fusion proteins to the nucleoplasm by fluorescence microscopy . A small C-proximal region containing 34 amino acid residues (residues 1369-1402) was found to contain full nuclear migration activities . Subsequent amino acid substitution experiments showed that a sequence of three positively charged amino acids (Lys1371-Arg1372-Arg1373) in this region are particularly important . Similar sequence has previously been defined as the nuclear localization signal of SV-40 large T antigen that also acts as a viral DNA helicase . Conservation of this motif was found in the C-terminal regions of the other RecQ type DNA helicases, including murine WS helicase, yeast sgs1 and rqh+1 and human Bloom syndrome DNA helicases.

J Cell Biol, 1998 Dec 14, 143(6), 1635 - 46
AIR-2: An Aurora/Ipl1-related protein kinase associated with chromosomes and midbody microtubules is required for polar body extrusion and cytokinesis in Caenorhabditis elegans embryos; Schumacher JM et al.; An emerging family of kinases related to the Drosophila Aurora and budding yeast Ipl1 proteins has been implicated in chromosome segregation and mitotic spindle formation in a number of organisms . Unlike other Aurora/Ipl1-related kinases, the Caenorhabditis elegans orthologue, AIR-2, is associated with meiotic and mitotic chromosomes . AIR-2 is initially localized to the chromosomes of the most mature prophase I-arrested oocyte residing next to the spermatheca . This localization is dependent on the presence of sperm in the spermatheca . After fertilization, AIR-2 remains associated with chromosomes during each meiotic division . However, during both meiotic anaphases, AIR-2 is present between the separating chromosomes . AIR-2 also remains associated with both extruded polar bodies . In the embryo, AIR-2 is found on metaphase chromosomes, moves to midbody microtubules at anaphase, and then persists at the cytokinesis remnant . Disruption of AIR-2 expression by RNA- mediated interference produces entire broods of one-cell embryos that have executed multiple cell cycles in the complete absence of cytokinesis . The embryos accumulate large amounts of DNA and microtubule asters . Polar bodies are not extruded, but remain in the embryo where they continue to replicate . The cytokinesis defect appears to be late in the cell cycle because transient cleavage furrows initiate at the proper location, but regress before the division is complete . Additionally, staining with a marker of midbody microtubules revealed that at least some of the components of the midbody are not well localized in the absence of AIR-2 activity . Our results suggest that during each meiotic and mitotic division, AIR-2 may coordinate the congression of metaphase chromosomes with the subsequent events of polar body extrusion and cytokinesis.

FEBS Lett, 1998 Nov 13, 439(1-2), 173 - 9
The Arabidopsis homologue of an eIF3 complex subunit associates with the COP9 complex; Karniol B et al.; The Arabidopsis COP9 complex is a multi-subunit repressor of photomorphogenesis which is conserved among multicellular organisms . Approximately 12 proteins copurify with the COP9 complex . Seven of these proteins are orthologues of subunits of the recently published mammalian COP9 complex . Four of the proteins show amino acid similarity to various subunits of the COP9 complex, eIF3 complex and 19S cap of the proteasome . We have studied one of these proteins in order to determine if it is a component of the COP9 complex . Arabidopsis p105 is highly similar to the p110 subunit of the human elF3 . The p105 gene is induced during photomorphogenesis, and RNA and protein analysis reveal different tissue accumulation patterns . p105 is found in a large protein complex . p105 interacts in yeast with both COP9 and FUS6, two known components of the COP9 complex . Our results indicate that p105 is not a component of the COP9 core complex, though it may interact with components of the complex.

J Virol, 1999 Jan, 73(1), 72 - 80
A novel heterogeneous nuclear ribonucleoprotein-like protein interacts with NS1 of the minute virus of mice; Harris CE et al.; NS1, the major nonstructural parvovirus protein of the minute virus of mice, is a multifunctional protein responsible for several aspects of viral replication . NS1 transactivates the P38 promoter (used to express the structural proteins), as well as its own strong promoter, P4 . To study the mechanism of activation and to map regions of NS1 responsible for transactivation, NS1 and various deletions of NS1 were cloned in frame with the GAL4DB and cotransfected into COS-7 and LA9 cells with a synthetic GAL4-responsive reporter plasmid . These studies showed NS1 can directly activate transcription through its 129 carboxyl-terminal amino acid residues . Any deletion from this region of the C terminus, even as few as 8 amino acids, completely abolishes transactivation . A yeast two-hybrid system used to identify protein-protein interactions demonstrated that NS1 is able to dimerize when expressed in yeast cells . However, only an almost complete NS11-638 bait was able to interact with the full-length NS1 . A two-hybrid screen identified a HeLa cell cDNA clone (NS1-associated protein 1 {NSAP1}) that interacts with NS11-276 and NS11-638 . An additional sequence was predicted from human EST (expressed sequence tag) data, and the cDNA was estimated to be at least 2,221 bp long, potentially encoding a 562-amino-acid protein product . A polyclonal antibody raised to a synthetic peptide within NSAP1 recognizes an approximately 65-kDa cellular protein . This NSAP1 cDNA has not previously been characterized, but the predicted protein sequence is 80% identical to the recently identified heterogeneous nuclear ribonucleoprotein (hnRNP) R (W . Hassfeld et al., Nucleic Acids Res . 26:439-445, 1998) . NSAP1 contains four ribonucleoprotein domains, as well as a highly repetitive C-terminal region . A closely related mouse cDNA (deduced from murine EST data) encodes a protein with only a single amino acid residue change from the human protein . NSAP1 is predicted to be a 65-kDa polynucleotide binding protein, and it likely functions in the regulation of splicing and/or transport of mRNAs from the nucleus.

Anim Behav, 1998 May, 55(5), 1271 - 9
The effect of complete versus incomplete information on odour discrimination in a parasitic wasp; Vet LEM et al.; We studied the function of learning in the parasitoid Leptopilina heterotoma by looking at discrimination of odour stimuli used in foraging for a host . To optimize the rate of encounters with hosts, these parasitoids are expected to assess the extent to which variation in host-substrate odours is reliably associated with variation in the presence of hosts, that is, substrate profitability . Where the association is reliable, parasitoids should attend to variation in odours and discriminate between them; where it is not, they should ignore it . We hypothesized that foraging decisions are based on the completeness of information the animal has about differences in substrate profitabilities . Our laboratory studies showed that discrimination and non-discrimination of odour stimuli are dynamic behavioural decisions that can be related to the degree of substrate variation and to an animal's informational state . In wind-tunnel studies, females learned to discriminate between odours from substrates that were qualitatively different, for example, between odours from apple and pear substrates or between yeast substrates with different C6 compounds added . They did not discriminate when differences were small (e.g . between odours from two apple varieties or between yeast patches with different concentrations of ethyl acetate), unless unrewarding experiences provided evidence of the absence of hosts in one of the substrates . Hence, we suggest that non-discrimination between odour stimuli in L . heterotoma is not a lack of ability to discriminate but a functional decision by the parasitoid .

Zhonghua Yi Xue Yi Chuan Xue Za Zhi, 1998 Dec 10, 15(6), 327 - 32
{Molecular cloning and characterization of novel protein kinase gene DYRK3}; Xia J et al.; OBJECTIVE: To isolate full length cDNA of a novel protein kinase and to deduce the protein kinase's classification position and functions . METHODS: cDNA libraries gDNA library was screened with a partial cDNA clone which is homologous to human protein kinase DYRK2 as probe . FISH mapping was performed . RESULTS: Two full length cDNAs of a novel protein kinase from human muscle cDNA library and human testis cDNA library were isolated . The full length cDNA from muscle has an open reading frame which is predicted to encode a protein of 588 amino acid residues and the cDNA from testis to encode a protein of 568 amino acid residues . CONCLUSION: Because the sequence from the 27th codon to the 3' end of the cDNA from muscle is identical to that from the 7th codon to the 3' end of the cDNA from testis, they should be different transcripts of the same gene . As the gene is highly homologous to human protein kinase DYRK2, the present authors termed the gene DYRK3 . DYRK3 is homologous to many serine/threonine protein kinases such as yeast Yak1, human Clk1, human Mnb, drosophila melanogaster Mnb and Cdk2 . DYRK3 should belong to the Clk family in CMGC group of serine/threonine protein kinase . DYRK3 has been mapped to chromosome 1q32 by FISH.

Cell, 1998 Nov 25, 95(5), 657 - 67
PIF3, a phytochrome-interacting factor necessary for normal photoinduced signal transduction, is a novel basic helix-loop-helix protein; Ni M et al.; The mechanism by which the phytochrome (phy) photoreceptor family transduces informational light signals to photoresponsive genes is unknown . Using a yeast two-hybrid screen, we have identified a phytochrome-interacting factor, PIF3, a basic helix-loop-helix protein containing a PAS domain . PIF3 binds to wild-type C-terminal domains of both phyA and phyB, but less strongly to signaling-defective, missense mutant-containing domains . Expression of sense or antisense PIF3 sequences in transgenic Arabidopsis perturbs photoresponsiveness in a manner indicating that PIF3 functions in both phyA and phyB signaling pathways in vivo . PIF3 localized to the nucleus in transient transfection experiments, indicating a potential role in controlling gene expression . Together, the data suggest that phytochrome signaling to photoregulated genes includes a direct pathway involving physical interaction between the photoreceptor and a transcriptional regulator.

J Am Acad Dermatol, 1998 Dec, 39(6), 944 - 50
Ketoconazole 2% shampoo in the treatment of tinea versicolor: a multicenter, randomized, double-blind, placebo-controlled trial; Lange DS et al.; BACKGROUND: Tinea versicolor is a common superficial fungal infection caused by a lipophilic yeast . This chronically recurring opportunistic infection is especially prevalent in tropical and semitropical regions . The topical short-term application of ketoconazole 2% shampoo may provide effective and safe therapy for tinea versicolor . OBJECTIVE: The purpose of this study was to evaluate the efficacy and safety of a single application (1 day) versus three daily applications (3 days) of ketoconazole 2% shampoo versus placebo shampoo in the treatment of mycologically confirmed tinea versicolor . METHODS: Three hundred twelve patients were included in the primary analyses for this 31-day study . Global evaluation scores were measured on days 10 and 31 with a 5-point scale (1 = healed to 5 = worsening), and a cellophane tape test was done at baseline and days 3, 10, and 31 . Efficacy was assessed by clinical response, defined as both a global evaluation score of 1 (healed) and a negative cellophane tape test on day 31 . Signs and symptoms of tinea versicolor (scaling, itching, erythema, hypopigmentation, hyperpigmentation) also were evaluated at baseline, day 10, and day 31 with a 4-point scale (0 = absent to 3 = severe) . RESULTS: Both regimens of ketoconazole shampoo were significantly (P < .001) more effective than placebo for rate of clinical response, global evaluation scores, and mycologic outcomes (cellophane tape test) . The clinical response rates at day 31 were 73%, 69%, and 5% for the 3-day ketoconazole, 1-day ketoconazole, and placebo groups, respectively . The difference in the efficacy of the two ketoconazole treatment regimens was not statistically significant . There were no significant differences between any of the treatment groups in the number of patients who experienced adverse events . No serious adverse events occurred and no patient withdrew from the trial prematurely because of an adverse event . CONCLUSION: Ketoconazole 2% shampoo, used as a single application or daily for 3 days, is safe and highly effective in the treatment of tinea versicolor.

Clin Genet, 1998 Nov, 54(5), 406 - 12
Isolation of a 370 kb YAC fragment spanning a translocation breakpoint at 3p14.1 associated with holoprosencephaly; Petek E et al.; Holoprosencephaly (HPE) is a common developmental defect involving the brain and face . HPE is extremely heterogeneous, some cases being associated with structural anomalies of the short arm of chromosome 3 . For a detailed characterization of a t(3;19)(p14.1;p13.1) breakpoint associated with HPE, we performed fluorescence in situ hybridization (FISH) analysis using yeast artificial chromosomes (YACs) mapped to the short arm of chromosome 3 from the Le Centre d'Etude du Polymorphisme Humain (CEPH) library . Three YACs mapped proximal, and one was located distal to the described breakpoint on chromosome 3 . One of the chromosome 3 'Mega-YACs' spanned the translocation breakpoint . From this chimeric YAC we generated a site specific probe of about 370 kb by digestion of the YAC-DNA, which will be assessed for gene alterations that could underlie HPE in this patient.

J Exp Med, 1998 Dec 7, 188(11), 2151 - 62
The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus; Matsuda F et al.; The complete nucleotide sequence of the 957-kb DNA of the human immunoglobulin heavy chain variable (VH) region locus was determined and 43 novel VH segments were identified . The region contains 123 VH segments classifiable into seven different families, of which 79 are pseudogenes . Of the 44 VH segments with an open reading frame, 39 are expressed as heavy chain proteins and 1 as mRNA, while the remaining 4 are not found in immunoglobulin cDNAs . Combinatorial diversity of VH region was calculated to be approximately 6,000 . Conservation of the promoter and recombination signal sequences was observed to be higher in functional VH segments than in pseudogenes . Phylogenetic analysis of 114 VH segments clearly showed clustering of the VH segments of each family . However, an independent branch in the tree contained a single VH, V4-44.1P, sharing similar levels of homology to human VH families and to those of other vertebrates . Comparison between different copies of homologous units that appear repeatedly across the locus clearly demonstrates that dynamic DNA reorganization of the locus took place at least eight times between 133 and 10 million years ago . One nonimmunoglobulin gene of unknown function was identified in the intergenic region.

Biochem J, 1998 Dec 15, 336 ( Pt 3), 711 - 7
Differential modulation of transcriptional activity of oestrogen receptors by direct protein-protein interactions with retinoid receptors; Song MR et al.; Control of oestradiol-responsive gene regulation by oestrogen receptors (ERs) may involve complex cross-talk with retinoic acid receptors (RARs) and retinoid X receptors (RXRs) . Recently, we have shown that ERalpha directly interacts with RARalpha and RXRalpha through their ligand binding domains (LBDs) . In the present work, we extend these results by showing that ERbeta binds similarly to RARalpha and RXRalpha but not to the glucocorticoid receptor, as demonstrated by the yeast two-hybrid tests and glutathione S-transferase pull-down assays . These direct interactions were also demonstrated in gel-shift assays, in which the oestrogen response element (ERE) binding by ERalpha was enhanced by the RXRalpha LBD but was abolished by the RARalpha LBD . In addition, we showed that RARalpha and RXRalpha bound the ERE as efficiently as ERalpha, suggesting that competition for DNA binding may affect the transactivation function of the ER . In transient transfection experiments, co-expression of RARalpha or RXRalpha, along with ERalpha or ERbeta, revealed differential modulation of the ERE-dependent transactivation, which was distinct from the results when each receptor alone was co-transfected . Importantly, when the LBD of RARalpha was co-expressed with ERalpha, transactivation of ERalpha on the ERE was repressed as efficiently as when wild-type RARalpha was co-expressed . Furthermore, liganded RARalpha or unliganded RXRalpha enhanced the ERalpha transactivation, suggesting the formation of transcriptionally active heterodimer complexes between the ER and retinoid receptors . Taken together, these results suggest that direct protein-protein interactions may play major roles in the determination of the biological consequences of cross-talk between ERs and RARalpha or RXRalpha.

Am J Hum Genet, 1998 Dec, 63(6), 1622 - 30
Mutation in PEX16 is causal in the peroxisome-deficient Zellweger syndrome of complementation group D; Honsho M et al.; Peroxisome-biogenesis disorders (PBDs), including Zellweger syndrome (ZS), are autosomal recessive diseases caused by a deficiency in peroxisome assembly as well as by a malfunction of peroxisomes, among which>10 genotypes have been identified . We have isolated a human PEX16 cDNA (HsPEX16) by performing an expressed-sequence-tag homology search on a human DNA database, by using yeast PEX16 from Yarrowia lipolytica and then screening the human liver cDNA library . This cDNA encodes a peroxisomal protein (a peroxin Pex16p) made up of 336 amino acids . Among 13 peroxisome-deficiency complementation groups (CGs), HsPEX16 expression morphologically and biochemically restored peroxisome biogenesis only in fibroblasts from a CG-D patient with ZS in Japan (the same group as CG-IX in the United States) . Pex16p was localized to peroxisomes through expression study of epitope-tagged Pex16p . One patient (PBDD-01) possessed a homozygous, inactivating nonsense mutation, C-->T at position 526 in a codon (CGA) for 176Arg, that resulted in a termination codon (TGA) . This implies that the C-terminal half is required for the biological function of Pex16p . PBDD-01-derived PEX16 cDNA was defective in peroxisome-restoring activity when expressed in the patient's fibroblasts . These results demonstrate that mutation in PEX16 is the genetic cause of CG-D PBDs.

Am J Hum Genet, 1998 Dec, 63(6), 1609 - 21
Mutations of SURF-1 in Leigh disease associated with cytochrome c oxidase deficiency; Tiranti V et al.; Leigh disease associated with cytochrome c oxidase deficiency (LD{COX-}) is one of the most common disorders of the mitochondrial respiratory chain, in infancy and childhood . No mutations in any of the genes encoding the COX-protein subunits have been identified in LD(COX-) patients . Using complementation assays based on the fusion of LD(COX-) cell lines with several rodent/human rho0 hybrids, we demonstrated that the COX phenotype was rescued by the presence of a normal human chromosome 9 . Linkage analysis restricted the disease locus to the subtelomeric region of chromosome 9q, within the 7-cM interval between markers D9S1847 and D9S1826 . Candidate genes within this region include SURF-1, the yeast homologue (SHY-1) of which encodes a mitochondrial protein necessary for the maintenance of COX activity and respiration . Sequence analysis of SURF-1 revealed mutations in numerous DNA samples from LD(COX-) patients, indicating that this gene is responsible for the major complementation group in this important mitochondrial disorder.

Immunol Lett, 1998 Oct, 63(3), 153 - 8
Tumor specific boosting of IL-2 induced NK activation by paraformaldehyde fixed tumor cells; Dhillon S et al.; NK activation in C57B1/6 mouse spleen cells was carried out with IL-2 in the presence or absence of paraformaldehyde fixed YAC tumor cells . Generation of anti-YAC cytolytic activity was markedly higher when activation was carried out in the presence of fixed tumor cells . In addition the cytotoxic effector cells generated were resistant to anti-Thy-1 + C treatment, indicating that the effector cells were not T lymphocytes . IL-2 activation of NK cells was compared When fixed YAC or EL4 tumor cells were added during the IL-2 activation phase, it was found that the addition of either of these tumor cells significantly boosted the levels of cytotoxic activity generated against both targets . Significantly higher anti-YAC cytotoxic activity was however generated when fixed YAC cells instead of EL4 cells were present during the activation phase . Similarly, significantly greater cytolytic activity was generated against EL4 cells, when fixed EL4 rather than YAC cells were present during the activation phase . Addition of paraformaldehyde fixed syngeneic or allogenic spleen cells instead of tumor cells, did not boost NK activation in response to IL-2, indicating that the boosting of NK activation did not result from exposure to alloantigens during the activation phase . These results indicate that an exposure to tumor cells during IL-2 activation phase, may boost the activation of NK cells in a tumor specific manner.

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