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 subst