J Biol Chem, 2002 May 31, 277(22), 19408 - 17 Epub 2002 Mar 21. Isolation and characterization of a human STAT1 gene regulatory element . Inducibility by interferon (IFN) types I and II and role of IFN regulatory factor-1; Wong LH et al.; The transcription factor STAT1 plays a pivotal role in signal transduction of type I and II interferons (IFNs) . STAT1 activation leads to changes in expression of key regulatory genes encoding caspases and cell cycle inhibitors . Deficient STAT1 expression in human cancer cells and virally mediated inhibition of STAT1 function have been associated with cellular resistance to IFNs and mycobacterial infection in humans . Thus, given the relative importance of STAT1, we isolated and characterized a human STAT1 intronic enhancer region displaying IFN-regulated activity . Functional analyses by transient expression identified a repressor region and type I and II IFN-inducible elements within the STAT1 enhancer sequence . A candidate IRF-E/GAS/IRF-E (IGI) sequence containing GAAANN nucleotide repeats was shown by gel shift assay to bind to IFN regulatory factor-1 (IRF-1), but not to IFN-stimulated gene factor-3 (ISGF-3) or STAT1-3 . An additional larger IGI-binding complex containing IRF-1 was identified . Mutation of the GAAANN repeats within the IGI DNA element eliminated IRF-1 binding and the IFN-regulated activity of the STAT1 intronic enhancer region . Transfection of the IFN-resistant MM96 cell line to express increased levels of IRF-1 protein also elevated STAT1, STAT2, and p48/IRF-9 expression and enhanced cellular responsiveness to IFN-beta . Reciprocating regulation between IRF-1 and STAT1 genes and encoded proteins indicates that an intracellular amplifier circuit exists controlling cellular responsiveness to the IFNs. Biochim Biophys Acta, 2002 Feb 13, 1589(1), 71 - 6 Identification of interaction between MEK2 and A-Raf-1; Yin XL et al.; Mitogen-activated protein (MAP) kinases are activated by dual-specificity kinases, termed MEKs . Using MEK2 as bait in yeast two-hybrid screening, besides c-Raf and KSR, A-Raf was identified as a novel partner that interacts with MEK2 . This interaction was confirmed by in vitro binding assay . Further investigation indicates that regions critical for this interaction were located between residues 255 and 606 that represent the kinase domain of A-Raf. Cell, 2002 Feb 22, 108(4), 523 - 31 A conserved mRNA export machinery coupled to pre-mRNA splicing; Reed R et al.; Recent advances have led to a new understanding of how mRNAs are exported from the nucleus to the cytoplasm . This process requires a heterodimeric mRNA export receptor that is part of an elaborate machinery conserved from yeast to humans . Export of mRNAs is coupled to upstream steps in gene expression, such as pre-mRNA splicing, and to downstream events, including nonsense-mediated decay. Cell, 2002 Feb 22, 108(4), 453 - 63 The RNA polymerase II machinery: structure illuminates function; Woychik NA et al.; Essential components of the eukaryotic transcription apparatus include RNA polymerase II, a common set of initiation factors, and a Mediator complex that transmits regulatory information to the enzyme . Insights into mechanisms of transcription have been gained by three-dimensional structures for many of these factors and their complexes, especially for yeast RNA polymerase II at atomic resolution. J Gen Virol, 2002 Apr, 83(Pt 4), 783 - 93 Multimerization reactions of coxsackievirus proteins 2B, 2C and 2BC: a mammalian two-hybrid analysis; de Jong AS et al.; Recently, homomultimerization and heteromultimerization reactions of the poliovirus P2 region proteins were investigated using a yeast two-hybrid approach (Cuconati et al., Journal of Virology 72, 1297-1307, 1998) . In this study, we investigated multimerization reactions of the 2B, 2C and 2BC proteins of the closely related coxsackie B3 virus (CBV3) using a mammalian two-hybrid system . This system allows the characterization of protein:protein interactions within a cellular environment that more closely mimics the native protein environment . Homomultimerization reactions were observed with the 2BC protein and, albeit weakly, with the 2B protein, but not with the 2C protein . To identify the determinants involved in the 2BC and 2B homomultimerization reactions, several mutants containing deletions or point mutations in the 2B region were tested . Disruption of the hydrophobic character of either the cationic amphipathic alpha-helix or the second hydrophobic domain of the 2B protein disturbed both the 2BC:2BC and the 2B:2B homomultimerization reactions . Disruption of either the cationic or the amphipathic character of the alpha-helix or deletion of the N-terminal 30 amino acids of the 2B protein, however, had no effect on the 2BC and 2B homomultimerization reactions . Heteromultimerization reactions were observed between proteins 2BC and 2B, and also between proteins 2BC and 2C, but not between the 2B and 2C proteins . The 2BC:2B and 2BC:2C heteromultimerization reactions were also mediated by hydrophobic determinants located in the amphipathic alpha-helix and the second hydrophobic domain . The nature of the interactions and their implications for the virus life-cycle are discussed. J Gen Virol, 2002 Apr, 83(Pt 4), 759 - 66 Hantavirus nucleocapsid protein interacts with the Fas-mediated apoptosis enhancer Daxx; Li XD et al.; Hantaviruses cause two severe diseases, haemorrhagic fever with renal syndrome in Eurasia and hantavirus pulmonary syndrome in the Americas . To understand more about the molecular mechanisms that lead to these diseases, the associations of Puumala virus nucleocapsid protein (PUUV-N) with cellular proteins were studied by yeast two-hybrid screening . Daxx, known as an apoptosis enhancer, was identified from a HeLa cDNA library and its interaction with PUUV-N was confirmed by GST pull-down assay, co-immunoprecipitation and co-localization studies . Furthermore, domains of interaction were mapped to the carboxyl-terminal region of 142 amino acids in Daxx and the carboxyl-terminal 57 residues in PUUV-N, respectively . In pepscan assays, the binding sites of Daxx to PUUV-N were mapped further to two lysine-rich regions, of which one overlaps the sequence of the predicted nuclear localization signal of Daxx . These data suggest a direct link between host cell machinery and a hantavirus structural component. Mol Biol Cell, 2002 Mar, 13(3), 1071 - 82 Role of adaptor complex AP-3 in targeting wild-type and mutated CD63 to lysosomes; Rous BA et al.; CD63 is a lysosomal membrane protein that belongs to the tetraspanin family . Its carboxyterminal cytoplasmic tail sequence contains the lysosomal targeting motif GYEVM . Strong, tyrosine-dependent interaction of the wild-type carboxyterminal tail of CD63 with the AP-3 adaptor subunit mu 3 was observed using a yeast two-hybrid system . The strength of interaction of mutated tail sequences with mu 3 correlated with the degree of lysosomal localization of similarly mutated human CD63 molecules in stably transfected normal rat kidney cells . Mutated CD63 containing the cytosolic tail sequence GYEVI, which interacted strongly with mu 3 but not at all with mu 2 in the yeast two-hybrid system, localized to lysosomes in transfected normal rat kidney and NIH-3T3 cells . In contrast, it localized to the cell surface in transfected cells of pearl and mocha mice, which have genetic defects in genes encoding subunits of AP-3, but to lysosomes in functionally rescued mocha cells expressing the delta subunit of AP-3 . Thus, AP-3 is absolutely required for the delivery of this mutated CD63 to lysosomes . Using this AP-3-dependent mutant of CD63, we have shown that AP-3 functions in membrane traffic from the trans-Golgi network to lysosomes via an intracellular route that appears to bypass early endosomes. Mol Biol Cell, 2002 Mar, 13(3), 817 - 29 Identification of a novel light intermediate chain (D2LIC) for mammalian cytoplasmic dynein 2; Grissom PM et al.; The diversity of dynein's functions in mammalian cells is a manifestation of both the existence of multiple dynein heavy chain isoforms and an extensive set of associated protein subunits . In this study, we have identified and characterized a novel subunit of the mammalian cytoplasmic dynein 2 complex . The sequence similarity between this 33-kDa subunit and the light intermediate chains (LICs) of cytoplasmic dynein 1 suggests that this protein is a dynein 2 LIC (D2LIC) . D2LIC contains a P-loop motif near its NH(2) terminus, and it shares a short region of similarity to the yeast GTPases Spg1p and Tem1p . The D2LIC subunit interacts specifically with DHC2 (or cDhc1b) in both reciprocal immunoprecipitations and sedimentation assays . The expression of D2LIC also mirrors that of DHC2 in a variety of tissues . D2LIC colocalizes with DHC2 at the Golgi apparatus throughout the cell cycle . On brefeldin A-induced Golgi fragmentation, a fraction of D2LIC redistributes to the cytoplasm, leaving behind a subset of D2LIC that is localized around the centrosome . Our results suggest that D2LIC is a bona fide subunit of cytoplasmic dynein 2 that may play a role in maintaining Golgi organization by binding cytoplasmic dynein 2 to its Golgi-associated cargo. Mol Biol Cell, 2002 Mar, 13(3), 782 - 94 Genomic analysis of homotypic vacuole fusion; Seeley ES et al.; Yeast vacuoles undergo fission and homotypic fusion, yielding one to three vacuoles per cell at steady state . Defects in vacuole fusion result in vacuole fragmentation . We have screened 4828 yeast strains, each with a deletion of a nonessential gene, for vacuole morphology defects . Fragmented vacuoles were found in strains deleted for genes encoding known fusion catalysts as well as 19 enzymes of lipid metabolism, 4 SNAREs, 12 GTPases and GTPase effectors, 9 additional known vacuole protein-sorting genes, 16 protein kinases, 2 phosphatases, 11 cytoskeletal proteins, and 28 genes of unknown function . Vacuole fusion and vacuole protein sorting are catalyzed by distinct, but overlapping, sets of proteins . Novel pathways of vacuole priming and docking emerged from this deletion screen . These include ergosterol biosynthesis, phosphatidylinositol (4,5)-bisphosphate turnover, and signaling from Rho GTPases to actin remodeling . These pathways are supported by the sensitivity of the late stages of vacuole fusion to inhibitors of phospholipase C, calcium channels, and actin remodeling . Using databases of yeast protein interactions, we found that many nonessential genes identified in our deletion screen interact with essential genes that are directly involved in vacuole fusion . Our screen reveals regulatory pathways of vacuole docking and provides a genomic basis for studies of this reaction. J Virol, 2002 Apr, 76(8), 3865 - 72 Oligomerization and cooperative RNA synthesis activity of hepatitis C virus RNA-dependent RNA polymerase; Wang QM et al.; The NS5B RNA-dependent RNA polymerase encoded by hepatitis C virus (HCV) plays a key role in viral replication . Reported here is evidence that HCV NS5B polymerase acts as a functional oligomer . Oligomerization of HCV NS5B protein was demonstrated by gel filtration, chemical cross-linking, temperature sensitivity, and yeast cell two-hybrid analysis . Mutagenesis studies showed that the C-terminal hydrophobic region of the protein was not essential for its oligomerization . Importantly, HCV NS5B polymerase exhibited cooperative RNA synthesis activity with a dissociation constant, K(d), of approximately 22 nM, suggesting a role for the polymerase-polymerase interaction in the regulation of HCV replicase activity . Further functional evidence includes the inhibition of the wild-type NS5B polymerase activity by a catalytically inactive form of NS5B . Finally, the X-ray crystal structure of HCV NS5B polymerase was solved at 2.9 A . Two extensive interfaces have been identified from the packing of the NS5B molecules in the crystal lattice, suggesting a higher-order structure that is consistent with the biochemical data. Int J Biochem Cell Biol, 2002 May, 34(5), 487 - 504 The novel human HUEL (C4orf1) protein shares homology with the DNA-binding domain of the XPA DNA repair protein and displays nuclear translocation in a cell cycle-dependent manner; Sim del LC et al.; We have previously isolated and characterized a novel human gene HUEL (C4orf1) that is ubiquitously expressed in a wide range of human fetal, adult tissues and cancer cell lines . HUEL maps to region 4p12-p13 within the short arm of chromosome 4 whose deletion is frequently associated with bladder and other carcinomas . Here we present the genomic organization, sizes and boundaries of exons and introns of HUEL . The GC-rich upstream genomic region and 5' untranslated region (UTR) together constitute a CpG island, a hallmark of housekeeping genes . The 3250 bp HUEL cDNA incorporates a 1704 bp ORF that translates into a hydrophilic protein of 568-amino acids (aa), detected as a band of approximately 70 kDa by Western blotting . We have isolated the murine homolog of HUEL which exhibits 89% nucleotide and 94% amino acid identity to its human counterpart . The HUEL protein shares significant homology with the minimal DNA-binding domain (DNA-BD) of the DNA repair protein encoded by the xeroderma pigmentosum group A (XPA) gene . Other notable features within HUEL include the putative nuclear receptor interaction motif, nuclear localization and export signals, zinc finger, leucine zipper and acidic domains . Mimosine-mediated cell cycle synchronization of PLC/PRF/5 liver cancer cells clearly portrayed translocation of HUEL into the nucleus specifically during the S phase of the cell cycle . Yeast two-hybrid experiments revealed interactions of HUEL with two partner proteins (designated HIPC and HIPB) bearing similarity to a mitotically phosphorylated protein and to reverse transcriptase . Co-immunoprecipitation assays validated the interaction between HUEL and HIPC proteins in mammalian cells . HUEL is likely to be an evolutionarily conserved, housekeeping gene that plays a role intimately linked with cellular replication, DNA synthesis and/or transcriptional regulation. J Anim Physiol Anim Nutr (Berl), 2002 Feb, 86(1-2), 36 - 41 A low-selenium diet increases thyroxine and decreases 3,5,3'triiodothyronine in the plasma of kittens; Yu S et al.; The effect of a low-selenium diet on thyroid hormone metabolism was investigated in growing kittens . Twelve specific-pathogen-free kittens with ages ranging from 16 to 18 weeks were divided into two groups of equal number with equal sex distribution in each group . One group was fed a yeast-based low-selenium diet (0.02 mg Se/kg diet) while the other group was fed the same diet supplemented with Na2SeO3 at 0.4 mg Se/kg diet for 8 weeks . Food intake, body weight and body weight gain were not affected by the low-Se diet during the study period . However, kittens given the low-Se diet had significantly reduced plasma selenium concentration and glutathione peroxidase activity . Plasma total thyroxine (T4) increased and total 3,5,3'triiodothyronine (T3) decreased significantly in kittens fed the low-Se diet at the end of the study . These results suggest that type I deiodinase in cats is a selenoprotein- or a selenium-dependent enzyme. Cell Microbiol, 2002 Mar, 4(3), 127 - 37 Linking fungal morphogenesis with virulence; Rooney PJ et al.; Pathogenic fungi have become an increasingly common cause of systemic disease in healthy people and those with impaired immune systems . Although a vast number of fungal species inhabit our planet, just a small number are pathogens, and one feature that links many of them is the ability to differentiate morphologically from mould to yeast, or yeast to mould . Morphological differentiation between yeast and mould forms has commanded attention for its putative impact on the pathogenesis of invasive fungal infections . This review explores the current body of evidence linking fungal morphogenesis and virulence . The topics addressed cover work on phase-locked fungal cells, expression of phase-specific virulence traits and modulation of host responses by fungal morphotypes . The effect of morphological differentiation on fungal interaction with host cells, immune modulation and the net consequence on pathogenesis of disease in animal model systems are considered . The evidence argues strongly that morphological differentiation plays a vital role in the pathogenesis of fungal infection, suggesting that factors associated with this conversion process represent promising therapeutic targets. J Med Chem, 2002 Mar 28, 45(7), 1535 - 42 Synthesis and biological evaluation of cycloalkylidene carboxylic acids as novel effectors of Ras/Raf interaction; Friese A et al.; The protooncogenes Ras and Raf play important roles in signal transduction pathways regulated by mitogen-activated protein kinases . Mutations of Ras that arrest the protein in its active state are frequently implicated in tumor formation . We used Ras and Raf proteins in the yeast two-hybrid system to search for natural or synthesized substances capable of modulating Ras/Raf interaction by specifically binding to one of the interacting partners . We found that cycloalkylidene carboxylic acids enhanced Ras/Raf interaction by acting on the cysteine-rich domain of Raf . Several analogues of the active substance 2-cyclohexylidene propanoic acid were synthesized and the importance of the semicyclic double bond in the stabilization of Ras/Raf interaction was demonstrated . Variation of the size and the substituents of the cyclic system as well as the length of the carboxylic acid resulted in enhanced Ras/Raf interaction. Anal Biochem, 2002 Apr 1, 303(1), 34 - 41 A method for functional mapping of protein-protein binding domain by preferential amplification of the shortest amplicon using PCR; Kawarasaki Y et al.; We have developed a novel method for rapid and empirical mapping of the protein interaction domain using a unique and atypical PCR-based amplification and a conventional yeast two-hybrid system . The modified PCR, designated as PASA-PCR, enables preferential amplification of the shortest amplicon from a complex expression library . PASA-PCR consists of reiterative cycles of denaturation of template DNAs and extremely abbreviated annealing/extension of primers to prevent their complete extension in a single cycle, followed by conventional amplification cycles . In PASA-PCR, the shortest (ranging from 400 to 1000 bp) amplicon is amplified almost exclusively from templates of various amplicon sizes . In addition, the frequency of in vitro recombination can be increased using low cooling rates (<0.5 degrees C/s) between the denaturation and annealing/extension steps, which was helpful in generating precisely trimmed protein-coding regions . Identification of Spc19-binding region of Spc34, which is a component of yeast's spindle pole body, was achieved by a combination of the yeast two-hybrid system and PASA-PCR . (c)2002 Elsevier Science (USA). Spectrochim Acta A Mol Biomol Spectrosc, 2002 Feb, 58(3), 457 - 65 Study on the resonance light scattering spectrum of berberine-cetyltrimethylammonium bromide system and the determination of nucleic acids at nanogram levels; Liu R et al.; The interaction of berberine with nucleic acid in the presence of cetyltrimethylammonium bromide (CTMAB) in aqueous solution has been studied by spectrophotometry and resonance light scattering (RLS) spectroscopy . At pH 7.30, the RLS signals of berberine were greatly enhanced by nucleic acid in the region of 300-600 nm characterized by four peaks at 324.0, 386.5, 416.5 and 465.0 nm . The binding properties were examined by using a Scatchard plot based on the measurement of enhanced RLS data at 416.5 nm . Under optimum conditions, the increase of RLS intensity of this system at 416.5 nm is proportional to the concentration of nucleic acid . The linear range is 7.5 x 10(-9)-7.5 x 10(-5) g ml(-1) for calf thymus DNA, 7.5 x 10(-9)-2.5 x 10(-5) g ml(-1) for herring sperm DNA, and 5.0 x 10(-9)-2.5 x 10(-5) g ml(-1) for yeast RNA . The detection limits (S/N = 3) are 2.1 ng ml(-1) for calf thymus DNA, 6.5 ng ml(-1) for herring sperm DNA and 3.5 ng ml(-1) for yeast RNA, respectively . Three synthetic samples were analyzed satisfactorily. Intensive Care Med, 2002 Mar, 28(3), 379 - 80 Epub 2001 Dec 18. Massive septic thrombus formation on a superior vena cava indwelling catheter following Torulopsis (Candida) glabrata fungemia; Gressianu MT et al.; Fungal endocarditis is an exceedingly rare complication of indwelling central venous catheters in adults . Here we describe what appears to be the first case of a right atrial thrombus superinfected with the yeast Torulopsis (Candida) glabrata and attached to an indwelling superior vena cava catheter that was not used for parenteral nutrition . A large vegetation-like mass adherent to the catheter tip was visualized by transesophageal echocardiography in a patient who presented with signs of septic pulmonary embolism . Following open-heart surgery, the definitive diagnosis was established by histopathologic examination of the surgical specimen. Clin Genet, 2002 Jan, 61(1), 54 - 61 Partial Xp duplication in a girl with dysmorphic features: the change in replication pattern of late-replicating dupX chromosome; Kokalj Vokac N et al.; In this paper we present the case of a girl at the age of 32 months with dysmorphic features, including general muscular hypotonia, developmental delay and mental retardation . The cytogenetic analysis revealed de novo partial duplication of Xp: 46,X,dup(X)(p11.23-->p22.33: :p11.23-->p22.33) . To characterize the duplication, X painting, Kallman (KAL), yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs) covering Xp11.23-->Xp22.33 region were used . Selective inactivation of the abnormal X chromosome using HpaII digestion of the AR gene was evident . After BrdU incorporation the abnormal X was late-replicating in all lymphocytes examined . There was one peculiar exception observed: the break-point region was consistently early replicating . The replicating pattern of this region corresponded to the active X chromosome . Methylation pattern of late replicating X chromosome was studied also using antibodies against 5-methylcytosine . The pattern corresponded to the normally inactive X chromosome, with the exception of the previously observed break-point region which revealed an early replicating pattern with strong fluorescent signal, similar to the pattern of the active X chromosome . The observed phenomenon could lead to the abnormal phenotype of the patient, with some normally inactive genes of the break-point region escaping the inactivation process . The abnormal clinical findings could also be due to tissue-dependent differences in the inactivation pattern. Biochem J, 2002 Apr 1, 363(Pt 1), 157 - 65 Nuclear receptor corepressor-dependent repression of peroxisome-proliferator-activated receptor delta-mediated transactivation; Krogsdam AM et al.; The nuclear receptor corepressor (NCoR) was isolated as a peroxisome-proliferator-activated receptor (PPAR) delta interacting protein using the yeast two-hybrid system . NCoR interacted strongly with the ligand-binding domain of PPAR delta, whereas interactions with the ligand-binding domains of PPAR gamma and PPAR alpha were significantly weaker . PPAR-NCoR interactions were antagonized by ligands in the two-hybrid system, but were ligand-insensitive in in vitro pull-down assays . Interaction between PPAR delta and NCoR was unaffected by coexpression of retinoid X receptor (RXR) alpha . The PPAR delta-RXR alpha heterodimer bound to an acyl-CoA oxidase (ACO)-type peroxisome-proliferator response element recruited a glutathione S-transferase-NCoR fusion protein in a ligand-independent manner . Contrasting with most other nuclear receptors, PPAR delta was found to interact equally well with interaction domains I and II of NCoR . In transient transfection experiments, NCoR and the related silencing mediator for retinoid and thyroid hormone receptor (SMRT) were shown to exert a marked dose-dependent repression of ligand-induced PPAR delta-mediated transactivation; in addition, transactivation induced by the cAMP-elevating agent forskolin was efficiently reduced to basal levels by NCoR as well as SMRT coexpression . Our results suggest that the transactivation potential of liganded PPAR delta can be fine-tuned by interaction with NCoR and SMRT in a manner determined by the expression levels of corepressors and coactivators. Yi Chuan Xue Bao, 2002 Feb, 29(2), 175 - 80 {Screening and detecting of proteins interaction with KyoT}; Li R et al.; To study the function of KyoT2 in vivo, two-hybrid yeast, purification of KyoT2 protein, preparation of antibody and GST-pulldown methods were used in the experiments . 42 clones were obtained after 5 x 10(6) clones were screened by four kinds of nutrition limitation and beta-galactosidase assay, 22 clones were obtained after restriction of positive clones . Finally, 13 genes were obtained by sequence assay . Two of these were RBP-Jk and PIAS1 . After they and KyoT2 changed vectors, negative two-hybrid yeast was finished . The result was positive; Using KyoT2 protein and antibody GST-pulldown of KyoT2 and RBP-Jk, KyoT2 and PIAS1 were done, the result was also positive . Therefore, KyoT2 interacted with RBP-Jk and PIAS1. J Cell Biol, 2002 Mar 18, 156(6), 1003 - 13 Epub 2002 Mar 18. Alpha-glucosidase I is required for cellulose biosynthesis and morphogenesis in Arabidopsis; Gillmor CS et al.; Novel mutations in the RSW1 and KNOPF genes were identified in a large-scale screen for mutations that affect cell expansion in early Arabidopsis embryos . Embryos from both types of mutants were radially swollen with greatly reduced levels of crystalline cellulose, the principal structural component of the cell wall . Because RSW1 was previously shown to encode a catalytic subunit of cellulose synthase, the similar morphology of knf and rsw1-2 embryos suggests that the radially swollen phenotype of knf mutants is largely due to their cellulose deficiency . Map-based cloning of the KNF gene and enzyme assays of knf embryos demonstrated that KNF encodes alpha-glucosidase I, the enzyme that catalyzes the first step in N-linked glycan processing . The strongly reduced cellulose content of knf mutants indicates that N-linked glycans are required for cellulose biosynthesis . Because cellulose synthase catalytic subunits do not appear to be N glycosylated, the N-glycan requirement apparently resides in other component(s) of the cellulose synthase machinery . Remarkably, cellular processes other than extracellular matrix biosynthesis and the formation of protein storage vacuoles appear unaffected in knf embryos . Thus in Arabidopsis cells, like yeast, N-glycan trimming is apparently required for the function of only a small subset of N-glycoproteins. Biochemistry, 2002 Mar 26, 41(12), 4059 - 69 Pentavalent ions dependency is a conserved property of adenosine kinase from diverse sources: identification of a novel motif implicated in phosphate and magnesium ion binding and substrate inhibition; Maj MC et al.; The catalytic activity of adenosine kinase (AK) from mammalian sources has previously been shown to exhibit a marked dependency upon the presence of pentavalent ions (PVI), such as phosphate (PO4), arsenate, or vanadate . We now show that the activity of AK from diverse sources, including plant, yeast, and protist species, is also markedly enhanced in the presence of PVI . In all cases, PO4 or other PVI exerted their effects primarily by decreasing the Km for adenosine and alleviating the inhibition caused by high concentrations of substrates . These results provide evidence that PVI dependency is a conserved property of AK and perhaps of the PfkB family of carbohydrate kinases which includes AK . On the basis of sequence alignments, we have identified a conserved motif NXXE within the PfkB family . The N and E of this motif make close contacts with Mg2+ and PO4 ions in the crystal structures of AK and bacterial ribokinase (another PfkB member which shows PVI dependency), implicating these residues in their binding . Site-directed mutagenesis of these residues in Chinese hamster AK have resulted in active proteins with greatly altered phosphate stimulation and substrate inhibition characteristics . The N239Q mutation leads to the formation of an active protein whose activity was not stimulated by PO4 or inhibited by high concentrations of adenosine or ATP . The activity of the E242D mutant protein was also not significantly altered in the presence of phosphate . Although PO4 had no effect on the KmAdenosine for this mutant, the KmATP, K(i)Adenosine, and K(i)ATP were significantly decreased . In contrast to these mutations, N239L or E242L mutant proteins showed greatly decreased activity with an altered Mg2+ requirement . These observations support the view that N239 and E242 play an important role in the binding of PO4 and Mg2+ ions required for the catalytic activity of adenosine kinase. Biochemistry, 2002 Mar 26, 41(12), 3968 - 76 Role of the low-affinity binding site in electron transfer from cytochrome C to cytochrome C peroxidase; Mei H et al.; The interaction of yeast iso-1-cytochrome c (yCc) with the high- and low-affinity binding sites on cytochrome c peroxidase compound I (CMPI) was studied by stopped-flow spectroscopy . When 3 microM reduced yCc(II) was mixed with 0.5 microM CMPI at 10 mM ionic strength, the Trp-191 radical cation was reduced from the high-affinity site with an apparent rate constant >3000 s(-1), followed by slow reduction of the oxyferryl heme with a rate constant of only 10 s(-1) . In contrast, mixing 3 microM reduced yCc(II) with 0.5 microM preformed CMPI *yCc(III) complex led to reduction of the radical cation with a rate constant of 10 s(-1), followed by reduction of the oxyferryl heme in compound II with the same rate constant . The rate constants for reduction of the radical cation and the oxyferryl heme both increased with increasing concentrations of yCc(II) and remained equal to each other . These results are consistent with a mechanism in which both the Trp-191 radical cation and the oxyferryl heme are reduced by yCc(II) in the high-affinity binding site, and the reaction is rate-limited by product dissociation of yCc(III) from the high-affinity site with apparent rate constant k(d) . Binding yCc(II) to the low-affinity site is proposed to increase the rate constant for dissociation of yCc(III) from the high-affinity site in a substrate-assisted product dissociation mechanism . The value of k(d) is <5 s(-1) for the 1:1 complex and >2000 s(-1) for the 2:1 complex at 10 mM ionic strength . The reaction of horse Cc(II) with CMPI was greatly inhibited by binding 1 equiv of yCc(III) to the high-affinity site, providing evidence that reduction of the oxyferryl heme involves electron transfer from the high-affinity binding site rather than the low-affinity site . The effects of CcP surface mutations on the dissociation rate constant indicate that the high-affinity binding site used for the reaction in solution is the same as the one identified in the yCc*CcP crystal structure. Antivir Chem Chemother, 2001 Sep, 12(5), 273 - 81 Peptidyl diazomethyl ketones inhibit the human rhinovirus 3C protease: effect on virus yield by partial block of P3 polyprotein processing; Murray MA et al.; The efficacy of a series of diazomethyl ketones (DMKs) was measured in rhinovirus-infected cultures and against the HRV14 3C protease . Their specificity and potency were confirmed against purified recombinant enzyme expressed in a yeast secretion system . An internally quenched fluorescent peptide substrate was used to assess the potency against the enzyme, obtaining a 50% inhibitory concentration (IC50) of 1 microM for both Z-L-F-Q-CHN2 and Z-V-L-F-Q-CHN2, while a lower affinity was observed for Z-F-Q-CHN2 . The tripeptide Z-L-F-Q-CHN2 blocked viral replication with an IC50 value of 30 microM as judged by the reduction in viral induced cytopathy of HeLa-H1 cells, as well as a marked reduction in viral plaque formation (50% effective concentration=20 microM) . Western blot analysis of viral proteins from infected cells indicates that this inhibitor works specifically by blocking viral polyprotein maturation, displaying a reduction of detectable 3C protease and an accumulation of the 3CD polypeptide . These results indicate that DMK inhibitors of the 3C protease have antiviral potency . Furthermore, the pattern of viral protein processing observed suggests that reducing the concentration of mature HRV 3C protease even in the presence of increased 3CD protein is sufficient to block proper viral processing and significantly reduce virus yield. APMIS, 2001 Nov, 109(11), 721 - 5 Histoplasma capsulatum var . capsulatum occurring in an HIV-positive Ghanaian immigrant to Italy . Identification of H . capsulatum DNA by PCR from paraffin sample; Rivasi F et al.; Histoplasmosis, which is highly endemic in the United States, is rare in Europe, usually imported but sometimes autochthonous . In Africa, histoplasmosis capsulati coexists with "African histoplasmosis", a characteristic skin infection caused by H . capsulatum var . duboisii . Histoplamosis due to H . capsulatum is one of the 12 secondary infections listed in the surveillance definitions of AIDS . We report the case of a 36-year-old black man with acquired immunodeficiency syndrome (AIDS) who was living in Italy but originally came from Ghana . Histoplasmosis was disseminated with fever and cutaneous manifestations . The diagnosis was demonstrated morphologically based on the presence of yeast, observed by light microscopy, in skin lesions and by identification of H . capsulatum var . capsulatum DNA by nested PCR from a paraffin sample . No clinical reports of histoplamosis capsulati in Ghana have been published until now . The present case stresses the role of immigration of subjects from outside Europe who have been infected in their native country. Folia Microbiol (Praha), 2001, 46(5), 413 - 6 Some nutritional factors influencing mevinolin production by Aspergillus terreus strain; Shindia AA; Aspergillus terreus produces the hypocholesterolemic compound mevinolin . Its growth and mevinolin production were affected by the composition of the culture medium . Both were at a maximum with glucose (6%) as the sole carbon source and in the presence of a mixture of yeast extract and sodium nitrate as nitrogen source . Influence of the concentration of some inorganic salts are also discussed. Curr Mol Med, 2001 Sep, 1(4), 447 - 55 Restoring the phenotype of fragile X syndrome: insight from the mouse model; Gantois I et al.; A mouse model for the fragile X syndrome, the most common form of inherited mental retardation, was generated a number of years ago . It shows characteristics compatible with the clinical symptoms of human patients . These include pathological changes such as macroorchidism, behavioral problems, and diminished visuo-spatial abilities . To investigate whether the fragile X syndrome is a potentially correctable disorder, several groups attempted to 'rescue' the knockout mutation by introduction of an intact copy of the FMR1 gene in the knockout mouse . Two different types of rescue mice have been created by injection of constructs based on FMR1 cDNA or on FMR1 genomic DNA . Several pathological, behavioral and cognitive function tests were performed on these two different rescue mouse lines to compare their characteristics with those of the knockout and control littermates . Each rescue line resembled the control in some aspects though neither of the 2 lines was a full 'rescue', e.g . resemble the control in all aspects investigated . Thus, rescue of some aspects of the phenotype has been achieved by introduction of FMR1 constructs in the fragile X knockout mice . The results implicate that, even if FMR1 production is cell type specific, the quantity of the FMRP expression is highly critical as overproduction may have a harmful effect. Vet Dermatol, 2002 Feb, 13(1), 7 - 13 Retrospective study: the presence of Malassezia in feline skin biopsies . A clinicopathological study; Mauldin EA et al.; Malassezia spp . dermatitis, a rare disorder in cats, has previously been associated with immune suppression and internal malignancies . This study evaluates the presence and importance of Malassezia spp . in feline biopsy specimens submitted for histopathological examination . Five hundred and fifty haematoxylin and eosin-stained skin biopsy specimens received for histopathological examination between January 1999 and November 2000 were reviewed . Fifteen (2.7%) submissions contained Malassezia organisms in the stratum corneum of the epidermis or follicular infundibulum . Eleven of 15 cats presented with an acute onset of multifocal to generalized skin lesions . All 11 cats were euthanized or died within 2 months of the onset of clinical signs . Seven cats had dermatopathological changes and clinical signs supportive of paraneoplastic alopecia, and three cats had an interface dermatitis suggestive of erythema multiforme or thymoma-associated dermatosis . Histopathological changes were nonspecific in one cat that was euthanized 2 weeks following onset of severe pruritus and alopecia . In three cats, Malassezia spp . were found in localized sites (two chin, one footpads) and appeared inconsequential to their overall health status . One cat had Malassezia spp . in association with cutaneous demodicosis . These findings suggest that Malassezia yeast in dermatopathological specimens from multifocal or generalized lesions should prompt a thorough clinical work-up for internal neoplasia. Oncogene, 2002 Mar 7, 21(11), 1717 - 26 Cdc25C interacts with PCNA at G2/M transition; Kawabe T et al.; Cdc25 activates maturation promoting factor (MPF) and promotes mitosis by removing the inhibitory phosphate from the Tyr-15 of Cdc2 in human cells . In this study, we searched the interacting protein(s) of human Cdc25C using the yeast two-hybrid screen and identified proliferating cell nuclear antigen (PCNA) as an interacting partner of Cdc25C . The interaction between Cdc25C and PCNA was confirmed in vitro and in vivo . Co-immunoprecipitation analyses using human T cell line, Jurkat, further revealed that Cdc25C interacted with PCNA transiently when cells began to enter mitosis . Immunofluorescence analysis also showed that Cdc25C and PCNA were transiently co-localized in the nucleus at the beginning of M phase . Together with the previous observations of the interaction between various cdc/cyclin and PCNA, our findings strongly suggested a potential role of PCNA at the G2 to M phase transition of cell cycle. Oncogene, 2002 Mar 7, 21(11), 1641 - 8 Tumour p53 mutations exhibit promoter selective dominance over wild type p53; Monti P et al.; The tumour suppressor gene p53 is frequently mutated in human cancer . Tumour derived p53 mutants are usually transcriptionally inactive, but some mutants retain the ability to transactivate a subset of p53 target genes . In addition to simple loss of function, some p53 mutants may be carcinogenic through a dominant negative mechanism . Aiming at a more general classification of p53 mutants into predictive functional categories it is important to determine (i) which p53 mutants are dominant, (ii) what features characterize dominant mutants and (iii) whether dominance is target gene specific . The ability of 71 p53 mutants to inhibit wild type p53 was determined using a simple yeast transcriptional assay . Approximately 30% of the mutants were dominant . They preferentially affect highly conserved amino acids (P<0.005), which are frequently mutated in tumours (P<0.005), and usually located near the DNA binding surface of the protein (P<0.001) . Different tumour-derived amino acid substitutions at the same codon usually have the same dominance phenotype . To determine whether the ability of p53 mutants to inhibit wild type p53 is target gene specific, the dominance towards p21, bax, and PIG3 binding sites was examined . Approximately 40% of the 45 mutants examined were dominant for the p21 (17/45) or PIG3 (20/45) responsive elements and 71% (32/45) were dominant for the bax responsive element . These differences are statistically significant (p21 vs bax, P<0.003; bax vs PIG3, P<0.02, Fisher's exact test) and defined a hierarchy of dominance . Finally, we extended the analysis to a group of mutants isolated in BRCA-associated tumours, some of which retained wild type level of transcription in yeast as well as in human cells, but show gain of function in transformation assays . Since transformation assays require transdominant inhibition of the endogenous wild type allele, one possible explanation for the behaviour of the BRCA-associated mutants is that they adopt conformations able to bind DNA alone but not in mixed tetramers with wild type p53 . The yeast data do not support this explanation, because all BRCA-associated mutants that behaved as wild type in transcription assay were recessive in dominance assays. J Cell Sci, 2002 Apr 1, 115(Pt 7), 1435 - 40 Essential role of human CDT1 in DNA replication and chromatin licensing; Rialland M et al.; Formation of pre-replicative complexes at origins is an early cell cycle event essential for DNA duplication . A large body of evidence supports the notion that Cdc6 protein, through its interaction with the origin recognition complex, is required for pre-replicative complex assembly by loading minichromosome maintenance proteins onto DNA . In fission yeast and Xenopus, this reaction known as the licensing of chromatin for DNA replication also requires the newly identified Cdt1 protein . We studied the role of hCdt1 protein in the duplication of the human genome by antibody microinjection experiments and analyzed its expression during the cell cycle in human non-transformed cells . We show that hCdt1 is essential for DNA replication in intact human cells, that it executes its function in a window of the cell cycle overlapping with pre-replicative complex formation and that it is necessary for the loading of minichromosome maintenance proteins onto chromatin . Intriguingly, we observed that hCdt1 protein, in contrast to other licensing factors, is already present in serum-deprived G0 arrested cells and its levels increase only marginally upon re-entry in the cell cycle. Eur J Biochem, 2002 Mar, 269(6), 1761 - 71 Differential effects of ergosterol and cholesterol on Cdk1 activation and SRE-driven transcription; Suarez Y et al.; Cholesterol is essential for cell growth and division, but whether this is just a consequence of its use in membrane formation or whether it also elicits regulatory actions in cell cycle machinery remains to be established . Here, we report on the specificity of this action of cholesterol in human cells by comparing its effects with those of ergosterol, a yeast sterol structurally similar to cholesterol . Inhibition of cholesterol synthesis by means of SKF 104976 in cells incubated in a cholesterol-free medium resulted in cell proliferation inhibition and cell cycle arrest at G2/M phase . These effects were abrogated by cholesterol added to the medium but not by ergosterol, despite that the latter was used by human cells and exerted similar homeostatic actions, as the regulation of the transcription of an SRE-driven gene construct . In contrast to cholesterol, ergosterol was unable to induce cyclin B1 expression, to activate Cdk1 and to resume cell cycle in cells previously arrested at G2 . This lack of effect was not due to cytotoxicity, as cells exposed to ergosterol remained viable and, upon supplementing with UCN-01, an activator of Cdk1, they progressed through mitosis . However, in the presence of suboptimal concentrations of cholesterol, ergosterol exerted synergistic effects on cell proliferation . This is interpreted on the basis of the differential action of these sterols, ergosterol contributing to cell membrane formation and cholesterol being required for Cdk1 activation . In summary, the action of cholesterol on G2 traversal is highly specific and exerted through a mechanism different to that used for cholesterol homeostasis, reinforcing the concept that cholesterol is a specific regulator of cell cycle progression in human cells. Br J Nutr, 2002 Jan, 87(1), 13 - 20 Bioavailability of selenium from raw or cured selenomethionine-enriched fillets of Atlantic salmon (Salmo salar) assessed in selenium-deficient rats; Ornsrud R et al.; The bioavailability of Se from raw and cured selenomethionine-enriched (Se-enriched) salmon fillets was assessed in Se-deficient male albino rats (Mol: Wist) . A low-Se Torula yeast feed was supplemented with 0, 50, 100, 150 or 200 microg Se/kg as sodium selenite or as Se from raw or cured Se-enriched salmon . The diets were fed to weanling rats for 10 and 30 d . Bioavailability of Se was assessed by metabolic balance, Se accumulation in femur, muscle, liver and plasma, and induction of Se-dependent glutathione peroxidase (EC 1.11.1.9; GSHpx) in plasma as response parameters . Except for the metabolic balance results, the slope-ratio method was used when calculating Se bioavailability from raw or cured Se-enriched fish fillets (test food) relative to sodium selenite (standard) . The data for fractional apparent absorption and fractional retention showed differences (P<0.05) among all three Se sources in the order raw salmon > cured salmon > selenite . At 10 d, Se from raw and cured Se-enriched fish fillets tended to be more bioavailable than selenite . This was supported by the observations for Se accumulation in femur and muscle and induction of GSHpx activity . At 30 d, all response parameters showed a higher bioavailability of Se from raw and cured Se-enriched fish fillets compared with selenite . Differences (P<0.05) in Se accumulation in muscle at 10 and 30d, and differences (P<0.05) in fractional apparent absorption and fractional retention suggested that curing salmon altered the utilisation of Se . The experimental results showed that enrichment of fish fillets with selenomethionine yields fillets with high Se bioavailability. Int J Oncol, 2002 Apr, 20(4), 739 - 44 Molecular cloning and characterization of bovine bgl-1, a novel family member of WD-40 repeat-containing lethal giant larvae tumor suppressor genes; Baek KH et al.; In this study, we have isolated a bovine homologue bgl-1 of lethal giant larvae (lgl) tumor suppressor oncogene from bovine brain by RT-PCR using primers designed based on the conserved sequences for lgl family members . The sequence analysis showed that the bgl-1 encodes a 1,036 amino acid polypeptide with the molecular weight of approximately 112 kDa containing a domain characteristic of WD-40 proteins . The amino acid sequence of bgl-1 showed a homology of 98.3 and 87.3% identity to that of mouse and human, respectively . Northern blot analysis showed that bgl-1 was highly expressed in brain, ovary and testis, with moderate expression in liver, uterus, lung and kidney . This suggests that the bgl-1 may play essential roles in each of these organs . The complementation analysis revealed that the bovine bgl-1 partially restored the Na+ tolerance in the absence of yeast lgl homologue, suggesting that bgl-1 is a bovine homologue of the lgl family. Curr Opin Genet Dev, 2002 Apr, 12(2), 156 - 61 Chromatin elongation factors; Svejstrup JQ; As RNA polymerase II leaves a gene promoter to transcribe the coding region, it faces a major obstacle - nucleosomes tightly wrapped into chromatin . Mechanisms to deal with this obstacle clearly exist in cells, as transcription through chromatin is very efficient in vivo, whereas nucleosomal templates pose a considerable problem for polymerase progression in reconstituted in vitro systems . Advances in our understanding of transcriptional elongation through chromatin have been made possible recently by the identification of several accessory factors that assist polymerase in the process . Insights into the function of these factors have been gained by a combination of yeast genetics and biochemical studies in mammalian systems. J Biol Chem, 2002 May 24, 277(21), 18494 - 500 Epub 2002 Mar 12. Elovl1 and p55Cdc genes are localized in a tail-to-tail array and are co-expressed in proliferating cells; Asadi A et al.; Elovl1 is a ubiquitously expressed gene, the product of which belongs to a highly conserved family of microsomal enzymes, which are involved in the formation of very long chain fatty acids and sphingolipids in yeast to man . To elucidate the structure and regulation of the Elovl gene we have isolated a lambda phage genomic DNA clone containing the entire mouse gene and found that Elovl1 consists of eight exons that are dispersed over 5.4 kb of genomic sequence . Interestingly, sequencing of the lambda clone to completion revealed that the insert contained a segment of the cell cycle gene p55Cdc directed in the opposite orientation . The genes are very tightly linked so that the 3'-end of the long mRNA species are complementary over a short stretch of nucleotides . Although both Elovl1 and p55Cdc are highly conserved genes, a BLAST search implies that the tail-to-tail arrangement has evolved in vertebrates . Despite the non-similar expression pattern in different tissues, mRNA analysis of the two genes disclosed simultaneous transcription during a proliferation-differentiation transition state, which suggests that the two genes may be regulated through a common bi-directional transcription mechanism under specific conditions. J Biol Chem, 2002 May 3, 277(18), 15221 - 4 Epub 2002 Mar 12. The PDZ proteins PICK1, GRIP, and syntenin bind multiple glutamate receptor subtypes . Analysis of PDZ binding motifs; Hirbec H et al.; Using sequence homology searches, yeast two-hybrid assays and glutathione S-transferase (GST)-pull-down approaches we have identified a series of glutamate receptor subunits that interact differentially with the PDZ proteins GRIP, PICK1, and syntenin . GST-pull-down experiments identified more interactions than detected by yeast two-hybrid assays . We report several receptor-protein interactions, strong ones include: (i) GRIP and syntenin with mGluR7a, mGluR4a, and mGluR6; (ii) PICK1 and GRIP with mGluR3; and (iii) syntenin with all forms of GluR1-4 and mGluR7b . We further characterized the novel mGluR7a-GRIP interaction found both in yeast two-hybrid and GST-pull-down assays and observed that mGluR7a localization overlapped with GRIP with in hippocampal neurons . The wide range of targets for PICK1, GRIP, and syntenin suggests they may represent a molecular mechanism that can concentrate and/or regulate a number of different receptors at a common site on a synapse . These data also suggest that the structural determinants involved in PDZ interactions are more complex than originally envisaged. Gene, 2002 Feb 6, 284(1-2), 31 - 40 Ermelin, an endoplasmic reticulum transmembrane protein, contains the novel HELP domain conserved in eukaryotes; Suzuki A et al.; We have cloned a cDNA encoding a novel protein referred to as ermelin from mouse C2 skeletal muscle cells . This protein contained six hydrophobic amino acid stretches corresponding to transmembrane domains, two histidine-rich sequences, and a sequence homologous to the fusion peptides of certain fusion proteins . Ermelin also contained a novel modular sequence, designated as HELP domain, which was highly conserved among eukaryotes, from yeast to higher plants and animals . All these HELP domain-containing proteins, including mouse KE4, Drosophila Catsup, and Arabidopsis IAR1, possessed multipass transmembrane domains and histidine-rich sequences . Ermelin was predominantly expressed in brain and testis, and induced during neuronal differentiation of N1E-115 neuroblastoma cells but downregulated during myogenic differentiation of C2 cells . The mRNA was accumulated in hippocampus and cerebellum of brain and central areas of seminiferous tubules in testis . Epitope-tagging experiments located ermelin and KE4 to a network structure throughout the cytoplasm . Staining with the fluorescent dye DiOC(6)(3) identified this structure as the endoplasmic reticulum . These results suggest that at least some, if not all, of the HELP domain-containing proteins are multipass endoplasmic reticulum membrane proteins with functions conserved among eukaryotes. Mamm Genome, 2002 Feb, 13(2), 102 - 7 Characterization of a novel gene adjacent to PAX6, revealing synteny conservation with functional significance; Kleinjan DA et al.; The human eye anomaly aniridia is normally caused by intragenic mutations of PAX6 . Several cases of aniridia are, however, associated with chromosomal rearrangements that leave the PAX6 gene intact . We have identified and characterized a novel gene, PAXNEB (C11orf19), downstream (telomeric) of PAX6 . Sequence analysis, including interspecies comparisons, show this gene to consist of 10 exons, with an unusually large final intron spanning 134 kb in human and 18 kb in Fugu . This intron is disrupted by each chromosomal rearrangement . The 2-kb PAXNEB transcript, encoding a 424-amino acid protein, is expressed in all cell lines tested . The homologous mouse cDNA is broadly expressed in mouse embryos . PAXNEB is highly conserved from mammals to fish, with some regions of the protein showing conservation to invertebrates, yeast, and plants . The possible role of PAXNEB in aniridia was assessed . Using a transgenic mouse model, we show that the aniridia phenotype of the chromosomal rearrangement cases is not due to the heterozygous loss of PAXNEB function. J Biol Chem, 2002 May 31, 277(22), 19897 - 904 Epub 2002 Mar 11. HIP1 and HIP12 display differential binding to F-actin, AP2, and clathrin . Identification of a novel interaction with clathrin light chain; Legendre-Guillemin V et al.; Huntingtin-interacting protein 1 (HIP1) and HIP12 are orthologues of Sla2p, a yeast protein with essential functions in endocytosis and regulation of the actin cytoskeleton . We now report that HIP1 and HIP12 are major components of the clathrin coat that interact but differ in their ability to bind clathrin and the clathrin adaptor AP2 . HIP1 contains a clathrin-box and AP2 consensus-binding sites that display high affinity binding to the terminal domain of the clathrin heavy chain and the ear domain of the AP2 alpha subunit, respectively . These consensus sites are poorly conserved in HIP12 and correspondingly, HIP12 does not bind to AP2 nor does it demonstrate high affinity clathrin binding . Moreover, HIP12 co-sediments with F-actin in contrast to HIP1, which exhibits no interaction with actin in vitro . Despite these differences, both proteins efficiently stimulate clathrin assembly through their central helical domain . Interestingly, in both HIP1 and HIP12, this domain binds directly to the clathrin light chain . Our data suggest that HIP1 and HIP12 play related yet distinct functional roles in clathrin-mediated endocytosis. EMBO J, 2002 Mar 15, 21(6), 1398 - 405 The Wilms' tumor gene Wt1 is required for normal development of the retina; Wagner KD et al.; The Wilms' tumor gene Wt1 is known for its important functions during genitourinary and mesothelial formation . Here we show that Wt1 is necessary for neuronal development in the vertebrate retina . Mouse embryos with targeted disruption of Wt1 exhibit remarkably thinner retinas than age-matched wild-type animals . A large fraction of retinal ganglion cells is lost by apoptosis, and the growth of optic nerve fibers is severely disturbed . Strikingly, expression of the class IV POU-domain transcription factor Pou4f2 (formerly Brn-3b), which is critical for the survival of most retinal ganglion cells, is lost in Wt1(-/-) retinas . Forced expression of Wt1 in cultured cells causes an up-regulation of Pou4f2 mRNA . Moreover, the Wt1(-KTS) splice variant can activate a reporter construct carrying 5'-regulatory sequences of the human POU4F2 . The lack of Pou4f2 and the ocular defects in Wt1(-/-) embryos are rescued by transgenic expression of a 280 kb yeast artificial chromosome carrying the human WT1 gene . Taken together, our findings demonstrate a continuous requirement for Wt1 in normal retina formation with a critical role in Pou4f2-dependent ganglion cell differentiation. Cancer Res, 2002 Mar 1, 62(5), 1496 - 502 Functional analysis of 44 mutant androgen receptors from human prostate cancer; Shi XB et al.; Mutations of the androgen receptor gene are believed to contribute to the androgen-independent growth of prostate cancer cells . To date, 56 missense mutations of the androgen receptor have been identified in human prostate cancer . The functional status of most of these mutants has not yet been investigated . To address their functional properties, we generated 44 androgen receptor mutants that have been identified in human prostate cancer and used a colorimetric yeast reporter assay to analyze their transactivational activities in response to seven different ligands . We found that these mutant androgen receptors exhibited diverse transactivational activity: seven (16%) showed loss of function, three (7%) had wild-type function, 14 (32%) had partial function, and 20 (45%) had gains of function . Five of 20 gain-of-function mutants had promiscuous activity, being transactivated by non-androgens . We also found that the combination of estradiol and progesterone at physiological concentrations weakly or moderately activated an additional seven mutant androgen receptors . Our findings provide essential information for understanding the role of mutant androgen receptors in prostate cancer. Cancer Res, 2002 Mar 1, 62(5), 1275 - 8 Mouse ING1 homologue, a protein interacting with A1, enhances cell death and is inhibited by A1 in mammary epithelial cells; Ha S et al.; We cloned mouse ING1 homologue (mINGh), an A1/Bfl-1-interacting protein, from mouse mammary glands using a yeast two-hybrid assay and unexpectedly found four splicing variants of mINGh by reverse transcription-PCR assay and sequence analysis . The alternative splicing variants were mINGh-S, mINGh-M, mINGh-L, and mINGh-L2 encoding 171, 248, 166, and 227 amino acids, respectively . Cell death of HC11 cells, induced by serum starvation, was enhanced by mINGhs, and the action of mINGhs was inhibited by A1 protein . These results indicate that A1 can inhibit cell death not only via the well known pathway related to the Bcl-2 family but also through direct interaction with mINGh in mammary epithelial cells. Vet Microbiol, 2002 Apr 22, 86(1-2), 95 - 102 Search for physical interaction between BICP0 of bovine herpesvirus-1 and p53 tumor suppressor protein; Saydam O et al.; The immediate-early (IE) protein BICP0 of bovine herpesvirus-1 (BHV-1) may have other functions besides transactivation of viral promoters . Recently, we observed that BICP0, delivered to cultured cells by a helpervirus-free amplicon system, forms spherical or doughnut-like structures in which the tumor suppressor protein p53 is sequestered . The objective was to determine whether BICP0 and p53 interact physically, we used both yeast and mammalian two-hybrid systems . As a bait plasmid, pVA3 which encodes a hybrid protein consisting of the Gal4 DNA binding domain fused to murine p53 was used . The BICP0 gene or its truncated versions were inserted into the prey plasmid pGAD424 . Bait and prey plasmids were cotransformed into yeast strain Y153, which has LacZ and HIS3 reporter genes under the control of Gal4 upstream activating sequence . After 4-6 days, colonies were stained for beta-galactosidase activity . In the mammalian two-hybrid system, pM-53 was used as a bait where truncated p53 fused to Gal4 DNA binding domain is expressed . The BICP0 gene was cloned into prey plasmid pVP16 . The interaction between p53 and SV40 T-antigen was evaluated as a positive control in both systems . Neither full-length BICP0 nor its truncated derivatives induced beta-galactosidase activity in yeast whereas the positive control turned blue under the same conditions . The mammalian two-hybrid system, in which chloramphenicol acetyltransferase (CAT) activity was used as a reporter, also failed to show an interaction between these two proteins . Co-localization of p53 with BICP0 in spherical structures is unlikely to result from a direct physical interaction between these two proteins . Mediation by additional cellular proteins may be required. SADJ, 2001 Dec, 56(12), 599 - 601 Identification of Candida dubliniensis in a HIV-positive South African population; Fisher JM et al.; Candida dubliniensis was identified as a distinctly separate species of the genus Candida in 1995 . Since then the yeast has attracted considerable interest due to its prevalence in HIV/AIDS patients and its ability to develop fluconazole resistance in HIV-seropositive individuals . Although C . dubliniensis has been identified in many centres around the world it has not yet been isolated in Africa . The purpose of this study was to identify C . dubliniensis in an HIV-positive population in the Western Cape, South Africa . A cohort of 50 tuberculosis patients co-infected with HIV was selected on admission to the Brooklyn Chest Hospital, Western Cape . The inclusion criteria for patients accepted for the study were: confirmed HIV seroconversion with a diagnosis of tuberculosis obtained from chest X-rays and sputum microscopy . C . dubliniensis was identified in 6 of the 50 patients accepted onto the study . The prevalence of C . dubliniensis in our study population was lower than that reported in similar North American and European studies . These results confirm the presence of C . dubliniensis in the South African HIV/AIDS population and indicate the urgent need for further investigations into the prevalence and pathogenesis of this clinically important species in both adult and paediatric HIV-positive patients. Bioorg Med Chem, 2002 May, 10(5), 1207 - 19 Coupling of isoprenoid triflates with organoboron nucleophiles: synthesis and biological evaluation of geranylgeranyl diphosphate analogues; Mu Y et al.; The Suzuki coupling reaction has been used to introduce a methyl group derived from commercially available methylboronic acid into a vinyl triflate . This has led to a concise synthesis of all-trans-geranylgeraniol, with the key step being the palladium-catalyzed, silver-mediated methylation of triflate to give ethyl geranylgeranoate . This coupling protocol has also been used to produce the novel geranylgeranyl diphosphate (GGPP) analogue 3-phenyl-3-desmethylgeranylgeranyl diphosphate (3-PhGGPP, ) . Our previously developed organocuprate coupling protocol has been used to introduce the cyclopropyl and tert-butyl moieties into the 3-position of vinyl triflate . The four GGPP analogues 3-vinyl-3-desmethylgeranylgeranyl diphosphate (3-vGGPP, ), 3-cyclopropyl-3-desmethylgeranylgeranyl diphosphate (3-cpGGPP, ), 3-tert-butyl-3-desmethyl-geranylgeranyl diphosphate (3-tbGGPP, ), and were then evaluated as potential inhibitors of recombinant yeast protein-geranylgeranyl transferase I (PGGTase I) . The potential mechanism-based inhibitors 3-vGGPP and 3-cpGGPP did not exhibit time-dependent inactivation of PGGTase I . Instead, both analogues were alternative substrates, in accord with the interaction of the corresponding farnesyl analogues 3-vFPP and 3-cpFPP with PFTase . The tert-butyl and phenyl analogues were not substrates, but were instead competitive inhibitors of PGGTase I . Note that all four of the GGPP analogues were bound less tightly by the enzyme than the natural substrate, in contrast to the behavior of the 3-substituted FPP analogues. Insect Biochem Mol Biol, 2002 Apr, 32(4), 477 - 86 cDNA cloning of calcineurin heterosubunits from the pheromone gland of the silkmoth, Bombyx mori; Yoshiga T et al.; Pheromone biosynthesis activating neuropeptide (PBAN) stimulates the step of fatty acyl reduction in the pheromone biosynthetic pathway of the silkmoth, Bombyx mori . It has been suggested that the intracellular signal transduction of PBAN in B . mori involves Ca(2+), calmodulin, and calcineurin (also known as protein phosphatase 2B) . We have cloned two cDNAs encoding calcineurin heterosubunits from a pheromone gland cDNA library of B . mori . The 2,996-bp clone predicts a 495-amino acid protein homologous to the catalytic subunit calcineurin A (CnA) with a molecular mass of 55,968 . The deduced amino acid sequence well conserves the calcineurin B (CnB)-binding domain and two subdomains, a calmodulin-binding and an autoinhibitory domain, showing 77-85% and 82% identities to the isoforms of Drosophila melanogaster CnA and human CnA, respectively . On the other hand, the 820-bp clone predicts a 170-amino acid protein homologous to the regulatory subunit CnB with a molecular mass of 19,357 . The deduced amino acid sequence well conserves four EF-hand type calcium-binding structures, showing 95% and about 85% identities to D . melanogaster CnB and mammalian CnBs, respectively . A yeast two-hybrid system has demonstrated the molecular interaction between B . mori CnA and CnB . Northern blot analyses revealed that both CnA and CnB genes were expressed in various larval and adult tissues of B . mori . Both transcripts detected in the pheromone gland three days before adult eclosion increased by the day before eclosion and the mRNA levels were found to be high even two days after adult eclosion . Immunohistochemical analysis has revealed that B . mori calcineurin is localized in the cytoplasm of the pheromone-producing cells. Mol Microbiol, 2001 Dec, 42(5), 1325 - 35 In vivo aggregation of the HET-s prion protein of the fungus Podospora anserina; Coustou-Linares V et al.; We have proposed that the {Het-s} infectious cytoplasmic element of the filamentous fungus Podospora anserina is the prion form of the HET-s protein . The HET-s protein is involved in a cellular recognition phenomenon characteristic of filamentous fungi and known as heterokaryon incompatibility . Under the prion form, the HET-s protein causes a cell death reaction when co-expressed with the HET-S protein, from which it differs by only 13 amino acid residues . We show here that the HET-s protein can exist as two alternative states, a soluble and an aggregated form in vivo . As shown for the yeast prions, transition to the infectious prion form leads to aggregation of a HET-s--green fluorescent protein (GFP) fusion protein . The HET-s protein is aggregated in vivo when highly expressed . However, we could not demonstrate HET-s aggregation at wild-type expression levels, which could indicate that only a small fraction of the HET-s protein is in its aggregated form in vivo in wild-type {Het-s} strains . The antagonistic HET-S form is soluble even at high expression level . A double amino acid substitution in HET-s (D23A P33H), which abolishes prion infectivity, suppresses in vivo aggregation of the GFP fusion . Together, these results further support the model that the {Het-s} element corresponds to an abnormal self-perpetuating aggregated form of the HET-s protein. Virology, 2002 Mar 1, 294(1), 141 - 50 Identification of a minimal HIV-1 gag domain sufficient for self-association; Zabransky A et al.; Gag polyprotein precursors play an essential role in the assembly of the HIV particle by polymerizing into a spherical shell at the plasma membrane . In order to define the domains within Gag responsible for this homotypic interaction, we have coupled the technology of the yeast two-hybrid system with the technology of a gene-based, semirandom library . By this method, we have identified a minimal region of Gag capable of efficient self-interaction . This region consists of the N-terminal portion of the nucleocapsid protein (NC), including the first zinc finger and the previously described interaction, or I, domain . In parallel with this randomized approach, individual HIV Gag domains, and combinations of these domains, were tested for potential homotypic and heterotypic interactions in the yeast two-hybrid system . Consistent with the results from the semirandom library screen, only combinations of species containing NC were strongly interacting . (C)2002 Elsevier Science (USA). Jpn J Pharmacol, 2001 Nov, 87(3), 214 - 25 A novel analgesic compound OT-7100 attenuates nociceptive responses in animal models of inflammatory and neuropathic hyperalgesia: a possible involvement of adenosinergic anti-nociception; Yasuda T et al.; We studied the effects of OT-7100 (5-n-butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo {1,5-a}pyrimidine), a novel analgesic compound, on the inhibitory action of adenosine on the contraction of guinea pig ileum and investigated the effects of OT-7100 on the nociceptive responses in animal models of inflammatory and peripheral neuropathic hyperalgesia and decreases spinal c-Fos expression . OT-7100 at 0.3 - 3 microM significantly enhanced the inhibitory effect of adenosine on the contraction of guinea pig ileum . The efficacy of OT-7100 (1, 3 or 10 mg/kg, p.o.) on hyperalgesia induced by yeast or substance P and in the Bennett model was significantly suppressed by coadministration of the adenosine A1 antagonist DPCPX (0.01 or 0.1 pmol/animal, i.t.), while OT-7100 without DPCPX significantly increased the nociceptive threshold in each rat model . OT-7100 (3, 10 and 30 mg/kg per day, p.o.) significantly inhibited the mechanical nociceptive threshold in the injured paw in the Chung model . OT-7100 (30 mg/kg, p.o.) significantly decreased the number of Fos-LI neurons in the spinal dorsal horn in the Bennett model . These finding suggest that OT-7100 inhibits hyperalgesia in these animal models possibly by enhancing adenosinergic neurotransmission in the dorsal horn, although we still lack direct evidence for it. Mol Cell Biol, 2002 Apr, 22(7), 2345 - 65 RACK1, an insulin-like growth factor I (IGF-I) receptor-interacting protein, modulates IGF-I-dependent integrin signaling and promotes cell spreading and contact with extracellular matrix; Hermanto U et al.; The insulin-like growth factor I (IGF-I) receptor (IGF-IR) is known to regulate a variety of cellular processes including cell proliferation, cell survival, cell differentiation, and cell transformation . IRS-1 and Shc, substrates of the IGF-IR, are known to mediate IGF-IR signaling pathways such as those of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K), which are believed to play important roles in some of the IGF-IR-dependent biological functions . We used the cytoplasmic domain of IGF-IR in a yeast two-hybrid interaction trap to identify IGF-IR-interacting molecules that may potentially mediate IGF-IR-regulated functions . We identified RACK1, a WD repeat family member and a Gbeta homologue, and demonstrated that RACK1 interacts with the IGF-IR but not with the closely related insulin receptor (IR) . In several types of mammalian cells, RACK1 interacted with IGF-IR, protein kinase C, and beta1 integrin in response to IGF-I and phorbol 12-myristate 13-acetate stimulation . Whereas most of RACK1 resides in the cytoskeletal compartment of the cytoplasm, transformation of fibroblasts and epithelial cells by v-Src, oncogenic IR or oncogenic IGF-IR, but not by Ros or Ras, resulted in a significantly increased association of RACK1 with the membrane . We examined the role of RACK1 in IGF-IR-mediated functions by stably overexpressing RACK1 in NIH 3T3 cells that expressed an elevated level of IGF-IR . RACK1 overexpression resulted in reduced IGF-I-induced cell growth in both anchorage-dependent and anchorage-independent conditions . Overexpression of RACK1 also led to enhanced cell spreading, increased stress fibers, and increased focal adhesions, which were accompanied by increased tyrosine phosphorylation of focal adhesion kinase and paxillin . While IGF-I-induced activation of IRS-1, Shc, PI3K, and MAPK pathways was unaffected, IGF-I-inducible beta1 integrin-associated kinase activity and association of Crk with p130(CAS) were significantly inhibited by RACK1 overexpression . In RACK1-overexpressing cells, delayed cell cycle progression in G(1) or G(1)/S was correlated with retinoblastoma protein hypophophorylation, increased levels of p21(Cip1/WAF1) and p27(Kip1), and reduced IGF-I-inducible Cdk2 activity . Reduction of RACK1 protein expression by antisense oligonucleotides prevented cell spreading and suppressed IGF-I-dependent monolayer growth . Our data suggest that RACK1 is a novel IGF-IR signaling molecule that functions as a positive mediator of cell spreading and contact with extracellular matrix, possibly through a novel IGF-IR signaling pathway involving integrin and focal adhesion signaling molecules. Mol Cell Biol, 2002 Apr, 22(7), 2318 - 28 Targeted mutagenesis of the Hira gene results in gastrulation defects and patterning abnormalities of mesoendodermal derivatives prior to early embryonic lethality; Roberts C et al.; The Hira gene encodes a nuclear WD40 domain protein homologous to the yeast transcriptional corepressors Hir1p and Hir2p . Using targeted mutagenesis we demonstrate that Hira is essential for murine embryogenesis . Analysis of inbred 129Sv embryos carrying the null mutation revealed an initial requirement during gastrulation, with many mutant embryos having a distorted primitive streak . Mutant embryos recovered at later stages have a range of malformations with axial and paraxial mesendoderm being particularly affected, a finding consistent with the disruption of gastrulation seen earlier in development . This phenotype could be partially rescued by a CD1 genetic background, although the homozygous mutation was always lethal by embryonic day 11, with death probably resulting from abnormal placentation and failure of cardiac morphogenesis. Mol Cell Biol, 2002 Apr, 22(7), 2136 - 46 Protein kinase A associates with HA95 and affects transcriptional coactivation by Epstein-Barr virus nuclear proteins; Han I et al.; HA95, a nuclear protein homologous to AKAP95, has been identified in immune precipitates of the Epstein-Barr virus (EBV) coactivating nuclear protein EBNA-LP from EBV-transformed lymphoblastoid cells (LCLs) . We now find that HA95 and EBNA-LP are highly associated in LCLs and in B-lymphoma cells where EBNA-LP is expressed by gene transfer . Binding was also evident in yeast two-hybrid assays . HA95 binds to the EBNA-LP repeat domain that is the principal coactivator of transcription . EBNA-LP localizes with HA95 and causes HA95 to partially relocalize with EBNA-LP in promyelocytic leukemia nuclear bodies . Protein kinase A catalytic subunit alpha (PKAcsalpha) is significantly associated with HA95 in the presence or absence of EBNA-LP . Although EBNA-LP is not a PKA substrate, HA95 or PKAcsalpha expression in B lymphoblasts specifically down-regulates the strong coactivating effects of EBNA-LP . The inhibitory effects of PKAcsalpha are reversed by coexpression of protein kinase inhibitor . PKAcsalpha also inhibits EBNA-LP coactivation with the EBNA-2 acidic domain fused to the Gal4 DNA binding domain . Furthermore, EBNA-LP- and EBNA-2-induced expression of the EBV oncogene, LMP1, is down-regulated by PKAcsalpha or HA95 expression in EBV-infected lymphoblasts . These experiments indicate that HA95 and EBNA-LP localize PKAcsalpha at nuclear sites where it can affect transcription from specific promoters . The role of HA95 as a scaffold for transcriptional regulation is discussed. J Cell Sci, 2002 Mar 15, 115(Pt 6), 1099 - 105 The PX domain: a new phosphoinositide-binding module; Ellson CD et al.; The PX domain, which until recently was an orphan domain, has emerged as the latest member of the phosphoinositide-binding module superfamily . Structural studies have revealed that it has a novel fold and identified key residues that interact with the bound phosphoinositide, enabling some prediction of phosphoinositide-binding specificity . Specificity for PtdIns(3)P appears to be the most common, and several proteins containing PX domains localise to PtdIns(3)P-rich endosomal and vacuolar structures through their PX domains: these include the yeast t-SNARE Vam7p, mammalian sorting nexins (involved in membrane trafficking events) and the Ser/Thr kinase CISK, which is implicated in cell survival . Additionally, phosphoinositide binding to the PX domains of p40(phox) and p47(phox) appears to play a critical role in the active assembly of the neutrophil oxidase complex. Biochem Biophys Res Commun, 2002 Mar 15, 291(5), 1166 - 72 ALG-2 interacts with the amino-terminal domain of annexin XI in a Ca(2+)-dependent manner; Satoh H et al.; The apoptosis-linked protein ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand protein family . ALG-2 forms a homodimer, a heterodimer with another penta-EF-hand protein, peflin, and a complex with its interacting protein, named AIP1 or Alix . By yeast two-hybrid screening using human ALG-2 as bait, we isolated a cDNA of a novel ALG-2-interacting protein, which turned out to be annexin XI . Deletion analysis revealed that ALG-2 interacted with the N-terminal domain of annexin XI (AnxN), which has an amino acid sequence similar to that of the C-terminal region of AIP1/Alix . Using recombinant biotin-tagged ALG-2 and the glutathione S-transferase (GST) fusion protein of AnxN, the direct interaction was analyzed by an ALG-2 overlay assay and by real-time interaction analysis with a surface plasmon resonance (SPR) biosensor . The dissociation constant (K(d)) was estimated to be approximately 70 nM . The Ca(2+)-dependent fluorescence change of ALG-2 in the presence of the hydrophobicity fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was inhibited by mixing with GST-AnxN, suggesting that the Pro/Gly/Tyr/Ala-rich hydrophobic region in AnxN masked the Ca(2+)-dependently exposed hydrophobic surface of ALG-2 . (C)2002 Elsevier Science (USA). Arch Biochem Biophys, 2002 Mar 1, 399(1), 66 - 72 The liver-specific human alpha(1)-microglobulin/bikunin precursor (AMBP) is capable of self-association; Tyagi S et al.; alpha-1-Microglobulin (A1M) and bikunin are two plasma glycoproteins encoded by an alpha-1-microglobulin/bikunin precursor (AMBP) gene . Despite their lack of any structural or functional relationship, both A1M and bikunin originate from AMBP cleavage by a furin-like protease that releases the two mature molecules . The AMBP gene maintains a tight control over its expression by a unique enhancer, which is controlled by several hepatocyte-enriched nuclear factors; however, the mechanisms of regulation of the intracellular levels of the AMBP protein are currently unknown . We report the ability of the AMBP protein to self-associate and form a dimer in a yeast environment using the yeast two-hybrid system and an in vitro dimerization assay . We also show that the A1M protein binds to its precursor protein, AMBP, whereas bikunin does not . This observation warrants further investigations for a dimerization-dependent intracellular control that AMBP may be involved in . The relevance of AMBP dimerization and its possible biological significance are postulated. Virology, 2001 Nov 25, 290(2), 224 - 36 The hepatitis C virus core protein interacts with NS5A and activates its caspase-mediated proteolytic cleavage; Goh PY et al.; Viral proteins interact with one another during viral replication, assembly, and maturation . Systematic interaction assays of the hepatitis C virus (HCV) proteins using the yeast two-hybrid method have uncovered a novel interaction between core and NS5A . This interaction was confirmed by in vitro binding assays, and coimmunoprecipitation in mammalian cells . Core and NS5A are also colocalized in COS-7 cells . Interestingly, NS5A is cleaved to give specific-size fragments, when core is coexpressed in mammalian cells . Overexpression of core produced many dying and rounded cells and effects such as DNA laddering and the truncation of poly(ADP-ribose) polymerase 1 (PARP1), both indicators of apoptosis . These observations led us to investigate the link between the induction of apoptosis by core and the cleavage of NS5A . The proteolysis of NS5A and these apoptotic events can be inhibited by caspase inhibitor, Z-VAD, indicating that core induces apoptosis and the cleavage of NS5A by caspases . In cells infected by the HCV, core may provide the intrinsic apoptotic signal, which produces truncated forms of NS5A . The biological function of core-NS5A interaction and the downstream effect of NS5A cleavage are discussed. J Biol Chem, 2002 May 17, 277(20), 18127 - 33 Epub 2002 Mar 06. The interaction of RGSZ1 with SCG10 attenuates the ability of SCG10 to promote microtubule disassembly; Nixon AB et al.; RGS proteins (regulators of G protein signaling) are a diverse family of proteins that act to negatively regulate signaling by heterotrimeric G proteins . Initially characterized as GTPase-activating proteins for Galpha subunits, recent data have implied additional functions for RGS proteins . We previously identified an RGS protein (termed RGSZ1) whose expression is quite specific to neuronal tissue (Glick, J . L., Meigs, T . E., Miron, A., and Casey, P . J . (1998) J . Biol . Chem . 273, 26008-26013) . In a continuing effort to understand the role of RGSZ1 in cellular signaling, the yeast two-hybrid system was employed to identify potential effector proteins of RGSZ1 . The microtubule-destabilizing protein SCG10 (superior cervical ganglia, neural specific 10) was found to directly interact with RGSZ1 in the yeast system, and this interaction was further verified using direct binding assays . Treatment of PC12 cells with nerve growth factor resulted in Golgi-specific distribution of SCG10 . A green fluorescent protein-tagged variant of RGSZ1 translocated to the Golgi complex upon treatment of PC12 cells with nerve growth factor, providing evidence that RGSZ1 and SCG10 interact in cells as well as in vitro . Analysis of in vitro microtubule polymerization/depolymerization showed that binding of RGSZ1 to SCG10 effectively blocked the ability of SCG10 to induce microtubule disassembly as determined by both turbidimetric and microscopy-based assays . These results identify a novel connection between RGS proteins and the cytoskeletal network that points to a broader role than previously envisioned for RGS proteins in regulating biological processes. J Biol Chem, 2002 May 24, 277(21), 19008 - 18 Epub 2002 Mar 06. Regulation of endocytosis of activin type II receptors by a novel PDZ protein through Ral/Ral-binding protein 1-dependent pathway; Matsuzaki T et al.; Using yeast two-hybrid screening, we have identified a mouse Postsynaptic density 95/Discs large/Zona occludens-1 (PDZ) protein that interacts with activin type II receptors (ActRIIs) . We named the protein activin receptor-interacting protein 2 (ARIP2) . ARIP2 was found to have one PDZ domain in the NH(2)-terminal region and interact specifically with ActRIIs among the receptors for the transforming growth factor beta family by the PDZ domain . Interestingly, overexpression of ARIP2 enhances endocytosis of ActRIIs and reduces activin-induced transcription in Chinese hamster ovary K1 cells . In addition, immunofluorescence co-localization studies indicated the direct involvement of ARIP2 in the intracellular translocation of ActRIIs by PDZ domain-mediated interaction . Moreover, we have identified that the COOH-terminal region of ARIP2 interacts with Ral-binding protein 1 (RalBP1) . RalBP1 is a potential effector protein of small GTP-binding protein Ral and regulates endocytosis of epidermal growth factor and insulin receptors . The studies using deletion mutants of RalBP1 and constitutively GTP and GDP binding forms of Ral indicate that ARIP2 regulates endocytosis of ActRIIs through the Ral/RalBP1-dependent pathway, and the GDP-GTP exchange of Ral is critical for this regulation. J Biol Chem, 2002 May 31, 277(22), 19673 - 8 Epub 2002 Mar 06. Identification of a karyopherin alpha 2 recognition site in PLAG1, which functions as a nuclear localization signal; Braem CV et al.; The activation of the pleomorphic adenoma gene 1 (PLAG1) is the most frequent gain-of-function mutation found in pleomorphic adenomas of the salivary glands . To gain more insight into the regulation of PLAG1 function, we searched for PLAG1-interacting proteins . Using the yeast two-hybrid system, we identified karyopherin alpha2 as a PLAG1-interacting protein . Physical interaction between PLAG1 and karyopherin alpha2 was confirmed by an in vitro glutathione S-transferase pull-down assay . Karyopherin alpha2 escorts proteins into the nucleus via interaction with a nuclear localization sequence (NLS) composed of short stretches of basic amino acids . Two putative NLSs were identified in PLAG1 . The predicted NLS1 (KRKR) was essential for physical interaction with karyopherin alpha2 in glutathione S-transferase pull-down assay, and its mutation resulted in decreased nuclear import of PLAG1 . Moreover, NLS1 was able to drive the nuclear import of the cytoplasmic protein beta-galactosidase . In contrast, predicted NLS2 of PLAG1 (KPRK) was not involved in karyopherin alpha2 binding nor in its nuclear import . The residual nuclear import of PLAG1 after mutation of the NLS1 was assigned to the zinc finger domain of PLAG1 . These observations indicate that the nuclear import of PLAG1 is governed by its zinc finger domain and by NLS1, a karyopherin alpha2 recognition site. J Biol Chem, 2002 May 3, 277(18), 15237 - 40 Epub 2002 Mar 06. Identification of a neuronal Cdk5 activator-binding protein as Cdk5 inhibitor; Ching YP et al.; Neuronal Cdc2-like kinase (Nclk) plays an important role in a variety of cellular processes, including neuronal cell differentiation, apoptosis, neuron migration, and formation of neuromuscular junction . The active kinase consists of a catalytic subunit, Cdk5, and an essential regulatory subunit, neuronal Cdk5 activator (p35(nck5a) or p25(nck5a)), which is expressed primarily in neurons of central nervous tissue . In our previous study using the yeast two-hybrid screening method, three novel p35(nck5a)-associated proteins were isolated . Here we show that one of these proteins, called C42, specifically inhibits the activation of Cdk5 by Nck5a . Co-immunoprecipitation data suggested that C42 and p35(nck5a) could form a complex within cultured mammalian cells . Deletion analysis has mapped the inhibitory domain of C42 to a region of 135 amino acids, which is conserved in Pho81, a yeast protein that inhibits the yeast cyclin-dependent protein kinase Pho85 . The Pho85.Pho80 kinase complex has been shown to be the yeast functional homologue of the mammalian Cdk5/p35(nck5a) kinase. Hypertension, 2002 Feb, 39(2 Pt 2), 695 - 8 Hypoxia inducible double plasmid system for myocardial ischemia gene therapy; Tang Y et al.; Coronary artery disease frequently involves repeated bouts of myocardial ischemia . To automatically up-regulate the cardioprotective transgenes under hypoxic ischemia, a "vigilant vector" gene therapy system was developed and tested in a rat embryonic myocardial cell line (H9c2) . In the vigilant vector, a hypoxia response element-incorporated promoter was used as a switch to turn on the gene expression in response to hypoxic signal . Furthermore, a novel double plasmid system was designed to elevate the potency of the vigilant vector . Instead of putting the promoter and the reporter gene in the same plasmid (single plasmid system), we separated them into two plasmids: the transactivator plasmid and reporter plasmid (double plasmid system) . The hypoxia response element (HRE)-incorporated promoter increased the expression of a chimeric transcription factor consisting of the yeast GAL4 DNA binding domain and the human nuclear (transcription) factor-kappaB (NF-kappaB) p65 activation domain . The powerful chimeric regulator binds specifically to the upstream activating sequence for GAL4 in the reporter plasmid and activates the transcription of the transgene . Our experiments showed that the HRE-mediated expression could quickly increase 2.08 +/- 0.75-fold within 6 hours of hypoxia and further augmented 7.12 +/- 1.52-fold when the hypoxia condition was prolonged to 24 hours . The hypoxia-inducible double plasmid system dramatically amplified the transgene expression under both hypoxia and normoxia by 412.79 +/- 185.27-fold and 205.35 +/- 65.44-fold, respectively, relative to the single plasmid system . From these results, we concluded that this hypoxia inducible double plasmid system could be used therapeutically to switch on genes that have proven beneficial effects in myocardial ischemia. Life Sci Space Res, 1964, 2, 261 - 6 The effect of low temperatures on the structure of enzymes; Voronov GT; The enzymes of the cold stable organisms possess lower levels of activation energy . A possibility for the adaptation of living creatures to low temperatures is finally associated with the structural alterations in proteins and enzymes . Polarographic studies on yeast alcohol dehydrogenase and on proteinase of B . subtilis at low temperatures have demonstrated that a lowering of temperature causes significant structural changes in the protein molecule. Proc Natl Acad Sci U S A, 2002 Mar 5, 99(5), 3147 - 52 Mycobacterium tuberculosis WhiB3 interacts with RpoV to affect host survival but is dispensable for in vivo growth; Steyn AJ et al.; Previous work established that the principal sigma factor (RpoV) of virulent Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, restores virulence to an attenuated strain containing a point mutation (Arg-515-->His) in the 4.2 domain of RpoV . We used the 4.2 domain of RpoV as bait in a yeast two-hybrid screen of an M . tuberculosis H37Rv library and identified a putative transcription factor, WhiB3, which selectively interacts with the 4.2 domain of RpoV in virulent strains but not with the mutated (Arg-515-->His) allele . Infection of mice and guinea pigs with a M . tuberculosis H37Rv whiB3 deletion mutant strain showed that whiB3 is not necessary for in vivo bacterial replication in either animal model . In contrast, an M . bovis whiB3 deletion mutant was completely attenuated for growth in guinea pigs . However, we found that immunocompetent mice infected with the M . tuberculosis H37Rv whiB3 mutant strain had significantly longer mean survival times as compared with mice challenged with wild-type M . tuberculosis . Remarkably, the bacterial organ burdens of both mutant and wild-type infected mice were identical during the acute and persistent phases of infection . Our results imply that M . tuberculosis replication per se is not a sufficient condition for virulence in vivo . They also indicate a different role for M . bovis and M . tuberculosis whiB3 genes in pathogenesis generated in different animal models . We propose that M . tuberculosis WhiB3 functions as a transcription factor regulating genes that influence the immune response of the host. Proc Natl Acad Sci U S A, 2002 Mar 5, 99(5), 3064 - 9 A prototypic platelet septin and its participation in secretion; Dent J et al.; Studies are presented characterizing platelet CDCrel-1, a protein expressed to high levels by megakaryocytes and belonging to a family of conserved proteins, termed septin . Septin filaments originally were identified in yeast as essential for budding but have become increasingly associated with processes in higher eukaryotic cells involving active membrane movement such as cytokinesis and vesicle trafficking . Direct proof of an in vivo function for septins in higher eukaryotes is limited to the characterization of the Drosophila septin, termed PNUT . We present studies identifying platelet CDCrel-1 as a protein kinase substrate in the presence of known platelet agonists . The immunopurification of CDCrel-1 revealed it to be part of a macromolecular complex containing a protein involved in platelet secretion, syntaxin 4 . Moreover, CDCrel-1 was localized in situ to areas surrounding platelet-storage granules . The relevance of CDCrel-1 to normal platelet function was established with the characterization of platelets from a CDCrel-1(Null) mouse . As compared with platelets from wild-type littermates, CDCrel-1(Null) platelets aggregate and release stored {14C}serotonin in the presence of subthreshold levels of collagen . These results provide new insights into the mechanisms regulating platelet secretion and identify platelet septins as a protein family contributing to membrane trafficking within the megakaryocyte and platelet. Proc Natl Acad Sci U S A, 2002 Mar 5, 99(5), 2959 - 64 The organization of Physcomitrella patensRAD51 genes is unique among eukaryotic organisms; Markmann-Mulisch U et al.; Genetic recombination pathways and genes are well studied, but relatively little is known in plants, especially in lower plants . To study the recombination apparatus of a lower land plant, a recombination gene well characterized particularly in yeast, mouse, and man, the RAD51 gene, was isolated from the moss Physcomitrella patens and characterized . Two highly homologous RAD51 genes were found to be present . Duplicated RAD51 genes have been found thus far exclusively in eukaryotes with duplicated genomes . Therefore the presence of two highly homologous genes suggests a recent genome duplication event in the ancestry of Physcomitrella . Comparison of the protein sequences to Rad51 proteins from other organisms showed that both RAD51 genes originated within the group of plant Rad51 proteins . However, the two proteins form a separate clade in a phylogenetic tree of plant Rad51 proteins . In contrast to RAD51 genes from other multicellular eukaryotes, the Physcomitrella genes are not interrupted by introns . Because introns are a common feature of Physcomitrella genes, the lack of introns in the RAD51 genes is unusual and may indicate the presence of an unusual recombination apparatus in this organism . The presence of duplicated intronless RAD51 genes is unique among eukaryotes . Studies of further members of this lineage are needed to determine whether this feature may be typical of lower plants. Proc Natl Acad Sci U S A, 2002 Mar 5, 99(5), 2824 - 9 Phosphatidylinositol-dependent actin filament binding by the SWI/SNF-like BAF chromatin remodeling complex; Rando OJ et al.; Recently, several chromatin remodeling complexes in yeast, Drosophila, and mammals have been shown to contain actin and actin-related proteins (arps) . However, the function of actin in these complexes is unclear . Here, we show that the mammalian SWI/SNF-like BAF complex binds to phosphatidylinositol 4,5-bisphosphate (PIP2) micelles and PIP2-containing mixed lipid vesicles, and that PIP2 binding allows the complex to associate with actin pointed ends and branch points . Actin binds to at least two distinct domains in the C terminus of the Brg1 protein, and interaction with only one of these domains is sensitive to PIP2 . Based on these findings, we propose a model for PIP2 activation of actin binding by relief of intramolecular capping of actin by Brg1. Trends Mol Med, 2002 Mar, 8(3), 121 - 6 New immunotherapeutic strategies to control vaginal candidiasis; Magliani W et al.; The widespread occurrence of mucosal infections caused by Candida, in particular recurrent vulvovaginal candidiasis among fertile-age women, together with the paucity of safe candidacidal antimycotics, have prompted a great number of investigations into the immunotherapy of candidal vaginitis . This article will discuss three different experimental approaches demonstrated to be potentially transferable to human disease: (1) the use of antibodies against well-defined cell-surface adhesins or enzymes; (2) the generation of yeast killer-toxin-like candidacidal anti-idiotypic antibodies and their engineered molecular derivatives (e.g . single chains, peptides); and (3) the generation of therapeutic vaccines and immunomodulators. Arthritis Res, 2002, 4(2), 134 - 8 Epub 2001 Nov 12. Autoantibodies directed to novel components of the PM/Scl complex, the human exosome; Brouwer R et al.; The autoantigenic polymyositis/scleroderma (PM/Scl) complex was recently shown to be the human homologue of the yeast exosome, which is an RNA-processing complex . Our aim was to assess whether, in addition to targeting the known autoantigens PM/Scl-100 and PM/Scl-75, autoantibodies also target recently identified components of the PM/Scl complex . The prevalence of autoantibodies directed to six novel human exosome components (hRrp4p, hRrp40p, hRrp41p, hRrp42p, hRrp46p, hCsl4p) was determined in sera from patients with idiopathic inflammatory myopathy (n = 48), scleroderma (n = 11), or the PM/Scl overlap syndrome (n = 10) . The sera were analyzed by enzyme-linked immunosorbent assays and western blotting using the affinity-purified recombinant proteins . Our results show that each human exosome component is recognized by autoantibodies . The hRrp4p and hRrp42p components were most frequently targeted . The presence of autoantibodies directed to the novel components of the human exosome was correlated with the presence of the anti-PM/Scl-100 autoantibody in the sera of patients with idiopathic inflammatory myopathy (IIM), as was previously found for the anti-PM/Scl-75 autoantibody . Other clear associations between autoantibody activities were not found . These results further support the conception that the autoimmune response may initially be directed to PM/Scl-100, whereas intermolecular epitope spreading may have caused the autoantibody response directed to the associated components. J Agric Food Chem, 2002 Mar 13, 50(6), 1548 - 52 Role of riboflavin in beer flavor instability: determination of levels of riboflavin and its origin in beer by fluorometric apoprotein titration; Duyvis MG et al.; A method for the quantitative determination of riboflavin levels in beer was developed . The method is based on the quenching of riboflavin fluorescence, which occurs when riboflavin binds to the aporiboflavin-binding protein from egg white . The method does not require any pretreatment of the beer before analysis, other than dilution, and proved to be simple, reliable, and sensitive . The lowest concentration that could be detected was approximately 10 nM riboflavin . The possible interference of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) with the determination of the riboflavin content of beer was excluded, because beer contains only a very small amount of FAD (0.03 microM) and no FMN . The riboflavin levels of the types and brands of beer investigated were in the range of 0.5-1.0 microM . The origin of the riboflavin in beer proved to be the malt . Hop and yeast hardly contributed to the riboflavin content of beer . Besides its use in the determination of riboflavin levels, the aporiboflavin-binding protein also provides a way to remove riboflavin from beer, which reduces the light sensitivity and the related lightstruck off-flavor formation in beer. Virology, 2002 Jan 20, 292(2), 198 - 210 Hepatitis C virus NS5A colocalizes with the core protein on lipid droplets and interacts with apolipoproteins; Shi ST et al.; The nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) has been shown to interact with a variety of cellular proteins and implicated in the regulation of cell growth, interferon resistance, and other cellular signaling pathways, but the role of NS5A in HCV pathogenesis has not been firmly established . To further characterize this multifunctional protein, we instigated the studies of the subcellular localization of NS5A in a hepatoma cell line . NS5A was localized to the perinuclear membrane structures, including the endoplasmic reticulum (ER) and the Golgi apparatus, by immunofluorescence staining and confocal microscopy . In addition, it was also associated with the surface of cytoplasmic globular structures when expressed alone or as a part of the NS3-5B polyprotein . Oil red O staining revealed that these globular structures were lipid droplets, where the HCV core protein was also localized . The association of NS5A with intracellular membrane was further confirmed by membrane flotation analysis . To determine whether NS5A interacts with any cellular lipid-binding protein, we performed yeast two-hybrid screening in both HepG2 and human liver cDNA libraries . Apolipoprotein A1 (apoA1), one of the protein components of high-density lipoprotein (HDL) particles, was identified by two independent screening processes . The interaction between NS5A and apoA1 was confirmed by both in vitro pull-down and in vivo coimmunoprecipitation experiments . Immunofluorescence staining revealed a significant colocalization of NS5A and apoA1 in the Golgi apparatus . Our results established an association of NS5A with lipid droplets and apoA1, suggesting that NS5A, together with the core protein, may play a role in the pathogenesis of the derangement of lipid metabolism, contributing to liver steatosis commonly observed in hepatitis C. J Trace Elem Med Biol, 2002, 16(1), 47 - 55 Parameters of dietary selenium and vitamin E deficiency in growing rabbits; Muller AS et al.; 4 x 5 growing female rabbits (New Zealand White) with an initial live weight of 610 +/- 62 g were fed a torula yeast based semisynthetic diet low in selenium (<0.03 mg/kg diet) and containing <2 mg alpha-tocopherol per kg (group I) . Group II received a vitamin E supplementation of 150 mg alpha-tocopherylacetate per kg diet, whereas for group III 0.40 mg Se as Na-selenite and for group IV both supplements were added . Selenium status and parameters of tissue damage were analyzed after 10 weeks on experiment (live weight 2,355 +/- 145 g) . Selenium depletion of the Se deficient rabbits (groups I and II) was indicated by a significantly lower plasma Se content (group I: 38.3 +/- 6.23 microg Se/mL plasma, group II: 42.6 +/- 9.77, group III: 149 +/- 33.4, group IV: 126 +/- 6.45) and a significantly lower liver Se content (group I: 89.4 +/- 18.2 microg/kg fresh matter, group II: 111 +/- 26.2) as compared to the Se supplemented groups III (983 +/- 204) and IV (926 +/- 73.9) . After 5 weeks on the experimental diets differences in the development of plasma glutathione peroxidase were observed . As compared to the initial status group (45.2 +/- 4.50) pGPx activity in mU/mg protein was decreased in group I (19.1 +/- 7.08), remained almost stable in the vitamin E supplemented group II (46.3 +/- 11.2) whereas an elevated enzyme activity was measured in the Se supplemented groups III (62.4 +/- 23.9) and IV (106 +/- 19.9) . In the rabbit organs investigated 10 weeks of Se deficiency caused a significant loss of Se dependent cellular glutathione peroxidase activity (GPx1) of 94% (liver), 80% (kidney), 50% (heart muscle) and 60% (musculus longissimus dorsi) in comparison to Se supplemented control animals . Damage of cellular lipids and proteins in the liver was due to either Se or vitamin E deficiency . However damage was most severe under conditions of a combined Se and vitamin E deficiency . It can be concluded that the activity of plasma glutathione peroxidase is a sensitive indicator of Se deficiency in rabbits . The loss of GPx1 activity indicates the selenium depletion in various rabbit organs . Both selenium and vitamin E are essential and highly efficient antioxidants which protect rabbits against lipid and protein oxidation. J Biol Chem, 2002 May 17, 277(20), 18111 - 7 Epub 2002 Mar 04. CXCR4/CCR5 down-modulation and chemotaxis are regulated by the proteasome pathway; Fernandis AZ et al.; Chemokines and their receptors play a critical role in host immune surveillance and are important mediators of human immunodeficiency virus (HIV) pathogenesis and inflammatory response . The chemokine receptors CCR5 and CXCR4, which act as co-receptors along with CD4 for HIV docking and entry, are down-modulated by their respective ligands, MIP-1beta/SDF-1alpha or by the HIV envelope protein, gp120 . We have studied the role of the proteasome pathway in the down-regulation of these receptors . Using the yeast and mammalian two-hybrid systems, we observed that the CCR5 receptor is constitutively associated with the zeta subunit of proteasome . Immunoprecipitation studies in CCR5 L1.2 cells revealed that this association was increased with MIP-1beta stimulation . The proteasome inhibitors, lactacystin and epoxomicin, attenuated MIP-1beta induced CCR5 down-modulation as detected by fluorescence-activated cell sorter analysis and confocal microscopy . The proteasome inhibitors also inhibited the SDF-1alpha and gp120 protein-induced down-modulation of the CXCR4 receptor in Jurkat cells . However, the inhibitors had no significant effect on the gp120-induced internalization of the CD4 receptor . These inhibitors also blocked cognate ligand-mediated chemotaxis but had no effect on SDF-1alpha-induced p44/42 MAP kinase or MIP-1beta-induced p38 kinase activities, thus indicating differential effects of the inhibitors on signaling mediated by these receptors . These results indicate that the CCR5 and CXCR4 receptor down-modulation mechanism and chemotaxis mediated by these receptors are dependent upon proteasome activity. J Biol Chem, 2002 Aug 16, 277(33), 30219 - 26 Epub 2002 Mar 04. Interaction between tyrosine kinase Etk and a RUN domain- and FYVE domain-containing protein RUFY1 . A possible role of ETK in regulation of vesicle trafficking; Yang J et al.; Etk/BMX tyrosine kinase is involved in regulation of various cellular processes including proliferation, differentiation, motility, and apoptosis . Through a yeast two-hybrid screening for the effectors of Etk, a new gene family designated as RUFY was identified . The RUFY gene family (RUFY1 and RUFY2) contains an N-terminal RUN domain and a C-terminal FYVE domain with two coiled-coil domains in-between . They appear to be homologues of a recently identified mouse Rabip4 (Cormant, M., Mari, M., Galmiche, A., Hofman, P., and Le Marchand-Brustel, Y . (2001) Proc . Natl . Acad . Sci . U . S . A . 98, 1637-1642) . RUFY proteins are localized predominantly to endosomes as evidenced by their co-localization with early endosome antigen marker (EEA1) . Etk interacts with RUFY1 through its SH3 and SH2 domains . RUFY1 is tyrosine-phosphorylated and appears to be a substrate of Etk . The RUFY1 mutant lacking the phosphorylation sites failed to go to the endosomes . Furthermore, overexpression of Etk in COS-1 and B82L cells resulted in increased plasma membrane localization of the epidermal growth factor receptor and delayed its induced endocytosis in COS-1 cells . The effects of Etk were blocked by the FYVE domain of RUFY1 . Interestingly, the FYVE domain of RUFY1 is targeted to the plasma membrane through an interaction between its proline-rich motif and the SH3 domain of Etk or possibly some other membrane-associated SH3 domain-containing protein(s), whereas the lipid binding activity of the FYVE domain is not required . Our data suggest that Etk may be involved in regulation of endocytosis through its interaction with an endosomal protein RUFY1. J Biol Chem, 2002 May 10, 277(19), 17112 - 6 Epub 2002 Feb 27. Altered amelogenin self-assembly based on mutations observed in human X-linked amelogenesis imperfecta (AIH1); Paine ML et al.; A hallmark of biological systems is a reliance on protein assemblies to perform complex functions . We have focused attention on mammalian enamel formation because it relies on a self-assembling protein complex to direct mineral habit . The principle protein of enamel is amelogenin, a 180-amino acid hydrophobic protein that self-assembles to form nanospheres . We have used independent technical methods, consisting of the yeast two-hybrid (Y2H) assay and surface plasmon resonance (SPR), to demonstrate the importance of amelogenin self-assembly domains . In addition, we have analyzed mutations in amelogenin observed in patients with amelogenesis imperfecta who demonstrate defects in enamel formation . Assessments of self-assembly of these mutant amelogenins by either SPR or Y2H assay yield concordant data . These data support the conclusion that the amelogenin amino-terminal self-assembly domain is essential to the creation of an enamel extracellular organic matrix capable of directing mineral formation . It also suggests that a pathway through which point mutations in the amelogenin protein can adversely impact on the formation of the enamel organ is by disturbing self-assembly of the organic matrix . These data support the utilization of the Y2H assay to search for protein interactions among extracellular matrix proteins that contribute to biomineralization and provide functional information on protein-protein and protein-mineral interactions. J Biol Chem, 2002 May 10, 277(19), 16968 - 75 Epub 2002 Feb 27. UDP-glucuronate decarboxylase, a key enzyme in proteoglycan synthesis: cloning, characterization, and localization; Moriarity JL et al.; UDP-glucuronate decarboxylase (UGD) catalyzes the formation of UDP-xylose from UDP-glucuronate . UDP-xylose is then used to initiate glycosaminoglycan biosynthesis on the core protein of proteoglycans . In a yeast two-hybrid screen with the protein kinase Akt (protein kinase B), we detected interactions with a novel sequence, which we cloned and expressed . The expressed protein displayed UGD activity but did not display the activities of homologous nucleotide sugar epimerases or dehydratases . We did not detect phosphorylation of UGD by Akt nor did we detect any influence of Akt on UGD activity . Effects of UGD on Akt kinase activity were also absent . Northern blot and Western blot analyses revealed the presence of UGD in multiple tissues and brain regions . Subcellular studies and histochemistry localized UGD protein to the perinuclear Golgi where xylosylation of proteoglycan core proteins is known to occur. Genes Dev, 2002 Mar 1, 16(5), 560 - 70 Involvement of the cohesin protein, Smc1, in Atm-dependent and independent responses to DNA damage; Kim ST et al.; Structural maintenance of chromosomes (SMC) proteins play important roles in sister chromatid cohesion, chromosome condensation, sex-chromosome dosage compensation, and DNA recombination and repair . Protein complexes containing heterodimers of the Smc1 and Smc3 proteins have been implicated specifically in both sister chromatid cohesion and DNA recombination . Here, we show that the protein kinase, Atm, which belongs to a family of phosphatidylinositol 3-kinases that regulate cell cycle checkpoints and DNA recombination and repair, phosphorylates Smc1 protein after ionizing irradiation . Atm phosphorylates Smc1 on serines 957 and 966 in vitro and in vivo, and expression of an Smc1 protein mutated at these phosphorylation sites abrogates the ionizing irradiation-induced S phase cell cycle checkpoint . Optimal phosphorylation of these sites in Smc1 after ionizing irradiation also requires the presence of the Atm substrates Nbs1 and Brca1 . These same sites in Smc1 are phosphorylated after treatment with UV irradiation or hydroxyurea in an Atm-independent manner, thus demonstrating that another kinase must be involved in responses to these cellular stresses . Yeast containing hypomorphic mutations in SMC1 and human cells overexpressing Smc1 mutated at both of these phosphorylation sites exhibit decreased survival following ionizing irradiation . These results demonstrate that Smc1 participates in cellular responses to DNA damage and link Smc1 to the Atm signal transduction pathway. Clin Exp Immunol, 2002 Feb, 127(2), 199 - 205 Immune response modulation by recombinant peptides expressed in virus-like particles; Svirshchevskaya EV et al.; Aspergillus fumigatus, a ubiquitous fungus, is implicated in the pathogenesis of a number of clinically different allergic diseases in man, including allergic bronchopulmonary aspergillosis . Peptide-based immunotherapy may offer an alternative treatment strategy for the management of allergic disease . The objective of this study was to alter the allergen-specific immune response using dominant T cell epitopes of a major A . fumigatus allergen, Asp f2, expressed in yeast as virus-like particles (VLP) . The T cell epitopes of Asp f2, recognized in mice with an H-2d background, were determined by producing T-cell hybridomas . Two dominant T cell epitopes, aa60--71 and aa235--249, were identified and expressed in a yeast VLP system . To induce t