Yeasts

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 tolerance VLP-peptides were injected subcutaneously into mice previously immunized with recombinant Asp f2 . The T cell immune response was abrogated totally in 3 weeks following a single injection of VLP but was restored 2 months later following intranasal antigen exposure . T-cell depletion resulted in the reduction of 20-30% of all antigen-specific immunoglobulin classes . Thus, recombinant peptides expressed in the VLP system can be used successfully in the modulation of Asp f2-induced immune response in mice, although a single administration is not sufficient to maintain a state of tolerance for a long period of time.

Hum Mol Genet, 2002 Mar 1, 11(5), 477 - 86
Human deafness dystonia syndrome is caused by a defect in assembly of the DDP1/TIMM8a-TIMM13 complex; Roesch K et al.; Mohr-Tranebjaerg syndrome (MTS/DFN-1) or deafness/dystonia syndrome results from a mutation in deafness/dystonia protein 1/translocase of mitochondrial inner membrane 8a (DDP1/TIMM8a) . DDP1/TIMM8a is similar to a family of yeast proteins in the mitochondrial intermembrane space which mediate the import and insertion of inner membrane proteins . We now show that TIMM8a assembles in a 70 kDa complex in the intermembrane space with TIMM13 . DDP1/TIMM8a is not detectable in fibroblasts derived from a patient with a missense mutation in the DDP1/TIMM8a gene; the point mutation results in cysteine-66 being changed to tryptophan-66 in the conserved 'twin CX(3)C' motif . The corresponding mutation in yeast translocase of inner membrane 8p (Tim8p) yields an unstable protein that does not assemble with yeast Tim13p . DDP1/TIMM8a, when expressed with TIMM13 in yeast mitochondria lacking the Tim8p-Tim13p complex, restores Tim23p import, and TIMM8a and TIMM13 can be cross-linked to the hTim23 import intermediate in rat and yeast mitochondria . In a similar manner to Tim8p, TIMM8a seemingly mediates the import of hTim23 . Deafness/dystonia syndrome thus may be caused by decreased levels of Tim23 in the mitochondrial inner membrane in affected tissues.

Gastroenterology, 2002 Mar, 122(3), 689 - 96
Cdx2 ectopic expression induces gastric intestinal metaplasia in transgenic mice; Silberg DG et al.; BACKGROUND & AIMS: Intestinal-type gastric cancer is often preceded by intestinal metaplasia in humans . The genetic events responsible for the transdifferentiation that occurs in intestinal metaplasia are not well understood . Cdx2, a transcription factor whose expression is normally limited to the intestine, has been detected in gastric intestinal metaplasia . Cdx2 induces differentiation of intestinal epithelial cells in vitro; therefore, we sought to establish whether a causal relationship exists between Cdx2 activation and intestinal metaplasia . METHODS: Cdx2 expression was directed to the gastric mucosa in transgenic mice using cis-regulatory elements of Foxa3 (Hnf3gamma) . Transgenic mice were analyzed for histologic and gene expression changes . RESULTS: Histologic examination of the gastric mucosa of the Foxa3/Cdx2 mice revealed the presence of alcian blue-positive intestinal-type goblet cells, a hallmark of intestinal metaplasia . In addition, Cdx2 induced the expression of intestine-specific genes . CONCLUSIONS: Gastric expression of Cdx2 alone was sufficient to induce intestinal metaplasia in mice . These mice represent a powerful tool to investigate the molecular mechanisms that promote intestinal metaplasia . Moreover, as gastric cancer in humans is often preceded by intestinal metaplasia, the phenotype described here strongly suggests involvement of Cdx2 in the initiation of the process leading to intestinal neoplasia of the gastric mucosa.

Plant J, 2002 Mar, 29(5), 661 - 78
Redistribution of membrane proteins between the Golgi apparatus and endoplasmic reticulum in plants is reversible and not dependent on cytoskeletal networks; Saint-Jore CM et al.; We have fused the signal anchor sequences of a rat sialyl transferase and a human galactosyl transferase along with the Arabidopsis homologue of the yeast HDEL receptor (AtERD2) to the jellyfish green fluorescent protein (GFP) and transiently expressed the chimeric genes in tobacco leaves . All constructs targeted the Golgi apparatus and co-expression with DsRed fusions along with immunolabelling of stably transformed BY2 cells indicated that the fusion proteins located all Golgi stacks . Exposure of tissue to brefeldin A (BFA) resulted in the reversible redistribution of ST-GFP into the endoplasmic reticulum . This effect occurred in the presence of a protein synthesis inhibitor and also in the absence of microtubules or actin filaments . Likewise, reformation of Golgi stacks on removal of BFA was not dependent on either protein synthesis or the cytoskeleton . These data suggest that ER to Golgi transport in the cell types observed does not require cytoskeletal-based mechanochemical motor systems . However, expression of an inhibitory mutant of Arabidopsis Rab 1b (AtRab1b(N121I) significantly slowed down the recovery of Golgi fluorescence in BFA treated cells indicating a role for Rab1 in regulating ER to Golgi anterograde transport.

Eur J Biochem, 2002 Mar, 269(5), 1474 - 83
Identification of the 19S regulatory particle subunits from the rice 26S proteasome; Shibahara T et al.; The 26S proteasome, a protein complex consisting of a 20S proteasome and a pair of 19S regulatory particles (RP), is involved in ATP-dependent proteolysis in eukaryotes . In yeast, the RP contains six different ATPase subunits and, at least, 11 non-ATPase subunits . In this study, we identified the rice homologs of yeast RP subunit genes from the rice expressed sequence tag (EST) library . The complete nucleotide sequences of the homologs for five ATPase subunits, OsRpt1, OsRpt2, OsRpt4, OsRpt5 and OsRpt6, and five non-ATPase subunits, OsRpn7, OsRpn8, OsRpn10, OsRpn11 and OsRpn12, and the partial sequences of one ATPase subunit, OsRpt3, and six non-ATPase subunits, OsRpn1, OsRpn2, OsRpn3, OsRpn5, OsRpn6 and OsRpn9, were determined . Gene homologs of four ATPase subunits, OsRpt1, OsRpt2, OsRpt4 and OsRpt5, and three non- ATPase subunits, OsRpn1, OsRpn2 and OsRpn9, were found to be encoded by duplicated genes . The rice RP was purified by immunoaffinity chromatography with a Protein A column immobilized antibody against rice 20S proteasome, and the subunit composition was determined . The homologs obtained from the rice EST library were identified as genes encoding subunits of RP purified from rice, including the both products of duplicated genes by using electrospray ionization quadrupole time-of-flight mass spectrometry . Post-translational modifications and processing in rice RP subunits were also identified . Various types of RP complex with different subunit compositions are present in rice cells, suggesting the multiple functions of rice proteasome.

Eur J Biochem, 2002 Mar, 269(5), 1373 - 81
Uncoupling of protein-3 induces an uncontrolled uncoupling of mitochondria after expression in muscle derived L6 cells; Guerini D et al.; The uncoupling proteins (UCPs) are thought to uncouple oxidative phosphorylation in the mitochondria and thus generate heat . One of the UCP isoforms, UCP3, is abundantly expressed in skeletal muscle, the major thermogenic tissue in humans . UCP3 has been overexpressed at high levels in yeast systems, where it leads to the uncoupling of cell respiration, suggesting that UCP3 may indeed be capable of dissipating the mitochondrial proton gradient . This effect, however, was recently shown to be a consequence of the high level of expression and incorrect folding of the protein and not to its intrinsic uncoupling activity . In the present study, we investigated the properties of UCP3 overexpressed in a relevant mammalian host system such as the rat myoblast L6 cell line . UCP3 was expressed in relatively low levels (< 1 microg x mg(-1) membrane protein) with the help of an adenovirus vector . Immunofluorescence microscopy of transduced L6 cells showed that UCP3 was expressed in more than 90% of the cells and that its staining pattern was characteristic for mitochondrial localization . The oxygen consumption of L6 cells under nonphosphorylating conditions increased concomitantly with the levels of UCP3 expression . However, uncoupling was associated with an inhibition of the maximal respiratory capacity of mitochondria and was not affected by purine nucleotides and free fatty acids . Moreover, recombinant UCP3 was resistant to Triton X-100 extraction under conditions that fully solubilize membrane bound proteins . Thus, UCP3 can be uniformly overexpressed in the mitochondria of a relevant muscle-derived cell line resulting in the expected increase of mitochondrial uncoupling . However, our data suggest that the protein is present in an incompetent conformation.

J Biol Chem, 2002 May 17, 277(20), 18098 - 105 Epub 2002 Feb 28.
A proton pump ATPase with testis-specific E1-subunit isoform required for acrosome acidification; Sun-Wada GH et al.; The vacuolar-type H(+)-ATPases (V-ATPases) are a family of multimeric proton pumps involved in a wide variety of physiological processes . We have identified two novel mouse genes, Atp6e1 and Atp6e2, encoding testis-specific (E1) and ubiquitous (E2) V-ATPase subunit E isoforms, respectively . The E1 transcript appears about 3 weeks after birth, corresponding to the start of meiosis, and is expressed specifically in round spermatids in seminiferous tubules . Immunohistochemistry with isoform-specific antibodies revealed that the V-ATPase with E1 and a2 isoforms is located specifically in developing acrosomes of spermatids and acrosomes in mature sperm . In contrast, the E2 isoform was expressed in all tissues examined and present in the perinuclear compartments of spermatocytes . The E1 isoform exhibits 70% identity with the E2, and both isoforms functionally complemented a null mutation of the yeast counterpart VMA4, indicating that they are bona fide V-ATPase subunits . The chimeric enzymes showed slightly lower K(m)(ATP) than yeast V-ATPase . Consistent with the temperature-sensitive growth of Deltavma4-expressing E1 isoform, vacuolar membrane vesicles exhibited temperature-sensitive coupling between ATP hydrolysis and proton transport . These results suggest that E1 isoform is essential for energy coupling involved in acidification of acrosome.

J Biol Chem, 2002 May 24, 277(21), 18961 - 6 Epub 2002 Feb 28.
Interactions of phocein with nucleoside-diphosphate kinase, Eps15, and Dynamin I; Baillat G et al.; Phocein, an intracellular protein interacting with striatin, bears a few homologies with the varsigma-subunits of clathrin adaptor proteins (Baillat, G., Moqrich, A., Castets, F., Baude, A., Bailly, Y., Benmerah, A., and Monneron, A . (2001) Mol . Biol . Cell 12, 663-673) . Using phocein as a bait in a yeast two-hybrid screen, we identified two novel interacting proteins, nucleoside-diphosphate kinase (NDPK) and Eps15 . Immunoprecipitation and pull-down experiments involving native and/or recombinant phocein and, respectively, NDPK and Eps15, biochemically validated their interactions . NDPK and Eps15 were recently shown to be functional neighbors of dynamin . Dynamin I is shown here to directly interact with NDPK through its C-terminal proline-rich domain, whereas recombinant phocein associates with native dynamin I . Immunocytochemical studies of rat embryonic hippocampal neurons demonstrated partial co-localization of phocein and dynamin I . Phocein thus appears to be a component of the complexes involved in some steps of the vesicular traffic machinery.

J Biochem (Tokyo), 2002 Mar, 131(3), 349 - 57
Characterization of the biological functions of a transcription factor, c-myc intron binding protein 1 (MIBP1); Fukuda S et al.; The c-myc intron binding protein 1 (MIBP1) is a gigantic zinc finger protein comprising 2,437 amino acids and belonging to the MHC binding protein (MBP) family . MIBP1 is suggested to be a transcription factor involved in various biological functions . We show here that MIBP1 represses c-myc transcription from the major promoter, P2 . Screening by the yeast two-hybrid system revealed that the MIBP1 protein interacts with the Ski-interacting protein (SKIP) . In vitro pull-down assays and in vivo co-immunoprecipitation experiments confirmed this interaction . The acidic region of MIBP1 was found to be the site of interaction with the N-terminal half of SKIP . In situ hybridization analysis using developing rat embryos revealed that the MIBP1 mRNA is highly expressed in post-mitotic neurons, but the expression in immature neuroepithelium is low . The expression of MIBP1 in adult rat brain is also predominantly in neuronal cells, indicating that MIBP1 is involved in the physiology of mature neuronal cells.

J Biochem (Tokyo), 2002 Mar, 131(3), 327 - 36
Gamma-adaptin interacts directly with Rabaptin-5 through its ear domain; Shiba Y et al.; In yeast two-hybrid screening using gamma1-adaptin, a subunit of the AP-1 adaptor complex of clathrin-coated vesicles derived from the trans-Golgi network (TGN), as bait, we found that it could interact with Rabaptin-5, an effector of Rab5 and Rab4 that regulates membrane docking with endosomes . Further two-hybrid analysis revealed that the interaction occurs between the ear domain of gamma1-adaptin and the COOH-terminal coiled-coil region of Rabaptin-5 . Pull down assay with a fusion protein between glutathione S-transferase and the ear domain of gamma1-adaptin and coimmunoprecipitation analysis revealed that the interaction occurs in vitro and in vivo . Immunocytochemical analysis showed that gamma1-adaptin and Rabaptin-5 colocalize to a significant extent on perinuclear structures, probably on recycling endosomes, and are redistributed into the cytoplasm upon treatment with brefeldin A . These results suggest that the gamma1-adaptin-Rabaptin-5 interaction may play a role in membrane trafficking between the TGN and endosomes.

J Biochem (Tokyo), 2002 Mar, 131(3), 307 - 11
The second largest subunit of mouse DNA polymerase epsilon, DPE2, interacts with SAP18 and recruits the Sin3 co-repressor protein to DNA; Wada M et al.; DNA polymerase epsilon is essential for cell viability and chromosomal DNA replication in budding yeast . In addition, DNA polymerase epsilon may be involved in DNA repair and cell-cycle checkpoint control . The enzyme consists of at least four subunits in mammalian cells as well as in yeast . The largest subunit of DNA polymerase epsilon is responsible for polymerase activity . To date, the functions of the other subunits have remained unknown . With a view to elucidating the functions of the second largest subunit of mouse DNA polymerase epsilon (DPE2), yeast two-hybrid screening was performed to identify mouse proteins that interact with this subunit . SAP18, a polypeptide associated with co-repressor protein Sin3, was identified as an interacting protein . A part of the N-terminal region of DPE2 (comprising amino acids 85-250) was found to be responsible for the interaction with SAP18 . The interaction induced repression of transcription in reporter plasmid assays, which was inhibited by trichostatin A . These results indicate that DPE2 may recruit histone deacetylase (HDAC) to the replication fork to modify the chromatin structure.

J Cell Sci, 2002 Mar 1, 115(Pt 5), 931 - 40
Kinesins klp5(+) and klp6(+) are required for normal chromosome movement in mitosis; West RR et al.; Proper mitotic chromosome segregation requires dynamic interactions between spindle microtubules and kinetochores . Here we demonstrate that two related fission yeast kinesins, klp5(+) and klp6(+), are required for normal chromosome segregation in mitosis . Null mutants frequently lack a normal metaphase chromosome alignment . Chromosome pairs move back and forth along the spindle for an extended period prior to sister chromatid separation, a phenotype reminiscent of the loss of CENP-E in metazoans . Ultimately, sister chromatids segregate, regardless of chromosome position along the spindle, and viable daughter cells are usually produced . The initiation of anaphase B is sometimes delayed, but the rate of spindle elongation is similar to wildtype . Despite a delay, anaphase B often begins before anaphase A is completed . The klp5Delta and klp6Delta null mutants are synthetically lethal with a deletion of the spindle assembly checkpoint gene, bub1(+), several mutants in components of the anaphase promoting complex, and a cold sensitive allele of the kinetochore and microtubule-binding protein, Dis1p . Klp5p-GFP and Klp6p-GFP localize to kinetochores from prophase to the onset of anaphase A, but relocalize to the spindle midzone during anaphase B . These data indicate that Klp5p and Klp6p are kinetochore kinesins required for normal chromosome movement in prometaphase.

J Cell Sci, 2002 Mar 1, 115(Pt 5), 899 - 911
The small GTPase Rab22 interacts with EEA1 and controls endosomal membrane trafficking; Kauppi M et al.; Rab22a is a small GTPase that is expressed ubiquitously in mammalian tissues and displays the highest sequence homology to Rab5 . In BHK-21 cells, overexpression of the wild-type Rab22a caused formation of abnormally large vacuole-like structures containing the early-endosomal antigen EEA1 but not Rab11, a marker of recycling endosomes or the late-endosomal/lysosomal markers LAMP-1 and lyso-bis-phosphatidic acid . In HeLa cells, overexpressed Rab22a was found on smaller EEA1-positive endosomes, but a portion of the protein was also found in the Golgi complex . Using the yeast two-hybrid system and a biochemical pull-down assay, the GTP-bound form of Rab22a was found to interact with the N-terminus of EEA1 . In HeLa cells overexpressing Rab22a or its mutants affected in the GTPase cycle, no significant changes were observed in the uptake of Alexa-transferrin . However, the GTPase-deficient Rab22a Q64L mutant caused a redistribution of transferrin-positive endosomes to the leading edges of cells and a fragmentation of the Golgi complex . In BHK cells, the Q64L mutant caused the accumulation of a fluid phase marker, TRITC-dextran, and a lysosomal hydrolase, aspartylglucosaminidase, in abnormal vacuole-like structures that contained both early and late endosome markers . Both the wild-type Rab22a and the Q64L mutant were found to interfere with the degradation of EGF . These results suggest that Rab22a may regulate the dynamic interactions of endosomal compartments and it may be involved in the communication between the biosynthetic and early endocytic pathways.

Nippon Eiseigaku Zasshi, 2002 Jan, 56(4), 615 - 21
{Heme metabolism in stress response}; Ogawa K; Heme and its metabolism fulfill significant roles in many homeostatic and adaptative reactions . For example, heme (protein) senses oxygen concentration to regulate hypoxia response genes such as erythropoietin, and free heme, a proxidant, controls levels of several oxidative stress response proteins as well as that of a few enzymes in the heme metabolic pathway . Heme oxygenase (HO) is the key enzyme in heme catabolism, which degrades heme to Fe, CO, and biliverdin . CO is known as a gaseous messenger in the vascular and nervous systems . Biliverdin is rapidly converted to bilirubin, whose antioxidative effect is proposed to protect cells against reactive oxygen species . HO-1, the inducible isozyme of HO, is induced not only by its substrate heme, but also NO, metals, hypoxia, and various other stimuli . Studies on HO-1 deficiency indicate that induction of HO-1 is essential to homeostasis, at least in humans . Heme response elements (HREs), which mediate the induction of HO-1 expression by heme, are identified in enhancer regions of the mouse HO-1 gene . HRE shares a nucleotide sequence with the Maf recognition element (MARE) . A transcriptional activator, Nrf2, has been shown to participate in HO-1 induction by several stimuli, including heme via HRE . A heme-binding transcription factor such as yeast Hap1 had been supposed to also exist in vertebrates, however, no such factor had been identified . Recently, we found that a mammalian transcription repressor, Bach1, directly binds heme, and that the DNA binding activity of Bach1 is negatively regulated by heme . Bach1 is capable of competing the binding to MARE with activators including Nrf2, therefore, HO-1 and other stress response genes bearing MARE may be regulated by heme via the MARE-binding transcription factors . Further analyses on the gene regulatory mechanism by heme would help us to understand the stress response system, especially against oxidative stress.

Plant Cell Physiol, 2002 Feb, 43(2), 230 - 8
Characterization of tobacco MADS-box genes involved in floral initiation; Jang S et al.; MADS-box genes encode regulatory factors that are involved at various stages in plant development . These genes function not only during early floral meristem identity, but also when the fate of floral organ primordia is determined in a later step . Here, we screened a floral bud cDNA library to isolate a tobacco MADS-box gene, NtMADS4, using the rice MADS-box gene, OsMADS1, as a probe . We previously reported that OsMADS1 plays a critical role in flower development in rice . Ectopic expression of NtMADS4 caused phenotypes of extremely early flowering as well as dwarfism . Plant MADS proteins have a K domain that mediates the formation of dimers . This dimerization appears to be an essential step for a functional protein complex . NtMADS11 was isolated as an interacting partner of NtMADS4 by yeast two-hybrid screening . The latter was included in the AGAMOUS-like 2 (AGL2) family whereas the former was categorized in the SQUAMOSA (SQUA) family . While the transcript of NtMADS4 was detectable only in reproductive organs, that of NtMADS11 was seen in both reproductive and vegetative organs . Expression levels were high for both genes during early developmental stages . Ectopic expression of NtMADS11 and OsMADS14 was able to rescue the floral organ defects seen in the strong ap1-1 mutant . Roles of NtMADS4 and NtMADS11 in the floral initiation are discussed.

EMBO J, 2002 Mar 1, 21(5), 1004 - 11
Role of the PLC-related, catalytically inactive protein p130 in GABA(A) receptor function; Kanematsu T et al.; The protein p130 was isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-delta1 but lacking PLC activity . We show that p130 plays an important role in signaling by the type A receptor for gamma-aminobutyric acid (GABA) . Yeast twohybrid screening identified GABARAP (GABA(A) receptor-associated protein), which is proposed to contribute to the sorting, targeting or clustering of GABA(A) receptors, as a protein that interacts with p130 . Furthermore, p130 competitively inhibited the binding of the gamma2 subunit of the GABA(A) receptor to GABARAP in vitro . Electrophysiological analysis revealed that the modulation of GABA-induced Cl- current by Zn2+ or diazepam, both of which act at GABA(A) receptors containing gamma subunits, is impaired in hippocampal neurons of p130 knockout mice . Moreover, behavioral analysis revealed that motor coordination was impaired and the intraperitoneal injection of diazepam induced markedly reduced sedative and antianxiety effects in the mutant mice . These results indicate that p130 is essential for the function of GABA(A) receptors, especially in response to the agents acting on a gamma2 subunit.

J Steroid Biochem Mol Biol, 2002 Jan, 80(1), 125 - 30
Antiestrogenic activities of Cimicifuga racemosa extracts; Zierau O et al.; Despite the wide use of extracts from the rhizome of black cohosh (Cimicifuga racemosa) for the treatment of menopausal complaints, surprisingly little is known on their potential estrogenic properties, e.g . on estrogen dependent gene transcription . In addition, available informations on the effects on cell proliferation are contradictory . We therefore, tested for estrogenic and antiestrogenic effects of Cimicifuga racemosa extracts on proliferation of MCF-7 cells and on gene expression using ethanolic and iso-propanolic extracts of this medical plant . Estrogenic properties of plant extracts could neither be detected in proliferation assays, nor on gene expression using an estradiol-inducible yeast assay or the estrogen-inducible MVLN cells . In contrast, in all three experimental systems Cimicifuga racemosa antagonized estradiol induced activities . Estradiol induced stimulation of proliferation was inhibited by a dosage >1 microg/ml of extract concentration, gene expression was suppressed by doses of 100-1000 microg/ml of Cimicifuga racemosa extracts . From these results we conclude, that extracts from the rhizome of Cimicifuga racemosa contain compounds with antiestrogenic properties.

J Steroid Biochem Mol Biol, 2002 Jan, 80(1), 49 - 60
Oestrogenic activity of parabens in MCF7 human breast cancer cells; Byford JR et al.; Parabens (4-hydroxybenzoic acid esters) have been recently reported to have oestrogenic activity in yeast cells and animal models . Since the human population is exposed to parabens through their widespread use as preservatives in foods, pharmaceuticals and cosmetics, we have investigated here whether oestrogenic activity of these compounds can also be detected in oestrogen-sensitive human cells . We report on the oestrogenic effects of four parabens (methylparaben, ethylparaben, n-propylparaben, n-butylparaben) in oestrogen-dependent MCF7 human breast cancer cells . Competitive inhibition of {3H}oestradiol binding to MCF7 cell oestrogen receptors could be detected at 1,000,000-fold molar excess of n-butylparaben (86%), n-propylparaben (77%), ethyl-paraben (54%) and methylparaben (21%) . At concentrations of 10(-6)M and above, parabens were are able to increase expression of both transfected (ERE-CAT reporter gene) and endogenous (pS2) oestrogen-regulated genes in these cells . They could also increase proliferation of the cells in monolayer culture, which could be inhibited by the antiestrogen ICI 182,780, indicating that the effects were mediated through the oestrogen receptor . However, no antagonist activity of parabens could be detected on regulation of cell proliferation by 17 beta-oestradiol at 10(-10)M . Molecular modelling has indicated the mode by which paraben molecules can bind into the ligand binding pocket of the crystal structure of the ligand binding domain (LBD) of the oestrogen receptor alpha (ERalpha) in place of 17beta-oestradiol; it has furthermore shown that two paraben molecules can bind simultaneously in a mode in which their phenolic hydroxyl groups bind similarly to those of the meso-hexoestrol molecule . Future work will need to address the extent to which parabens can accumulate in hormonally sensitive tissues and also the extent to which their weak oestrogenic activity can add to the more general environmental oestrogen problem.

Reproduction, 2002 Feb, 123(2), 235 - 42
Oestrogenic activity of the hop phyto-oestrogen, 8-prenylnaringenin; Milligan S et al.; The female flowers of the hop plant (hop cones) are used as a preservative and as a flavouring agent in beer . A novel phyto-oestrogen, 8-prenylnaringenin, was recently identified in hops and this study was undertaken to characterize the oestrogenic activity of this compound using a combination of in vitro and in vivo assays . Natural and semi-synthetic 8-prenylnaringenin showed similar bioactivities both in a yeast screen transfected with the human oestrogen receptor and in oestrogen-responsive human Ishikawa Var-I cells . 8-Prenylnaringenin showed comparable binding activity to both oestrogen receptor isoforms (ER alpha and ER beta) . 8-Prenylnaringenin extracted from hops contains similar amounts of both (R)- and (S)- enantiomers, indicating that the compound is normally formed non-enzymatically . Both enantiomers showed similar bioactivity in vitro and similar binding characteristics to ER alpha and ER beta . The oestrogenic activity of 8-prenyl-naringenin in vitro was greater than that of established phyto-oestrogens such as coumestrol, genistein and daidzein . The high oestrogenic activity was confirmed in an acute in vivo test using uterine vascular permeability as an end point . When the compound was given to ovariectomized mice in their drinking water, oestrogenic stimulation of the vaginal epithelium required concentrations of 100 mug ml(-1) (about 500-fold greater than can be found in any beer).

Biochem Biophys Res Commun, 2002 Mar 8, 291(4), 732 - 7
Gurken, a TGF-alpha-like protein involved in axis determination in Drosophila, directly binds to the EGF-receptor homolog Egfr; Shmueli A et al.; The establishment of axial polarity in the Drosophila egg and embryo depends on intercellular communication between two cell types in the ovary, the germline, and the soma . The genes gurken and egfr encode two essential players of this communication pathway . Gurken protein, a TGF-alpha-like molecule, is expressed in the germline, while the EGF-receptor homolog, Egfr, is expressed in the somatic cells of the ovary . Using the yeast two-hybrid system we show here, for the first time, that Gurken protein directly binds to the extracellular domain of Egfr . This direct physical association requires the presence of an intact EGF motif within Gurken protein . Furthermore, we provide evidence that this characteristic motif may be sufficient for interaction with the receptor, at list in vitro . Our results firmly establish Gurken as the germline ligand of Drosophila Egfr.

J Infect Dis, 2002 Mar 1, 185(5), 573 - 83 Epub 2002 Feb 14.
Variability of the nonstructural 5A protein of hepatitis C virus type 3a isolates and relation to interferon sensitivity; Castelain S et al.; The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) genotype 1 is thought to interact with several cellular proteins, including the double-stranded RNA-dependent protein kinase (PKR) induced by interferon (IFN) . The PKR-binding domain (PKR-BD; aa 2209-2274), including the IFN sensitivity-determining region (aa 2209-2248) and other regions, could be linked to IFN resistance . Thus, the entire NS5A sequence of 27 isolates of HCV genotype 3a was investigated in relation to the clinical response to IFN . The NS5A 3a protein presented a low variability with some specific variable regions . Differential analysis between IFN-resistant and -sensitive isolates identified 5 regions in NS5A, 2 of them inside the PKR-BD and another around the variable 3 region . However, using the yeast growth suppression assay, no interaction was found between 5 resistant NS5A 3a proteins and PKR . Some amino acid changes of the NS5A protein of genotype 3a seemed to relate to IFN resistance independently of the PKR pathway.

Sci STKE . 1999 Sep 28;1999(1):PE1.
Signal transduction by MAP kinases: regulation by phosphorylation-dependent switches; Whitmarsh AJ et al.; The kinases of mitogen-activated protein (MAP) kinase cascades transmit signals through sequential phosphorylation and activation of the enzymes . However, recent evidence indicates that protein-protein interactions between the kinases themselves or with substrates or other components are also a critical means of regulation . Whitmarsh and Davis summarize these findings with emphasis on new evidence from yeast that, when phosphorylated, a MAP kinase kinase actually switches from a negative regulator that binds to and inhibits its target MAP kinase to a positive regulator of that same enzyme.

Mol Cell Biol, 2002 Mar, 22(6), 1858 - 67
The Rab27a/granuphilin complex regulates the exocytosis of insulin-containing dense-core granules; Yi Z et al.; Recently, we identified and characterized a novel protein, granuphilin, whose domain structure is similar to that of the Rab3 effector protein rabphilin3 (J . Wang, T . Takeuchi, H . Yokota, and T . Izumi, J . Biol . Chem . 274:28542-28548, 1999) . Screening its possible Rab partner by a yeast two-hybrid system revealed that an amino-terminal zinc-finger domain of granuphilin interacts with Rab27a . Granuphilin preferentially bound to the GTP form of Rab27a . Formation of the Rab27a/granuphilin complex was readily detected in the pancreatic beta cell line MIN6 . Moreover, the tissue distributions of Rab27a and granuphilin are remarkably similar: both had significant and specific expression in pancreatic islets and in pituitary tissue, but no expression was noted in the brain . Analyses by immunofluorescence, immunoelectron microscopy, and sucrose density gradient subcellular fractionation showed that Rab27a and granuphilin are localized on the membrane of insulin granules . These findings suggest that granuphilin functions as a Rab27a effector protein in beta cells . Overexpression of wild-type Rab27a and its GTPase-deficient mutant significantly enhanced high K(+)-induced insulin secretion without affecting basal insulin release . Although Rab3a, another exocytotic Rab protein, has some similarities with Rab27a in primary sequence, intracellular distribution, and affinity toward granuphilin, overexpression of Rab3a caused different effects on insulin secretion . These results indicate that Rab27a is involved in the regulated exocytosis of conventional dense-core granules possibly through the interaction with granuphilin, in addition to its recently identified role in lysosome-related organelles.

Mol Cell Biol, 2002 Mar, 22(6), 1714 - 22
Ral-GTPase influences the regulation of the readily releasable pool of synaptic vesicles; Polzin A et al.; The Ral proteins are members of the Ras superfamily of GTPases . Because they reside in synaptic vesicles, we used transgenic mice expressing a dominant inhibitory form of Ral to investigate the role of Ral in neurosecretion . Using a synaptosomal secretion assay, we found that while K(+)-evoked secretion of glutamate was normal, protein kinase C-mediated enhancement of glutamate secretion was suppressed in the mutant mice . Since protein kinase C effects on secretion have been shown to be due to enhancement of the size of the readily releasable pool of synaptic vesicles docked at the plasma membrane, we directly measured the refilling of this readily releasable pool of synaptic vesicles after Ca(2+)-triggered exocytosis . Refilling of the readily releasable pool was suppressed in synaptosomes from mice expressing dominant inhibitory Ral . Moreover, we found that protein kinase C and calcium-induced phosphorylation of proteins thought to influence synaptic vesicle function, such as MARCKS, synapsin, and SNAP-25, were all reduced in synaptosomes from these transgenic mice . Concomitant with these studies, we searched for new functions of Ral by detecting proteins that specifically bind to it in cells . Consistent with the phenotype of the transgenic mice described above, we found that active but not inactive RalA binds to the Sec6/8 (exocyst) complex, whose yeast counterpart is essential for targeting exocytic vesicles to specific docking sites on the plasma membrane . These findings demonstrate a role for Ral-GTPase signaling in the modulation of the readily releasable pool of synaptic vesicles and suggest the possible involvement of Ral-Sec6/8 (exocyst) binding in modulation of synaptic strength.

Curr Biol, 2002 Feb 19, 12(4), 277 - 87
The Caenorhabditis elegans Skp1-related gene family: diverse functions in cell proliferation, morphogenesis, and meiosis; Nayak S et al.; BACKGROUND: The SCF ubiquitin-ligase complex targets the ubiquitin-mediated degradation of proteins in multiple dynamic cellular processes . A key SCF component is the Skp1 protein that functions within the complex to link the substrate-recognition subunit to a cullin that in turn binds the ubiquitin-conjugating enzyme . In contrast to yeast and humans, Caenorhabditis elegans contains multiple expressed Skp1-related (skr) genes . RESULTS: The 21 Skp1-related (skr) genes in C . elegans form one phylogenetic clade, suggesting that a single ancestral Skp1 gene underwent independent expansion in C . elegans . The cellular and developmental functions of the 21 C . elegans skr genes were probed by dsRNA-mediated gene inactivation (RNAi) . The RNAi phenotypes of the skr genes fall into two classes . First, the highly similar skr-7, -8, -9, and -10 genes are required for posterior body morphogenesis, embryonic and larval development, and cell proliferation . Second, the related skr-1 and -2 genes are required for the restraint of cell proliferation, progression through the pachytene stage of meiosis, and the formation of bivalent chromosomes at diakinesis . CUL-1 was found to interact with SKR-1, -2, -3, -7, -8, and -10 in the yeast two-hybrid system . Interestingly, SKR-3 could interact with both CUL-1 and its close paralog CUL-6 . CONCLUSIONS: Members of the expanded skr gene family in C . elegans perform critical functions in regulating cell proliferation, meiosis, and morphogenesis . The finding that multiple SKRs are able to bind cullins suggests an extensive set of combinatorial SCF complexes.

Curr Biol, 2002 Feb 19, 12(4), 267 - 75
Multiple Skp1-related proteins in Caenorhabditis elegans: diverse patterns of interaction with Cullins and F-box proteins; Yamanaka A et al.; BACKGROUND: The ubiquitin-proteasome pathway of proteolysis controls the abundance of specific regulatory proteins . The SCF complex is a type of ubiquitin-protein ligase (E3) that contributes to this pathway in many biological systems . In yeast and mammals, the SCF complex consists of common components, including Skp1, Cdc53/Cul1, and Rbx1, as well as variable components known as F-box proteins . Whereas only one functional Skp1 gene is present in the human genome, the genome of Caenorhabditis elegans has now been shown to contain at least 21 Skp1-related (skr) genes . The biochemical properties, expression, and function of the C . elegans SKR proteins were examined . RESULTS: Of the 17 SKR proteins examined, eight (SKR-1, -2, -3, -4, -7, -8, -9, and -10) were shown to interact with C . elegans CUL1 by yeast two-hybrid analysis or a coimmunoprecipitation assay in mammalian cells . Furthermore, SKR proteins exhibited diverse binding specificities for C . elegans F-box proteins . The tissue specificity of expression of the CUL1-interacting SKR proteins was also varied . Suppression of skr-1 or skr-2 genes by double-stranded RNA interference resulted in embryonic death, whereas that of skr-7, -8, -9, or -10 was associated with slow growth and morphological abnormalities . CONCLUSIONS: The multiple C . elegans SKR proteins exhibit marked differences in their association with Cullins and F-box proteins, in tissue specificity of expression, and in phenotypes associated with functional suppression by RNAi . At least eight of the SKR proteins may, like F-box proteins, act as variable components of the SCF complex in C . elegans.

Zhonghua Yu Fang Yi Xue Za Zhi, 1999 May, 33(3), 156 - 7
{Component in diphtheria-pertussis-tetanus-hepatitis B vaccine}; Xie D et al.; OBJECTIVE: To study immunogenecity of yeast-derived recombinant hepatitis B (YHB) component in diphtheria-pertussis-tetanus-YHB vaccine (DPTw-YHB) . METHODS: Immunogenecity of tetra-valent DTPw-YHB vaccine and mono-valent recombinant YHB vaccine, and that of the tetra-valent vaccine with varied YHB component were compared . The efficiency and stability of recombinant YHB in the tetra-valent vaccine stored at 2 - 8 degrees C for 18 months were determined . RESULTS: The efficiency of recombinant YHB in the tetra-valent vaccine enhanced significantly in mice, as compared with that of mono-valent recombinant YHB vaccine, with an average mouse ED(50) of 1:2.0 - 1:3.1 . There was no significant difference in efficiency of the tetra-valent vaccine with varied recombinant YHB component . Recombinant YHBin the DPTw-YHB tetra-valent vaccine still kept good stability stored at 2 - 8 degrees C for 18 months . CONCLUSION: Recombinant YHB in the tetra-valent vaccine was more immunogenic than the mono-valent YHB vaccine . No interference and inhibition of DPTw to recombinant YHB was found, indicating good compatibility between DPTw and YHB.

Genomics, 2002 Mar, 79(3), 423 - 31
NAPP2, a peroxisomal membrane protein, is also a transcriptional corepressor; Gavva NR et al.; Nuclear factor-erythroid number 2 (NF-E2) is a positive regulatory, DNA binding transcription factor for gene expression in erythroid and megakaryocytic cells . To further understand the mechanisms of NF-E2 function, we used expression cloning to identify coregulators interacting with the erythroid-specific subunit of NF-E2, p45 . We have isolated a protein, NAPP2, which contains an aspartic-acid- and glutamic-acid-rich region and a nuclear localization signal . The gene encoding NAPP2, PEX14, is located on chromosome 1p36 and is ubiquitously expressed . The domains of interaction in vitro and in vivo between p45 and NAPP2 were mapped by a yeast two-hybrid system and cotransfection experiments . In mammalian cell culture, ectopically expressed NAPP2 inhibited p45-directed transcriptional activation . Furthermore, NAPP2 functions as a corepressor and interacts specifically with histone deacetylase l (HDAC1), but not HDAC2 or HDAC3 . NAPP2 is thus potentially a negative coregulator of NF-E2 . NAPP2 is identical to PEX14, an integral membrane protein essential for protein docking onto the peroxisomes . These studies have identified a novel, bifunctional protein capable of acting as a transcriptional corepressor and a polypeptide transport modulator . They also suggest that NF-E2 may function both positively and negatively in the transcription regulation of specific erythroid and megakaryocytic genes.

Genomics, 2002 Mar, 79(3), 274 - 7
Physical and transcript map of a 2-Mb region in Xp22.1 containing candidate genes for X-linked mental retardation and short stature; Zhang S et al.; Genetic loci for several diseases, including X-linked nonspecific mental retardation and short stature, have been mapped to Xp22.1 . In spite of the recent publications of two draft sequences for the human genome, this region seems to be largely unmapped and unsequenced . Here we report an integrated physical and transcript map of approximately 2-Mb from DXS8004 to DXS365 . Using sequence tagged site (STS)-content mapping and chromosome walking, we assembled a genomic clone contig of 54 BACs and one cosmid with an estimated 4.5-fold coverage of this region . The minimum tiling path consists of 23 BACs and one cosmid . Onto this contig, we mapped 30 new STSs derived from the unique end-sequences of the BACs, three expressed sequence tags, five genes, and seven CpG islands . This integrated map provides a unique resource for the positional cloning of candidate disease genes mapping to Xp22.1 and is therefore of value for the completion of the genomic sequence of this region.

Plant J, 2002 Jan, 29(2), 193 - 202
An ankyrin repeat-containing protein plays a role in both disease resistance and antioxidation metabolism; Yan J et al.; The Arabidopsis ankyrin repeat-containing protein AKR2 was identified as a GF14(lambda)-interacting protein in a yeast two-hybrid screening (GF14(lambda) is a 14-3-3 protein) . Reduced expression of AKR2 by using the antisense technique results in small necrotic areas in leaves accompanied by higher production of H2O2, similar to the hypersensitive response to pathogen infection in plant disease resistance . Transcripts of genes encoding pathogen-induced protein PR-1 (pathogen-related protein 1) and stress-responsive protein GST6 (glutathione S-transferase 6) are increased in antisense plants . The resistance to a bacterial pathogen infection was also increased by at least 10-fold in antisense plants . AKR2 also interacts with another GF14(lambda)-interacting protein, the ascorbate peroxidase 3 that scavenges H2O2 in plant cells . These data suggest that AKR2 may be a negative regulator of PR-1 expression, and is probably involved in the regulation of antioxidation metabolism that is shared by both disease resistance and stress responses.

Nucleic Acids Res, 2002 Mar 1, 30(5), 1163 - 8
Interaction generality, a measurement to assess the reliability of a protein-protein interaction; Saito R et al.; Here we introduce the 'interaction generality' measure, a new method for computationally assessing the reliability of protein-protein interactions obtained in biological experiments . This measure is basically the number of proteins involved in a given interaction and also adopts the idea that interactions observed in a complicated interaction network are likely to be true positives . Using a group of yeast protein-protein interactions identified in various biological experiments, we show that interactions with low generalities are more likely to be reproducible in other independent assays . We constructed more reliable networks by eliminating interactions whose generalities were above a particular threshold . The rate of interactions with common cellular roles increased from 63% in the unadjusted estimates to 79% in the refined networks . As a result, the rate of cross-talk between proteins with different cellular roles decreased, enabling very clear predictions of the functions of some unknown proteins . The results suggest that the interaction generality measure will make interaction data more useful in all organisms and may yield insights into the biological roles of the proteins studied.

J Virol, 2002 Mar, 76(6), 3065 - 71
Pseudorabies virus UL36 tegument protein physically interacts with the UL37 protein; Klupp BG et al.; The UL36 open reading frame encoding the tegument protein ICP1/2 represents the largest open reading frame in the genome of herpes simplex virus type 1 (HSV-1) . Polypeptides homologous to the HSV-1 UL36 protein are present in all subfamilies of HERPESVIRIDAE: We sequenced the UL36 gene of the alphaherpesvirus pseudorabies virus (PrV) and prepared a monospecific polyclonal rabbit antiserum against a bacterial glutathione S-transferase (GST)-UL36 fusion protein for identification of the protein . The antiserum detected a >300-kDa protein in PrV-infected cells and in purified virions . Interestingly, in coprecipitation analyses using radiolabeled infected-cell extracts, the anti-UL36 serum reproducibly coprecipitated the UL37 tegument protein, and antiserum directed against the UL37 protein coprecipitated the UL36 protein . This physical interaction could be verified using yeast two-hybrid analysis which demonstrated that the UL37 protein interacts with a defined region within the amino-terminal part of the UL36 protein . By use of immunogold labeling, capsids which accumulate in the cytoplasm in the absence of the UL37 protein (B . G . Klupp, H . Granzow, E . Mundt, and T . C . Mettenleiter, J . Virol . 75:8927-8936, 2001) as well as wild-type intracytoplasmic and extracellular virions were decorated by the anti-UL36 antiserum, whereas perinuclear primary enveloped virions were not . We postulate that the physical interaction of the UL36 protein, which presumably constitutes the innermost layer of the tegument (Z . Zhou, D . Chen, J . Jakana, F . J . Rixon, and W . Chiu, J . Virol . 73:3210-3218, 1999), with the UL37 protein is an important early step in tegumentation during virion morphogenesis in the cytoplasm.

J Virol, 2002 Mar, 76(6), 2871 - 80
Identification of temperature-sensitive mutations in the phosphoprotein of respiratory syncytial virus that are likely involved in its interaction with the nucleoprotein; Lu B et al.; The phosphoprotein (P) of human respiratory syncytial virus (RSV) is an essential component of the viral RNA polymerase, along with the large polymerase (L), nucleocapsid (N), and M2-1 proteins . By screening a randomly mutagenized P gene cDNA library, two independent mutations, one with a substitution of glycine at position 172 by serine (G172S) and the other with a substitution of glutamic acid at position 176 by glycine (E176G), were identified to result in the loss of N-P interaction at 37 degrees C in the yeast two-hybrid assay . Both P mutants exhibited greatly reduced activity in supporting the replication and transcription of an RSV minigenome replicon at 37 and 39 degrees C . The G172S and E176G mutations were introduced individually into the RSV A2 (rA2) antigenomic cDNA, and recombinant viruses, rA2-P172 and rA2-P176, were obtained . Both viruses replicate as well as wild-type A2 virus in both Vero and HEp-2 cells at 33 degrees C, but each mutant virus exhibited temperature-sensitive replication in both cell lines . rA2-P176 is more temperature sensitive than rA2-P172 . Coimmunoprecipitation of the N protein with each P mutant from virus-infected cells demonstrates that N-P interaction is impaired at 37 degrees C . In addition, the levels of replication of rA2-P172 and rA2-P176 in the lungs of mice and cotton rats were reduced . As is the case with the in vitro assays, rA2-P176 is more restricted in replication in the lower respiratory tract of mice and cotton rats than rA2-P172 . During in vitro passage at 37 degrees C, the E176G mutation in rA2-P176 was rapidly changed from glycine to predominantly aspartic acid; mutations to cysteine or serine were also detected . All of the revertants lost the temperature-sensitive phenotype . To analyze the importance of the amino acids in the region from positions 161 to 180 for the P protein function, additional mutations were introduced and their functions were analyzed in vitro . A double mutant containing both G172S and E176G changes in the P gene, substitution of the three charged residues at positions 174 to 176 by alanine, and a deletion of residues from positions 161 to 180 completely abolished the P protein function in the minigenome assay . Thus, the amino acids at positions 172 and 176 and the adjacent charged residues play critical roles in the function of the P protein.

Development, 2002 Feb, 129(4), 957 - 64
Manipulation of leaf shape by modulation of cell division; Wyrzykowska J et al.; The role of cell division as a causal element in plant morphogenesis is debatable, with accumulating evidence supporting the action of cell division-independent mechanisms . To directly test the morphogenic function of cell division, we have utilised a microinduction technique to locally and transiently manipulate the expression in transgenic plants of two genes encoding putative effectors of the cell cycle, a tobacco A-type cyclin and a yeast cdc25 . The results show that local expression of these genes leads to modulation of cell division patterns . Moreover, whereas altered cell division in the apical meristem had no influence on organogenesis, local induction of cell proliferation on the flanks of young leaf primordia led to a dramatic change in lamina development and, thus, leaf shape . These data indicate that the role of cell division in plant morphogenesis is context dependent and identify cell division in the leaf primordium as a potential target for factors regulating leaf shape.

Blood, 2002 Mar 1, 99(5), 1794 - 801
Macrophage functional maturation and cytokine production are impaired in C/EBP epsilon-deficient mice; Tavor S et al.; Members of the CCAAT/enhancer-binding protein (C/EBP) family are involved in the regulation of cellular differentiation and function of many tissues . Unlike the other members of the family, C/EBP epsilon expression is restricted to granulocytes, macrophages, and lymphocytes . C/EBP epsilon is highly conserved between human and rodents and is essential for terminal granulopoiesis in both species . To study the role that C/EBP epsilon plays in macrophages, wild-type and C/EBP epsilon-deficient (-/-) murine macrophages obtained from thioglycollate-elicited peritoneal lavages and differentiated bone marrow cells were compared . Although macrophage development occurred in both types of mice, the C/EBP epsilon -/- cells had a lower expression of macrophage markers and a morphologic and ultrastructural appearance of immaturity . Phagocytic function, measured by calculating the percentage of internalized opsonized fluorescein isothiocyanate (FITC)-labeled yeast, was significantly impaired in the C/EBP epsilon -/- macrophages compared with their wild-type counterparts . Furthermore, the differential expression of 26 macrophage-specific genes between wild-type and C/EBP-/- mice was analyzed . A subset of genes involved in differentiation, immune, and inflammatory responses was found down-regulated in the C/EBP-/- macrophages . Taken together, this study implicates the C/EBP epsilon gene as an important transcription factor required for normal function and development of macrophages.

Zhonghua Yu Fang Yi Xue Za Zhi, 2000 Jan, 34(1), 44 - 6
{Characteristics of producing-fumonisin and dimorphic fungus of fusarium moniliforme}; Wang Z et al.; OBJECTIVE: To study the carcinogenesis of fusarium moniliforme and its fumonisins in animals . METHODS: The producing-fumonisin and dimorphic fungus pathogenicity of F . moniliforme from Zhejiang, China were identified and characterized . RESULTS: The F . moniliforme isolated from foodstuffs was shown to produces fumonisin B(1) > 400 microg/g in medium by IC-ELISA . Ten foodstuffs samples showed (2% - 100%) detection rate of F . moniliforme and contained fumonisin B(1) between (3.7 - 143.1) microg/g . The strains of F . moniliforme formed yeast-like colony in Sabouraud's agar plates contained 9% NaCl at 37degrees C incubator and budding multiplication mostly . In blood agar plates the strains of F . moniliforme showed grass green haemolytic reactions . CONCLUSION: It is important to carry out (or to perform) oulepidemiologicol survey of F . moniliforme as a primary pathogenic fungus, as well as the mycotoxins of F . moniliforme in vitro.

Oral Microbiol Immunol, 2002 Feb, 17(1), 60 - 4
Candida attachment to oral epithelium; Vitkov L et al.; Inflamed oral mucosa biopsies from patients with thrush and high candidal density were observed in a transmission electron microscope (TEM) using ultra-histochemical staining with ruthenium red for glycocalyx visualization . Fimbriae comprising the glycocalyx and enabling yeast adhesion to epithelial cells were clearly visualized by ruthenium red . All internalized portions of the yeast walls were devoid of glycocalyx, indicating that the growing tips of the hyphae mechanically penetrated the host cells . The attachment of Candida occurred in two ways: by fimbria-mediated adhesion enabling colonization of the epithelial surface, and by invasion of the superficial epithelial cells via hyphae . As the interaction between adhesin receptors and adhesins stimulates the production of proinflammatory cytokines, Candida adhesion itself is assumed to induce mucosal inflammation.

Oral Microbiol Immunol, 2002 Feb, 17(1), 44 - 9
Molecular characterization of Candida spp . isolated from the oral cavities of patients from diverse clinical settings; Al-Karaawi ZM et al.; Infections by Candida spp . have increased in medical importance over the past few decades . Our understanding of species identification, commensalisms, pathogenicity, person-to-person spread, and the development of antifungal resistance within specific strains has been greatly enhanced by the utilization of molecular epidemiological methodology . The aim of the current research was to assess the quantity, species and molecular characterization of oral yeast isolates from well-defined cohorts of immunocompetent patients from a diverse range of clinical settings . Oral rinse samples were assessed for the growth of yeast and degree of colonization . Isolates were defined to the species level by both phenotypic and molecular methods and strains were further genotypically subtyped . Significant variation was shown to exist in the number, species and genotypic subgroups of yeast isolated from the oral cavity in different patient groups . This variation could be attributed to the local oral conditions unique to these patient groups.

Mol Cell Neurosci, 2002 Feb, 19(2), 292 - 306
Protein kinase C delta (PKCdelta) is required for protein tyrosine phosphatase mu (PTPmu)-dependent neurite outgrowth; Rosdahl JA et al.; Protein tyrosine phosphatase mu (PTPmu) is an adhesion molecule in the immunoglobulin superfamily and is expressed in the developing nervous system . We have shown that PTPmu can promote neurite outgrowth of retinal ganglion cells and it regulates neurite outgrowth mediated by N-cadherin (S . M . Burden-Gulley and S . M . Brady-Kalnay, 1999, J . Cell Biol . 144, 1323-1336) . We previously demonstrated that PTPmu binds to the scaffolding protein RACK1 in yeast and mammalian cells (T . Mourton et al., 2001, J . Biol . Chem . 276, 14896-14901) . RACK1 is a receptor for activated protein kinase C (PKC) . In this article, we demonstrate that PKC is involved in PTPmu-dependent signaling . PTPmu, RACK1, and PKCdelta exist in a complex in cultured retinal cells and retinal tissue . Using pharmacologic inhibition of PKC, we demonstrate that PKCdelta is required for neurite outgrowth of retinal ganglion cells on a PTPmu substrate . These results suggest that PTPmu signaling via RACK1 requires PKCdelta activity to promote neurite outgrowth . (C)2002 Elsevier Science (USA).

Plant Mol Biol, 2002 Jan, 48(1-2), 173 - 82
Gene replacement by homologous recombination in plants; Puchta H; After the elucidation of the sequence of the yeast genome a major effort was started to elucidate the biological function of all open reading frames of this organisms by targeted gene replacement via homologous recombination . The establishment of the complete sequence of the genome of Arabidopsis thaliana would principally allow a similar approach . However, over the past dozen years all attempts to establish an efficient gene targeting technique in flowering plants were in the end not successful . In contrast, in Physcomitrella patens an efficient gene targeting procedure has been set up, making the moss a valuable model system for plant molecular biologists . But also for flowering plants recently several new approaches--some of them based on the availability of the genomic sequence of Arabidopsis--were initiated that might finally result on the set up of a general applicable technique . Beside the production of hyper-recombinogenic plants either via expression or suppression of specific gene functions or via undirected mutagenesis, the application of chimeric oligonucleotides might result in major progress.

Plant Mol Biol, 2002 Jan, 48(1-2), 133 - 41
Large-scale plant proteomics; Kersten B et al.; Large-scale and high throughput approaches increasingly play an essential role in the study of biological systems, which are per se highly complex . Therefore, they need to be examined by these extensive methods to receive information about the large genomic and proteomic networks . In plant biology, this purpose has a strong support through the accessability of the complete genome sequence of the model plant Arabidopsis thaliana . This brief review intends to focus on the basics and the state-of-the-art of these high-throughput technologies and their application to plant proteomics . It describes protein microarrays, the use of antibodies, 2-DE and MS methods and the yeast two hybrid system, which are emerging as the major technologies for plant proteomics.

Biochem Genet, 2001 Dec, 39(11-12), 369 - 77
Cloning, tissue expression pattern, and chromosome location of a novel human gene BRI3BP; Lin L et al.; A novel cDNA fragment was identified from a human fetal brain cDNA library by using the coding sequence of human BRI3 gene (Accession No . NM015379) as bait in a yeast two-hybrid screening . Then by 5'-RACE (rapid amplification of cDNA end) and electronic hybridization, we obtained a 1.9 kb contig which consists of a novel gene . It was designated as BRI3BP by the HUGO Nomenclature Committee . It contains an open reading frame encoding 251 amino acids . The calculated molecular weight of the deduced protein is 27.8 kU . The predicted isoelectric point is 9.48 . Northern hybridization showed its mRNA was highly expressed in brain, kidney, and liver . By RH mapping, the BRI3BP gene was mapped to human chromosome 12q24.2-qter

Acta Derm Venereol, 2001 Nov-Dec, 81(6), 418 - 22
Serum IgE reactivity to Malassezia furfur extract and recombinant M . furfur allergens in patients with atopic dermatitis; Zargari A et al.; IgE reactivity to the opportunistic yeast Malassezia furfur can be found in patients with atopic dermatitis (AD) . We have previously cloned and expressed 6 recombinant allergens (rMal f 1, rMal f 5-9) from M . furfur . In the present study, we used ImmunoCAP to investigate whether these rMal f allergens can be useful in the diagnosis of M . furfur-associated AD compared with the M . furfur extract . A total of 156 adult patients with a clinical diagnosis of AD participated in the study . Sixty-four percent had increased total serum IgE levels, 79% had specific IgE antibodies to common inhalant allergens and 47% had IgE antibodies to M . furfur extract . IgE antibodies to any of the rMal f allergens were detected among 86 (55%) of the patients, 14 (16%) of whom did not react to the M . furfur extract . Any individual rMal f allergen detected between 32% and 89% of the patients ImmunoCAP-positive to the M . furfur extract, with the highest sensitivity for rMal f 9 . Therefore, a couple of individual rMal f allergens can improve the diagnosis of M . furfur-associated IgE allergies in patients with AD.

Planta Med, 2002 Feb, 68(2), 164 - 7
Composition and antifungal activity of the essential oil of Solidago chilensis; Vila R et al.; Volatile constituents of the essential oils from leaves and inflorescences of Solidago chilensis Meyen were analyzed by GC-FID, GC-MS and 13C-NMR and thirty-six different compounds were identified . Pumiloxide, an unusual labdane diterpene, was found to be one of the major components in both oils (15.3 % and 12.3 %, respectively) . Other important constituents were limonene and several sesquiterpenes, mainly gamma-cadinene . The antifungal activity of the leaf oil was assayed against five different strains of filamentous fungi and one yeast . Paper disk agar diffusion test showed human pathogenic dermatophytes to be the most sensitive.

Science, 2002 Mar 15, 295(5562), 2084 - 8 Epub 2002 Feb 21.
Structural insights into group II intron catalysis and branch-site selection; Zhang L et al.; Group II self-splicing introns catalyze autoexcision from precursor RNA transcripts by a mechanism strikingly similar to that of the spliceosome, an RNA-protein assembly responsible for splicing together the protein-coding parts of most eukaryotic pre-mRNAs . Splicing in both cases initiates via nucleophilic attack at the 5' splice site by the 2' OH of a conserved intron adenosine residue, creating a branched (lariat) intermediate . Here, we describe the crystal structure at 3.0 A resolution of a 70-nucleotide RNA containing the catalytically essential domains 5 and 6 of the yeast ai5gamma group II self-splicing intron, revealing an unexpected two-nucleotide bulged structure around the branch-point adenosine in domain 6.

J Nat Prod, 2002 Feb, 65(2), 170 - 4
Bioactive saponins from Acacia tenuifolia from the suriname rainforest; Seo Y et al.; Bioassay-guided fractionation of the MeOH extract of Acacia tenuifolia using the engineered yeast strains 1138, 1140, 1353, and Sc7 as the bioassay tool resulted in the isolation of the three new saponins 3, 5, and 6 and the three known saponins 1, 2, and 4 . The structures of the new compounds were established on the basis of HRMS, 1D and 2D NMR spectral data on the intact saponins, and GC-MS analyses of the sugars . Compounds 1,2 and 5,6 showed cytotoxicity against mammalian cell lines.

Oncogene, 2002 Feb 21, 21(9), 1381 - 90
The putative tumor suppressor RASSF1A homodimerizes and heterodimerizes with the Ras-GTP binding protein Nore1; Ortiz-Vega S et al.; Nore and RASSF1A are noncatalytic proteins that share 50% identity over their carboxyterminal 300 AA, a segment that encompasses a putative Ras-Rap association (RA) domain . RASSF1 is expressed as several splice variants, each of which contain an RA domain, however the 340 AA RASSF1A, but not the shorter RASSF1C variant, is a putative tumor suppressor . Nore binds to Ras and several Ras-like GTPases in a GTP dependent fashion however neither RASSF1 (A or C) or the C . elegans Nore/RASSF1 homolog, T24F1.3 exhibit any interaction with Ras or six other Ras-like GTPases in a yeast two-hybrid expression assay . A low recovery of RASSF1A (but not RASSF1C) in association with RasG12V is observed however on transient expression in COS cells . Nore and RASSF1A can each efficiently homodimerize and heterodimerize with each other through their nonhomologous aminoterminal segments . Recombinant RASSF1C exhibits a much weaker ability to homodimerize or heterodimerize; thus the binding of RASSF1C to Nore is very much less than the binding of RASSF1A to Nore . The association of RASSF1A with RasG12V in COS cells appears to reflect the heterodimerization of RASSF1A with Nore, inasmuch the recovery of RASSF1A with RasG12V is increased by concurrent expression of full length Nore, and abolished by expression of Nore deleted of its RA domain . The preferential ability of RASSF1A to heterodimerize with Nore and thereby associate with Ras-like GTPases may be relevant to its putative tumor suppressor function.

Cytogenet Cell Genet, 2001, 94(3-4), 137 - 41
The pericentromeric region of human chromosome 11: evidence for a chromosome-specific duplication; Zhang J et al.; We have identified a chromosome duplication in the pericentromeric region of human chromosome 11 located in 11p11 and 11q14 . A detailed physical map of each duplicated region was generated to describe the nature of the duplication, the involvement at the centromere and to resolve the correct maps . All clones were evaluated to ensure they were representative of their genetic origin . The order of clones, based on their marker content, as well as the distance covered was determined by SEGMAP . Each duplication encompasses more than 1 Mb of DNA and appears to be chromosome 11 specific . Ten STS markers were mapped within each duplication . Comparative sequence analysis along the duplication identified 35 nucleotide changes in 2,036 bp between the two copies, suggesting the duplication occurred over 14 million years ago . A suggested organization of the pericentromeric region, including the duplications and alpha-related repetitive sequences, is presented .

J Biol Chem, 2002 May 3, 277(18), 16139 - 46 Epub 2002 Feb 20.
AUF1 Is a bcl-2 A + U-rich element-binding protein involved in bcl-2 mRNA destabilization during apoptosis; Lapucci A et al.; We previously identified a conserved A + U-rich element (ARE) in the 3'-untranslated region of bcl-2 mRNA . We have also recently demonstrated that the bcl-2 ARE interacts with a number of ARE-binding proteins (AUBPs) whose pattern changes during apoptosis in association with bcl-2 mRNA half-life reduction . Here we show that the AUBP AUF1 binds in vitro to bcl-2 mRNA . The results obtained in a yeast RNA three-hybrid system have demonstrated that the 1-257-amino acid portion of p37 AUF1 (conserved in all isoforms), containing the two RNA recognition motifs, also binds to the bcl-2 ARE in vivo . UVC irradiation-induced apoptosis results in an increase of AUF1 . Inhibition of apoptosis by a general caspase inhibitor reduces this increase by 2-3-fold . These results indicate involvement of AUF1 in the ARE/AUBP-mediated modulation of bcl-2 mRNA decay during apoptosis.

Mycoses, 2002 Feb, 45(1-2), 19 - 21
Antifungal activity of Crotalus durissus cumanensis venom; Magaldi S et al.; The susceptibility to Crotalus venom of 14 yeast and 10 mould fungal isolates was assessed . This venom was tested in a standardized well diffusion test, using 400 microg/20 microl well . The percentage of susceptibility to yeast isolates was 78.6% (> 8 mm); that for filamentous isolates was 50% (> 8 mm).

Genes Cells, 2002 Jan, 7(1), 19 - 27
Coaction of DNA topoisomerase IIIalpha and a RecQ homologue during the germ-line mitosis in Caenorhabditis elegans; Kim YC et al.; BACKGROUND: Among the four RecQ homologues predicted from the Caenorhabditis elegans genomic DNA sequence, T04A11.6 is most similar to Bloom syndrome's protein in humans . To investigate a possible interaction of the protein with topoisomerase IIIalpha (TOP3alpha), as observed between TOP3 and RecQ homologues in yeast and human, the top3alpha gene expression was suppressed by RNA interference (RNAi) in the him-6(e1104) C . elegans strain which is mutated in T04A11.6 (F . Mueller & C . Wicky, personal communication) . RESULTS: Germ cells in the gonads of the progeny him-6(e1104);top3alpha(RNAi) showed severe chromosomal abnormalities and were arrested during mitosis with a subsequent failure in meiotic entry . Most of the aberrant chromosomes were stained by the TUNEL assay but not by the SYTO12 dye, suggesting extensive DNA breaks not associated with apoptosis . The phenotypes in the germ cells of him-6(e1104);top3alpha(RNAi) were also observed in the progeny produced by double RNA interference of the top3alpha and him-6 gene expression, though at a reduced level . The over-expressed TOP3alpha and Him-6 proteins showed specific physical interaction in vitro, in agreement with the genetic interaction in C . elegans . CONCLUSION: In C . elegans, TOP3alpha and the RecQ homologue (T04A11.6) contribute to genome stability during germ-line mitosis, probably by acting in a complex.

Eur J Biochem, 2002 Jan, 269(2), 638 - 49
Dematin interacts with the Ras-guanine nucleotide exchange factor Ras-GRF2 and modulates mitogen-activated protein kinase pathways; Lutchman M et al.; Erythroid dematin is a major component of red blood cell junctional complexes that link the spectrin-actin cytoskeleton to the overlying plasma membrane . Transcripts of dematin are widely distributed including human brain, heart, lung, skeletal muscle, and kidney . In vitro, dematin binds and bundles actin filaments in a phosphorylation-dependent manner . The primary structure of dematin consists of a C-terminal domain homologous to the 'headpiece' domain of villin, an actin-binding protein of the brush border cytoskeleton . Except filamentous actin, no other binding partners of dematin have been identified . To investigate the physiological function of dematin, we employed the yeast two-hybrid assay to identify dematin-interacting proteins in the adult human brain . Here, we show that dematin interacts with the guanine nucleotide exchange factor Ras-GRF2 by yeast two-hybrid assay, and this interaction is further confirmed by blot overlay, surface plasmon resonance, co-transfection, and co-immunoprecipitation assays . Human Ras-GRF2 is expressed in a variety of tissues and, similar to other guanine nucleotide exchange factors (GEFs), displays anchorage independent growth in soft agar . Co-transfection and immunoblotting experiments revealed that dematin blocks transcriptional activation of Jun by Ras-GRF2 and activates ERK1 via a Ras-GRF2 independent pathway . Because much of the present evidence has centered on the identification of the Rho family of GTPases as key regulators of the actin cytoskeleton, the direct association between dematin and Ras-GRF2 may provide an alternate mechanism for regulating the activation of Rac and Ras GTPases via the actin cytoskeleton.

Eur J Biochem, 2002 Jan, 269(2), 538 - 45
NBR1 interacts with fasciculation and elongation protein zeta-1 (FEZ1) and calcium and integrin binding protein (CIB) and shows developmentally restricted expression in the neural tube; Whitehouse C et al.; NBR1 (named as next to BRCA1) was originally cloned as a candidate gene for the ovarian cancer antigen CA125, using expression cloning with the anti-CA125 Ig, OC125 . NBR1 has been of interest due to its position close to BRCA1, although no involvement in breast or ovarian cancer has been demonstrated . Recently, the antigen CA125 has been cloned, and identified as a new mucin, MUC16, entirely different from NBR1 . The function of NBR1 remains unknown . To investigate its function, a yeast two-hybrid study was performed to identify interacting protein partners that may reflect a biological role for this protein . Here, we show that NBR1 interacts with two proteins; fasciculation and elongation protein zeta-1 (FEZ1), a PKCzeta interacting protein, and calcium and integrin binding protein (CIB), which is associated with polo-like kinases Fnk/Snk and the Alzheimer's disease presenilin 2 protein . Co-transfection of FEZ1 and NBR1 showed overlapping localization in the cytoplasm, whereas coexpression of NBR1 and CIB resulted in a shift of CIB protein expression from the nucleus to the perinuclear compartment . FEZ1 is highly expressed in the brain and in situ hybridization analysis of Nbr1 showed that its expression is also regulated in the murine brain during development . These data suggest that NBR1 may function, through interaction with CIB and FEZ1 in cell signalling pathways, with a developmentally restricted expression suggesting a possible role in neural development.

Exp Cell Res, 2002 Mar 10, 274(1), 1 - 8
Evidence for repeat-induced gene silencing in cultured Mammalian cells: inactivation of tandem repeats of transfected genes; McBurney MW et al.; Foreign DNA can be readily integrated into the genomes of mammalian embryonic cells by retroviral infection, DNA microinjection, and transfection protocols . However, the transgenic DNA is frequently not expressed or is expressed at levels far below expectation . In a number of organisms such as yeast, plants, Drosophila, and nematodes, silencing of transfected genes is triggered by the interaction between adjacent or dispersed copies of genes of identical sequence . We set out to determine whether a mechanism similar to repeat-induced gene silencing (RIGS) is responsible for the silencing of transgenes in murine embryonal carcinoma stem cells . We compared the expression of identical reporter gene constructs in cells carrying single or multiple copies and found that the level of expression per integrated copy was more than 10-fold higher in single-copy integrants . In cells carrying tandem copies of the transgene, many copies were methylated and clones frequently failed to express both copies of near-identical integrated alleles . Addition of extra copies of the reporter gene coding sequence reduced the level of expression from the same reporter driven by a eukaryotic promoter . We also found that inhibitors of histone deacetylase such as trichostatin A forestall the silencing of multicopy transgenes, suggesting that chromatin mediates the silencing of transfected genes . This evidence is consistent with the idea that RIGS does occur in mammalian embryonic stem cells although silencing of single-copy transgenes also occurs, suggesting that RIGS is only one of the mechanisms responsible for triggering transgene silencing .

Mol Biol Cell, 2002 Feb, 13(2), 670 - 82
Identification and characterization of ART-27, a novel coactivator for the androgen receptor N terminus; Markus SM et al.; The androgen receptor (AR) is a ligand-regulated transcription factor that stimulates cell growth and differentiation in androgen-responsive tissues . The AR N terminus contains two activation functions (AF-1a and AF-1b) that are necessary for maximal transcriptional enhancement by the receptor; however, the mechanisms and components regulating AR transcriptional activation are not fully understood . We sought to identify novel factors that interact with the AR N terminus from an androgen-stimulated human prostate cancer cell library using a yeast two-hybrid approach designed to identify proteins that interact with transcriptional activation domains . A 157-amino acid protein termed ART-27 was cloned and shown to interact predominantly with the AR(153-336), containing AF-1a and a part of AF-1b, localize to the nucleus and increase the transcriptional activity of AR when overexpressed in cultured mammalian cells . ART-27 also enhanced the transcriptional activation by AR(153-336) fused to the LexA DNA-binding domain but not other AR N-terminal subdomains, suggesting that ART-27 exerts its effect via an interaction with a defined region of the AR N terminus . ART-27 interacts with AR in nuclear extracts from LNCaP cells in a ligand-independent manner . Interestingly, velocity gradient sedimentation of HeLa nuclear extracts suggests that native ART-27 is part of a multiprotein complex . ART-27 is expressed in a variety of human tissues, including sites of androgen action such as prostate and skeletal muscle, and is conserved throughout evolution . Thus, ART-27 is a novel cofactor that interacts with the AR N terminus and plays a role in facilitating receptor-induced transcriptional activation.

Mol Biol Cell, 2002 Feb, 13(2), 579 - 92
Interactions of elongation factor 1alpha with F-actin and beta-actin mRNA: implications for anchoring mRNA in cell protrusions; Liu G et al.; The targeting of mRNA and local protein synthesis is important for the generation and maintenance of cell polarity . As part of the translational machinery as well as an actin/microtubule-binding protein, elongation factor 1alpha (EF1alpha) is a candidate linker between the protein translation apparatus and the cytoskeleton . We demonstrate in this work that EF1alpha colocalizes with beta-actin mRNA and F-actin in protrusions of chicken embryo fibroblasts and binds directly to F-actin and beta-actin mRNA simultaneously in vitro in actin cosedimentation and enzyme-linked immunosorbent assays . To investigate the role of EF1alpha in mRNA targeting, we mapped the two actin-binding sites on EF1alpha at high resolution and defined one site at the N-terminal 49 residues of domain I and the other at the C-terminal 54 residues of domain III . In vitro actin-binding assays and localization in vivo of recombinant full-length EF1alpha and its various truncates demonstrated that the C terminus of domain III was the dominant actin-binding site both in vitro and in vivo . We propose that the EF1alpha-F-actin complex is the scaffold that is important for beta-actin mRNA anchoring . Disruption of this complex would lead to delocalization of the mRNA . This hypothesis was tested by using two dominant negative polypeptides: the actin-binding domain III of EF1alpha and the EF1alpha-binding site of yeast Bni1p, a protein that inhibits EF1alpha binding to F-actin and also is required for yeast mRNA localization . We demonstrate that either domain III of EF1alpha or the EF1alpha-binding site of Bni1p inhibits EF1alpha binding to beta-actin mRNA in vitro and causes delocalization of beta-actin mRNA in chicken embryo fibroblasts . Taken together, these results implicate EF1alpha in the anchoring of beta-actin mRNA to the protrusion in crawling cells.

J Biol Chem, 2002 May 3, 277(18), 15985 - 91 Epub 2002 Feb 19.
A novel zinc finger protein interacts with receptor-interacting protein (RIP) and inhibits tumor necrosis factor (TNF)- and IL1-induced NF-kappa B activation; Chen D et al.; Receptor-interacting protein (RIP) is a serine/threonine protein kinase that is critically involved in tumor necrosis factor receptor-1 (TNF-R1)-induced NF-kappa B activation . In a yeast two-hybrid screening for potential RIP-interacting proteins, we identified ZIN (zinc finger protein inhibiting NF-kappa B), a novel protein that specifically interacts with RIP . ZIN contains four RING-like zinc finger domains at the middle and a proline-rich domain at the C terminus . Overexpression of ZIN inhibits RIP-, IKK beta-, TNF-, and IL1-induced NF-kappa B activation in a dose-dependent manner in 293 cells . Domain mapping experiments indicate that the RING-like zinc finger domains of ZIN are required for its interaction with RIP and inhibition of RIP-mediated NF-kappa B activation . Overexpression of ZIN also potentiates RIP- and TNF-induced apoptosis . Moreover, immunofluorescent staining indicates that ZIN is a cytoplasmic protein and that it colocalizes with RIP . Our findings suggest that ZIN is an inhibitor of TNF- and IL1-induced NF-kappa B activation pathways.

Hum Mol Genet, 2002 Feb 15, 11(4), 389 - 96
KRIT1 association with the integrin-binding protein ICAP-1: a new direction in the elucidation of cerebral cavernous malformations (CCM1) pathogenesis; Zawistowski JS et al.; Mutations in KRIT1, a protein initially identified based on a yeast two-hybrid interaction with the RAS-family GTPase RAP1A, are responsible for the development of the inherited vascular disorder cerebral cavernous malformations (CCM1) . As the function of the KRIT1 protein and its role in CCM pathogenesis remain unknown, we performed yeast two-hybrid screens to identify additional protein binding partners . A fragment containing the N-terminal 272 amino acid residues of KRIT1, a region lacking similarity to any known protein upon database searches, was used as bait . From parallel screens of human fetal brain and HeLa cDNA libraries, we obtained multiple independent isolates of human integrin cytoplasmic domain-associated protein-1 (ICAP-1) as interacting clones . The interaction of KRIT1 and ICAP-1 was confirmed by GST-KRIT1 trapping of endogenous ICAP-1 from 293T cells . The alpha isoform of ICAP-1 is a 200 amino acid serine/threonine-rich phosphoprotein which binds the cytoplasmic tail of beta1 integrins . We show that mutagenesis of the N-terminal KRIT1 NPXY amino acid sequence, a motif critical for ICAP-1 binding to beta1 integrin molecules, completely abrogates the KRIT1/ICAP-1 interaction . The interaction between ICAP-1 and KRIT1, and the presence of a FERM domain in the latter, suggest that KRIT1 might be involved in the bidirectional signaling between integrin molecules and the cytoskeleton . Furthermore, these data suggest that KRIT1 might affect cell adhesion processes via integrin signaling in CCM1 pathogenesis.

Trends Cell Biol, 2002 Jan, 12(1), 21 - 7
Motor-cargo interactions: the key to transport specificity; Karcher RL et al.; Eukaryotic cells organize their cytoplasm by moving different organelles and macromolecular complexes along microtubules and actin filaments . These movements are powered by numerous motor proteins that must recognize their respective cargoes in order to function . Recently, several proteins that interact with motors have been identified by yeast two-hybrid and biochemical analyses, and their roles in transport are now being elucidated . In several cases, analysis of the binding partners helped to identify new transport pathways, new types of cargo, and transport regulated at the level of motor-cargo binding . We discuss here how different motors of the kinesin, dynein and myosin families recognize their cargo and how motor-cargo interactions are regulated.

Biochim Biophys Acta, 2002 Jan 30, 1542(1-3), 160 - 72
Monomeric alcohol oxidase is preferentially digested by a novel protease from Candida boidinii; Stewart MQ et al.; A protease activity has been partially purified from peroxisomal matrix fractions of the methylotrophic yeast Candida boidinii . The enzyme migrates as a single peak on a sucrose velocity gradient with an apparent native molecular mass of approximately 80-90 kDa . Activity can be recovered from nonreducing sodium dodecyl sulfate gels as a approximately 20 kDa species, suggesting it is an oligomer . The protein exhibits chymotrypsin-like activity and cleaves the model compound suc-L-L-V-Y-AMC . Additionally, monomers of alcohol oxidase (AO), an abundant protein of C . boidinii peroxisomes, generated in vitro or in pulse-radiolabeled cells, are preferentially sensitive to degradation by the protease . Sensitivity is lost over time in vivo as AO folds and matures into octamers, suggesting that the protease may be involved in these processes.

Biochim Biophys Acta, 2002 Jan 30, 1542(1-3), 41 - 56
Characterization of ubiquilin 1, an mTOR-interacting protein; Wu S et al.; The mTOR protein kinase is known to control cell cycle progression and cell growth through regulation of translation, transcription, membrane traffic and protein degradation . Known interactions of mTOR do not account for the multiple functions of this protein . Using a non-catalytic segment of mTOR (1-670) as bait in a yeast two-hybrid screen for interacting proteins, ubiquilin 1 (NM013438) was identified . Ubiquilin 1 is a member of a phylogenetically conserved gene family of unknown function, characterized by an N-terminal ubiquitin-like (Ubq) domain, a C-terminal ubiquitin associated (Uba) domain and a central region containing numerous NPXvar phi motifs (X, any; phi, hydrophobic amino acid) . GST-ubiquilin 1 binds specifically to FLAG-mTOR (residues 1-670) in mammalian cells; residues 570-670 of mTOR and 226-323 of ubiquilin 1 are required for this interaction . Both mTOR and ubiquilin immunoreactivity appear as fine speckles throughout the cytoplasm; significant colocalization with cytoskeletal elements, early endosomes or proteasomes is not observed . As assessed by cell fractionation, mTOR is predominantly associated with low density membranes, along with 10% of ubiquilin 1 . Ubiquilin 1 is a rapamycin-insensitive phosphoprotein . Overexpression of ubiquilin 1 does not alter the kinase activity of cotransfected mTOR or the phosphorylation of the mTOR target, p70 S6 kinase, in the presence or absence of rapamycin . Our data suggest that we have identified a novel mTOR interactor, ubiquilin 1 . The biological significance of this, presumably membrane based, interaction, requires further study.

Cell, 2002 Feb 8, 108(3), 345 - 56
Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1; Bernier-Villamor V et al.; E2 enzymes catalyze attachment of ubiquitin and ubiquitin-like proteins to lysine residues directly or through E3-mediated reactions . The small ubiquitin-like modifier SUMO regulates nuclear transport, stress response, and signal transduction in eukaryotes and is essential for cell-cycle progression in yeast . In contrast to most ubiquitin conjugation, the SUMO E2 enzyme Ubc9 is sufficient for substrate recognition and lysine modification of known SUMO targets . Crystallographic analysis of a complex between mammalian Ubc9 and a C-terminal domain of RanGAP1 at 2.5 A reveals structural determinants for recognition of consensus SUMO modification sequences found within SUMO-conjugated proteins . Structure-based mutagenesis and biochemical analysis of Ubc9 and RanGAP1 reveal distinct motifs required for substrate binding and SUMO modification of p53, IkappaBalpha, and RanGAP1.

Virology, 2002 Feb 1, 293(1), 103 - 17
A new cellular factor recognizes E2 binding sites of papillomaviruses which mediate transcriptional repression by E2; Boeckle S et al.; Repression of transcription by the full-length E2 protein of papillomaviruses (PV) seems to occur when the E2 binding sites and those of positively acting cellular factors overlap . Previously, we showed that RUNX1 (formerly called CBF) binds to the repression-mediating E2 binding site P2 of human PV type 8 (HPV8) . By a yeast one-hybrid system we could identify an unknown protein binding also to P2, tentatively called PBF (papillomavirus binding factor) . PBF recognizes the sequence CCGG, which represents the 3' half of the E2 binding site just adjacent to the RUNX1 motif . PBF also binds to the repression-mediating E2 BS-1 in BPV1, which is conserved to P2 of HPV8 . Point mutations destroying PBF binding to HPV8 P2 and BPV-1 E2 BS-1 in vitro reduce promoter activity in corresponding reporter constructs . Our results suggest that PBF might play a role in transcription of PV genes and in E2-mediated repression.

Matrix Biol, 2002 Mar, 21(2), 207 - 14
The PDZ domain of TIP-2/GIPC interacts with the C-terminus of the integrin alpha5 and alpha6 subunits; El Mourabit H et al.; Different cDNA libraries were screened by the yeast two-hybrid system using as a bait the cytoplasmic sequence of integrin alpha6A or alpha6B subunits . Surprisingly, the same PDZ domain-containing protein, TIP-2/GIPC, was isolated with either of the variants, although their sequences are different . Direct interaction assays with the cytoplasmic domain of the integrin alpha1--7 subunits revealed that in addition to alpha6A and alpha6B, TIP-2/GIPC reacted also with alpha5, but not other alpha integrin subunits . The specificity of the interaction was confirmed by in vitro protein binding assays with purified peptides corresponding to integrin cytoplasmic domains . Further analysis with either truncation fragments of TIP-2/GIPC or mutated integrin cytoplasmic domains indicated that the interaction occurs between the PDZ domain of TIP-2/GIPC and a consensus PDZ domain-binding sequence, SDA, present at the C-terminus of the integrin alpha5 and alpha6A subunits . The integrin alpha6B subunit terminates with a different sequence, SYS, which may represent a new PDZ domain-binding motif.

FEBS Lett, 2002 Feb 13, 512(1-3), 308 - 12
Induction of mammalian cell death by a plant Bax inhibitor; Yu LH et al.; Arabidopsis thaliana AtBI-1 is an orthologue of mammalian Bax inhibitor-1 capable of suppressing Bax-induced cell death in yeast as well as mammalian cells . Here we investigated whether or not AtBI-1 suppresses Bax-induced cell death using human fibrosarcoma HT1080 cells . Surprisingly, AtBI-1 did not block Bax-induced cell death, but it triggered apoptotic cell death in mammalian cells . The proapoptotic effect of AtBI-1 was blocked by the X-linked caspase inhibitor XIAP, suggesting that the cell death caused by AtBI-1 is similar to that caused by Bax.

Plant J, 2002 Jan, 29(2), 203 - 13
RNA splicing in higher plant mitochondria: determination of functional elements in group II intron from a chimeric cox II gene in electroporated wheat mitochondria; Farre JC et al.; Higher plant mitochondria mainly contain group II introns presenting a secondary structure with six helical domains linked to a central hub . Experimental evidence of functional elements in higher plant mitochondria introns is limited since they are unable to undergo self-splicing and the definition of functional domains is based on data obtained from yeast autocatalytic introns . Here we study the role of putative functional elements required for the splicing reaction . The exon-binding and intron-binding sites (EBS and IBS, respectively), and the domain 6, which is involved in lariat formation, were analysed by site-directed mutagenesis and transient expression in electroporated mitochondria . The data presented here demonstrate the role of EBS1-IBS1 and EBS2-IBS2 interactions and reveal a new secondary-structure interaction . The role of the C to U editing conversion in the IBS1 motif is discussed.

Biochemistry, 2002 Feb 26, 41(8), 2844 - 9
Gene expression, mutation, and structure-function relationship of scorpion toxin BmP05 active on SK(Ca) channels; Wu JJ et al.; Four peptide inhibitors of small-conductance Ca(2+)-activated, apamin-sensitive K(+) channels (SK(Ca)) have been isolated from the venom of the Chinese scorpion Buthus martensi, named BmP01, BmP02, BmP03, and BmP05, respectively {Romi-Lebrun, R . (1997) Eur . J . Biochem . 245, 457-464} . Among them BmP05 with 31 amino acid residues has been intensively studied due to its most potent toxicity . To investigate the structure-function relationship of BmP05, its wild type and seven mutants (their C-termini unamidated) were successfully expressed in the yeast secretion system and purified with a high yield over 8 mg/L . Their toxicity to mice and electrophysiological activity on the K(+) currents (SK(Ca) and Kv) in rat adrenal chromaffin cells were measured and compared . The results indicated the following: (1) As a selective antagonist against SK(Ca), 1 microM rBmP05 is equivalent to 0.2 microM apamin, and its IC(50) is 0.92 microM . (2) The basic residues Lys and Arg located at positions 6 and 13 in the N-terminal alpha-helix region are essential and synergetic in the interaction of the toxin with SK(Ca) . (3) Disruption of the alpha-helix by mutation of Gln at position 9 with Pro results in almost total loss of toxicity . (4) The C-terminal residue His31 plays an auxiliary role in the interaction of the toxin with SK(Ca) . (5) The beta-turn connecting two beta-sheets near the C-terminal part is responsible for the specificity of the toxin to the different subtypes of K(+) channels.

Biochemistry, 2002 Feb 26, 41(8), 2684 - 93
Cation-induced stabilization of the engineered cation-binding loop in cytochrome c peroxidase (CcP); Bhaskar B et al.; We have previously shown that the K(+) site found in the proximal heme pocket of ascorbate peroxidase (APX) could be successfully engineered into the closely homologous cytochrome c peroxidase (CcP) {Bonagura et al., (1996) Biochemistry 35, 6107-6115; Bonagura et al . (1999) Biochemistry 38, 5538-5545} . In addition, specificity could be switched to binding Ca(2+) as found in other peroxidases {Bonagura et al . (1999) J . Biol . Chem . 274, 37827-37833} . The introduction of a proximal cation-binding site also promotes conversion of the Trp191 containing cation-binding loop from a "closed" to an "open" conformer . In the present study we have changed a crucial hinge residue of the cation-binding loop, Asn195, to Pro which stabilizes the loop, albeit, only in the presence of bound K(+) . The crystal structure of this mutant, N195PK2, has been refined to 1.9 A . As predicted, introduction of this crucial hinge residue stabilizes the cation-binding loop in the presence of the bound K(+) . As in earlier work, the characteristic EPR signal of Trp191 cation radical becomes progressively weaker with increasing {K(+)} and the lifetime of the Trp191 radical also has been considerably shortened in this mutant . This mutant CcP exhibits reduced enzyme activity, which could be titrated to lower levels with increasing {K(+)} when horse heart cytochrome c is the substrate . However, with yeast cytochrome c as the substrate, the mutant was as active as wild-type at low ionic strength, but 40-fold lower at high ionic strength . We attribute this difference to a change in the rate-limiting step as a function of ionic strength when yeast cytochrome c is the substrate.

Oncogene, 2002 Feb 7, 21(7), 1123 - 9
Endonuclein is a cell cycle regulated WD-repeat protein that is up-regulated in adenocarcinoma of the pancreas; Honore B et al.; The transcript encoding endonuclein, the human homolog of yeast PWP1, was previously found up-regulated in pancreatic cancer tissue . By immunohistochemistry we detected a ubiquitous presence in several tissues examined: skin, liver, thyroid gland, heart muscle, neurons, kidney, bladder, pancreas, adrenal gland, ovary, uterus, testis and prostate gland . We especially noticed that normal pancreatic exocrine cells exhibited low protein levels while pancreatic adenocarcinoma cells revealed high levels . We found a heterogeneous subcellular distribution, especially with varying nuclear levels . In proliferating cells endonuclein protein expression and localization was cell cycle dependent, with increasing levels and nuclear focusing during the interphase toward mitosis . Ultrastructural analysis revealed ER and nuclear localization . Endonuclein contains five WD-repeats, indicating a putative role in crucial regulatory activities in the nucleus as well as in the ER.

Oncogene, 2002 Jan 24, 21(5), 844 - 8
Inhibition of human endothelial cell proliferation by ShIF, a vacuolar H(+)-ATPase-like protein; Tulin EE et al.; ShIF is a bone marrow stroma cell-derived factor originally identified to support proliferation of bone marrow cells in vitro . This protein shares high sequence homology to the yeast vacuolar H(+)-ATPase subunit, Vph1p, and the 116 kDa proton pump of the rat and bovine synaptic vesicle, Vpp1 . We examined the function of ShIF in the proliferation of human umbilical vein endothelial cells (HUVEC) . ShIF inhibited HUVEC proliferation in a dose-dependent manner . Recombinant ShIF added at 10 and 20 ng/ml inhibited HUVEC proliferation by 21.6 and 44.3%, respectively and increasing the concentration of ShIF to 100 ng/ml inhibited proliferation by as much as 55.5% . When HUVEC cells were cultured at various concentrations of ShIF in the presence of anti-ShIF antibody, the inhibitory effects of ShIF to HUVEC proliferation were abrogated by 89-91% indicating that the activity of ShIF to HUVEC was specific . HUVEC cultured in the presence of ShIF and bafilomycin, a specific inhibitor of ATPase, resulted to a 90% growth inhibition . Thus, ShIF may act as an antagonist to the ATPase complex by disrupting the production of cellular ATP thereby decreasing the ability of HUVEC to proliferate.

Oncogene, 2002 Jan 21, 21(4), 611 - 8
ATM function and telomere stability; Pandita TK; Accumulation of DNA damage has been associated with the onset of senescence and predisposition to cancer . The gene responsible for ataxia telangiectasia (A-T) is ATM (ataxia-telangiectasia mutant), a master controller of cellular pathways and networks, orchestrating the responses to a specific type of DNA damage: the double strand break . Based on the homology of the human ATM gene to the TEL1, MEC1 and rad3 genes of yeast, it has now been demonstrated that mutations in ATM lead to defective telomere maintenance in mammalian cells . While ATM has both nuclear and cytoplasmic functions, this review will focus on its roles in telomere metabolism and how ATM and telomeres serve as controllers of cellular responses to DNA damage.

Nat Genet, 2002 Mar, 30(3), 329 - 34 Epub 2002 Feb 19.
Higher-order structure in pericentric heterochromatin involves a distinct pattern of histone modification and an RNA component; Maison C et al.; Post-translational modification of histone tails is thought to modulate higher-order chromatin structure . Combinations of modifications including acetylation, phosphorylation and methylation have been proposed to provide marks recognized by specific proteins . This is exemplified, in both mammalian cells and fission yeast, by transcriptionally silent constitutive pericentric heterochromatin . Such heterochromatin contains histones that are generally hypoacetylated and methylated by Suv39h methyltransferases at lysine 9 of histone H3 (H3-K9) . Each of these modification states has been implicated in the maintenance of HP1 protein-binding at pericentric heterochromatin, in transcriptional silencing and in centromere function . In particular, H3-K9 methylation is thought to provide a marking system for the establishment and maintenance of stably repressed regions and heterochromatin subdomains . To address the question of how these two types of modifications, as well as other unidentified parameters, function to maintain pericentric heterochromatin, we used a combination of histone deacetylase inhibitors, RNAse treatments and an antibody raised against methylated branched H3-K9 peptides . Our results show that both H3-K9 acetylation and methylation can occur on independent sets of H3 molecules in pericentric heterochromatin . In addition, we identify an RNA- and histone modification-dependent structure that brings methylated H3-K9 tails together in a specific configuration required for the accumulation of HP1 proteins in these domains.

J Neurosci, 2002 Feb 15, 22(4), 1280 - 9
Tamalin, a PDZ domain-containing protein, links a protein complex formation of group 1 metabotropic glutamate receptors and the guanine nucleotide exchange factor cytohesins; Kitano J et al.; In this investigation, we report identification and characterization of a 95 kDa postsynaptic density protein (PSD-95)/discs-large/ZO-1 (PDZ) domain-containing protein termed tamalin, also recently named GRP1-associated scaffold protein (GRASP), that interacts with group 1 metabotropic glutamate receptors (mGluRs) . The yeast two-hybrid system and in vitro pull-down assays indicated that the PDZ domain-containing, amino-terminal half of tamalin directly binds to the class I PDZ-binding motif of group 1 mGluRs . The C-terminal half of tamalin also bound to cytohesins, the members of guanine nucleotide exchange factors (GEFs) specific for the ADP-ribosylation factor (ARF) family of small GTP-binding proteins . Tamalin mRNA is expressed predominantly in the telencephalic region and highly overlaps with the expression of group 1 mGluR mRNAs . Both tamalin and cytohesin-2 were enriched and codistributed with mGluR1a in postsynaptic membrane fractions . Importantly, recombinant and native mGluR1a/tamalin/cytohesin-2 complexes were coimmunoprecipitated from transfected COS-7 cells and rat brain tissue, respectively . Transfection of tamalin and mutant tamalin lacking a cytohesin-binding domain caused an increase and decrease in cell-surface expression of mGluR1a in COS-7 cells, respectively . Furthermore, adenovirus-mediated expression of tamalin and dominant-negative tamalin facilitated and reduced the neuritic distribution of endogenous mGluR5 in cultured hippocampal neurons, respectively . The results indicate that tamalin plays a key role in the association of group 1 mGluRs with the ARF-specific GEF proteins and contributes to intracellular trafficking and the macromolecular organization of group 1 mGluRs at synapses.

Genes Dev, 2002 Feb 15, 16(4), 452 - 66
IRE1-mediated unconventional mRNA splicing and S2P-mediated ATF6 cleavage merge to regulate XBP1 in signaling the unfolded protein response; Lee K et al.; All eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) by signaling an adaptive pathway termed the unfolded protein response (UPR) . In yeast, a type-I ER transmembrane protein kinase, Ire1p, is the proximal sensor of unfolded proteins in the ER lumen that initiates an unconventional splicing reaction on HAC1 mRNA . Hac1p is a transcription factor required for induction of UPR genes . In higher eukaryotic cells, the UPR also induces site-2 protease (S2P)-mediated cleavage of ER-localized ATF6 to generate an N-terminal fragment that activates transcription of UPR genes . To elucidate the requirements for IRE1alpha and ATF6 for signaling the mammalian UPR, we identified a UPR reporter gene that was defective for induction in IRE1alpha-null mouse embryonic fibroblasts and S2P-deficient Chinese hamster ovary (CHO) cells . We show that the endoribonuclease activity of IRE1alpha is required to splice XBP1 (X-box binding protein) mRNA to generate a new C terminus, thereby converting it into a potent UPR transcriptional activator . IRE1alpha was not required for ATF6 cleavage, nuclear translocation, or transcriptional activation . However, ATF6 cleavage was required for IRE1alpha-dependent induction of UPR transcription . We propose that nuclear-localized IRE1alpha and cytoplasmic-localized ATF6 signaling pathways merge through regulation of XBP1 activity to induce downstream gene expression . Whereas ATF6 increases the amount of XBP1 mRNA, IRE1alpha removes an unconventional 26-nucleotide intron that increases XBP1 transactivation potential . Both processing of ATF6 and IRE1alpha-mediated splicing of XBP1 mRNA are required for full activation of the UPR.

Neurochem Int, 2002 May, 40(6), 553 - 7
Spinocerebellar ataxias due to mitochondrial defects; Kaplan J; A number of ataxias have been shown to result from defects in mitochondrial function . The genes responsible for Friedreich ataxia (FRDA) and for X-linked sideroblastic anemia with ataxia are nuclear genes that encode mitochondrial proteins . These genes, which are highly conserved in species as diverse as humans and yeast, play a role in mitochondrial iron metabolism and in the formation of iron-sulfur clusters . Defects in vitamin E metabolism, due to mutations in tocopherol transfer protein (TTP), also result in ataxia . It is hypothesized that the biochemical feature common to these ataxias is increased oxidant damage either through increased oxidants or decreased anti-oxidants.

Lett Appl Microbiol, 2002, 34(2), 114 - 8
Nisin and pediocin production on mussel-processing waste supplemented with glucose and five nitrogen sources; Guerra NP et al.; AIMS: Optimization of bacteriocin production by L . lactis subsp . lactis CECT 539 and Ped . acidilactici NRRL B-5627 on mussel-processing wastes . METHODS AND RESULTS: The concentrations of glucose and five nitrogen sources that optimize nisin and pediocin production were determined using a second order orthogonal factorial design . NH4Cl, glycine and glutamic acid were poor nitrogen sources . Enhanced pediocin and nisin productions were achieved in a medium supplemented with yeast extract or Bacto casitone . CONCLUSIONS: Mussel-processing wastes could be successfully used as a culture medium for bacteriocin production at low production cost . SIGNIFICANCE AND IMPACT OF THE STUDY: Average optimization led to a threefold increase in pediocin production (from 322 to 934 BU ml(-1)) and in nisin production (from 32 to 100 BU ml(-1)) when compared with the unsupplemented medium . For this reason, mussel-processing waste warrants further investigation due to its potential use as a cheap culture medium for upscaling bacteriocin production.

Fungal Genet Biol, 2002 Mar, 35(2), 81 - 97
Histoplasma capsulatum molecular genetics, pathogenesis, and responsiveness to its environment; Woods JP; Histoplasma capsulatum is a thermally dimorphic ascomycete that is a significant cause of respiratory and systemic disease in mammals including humans, especially immunocompromised individuals such as AIDS patients . As an environmental mold found in the soil, it is a successful member of a competitive polymicrobial ecosystem . Its host-adapted yeast form is a facultative intracellular pathogen of mammalian macrophages . H . capsulatum faces a variety of environmental changes during the course of infection and must survive under harsh conditions or modulate its microenvironment to achieve success as a pathogen . Histoplasmosis may be considered the fungal homolog of the bacterial infection tuberculosis, since both H . capsulatum and Mycobacterium tuberculosis exploit the macrophage as a host cell and can cause acute or persistent pulmonary and disseminated infection and reactivation disease . The identification and functional analysis of biologically or pathogenically important H . capsulatum genes have been greatly facilitated by the development of molecular genetic experimental capabilities in this organism . This review focuses on responsiveness of this fungus to its environment, including differential expression of genes and adaptive phenotypic traits.

Environ Toxicol, 2002 Feb, 17(1), 80 - 6
Acute toxicity, mutagenicity, and estrogenicity of bisphenol-A and other bisphenols; Chen MY et al.; Although abundant data are available on the toxicity of bisphenol-A (2,2-bis (4-hydroxydiphenyl)propane; BPA), little is known about the toxicities of the structurally similar compounds, namely bisphenols (BPs) . A variety of BPs were examined for their acute toxicity against Daphnia magna, mutagenicity, and estrogenic activity using the Daphtoxkit (Creasel Ltd.), the umu test system, and the yeast two-hybrid system, respectively, in comparison with BPA . BPA was moderately toxic to D . magna (48-h EC50 was 10 mg/l) according to the current U.S . EPA acute toxicity evaluation standard, and it was weakly estrogenic with 5 orders of magnitude lower activity than that of the natural estrogen 17 beta-estradiol in the yeast screen, while no mutagenicity was observed . All seven BPs tested here showed moderate to slight acute toxicity, no mutagenicity, and weak estrogenic activity as well as BPA . Some of the BPs showed considerably higher estrogenic activity than BPA, and others exhibited much lower activity . Among the tested BPs, two compounds, i.e., bisphenol-S (bis(4-hydroxydiphenyl)sulfone) and bis(4-hydroxyphenyl)sulfide, have never been reported for their estrogenic activity previously.

J Biol Chem, 2002 Apr 26, 277(17), 14681 - 7 Epub 2002 Feb 14.
The calmodulin-binding domain of the catalytic gamma subunit of phosphorylase kinase interacts with its inhibitory alpha subunit: evidence for a Ca2+ sensitive network of quaternary interactions; Rice NA et al.; Chemical cross-linking as a probe of conformation has consistently shown that activators, including Ca(2+) ions, of the (alphabetagammadelta)(4) phosphorylase kinase holoenzyme (PhK) alter the interactions between its regulatory alpha and catalytic gamma subunits . The gamma subunit is also known to interact with the delta subunit, an endogenous molecule of calmodulin that mediates the activation of PhK by Ca(2+) ions . In this study, we have used two-hybrid screening and chemical cross-linking to dissect the regulatory quaternary interactions involving these subunits . The yeast two-hybrid system indicated that regions near the C termini of the gamma (residues 343-386) and alpha (residues 1060-1237) subunits interact . The association of this region of alpha with gamma was corroborated by the isolation of a cross-linked fragment of alpha containing residues 1015-1237 from an alpha-gamma dimer that had been formed within the PhK holoenzyme by formaldehyde, a nearly zero-length cross-linker . Because the region of gamma that we found to interact with alpha has previously been shown to contain a high affinity binding site for calmodulin (Dasgupta, M., Honeycutt, T., and Blumenthal, D . K . (1989) J . Biol . Chem . 264, 17156-17163), we tested the influence of Ca(2+) on the conformation of the alpha subunit and found that the region of alpha that interacts with gamma was, in fact, perturbed by Ca(2+) . The results herein support the existence of a Ca(2+)-sensitive communication network among the delta, gamma, and alpha subunits, with the regulatory domain of gamma being the primary mediator . The similarity of such a Ca(2+)-dependent network to the interactions among troponin C, troponin I, and actin is discussed in light of the known structural and functional similarities between troponin I and the gamma subunit of PhK.

BMC Cell Biol . 2002;3(1):2 . Epub 2002 Jan 22.
Latent transforming growth factor beta-binding protein-3 and fibulin-1C interact with the extracellular domain of the heparin-binding EGF-like growth factor precursor; Brooke JS et al.; BACKGROUND: The membrane-bound cell-surface precursor and soluble forms of heparin-binding epidermal growth factor-like growth factor (HB-EGF) contribute to many cellular developmental processes . The widespread occurrence of HB-EGF in cell and tissue types has led to observations of its role in such cellular and tissue events as tumor formation, cell migration, extracellular matrix formation, wound healing, and cell adherence . Several studies have reported the involvement of such extracellular matrix proteins as latent transforming growth factor beta-binding protein, TGF-beta, and fibulin-1 in some of these processes . To determine whether HB-EGF interacts with extracellular matrix proteins we used the extracellular domain of proHB-EGF in a yeast two-hybrid system to screen a monkey kidney cDNA library . cDNA clones containing nucleotide sequences encoding domains of two proteins were obtained and their derived amino acid sequences were evaluated . RESULTS: From approximately equal to 3 x 10(6) screened monkey cDNA clones, cDNA clones were recovered that contained nucleotide sequences encoding domains of the monkey latent transforming growth factor-beta binding protein-3 (MkLTBP-3) and fibulin-1C protein . The amino acid sequence derived from the MkLTBP-3 gene shared 98.6% identity with human LTBP-3 and 86.7% identity with mouse LTBP-3 amino acid sequences . The amino acid sequence derived from the monkey fibulin-1C gene shared 97.2% identity with human fibulin-1C . Yeast two-hybrid screens indicate that LTBP-3 and fibulin-1C interact with proHB-EGF through their calcium-binding EGF-like modules . CONCLUSIONS: The interactions of the extracellular domain of proHB-EGF with LTBP-3 and fibulin-1C suggest novel functions for HB-EGF between cell and tissue surfaces.

Plant J, 2002 Feb, 29(4), 465 - 73
Two distinct high-affinity sulfate transporters with different inducibilities mediate uptake of sulfate in Arabidopsis roots; Yoshimoto N et al.; Sulfate transporters present at the root surface facilitate uptake of sulfate from the environment . Here we report that uptake of sulfate at the outermost cell layers of Arabidopsis root is associated with the functions of highly and low-inducible sulfate transporters, Sultr1;1 and Sultr1;2, respectively . We have previously reported that Sultr1;1 is a high-affinity sulfate transporter expressed in root hairs, epidermal and cortical cells of Arabidopsis roots, and its expression is strongly upregulated in plants deprived of external sulfate . A novel sulfate transporter gene, Sultr1;2, identified on the BAC clone F28K19 of Arabidopsis, encoded a polypeptide of 653 amino acids that is 72.6% identical to Sultr1;1 and was able to restore sulfate uptake capacity of a yeast mutant lacking sulfate transporter genes (K(m) for sulfate = 6.9 +/- 1.0 microm) . Transgenic Arabidopsis plants expressing the fusion gene construct of the Sultr1;2 promoter and green fluorescent protein (GFP) showed specific localization of GFP in the root hairs, epidermal and cortical cells of roots, and in the guard cells of leaves, suggesting that Sultr1;2 may co-localize with Sultr1;1 in the same cell layers at the root surface . Sultr1;1 mRNA was abundantly expressed under low-sulfur conditions (50-100 microm sulfate), whereas Sultr1;2 mRNA accumulated constitutively at high levels under a wide range of sulfur conditions (50-1500 microm sulfate), indicating that Sultr1;2 is less responsive to changes in sulfur conditions . Addition of selenate to the medium increased the level of Sultr1;1 mRNA in parallel with a decrease in the internal sulfate pool in roots . The level of Sultr1;2 mRNA was not influenced under these conditions . Antisense plants of Sultr1;1 showed reduced accumulation of sulfate in roots, particularly in plants treated with selenate, suggesting that the inducible transporter Sultr1;1 contributes to the uptake of sulfate under stressed conditions.

Plant J, 2002 Feb, 29(4), 405 - 15
Condensin and cohesin knockouts in Arabidopsis exhibit a titan seed phenotype; Liu Cm CM et al.; The titan (ttn) mutants of Arabidopsis exhibit striking alterations in chromosome dynamics and cell division during seed development . Endosperm defects include aberrant mitoses and giant polyploid nuclei . Mutant embryos differ in cell size, morphology and viability, depending on the locus involved . Here we demonstrate that three TTN genes encode chromosome scaffold proteins of the condensin (SMC2) and cohesin (SMC1 and SMC3) classes . These proteins have been studied extensively in yeast and animal systems, where they modulate chromosome condensation, chromatid separation, and dosage compensation . Arabidopsis contains single copies of SMC1 and SMC3 cohesins . We used forward genetics to identify duplicate T-DNA insertions in each gene . These mutants (ttn7 and ttn8) have similar titan phenotypes: giant endosperm nuclei and arrested embryos with a few small cells . A single SMC2 knockout (ttn3) was identified and confirmed by molecular complementation . The weak embryo phenotype observed in this mutant may result from expression of a related gene (AtSMC2) with overlapping functions . Further analysis of titan mutants and the SMC gene family in Arabidopsis should provide clues to chromosome mechanics in plants and insights into the regulation of nuclear activity during endosperm development.

Eur J Biochem, 2002 Feb, 269(3), 909 - 14
Identification of syntaxin-1A sites of phosphorylation by casein kinase I and casein kinase II; Dubois T et al.; Casein kinases I (CKI) are serine/threonine protein kinases widely expressed in a range of eukaryotes including yeast, mammals and plants . They have been shown to play a role in diverse physiological events including membrane trafficking . CKI alpha is associated with synaptic vesicles and phosphorylates some synaptic vesicle associated proteins including SV2 . In this report, we show that syntaxin-1A is phosphorylated in vitro by CKI on Thr21 . Casein kinase II (CKII) has been shown previously to phosphorylate syntaxin-1A in vitro and we have identified Ser14 as the CKII phosphorylation site, which is known to be phosphorylated in vivo . As syntaxin-1A plays a key role in the regulation of neurotransmitter release by forming part of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, we propose that CKI may play a role in synaptic vesicle exocytosis.

Biochem Biophys Res Commun, 2002 Feb 22, 291(2), 385 - 93
Titin mutations as the molecular basis for dilated cardiomyopathy; Itoh-Satoh M et al.; Dilated cardiomyopathy (DCM) is a heterogeneous cardiac disease characterized by ventricular dilatation and systolic dysfunction . Recent genetic studies have revealed that mutations in genes for cardiac sarcomere components lead to DCM . The cardiac sarcomere consists of thick and thin filaments and a giant protein, titin . Because one of the loci of familial DCM was mapped to the region of the titin gene, we searched for titin mutations in the patients and identified four possible disease-associated mutations . Two mutations, Val54Met and Ala743Val, were found in the Z-line region of titin and decreased binding affinities of titin to Z-line proteins T-cap/telethonin and alpha-actinin, respectively, in yeast two-hybrid assays . The other two mutations were found in the cardiac-specific N2-B region of titin and one of them was a nonsense mutation, Glu4053ter, presumably encoding for a truncated nonfunctional molecule . These observations suggest that titin mutations may cause DCM in a subset of the patients . (c)2002 Elsevier Science (USA).

Biochem Biophys Res Commun, 2002 Feb 22, 291(2), 305 - 12
Functional redundancy in the myotubularin family; Laporte J et al.; Myotubularin-related genes define a novel highly conserved family of eukaryotic proteins of at least 11 human members . The hMTM1 gene that codes for myotubularin is mutated in X-linked myotubular myopathy, a severe congenital disease . Recently, we and others have characterized myotubularin as a potent and specific phosphatidylinositol 3-phosphate 3-phosphatase . In the present study we investigated the lipid phosphatase activity and the subcellular localization of two other members of the family, hMTMR2 protein that is mutated in the demyelinating neuropathy Charcot-Marie-Tooth type 4B and the FYVE-finger containing hMTMR3 protein . Our results show that both proteins are potent phosphatidylinositol 3-phosphate 3-phosphatases either in vitro or in yeast where they interfered with vesicular trafficking . Their localization is mainly cytoplasmic, with however strong labeling of Rac-inducible plasma membrane ruffles . The fact that the ubiquitously expressed hMTM1 and hMTMR2 genes are involved in different pathologies indicates that despite their shared enzymatic activity, they are not functionally redundant, at least in certain cell types . This might be explained by subtle differences in expression and/or in recruitment and regulation at their specific site of action . (c)2002 Elsevier Science (USA).

J Trace Elem Med Biol, 2001, 15(4), 201 - 8
Selenium and glutathione levels, and glutathione peroxidase activities in blood components of uremic patients on hemodialysis supplemented with selenium and treated with erythropoietin; Zachara BA et al.; Patients with chronic renal failure (CRF) often have reduced concentrations of selenium (Se) and lowered activities of glutathione peroxidase (GSH-Px) in blood components . The kidney is a major source of plasma GSH-Px . We measured Se and glutathione levels in blood components and red cell and plasma GSH-Px activities in 58 uremic patients on regular (3 times a week) hemodialysis (HD) . The dialyzed patients were divided in 4 subgroups and were supplemented for 3 months with: 1) placebo (bakers yeast), 2) erythropoietin (EPO; 3 times a week with 2,000 U after each HD session), 3) Se-rich yeast (300 microg 3 times a week after each HD), and 4) Se-rich yeast plus EPO in doses as above . The results were compared with those for 25 healthy subjects . The Se concentrations and GSH-Px activities in the blood components of dialyzed uremic patients were significantly lower compared with the control group . Treatment of the HD patients with placebo and EPO only did not change the parameters studied . The treatment with Se as well as with Se and EPO caused an increase in Se levels and red cell GSH-Px activity . Plasma GSH-Px activity, however, increased only slowly or did not change after treatment with Se and with Se plus EPO . In the group treated with Se plus EPO the element concentration in blood components was higher compared with the group supplemented with Se alone . The weak or absence of response in plasma GSH-Px activity to Se supply indicates that the impaired kidney of uremic HD patients has reduced possibilities to synthesize this enzyme.

Mamm Genome, 2001 Nov, 12(11), 830 - 6
Rapid decrease of RNA level of a novel mouse mitochondria solute carrier protein (Mscp) gene at 4-5 weeks of age; Li QZ et al.; We cloned a novel mouse gene that encodes a protein with homology to the mitochondria solute carrier proteins (Mscp) . The major full-length Mscp transcript contains 4112 bp of cDNA and a deduced protein of 338 amino acids . The Mscp protein shares 50%, 40%, and 39% sequence identity with the C . elegans hypothetical protein T26089 and the yeast mitochondria carrier proteins MRS3 and MRS4, respectively . It also showed homology with the uncoupling proteins (UCP1, UCP2, and UCP3; 22%, 24%, and 29% identity, respectively) . The protein has six transmembrane domains and three mitochondria energy-transfer protein signature motifs, which are conserved among all the members of mitochondria carrier protein family . Northern analysis indicated that the Mscp gene is highly expressed in the spleen . Using cDNA microarray and Northern analysis, we have shown a significant decrease of the splenic Mscp mRNA levels around 4-5 weeks of age in several mouse strains including C57BL/6J, nonobese diabetic (NOD), and several NOD-congenic mice . These results suggest that the Mscp gene is decreased during splenic lymphocyte maturation in these mice.

J Biol Chem, 2002 May 3, 277(18), 15317 - 24 Epub 2002 Feb 13.
Chlorella virus RNA triphosphatase . Mutational analysis and mechanism of inhibition by tripolyphosphate; Gong C et al.; Chlorella virus RNA triphosphatase (cvRtp1) is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, poxviruses, and baculoviruses . The primary structure of cvRtp1 is more similar to that of the yeast RNA triphosphatase Cet1 than it is to the RNA triphosphatases of other DNA viruses . To evaluate the higher order structural similarities between cvRtp1 and the fungal enzymes, we performed an alanine scan of individual residues of cvRtp1 that were predicted, on the basis of the crystal structure of Cet1, to be located at or near the active site . Twelve residues (Glu(24), Glu(26), Asp(64), Arg(76), Lys(90), Glu(112), Arg(127), Lys(129), Arg(131), Asp(142), Glu(163), and Glu(165)) were deemed essential for catalysis by cvRtp1, insofar as their replacement by alanine reduced phosphohydrolase activity to <5% of the wild-type value . Structure-activity relationships were elucidated by introducing conservative substitutions at the essential positions . The mutational results suggest that the active site of cvRtp1 is likely to adopt a tunnel fold like that of Cet1 and that a similar constellation of side chains within the tunnel is responsible for metal binding and reaction chemistry . Nonetheless, there are several discordant mutational effects in cvRtp1 versus Cet1, which suggest that different members of the phosphohydrolase family vary in their reliance on certain residues within the active site tunnel . We found that tripolyphosphate and pyrophosphate were potent competitive inhibitors of cvRtp1 (K(i) = 0.6 microm tripolyphosphate and 2.4 microm pyrophosphate, respectively), whereas phosphate had little effect . cvRtp1 displayed a weak intrinsic tripolyphosphatase activity (3% of its ATPase activity) but was unable to hydrolyze pyrophosphate.

J Biol Chem, 2002 Apr 26, 277(17), 15113 - 23 Epub 2002 Feb 13.
Physical interaction of p73 with c-Myc and MM1, a c-Myc-binding protein, and modulation of the p73 function; Watanabe K et al.; p73 shares high sequence homology with the tumor suppressor p53 . Like p53, ectopic overexpression of p73 induces cell cycle arrest and/or apoptosis, and these biological activities are linked to its sequence-specific transactivation function . The COOH-terminal region of p73 is unique and has a function to modulate DNA-binding ability and transactivation activity . To identify and characterize cellular proteins that interact with the COOH-terminal region of p73 alpha and regulate its activity, we employed a yeast-based two-hybrid screen with a human fetal brain cDNA library . We found MM1, a nuclear c-Myc-binding protein, was associated with p73 alpha in both yeast two-hybrid and in vitro pull-down assays . In mammalian cells, MM1 co-immunoprecipitated with p73 alpha, whereas p73 beta and tumor suppressor p53 did not interact with MM1 . Overexpression of MM1 in p53-deficient osteosarcoma SAOS-2 cells enhanced the p73 alpha-dependent transcription from the p53/p73-responsive Bax and PG13 promoters, whereas p73 beta- and p53-mediated transcriptional activation was unaffected in the presence of MM1 . MM1 also stimulated the p73 alpha-mediated growth suppression in SAOS-2 cells . More importantly, we found that c-Myc was physically associated with p73 alpha and significantly impaired the transcriptional activity of p73 alpha on Bax and p21(waf1) promoters . Expression of MM1 strongly reduced the c-Myc-mediated inhibitory activity on p73 alpha . These results suggest that MM1 may act as a molecular partner for p73 to prevent the c-Myc-mediated inhibitory effect on its activity.

Nutrition, 2002 Feb, 18(2), 201 - 4
A pilot study of the safety and efficacy of cholestin in treating HIV-related dyslipidemia; Keithley JK et al.; OBJECTIVE: We collected preliminary safety and efficacy data on the effects of Cholestin, a statin-containing dietary supplement, in individuals with dsylipidemia related to human immunodeficiency virus . METHODS: Fourteen adults with dsylipidemia related to human immunodeficiency virus characterized by hypercholesterolemia, hypertriacylglycerolemia, or both participated in a randomized, double-blind, placebo-controlled pilot study in an infectious disease clinic based in an academic medical center . Participants were randomly assigned to receive 1.2 g of Cholestin twice daily (n = 7) or placebo (n = 7) for 8 wk . The main outcome measures were safety (hepatic function tests, plasma human immunodeficiency virus-1 RNA levels, CD4(+) cell counts, adverse effects) and efficacy (fasting serum cholesterol: total, high- and low-density lipoproteins, and fasting serum triacylglycerols) . Safety and efficacy outcomes were evaluated at 2- and 8-wk intervals . RESULTS: Twelve participants (n = 6 per group) completed the 8-wk treatment protocol . After 8 wk of treatment with Cholestin, there were significant declines from baseline in mean (+/- standard error of the mean) fasting total cholesterol (-30.8 +/- 8.8 versus 7.7 +/- 5.6; P = 0.01) and low-density lipoprotein cholesterol (-32.2 +/- 7.2 versus 26.3 +/- 14.2; P = 0.01) versus placebo . Moreover, the decline in fasting total cholesterol was significant (-40.2 +/- 4.8 versus 2.8 +/- 11.9; P = 0.006) after 2 wk of therapy, at which time the low-density lipoprotein cholesterol approached significance (-30.2 +/- 7.4 versus 4.4 +/- 15.2; P = 0.068) . High-density lipoprotein cholesterol and triacylglycerol levels did not change at either time point . No adverse effects were seen with Cholestin.CONCLUSIONS: Cholestin may safely lower total and low-density lipoprotein cholesterol in patients with dsylipidemia related to human immunodeficiency virus . Larger and longer-term trials of this approach are warranted.

Biol Chem, 2001 Dec, 382(12), 1649 - 62
Mig-6 is a negative regulator of the epidermal growth factor receptor signal; Hackel PO et al.; In contrast to signal generation and transmission, the mechanisms and molecules that negatively regulate receptor tyrosine kinase (RTK) signaling are poorly understood . Here we characterize Mig-6 as a novel negative feedback regulator of the epidermal growth factor receptor (EGFR) and potential tumor suppressor . Mig-6 was identified in a yeast two-hybrid screen with the kinase active domain of the EGFR as bait . Upon EGF stimulation Mig-6 binds to the EGFR involving a highly acidic region between amino acids 985-995 . This interaction is kinase activity-dependent, but independent of tyrosine 992 . Mig-6 overexpression results in reduced activation of the mitogenactivated protein kinase ERK2 in response to EGF, but not FGF or PDGF, stimulation and in enhanced receptor internalization without affecting the rate of degradation . The induction of Mig-6 mRNA expression in response to EGF, but not FGF, indicates the existence of a negative regulatory feedback loop . Consistent with these findings, a possible role as tumor suppressor is indicated by Mig-6-mediated inhibition of EGFR overexpression-induced transformation of Rati cells.

J Gen Virol, 2002 Mar, 83(Pt 3), 511 - 6
Topors, a p53 and topoisomerase I binding protein, interacts with the adeno-associated virus (AAV-2) Rep78/68 proteins and enhances AAV-2 gene expression; Weger S et al.; The adeno-associated virus type 2 (AAV-2) Rep proteins are essential for AAV DNA replication and regulation of AAV gene expression . We have identified a cellular protein interacting with Rep78 and Rep68 in yeast two-hybrid analysis and in GST pull-down assays . This protein has recently been described as both a p53 (p53BP3) and a topoisomerase I interacting protein (Topors) . It contains an arginine/serine-rich domain, a RING finger domain and five PEST sequences . A minimal sequence sufficient for interaction with Rep was mapped to Topors amino acids 871 to 917 . We show that the same region is also involved in the interaction with p53 . Rep sequences involved in interaction with Topors were mapped to Rep amino acids 172 to 481 . Overexpression of Topors stimulated AAV gene expression in the absence of helper virus, suggesting a function of Topors as a transcriptional regulator.

Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 2124 - 8 Epub 2002 Feb 12.
Characterization of the Met326Ile variant of phosphatidylinositol 3-kinase p85alpha; Almind K et al.; Phosphatidylinositol 3-kinase is a key step in the metabolic actions of insulin . Two amino acid substitutions have been identified in the gene for the regulatory subunit of human p85alpha, Met-326Ile, and Asn-330Asp, and the former has been associated with alterations in glucose/insulin homeostasis . When the four human p85alpha proteins were expressed in yeast, a 27% decrease occurred in the level of protein expression of p85alpha(Ile/Asp) (P = 0.03) and a 43% decrease in p85alpha(Ile/Asn) (P = 0.08) as compared with p85alpha(Met/Asp) . Both p85alpha(Ile/Asp) and p85alpha(Ile/Asn) also exhibited increased binding to phospho-insulin receptor substrate-1 by 41% and 83%, respectively (P < 0.001), as compared with p85alpha(Met/Asp) . The expression of p85alpha(Ile) was also slightly decreased and the binding to insulin receptor substrate-1 slightly increased in brown preadipocytes derived from p85alpha knockout mice . Both p85alpha(Met) and p85alpha(Ile) had similar effects on AKT activity and were able to reconstitute differentiation of the preadipocytes, although the triglyceride concentration in fully differentiated adipocytes and insulin-stimulated 2-deoxyglucose uptake were slightly lower than in adipocytes expressing p85alpha(Met) . Thus, the Met-326Ile variant of p85alpha is functional for intracellular signaling and adipocyte differentiation but has small alterations in protein expression and activity that could play a role in modifying insulin action.

Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 1910 - 4 Epub 2002 Feb 12.
Human DINB1-encoded DNA polymerase kappa is a promiscuous extender of mispaired primer termini; Washington MT et al.; Both in yeast and humans, DNA polymerase (Pol) (eta) functions in the error-free replication of UV-damaged DNA, and Pol(eta) has the unique ability to efficiently replicate through a cis-syn thymine-thymine (T-T) dimer by inserting two As opposite the two Ts of the dimer . Although human DINB1-encoded Pol(kappa) belongs to the same protein family as Pol(eta), Pol(kappa) shows no ability to bypass this DNA lesion and its biological function has remained unclear . Here, we examine Pol(kappa) for its ability to extend from primer-terminal mispairs opposite nondamaged and damaged DNA templates . We find that Pol(kappa) is a promiscuous extender of primer-terminal mispairs opposite nondamaged DNA templates, and interestingly, it is also very efficient at extending from a G opposite the 3'T of a T-T dimer . These observations provide biochemical evidence for a role of Pol(kappa) in the extension of mismatched base pairs during normal DNA replication, and in addition, they implicate Pol(kappa) in the mutagenic bypass of T-T dimers . In its proficient mismatch extension ability, Pol(kappa) is more similar to the unrelated DNA polymerase zeta than it is to the phylogenetically related Pol(eta) or Pol(iota) . Thus, in humans, Pol(kappa) would compete with Pol(zeta) for the extension of mismatched base pairs on damaged and undamaged DNAs.

Nucleic Acids Res, 2002 Feb 15, 30(4), 1046 - 55
Functional dissection of the ParB homologue (KorB) from IncP-1 plasmid RK2; Lukaszewicz M et al.; Active partitioning of low-copy number plasmids requires two proteins belonging to the ParA and ParB families and a cis-acting site which ParB acts upon . Active separation of clusters of plasmid molecules to the defined locations in the cell before cell division ensures stable inheritance of the plasmids . The central control operon of IncP-1 plasmids codes for regulatory proteins involved in the global transcriptional control of operons for vegetative replication, stable maintenance and conjugative transfer . Two of these proteins, IncC and KorB, also play a role in active partitioning, as the ParA and ParB homologues, respectively . Here we describe mapping the regions in KorB responsible for four of its different functions: dimerisation, DNA binding, repression of transcription and interaction with IncC . For DNA binding, amino acids E151 to T218 are essential, while repression depends not only on DNA binding but, additionally, on the adjacent region amino acids T218 to R255 . The C-terminus of KorB is the main dimerisation domain but a secondary oligomerisation region is located centrally in the region from amino acid I174 to T218 . Using three different methods (potentiation of transcriptional repression, potentiation of DNA binding and activation in the yeast two-hybrid system) we identify this region as also responsible for interactions with IncC . This IncC-KorB contact differs in location from the ParA-ParB/SopA-SopB interactions in P1/F but is similar to these systems in lying close to a masked oligomerisation determinant.

Nucleic Acids Res, 2002 Feb 15, 30(4), 1009 - 15
Involvement of Rad51C in two distinct protein complexes of Rad51 paralogs in human cells; Liu N et al.; Genetic studies in rodent and chicken mutant cell lines have suggested that Rad51 paralogs (XRCC2, XRCC3, Rad51B/Rad51L1, Rad51C/Rad51L2 and Rad51D/Rad51L3) play important roles in homologous recombinational repair of DNA double-strand breaks and in maintaining chromosome stability . Previous studies using yeast two- and three-hybrid systems have shown interactions among these proteins, but it is not clear whether these interactions occur simultaneously or sequentially in vivo . By utilizing immunoprecipitation with extracts of human cells expressing epitope-tagged Rad51 paralogs, we demonstrate that XRCC2 and Rad51D, while stably interacting with each other, co-precipitate with Rad51C but not with XRCC3 . In contrast, Rad51C is pulled down with XRCC3, whereas XRCC2 and Rad51D are not . In addition, Rad51B could be pulled down with Rad51C and Rad51D, but not with XRCC3 . These results suggest that Rad51C is involved in two distinct in vivo complexes: Rad51B-Rad51C-Rad51D-XRCC2 and Rad51C-XRCC3 . In addition, we demonstrate that Rad51 co-precipitates with XRCC3 but not with XRCC2 or Rad51D, suggesting that Rad51 can be present in an XRCC3-Rad51C-Rad51 complex . These complexes may act as functional units and serve accessory roles for Rad51 in the presynapsis stage of homologous recombinational repair.

Nucleic Acids Res, 2002 Feb 15, 30(4), 1001 - 8
Interactions involving the Rad51 paralogs Rad51C and XRCC3 in human cells; Wiese C et al.; Homologous recombinational repair of DNA double-strand breaks and crosslinks in human cells is likely to require Rad51 and the five Rad51 paralogs (XRCC2, XRCC3, Rad51B/Rad51L1, Rad51C/Rad51L2 and Rad51D/Rad51L3), as has been shown in chicken and rodent cells . Previously, we reported on the interactions among these proteins using baculovirus and two- and three-hybrid yeast systems . To test for interactions involving XRCC3 and Rad51C, stable human cell lines have been isolated that express (His)6-tagged versions of XRCC3 or Rad51C . Ni2+-binding experiments demonstrate that XRCC3 and Rad51C interact in human cells . In addition, we find that Rad51C, but not XRCC3, interacts directly or indirectly with Rad51B, Rad51D and XRCC2 . These results argue that there are at least two complexes of Rad51 paralogs in human cells (Rad51C-XRCC3 and Rad51B-Rad51C-Rad51D-XRCC2), both containing Rad51C . Moreover, Rad51 is not found in these complexes . X-ray treatment did not alter either the level of any Rad51 paralog or the observed interactions between paralogs . However, the endogenous level of Rad51C is moderately elevated in the XRCC3-overexpressing cell line, suggesting that dimerization between these proteins might help stabilize Rad51C.

J Biol Chem, 2002 May 31, 277(22), 19783 - 91 Epub 2002 Feb 12.
The insulin-sensitive glucose transporter, GLUT4, interacts physically with Daxx . Two proteins with capacity to bind Ubc9 and conjugated to SUMO1; Lalioti VS et al.; In this study we have used the yeast two-hybrid system to identify proteins that interact with the carboxyl-cytoplasmic domain (residues 464-509) of the insulin-sensitive glucose transporter GLUT4 (C-GLUT4) . Using as bait C-GLUT4, we have isolated the carboxyl domain of Daxx (C-Daxx), the adaptor protein associated with the Fas and the type II TGF-beta (TbetaRII) receptors (1,2 ) . The two-hybrid interaction between C-GLUT4 and C-Daxx is validated by the ability of in vitro translated C-GLUT4 to interact with in vitro translated full-length Daxx and C-Daxx . C-Daxx does not interact with the C-cytoplasmic domain of GLUT1, the ubiquitous glucose transporter homologous to GLUT4 . Replacement of alanine and serine for the dileucine pair (Leu(489)-Leu(490)) critical for targeting GLUT4 from the trans-Golgi network to the perinuclear intracellular store as well as for its surface internalization by endocytosis inhibits 2-fold the interaction of C-GLUT4 with Daxx . Daxx is pulled down with GLUT4 immunoprecipitated from lysates of 3T3-L1 fibroblasts stably transfected with GLUT4 and 3T3-L1 adipocytes expressing physiological levels of the two proteins . Similarly, GLUT4 is recovered with anti-Daxx immunoprecipitates . Using an established cell fractionation procedure we present evidence for the existence of two distinct intracellular Daxx pools in the nucleus and low density microsomes . Confocal immunofluorescence microscopy studies localize Daxx to promyelocytic leukemia nuclear bodies and punctate cytoplasmic structures, often organized in strings and underneath the plasma membrane . Daxx and GLUT4 are SUMOlated as shown by their reaction with an anti-SUMO1 antibody and by the ability of this antibody to pull down Daxx and GLUT4.

Mol Cell Biol, 2002 Mar, 22(5), 1513 - 25
Identification of Akt association and oligomerization domains of the Akt kinase coactivator TCL1; Kunstle G et al.; Serine/threonine kinase Akt/protein kinase B, the cellular homologue of the transforming viral oncogene v-Akt, plays a central role in the regulation of cell survival and proliferation . We have previously demonstrated that the proto-oncogene TCL1 is an Akt kinase coactivator . TCL1 binds to Akt and mediates the formation of oligomeric TCL1-Akt high-molecular-weight protein complexes in vivo . Within these protein complexes, Akt is preferentially phosphorylated and activated . The MTCP1/TCL1/TCL1b oncogene activation is the hallmark of human T-cell prolymphocytic leukemia (T-PLL), a form of adult leukemia . In the present study, using a PCR-generated random TCL1 library combined with a yeast two-hybrid screening detecting loss of interaction, we identified D16 and I74 as amino acid residues mediating the association of TCL1 with Akt . Based on molecular modeling, we determined that the beta C-sheet of TCL1 is essential for TCL1 homodimerization . Studies with mammalian overexpression systems demonstrated that both Akt association and oligomerization domains of TCL1 are distinct functional domains . In vitro kinase assays and overexpression experiments in mammalian cells demonstrated that both TCL1-Akt interaction and oligomerization of TCL1 were required for TCL1-induced Akt activation and substrate phosphorylation . Assays for mitochondrial permeability transition, nuclear translocation, and cell recovery demonstrated that both Akt association and homodimerization of TCL1 are similarly needed for the full function of TCL1 as an Akt kinase coactivator in vivo . The results demonstrate the structural basis of TCL1-induced activation of Akt, which causes human T-PLL.

J Biol Chem, 2002 May 3, 277(18), 15819 - 27 Epub 2002 Feb 11.
Galectin-3 translocates to the perinuclear membranes and inhibits cytochrome c release from the mitochondria . A role for synexin in galectin-3 translocation; Yu F et al.; Galectin-3 is a multifunctional oncogenic protein found in the nucleus and cytoplasm and also the extracellular milieu . Although recent studies demonstrated an anti-apoptotic activity of galectin-3, neither the functional site nor the mechanism of how galectin-3 regulates apoptosis is known . In this study, we examined the subcellular localization of galectin-3 during apoptosis and investigated its anti-apoptotic actions . We report that galectin-3 translocates to the perinuclear membrane following a variety of apoptotic stimuli . Confocal microscopy and biochemical analysis revealed that galectin-3 is enriched in the mitochondria and prevents mitochondrial damage and cytochrome c release . Using a yeast two-hybrid system, we screened for galectin-3-interacting proteins that regulate galectin-3 localization and anti-apoptotic activity . Synexin, a Ca(2+)- and phospholipid-binding protein, was one of the proteins identified . We confirmed direct interaction between galectin-3 and synexin by glutathione S-transferase pull-down assay in vitro . We showed that galectin-3 failed to translocate to the perinuclear membranes when expression of synexin was down-regulated using an oligodeoxyribonucleotide complementary to the synexin mRNA, suggesting a role for synexin in galectin-3 trafficking . Furthermore, synexin down-regulation abolished anti-apoptotic activity of galectin-3 . Taken together, these results suggest that synexin mediates galectin-3 translocation to the perinuclear mitochondrial membranes, where it regulates mitochondrial integrity critical for apoptosis regulation.

Nat Cell Biol, 2002 Mar, 4(3), 222 - 31
Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex; Fujita Y et al.; In epithelial cells, tyrosine kinases induce the tyrosine phosphorylation and ubiquitination of the E-cadherin complex, which induces endocytosis of E-cadherin . With a modified yeast 2-hybrid system, we isolated Hakai, an E-cadherin binding protein, which we have identified as an E3 ubiquitin-ligase . Hakai contains SH2, RING, zinc-finger and proline-rich domains, and interacts with E-cadherin in a tyrosine phosphorylation-dependent manner, inducing ubiquitination of the E-cadherin complex . Expression of Hakai in epithelial cells disrupts cell--cell contacts and enhances endocytosis of E-cadherin and cell motility . Through dynamic recycling of E-cadherin, Hakai can thus modulate cell adhesion, and could participate in the regulation of epithelial--mesenchymal transitions in development or metastasis.

J Clin Endocrinol Metab, 2002 Feb, 87(2), 752 - 7
Identification of the 49-kDa autoantigen associated with lymphocytic hypophysitis as alpha-enolase; O'Dwyer DT et al.; Lymphocytic hypophysitis is part of the spectrum of organ-specific autoimmune diseases, and although its histopathology is well documented, its pathogenesis is unclear . Serum autoantibodies directed against a 49-kDa cytosolic protein are detected by immunoblotting in 70% of patients with biopsy-proven lymphocytic hypophysitis . Here we report the purification and identification of this first target autoantigen in lymphocytic hypophysitis . The autoantigen has a molecular mass of 49 kDa, a cytosolic localization, and a ubiquitous tissue distribution . The 49-kDa protein was purified from monkey brain and human placental cytosol . Limited amino acid sequencing after proteolytic digestion of the human placental protein showed identity with alpha-enolase . The identification was confirmed using sera from patients with pituitary autoimmunity, which strongly reacted with recombinant human alpha-enolase and yeast enolase, but not with rabbit muscle beta- enolase . This indicates that the immunoreactive epitopes are largely conserved from yeast to human, but are not present in beta-enolase . alpha-Enolase autoantibodies are not specific to pituitary autoimmune disease and have been reported in other autoimmune diseases . However, this study is the first to indicate a role for alpha-enolase as an autoantigen in lymphocytic hypophysitis.

J Biol Chem, 2002 Apr 19, 277(16), 13873 - 82 Epub 2002 Feb 08.
MRK, a mixed lineage kinase-related molecule that plays a role in gamma-radiation-induced cell cycle arrest; Gross EA et al.; Mitogen-activated protein (MAP) kinase pathways are three-kinase modules that mediate diverse cellular processes and have been highly conserved among eukaryotes . By using a functional complementation screen in yeast, we have identified a human MAP kinase kinase kinase (MAPKKK) that shares homology with members of the mixed lineage kinase (MLK) family and therefore was called MRK (MLK-related kinase) . We report the structure of the MRK gene, from which are generated two splice forms of MRK, MRK-alpha and MRK-beta, encoding for proteins of 800 and 456 amino acids, respectively . By using a combination of solid phase protein kinase assays, transient transfections in cells, and analysis of endogenous proteins in stably transfected Madin-Darby canine kidney cells, we found that MRK-beta preferentially activates ERK6/p38gamma via MKK3/MKK6 and JNK through MKK4/MKK7 . We also show that expression of wild type MRK increases the cell population in the G(2)/M phase of the cell cycle, whereas dominant negative MRK attenuates the G(2) arrest caused by gamma-radiation . In addition, exposure of cells to gamma-radiation induces MRK activity . These data suggest that MRK may mediate gamma-radiation signaling leading to cell cycle arrest and that MRK activity is necessary for the cell cycle checkpoint regulation in cells.

Environ Health Perspect, 2002 Feb, 110(2), A88 - 91
Delitto Perfetto: foreign DNA disappears without a trace; Medlin J; A group of NIEHS scientists has recently perfected a new in vivo gene modification technique that enables scientists to quickly generate site-directed mutations onto specific regions of the yeast genome . The researchers were able to create genetic mutations by inserting foreign DNA into host DNA without leaving a trace of the foreign DNA . Dubbed "Delitto Perfetto," Italian for "perfect murder," the new technique leaves behind no clue that foreign DNA was introduced to engineer the desired genetic changes . It could transform the way genetic researchers analyze how genes function and respond to human disease and environmental influences.

J Cell Biochem, 2002, 84(4), 795 - 802
Identification of transcription factor in the promoter region of rat regucalcin gene: binding of nuclear factor I-A1 to TTGGC motif; Misawa H et al.; Hepatic nuclear protein has been reported to bind specifically to the TTGGC sequence of the rat regucalcin gene promoter region in stimulating the promoter activity (Misawa and Yamaguchi {2000} Biochem . Biophys . Res . Commun . 279: 275-281) . The present study was undertaken to identify transcription factor, which binds to TTGGC motif in the rat regucalcin gene promoter, using the yeast one-hybrid system . The sequence between -525 and -504, which has been defined as a functional promoter element II-b, was used as bait to screen a rat liver cDNA library . Two cDNA clones were identified as a nuclear factor I-A1 (NF1-A1) . The results of gel mobility shift assay and mutation analysis using recombinant NF1-A1 protein showed that this protein could specifically bind to TTGGC motif of the II-b oligonucleotide in promoter region . The expression of NF1-A1 mRNA was found in the liver, kidney, heart, spleen, and brain of rats . This study demonstrates that NF1-A1 is a transcription factor in stimulating the rat regucalcin gene promoter activity .

Bioessays, 2002 Feb, 24(2), 166 - 74
Histone ubiquitination: a tagging tail unfolds?
Jason LJ, Moore SC, Lewis JD, Lindsey G, Ausio J.
Despite the fact that histone H2A ubiquitination affects about 10-15% of this histone in most eukaryotic cells, histone ubiquitination is among one of the less-well-characterized post-translational histone modifications . Nevertheless, some important observations have been made in recent years . Whilst several enzymes had been known to ubiquitinate histones in vitro, recent studies in yeast have led to the unequivocal identification of the enzyme responsible for this post-translational modification in this organism . A strong functional co-relation to meiosis and spermiogenesis has also now been well documented, although its participation in other functional aspects of chromatin metabolism, such as transcription or DNA repair, still remains rather speculative and controversial . Because of its nature, histone ubiquitination represents the most bulky structural change to histones and as such it would be expected to exert an important effect on chromatin structure . Past and recent structural studies, however, indicate a surprising lack of effect of (H2A/H2B) ubiquitination on nucleosome architecture and of uH2A on chromatin folding . These results suggest that this modification may serve as a signal for recognition by functionally relevant trans-acting factors and/or operate synergistically in conjunction with other post-translational modifications such as for instance acetylation .

Prev Cardiol, 2000 Spring, 3(2), 83 - 87
Current concepts in optimum nutrition for cardiovascular disease; Platt R; For the past decade, nutritionists have focused on consensus guidelines (National Cholesterol Education Program) to reduce dietary saturated fatty acids, cholesterol, and excess body weight . However, researchers are looking at other ways that diet may influence the progression of cardiovascular disease, including lipoprotein oxidation, thrombosis progression, cardiac arrhythmia, and medication interaction . Some areas of investigation include the role of various fatty acids and supplements-in the form of vitamins, minerals, herbs, and functional foods-as well as traditional foods and diets from other parts of the world . This review outlines some of the new and relevant nutritional approaches including: specific fatty acids (omega 3, monounsaturated and trans fatty acids), dietary supplements (herbs, antioxidants, vitamins C and E, Coenzyme Q10, B vitamins and homocysteine, L-arginine, Chinese red yeast rice, garlic, soy, flax seed, and dietary fiber), food and drink (tea, nuts, plant-sterol and stanol-ester-containing spreads, alcohol, and grapefruit juice), and the Mediterranean diet . (c) 2000 by CHF, Inc.

J Chromatogr A, 2002 Jan 25, 944(1-2), 189 - 201
Optimization of azeotropic protein separations in gradient and isocratic ion-exchange simulated moving bed chromatography; Houwing J et al.; The separation of dilute binary mixtures of proteins by salt aided ion-exchange simulated moving bed (SMB) chromatography is optimized with respect to throughput, desorbent consumption and salt consumption . The optimal flow-rate ratios are analytically determined via an adopted "triangle theory" . Azeotropic phenomena are included in this procedure . The salt concentrations in the feed and recycled liquid are subsequently determined by numerical optimization . The azeotropic separation of bovine serum albumin and a yeast protein is used to illustrate the procedure . Gradient operation of the SMB is generally preferred over isocratic operation . A feed of azeotropic salt concentration can only be separated in a gradient SMB . Desorbent and salt consumption are always lower in gradient than in isocratic SMB chromatography.

Am J Physiol Renal Physiol, 2002 Mar, 282(3), F424 - 30
Interaction with grp58 increases activity of the thiazide-sensitive Na-Cl cotransporter; Wyse B et al.; The thiazide-sensitive sodium-chloride cotransporter (NCC) is expressed by distal convoluted tubule cells of the mammalian kidney . We used yeast two-hybrid screening to identify that glucose-regulated protein 58 (grp58), a protein induced by glucose deprivation, binds to the COOH terminus of the NCC . Immunoprecipitation of rat kidney cortex homogenates using a guinea pig anti-NCC antibody confirmed that grp58 associates with the NCC in vivo . Northern blots indicated that grp58 is highly expressed in rat kidney cortex . Immunofluorescence showed that grp58 protein abundance in kidney is highest in epithelial cells of the distal nephron, where it colocalizes with NCC near the apical membrane . To determine whether this interaction has a functional significance, NCC and grp58 cRNA were coexpressed in Xenopus laevis oocytes . In oocytes overexpressing grp58, sodium uptake was increased compared with control . Because oocytes express endogenous grp58, antisense experiments were performed to evaluate whether endogenous grp58 affected NCC activity in oocytes . Sodium uptake was lower in oocytes injected with both antisense grp58 cRNA and sense NCC compared with sense NCC oocytes . Western blot analysis did not show any effect of grp58 expression on processing of the NCC . These data indicate a novel, functionally important interaction between grp58 and the NCC in rat kidney cortex.

Appl Microbiol Biotechnol, 2002 Jan, 58(1), 67 - 72
Evaluation of an endo-beta-mannanase produced by Streptomyces ipomoea CECT 3341 for the biobleaching of pine kraft pulps; Montiel MD et al.; An endo-beta-mannanase (EC 3.2.1.78) from Streptomyces ipomoea CECT 3341 was purified and applied to the biobleaching of pine kraft pulps . The maximum level of endo-beta-mannanase activity (0.6 units ml(-1)) was achieved after 4 days of growth in a medium containing locust bean gum and yeast extract . Zymograms revealed mannanase bands (Man) with high and low electrophoretic mobility on the second and seventh days of incubation (Man1, Man3) and three bands of high, medium and low mobility from the third to sixth days of growth (Man1, Man2, Man3) . Although these exhibited different molecular masses, their amino-terminal sequences were identical . The action of proteases detected in the culture supernatant could be responsible for such events, suggesting that only one endo-beta-mannanase is produced by S . ipomoea . The purified Man3 exhibited a molecular mass of 40 kDa, an isoelectric point of 4.0 and an optimal temperature and pH reaction of 55 degrees C and 7.5, respectively . It was strongly inhibited by Ag+, Hg2+, Al3+ and Fe3+, and was strongly activated by Mn2+ . The ability of the purified endo-beta-mannanase to improve the bleachability of pine kraft pulp, when applied with alkaline extraction, was demonstrated by an increase in the pulp brightness (1.7%, using the International Standards Organisation's test) and an absence of variations in the viscosity values . A relationship between the increase in pulp brightness and the presence of manganese in the pulps could be established.

Genomics, 2002 Feb, 79(2), 177 - 85
Evolution of the regulators of G-protein signaling multigene family in mouse and human; Sierra DA et al.; The regulators of G-protein signaling (RGS) proteins are important regulatory and structural components of G-protein coupled receptor complexes . RGS proteins are GTPase activating proteins (GAPs) of Gi-and Gq-class Galpha proteins, and thereby accelerate signaling kinetics and termination . Here, we mapped the chromosomal positions of all 21 Rgs genes in mouse, and determined human RGS gene structures using genomic sequence from partially assembled bacterial artificial chromosomes (BACs) and Celera fragments . In mice and humans, 18 of 21 RGS genes are either tandemly duplicated or tightly linked to genes encoding other components of G-protein signaling pathways, including Galpha, Ggamma, receptors (GPCR), and receptor kinases (GPRK) . A phylogenetic tree revealed seven RGS gene subfamilies in the yeast and metazoan genomes that have been sequenced . We propose that similar systematic analyses of all multigene families from human and other mammalian genomes will help complete the assembly and annotation of the human genome sequence.

Biochem Biophys Res Commun, 2002 Feb 15, 291(1), 85 - 90
Identification of rat EMAP, a delta-glutamate receptor binding protein; Ly CD et al.; While most subtypes of glutamate receptors have been studied extensively, less is known about the delta-glutamate receptors, delta1 and delta2 . Although neither forms functional channels when expressed in heterologous cells, genetic analyses have demonstrated the physiological significance of delta2 . We used the cytosolic C-terminus of the delta2 glutamate receptor subunit in a yeast two-hybrid screen of a rat brain cDNA library to identify delta-glutamate receptor binding proteins . We isolated rat EMAP, the rat homolog of a microtubule-associated protein initially isolated and characterized in echinoderms . Rat EMAP contains 10 WD-repeats, which are domains important for mediating protein-protein interactions in a wide variety of proteins . Rat EMAP binds to delta-glutamate receptor subunits within a 50-amino-acid segment of the delta C-terminus . It is widely expressed in both brain and peripheral tissues, including high expression in brainstem and enrichment in the postsynaptic density . (c)2002 Elsevier Science (USA).

Plant Cell Physiol, 2002 Jan, 43(1), 99 - 107
Arabidopsis GARP transcriptional activators interact with the Pro-rich activation domain shared by G-box-binding bZIP factors; Tamai H et al.; The Pro-rich regions, found in a subset of plant bZIP transcription factors, including G-box-binding factors (GBFs) of Arabidopsis thaliana, are thought to be deeply involved in transcriptional regulation . However, the molecular mechanisms of the Pro-rich region-mediated transcriptional regulation are still largely unknown . Here we report evidence showing that two closely related Arabidopsis proteins, designated GPRI1 and GPRI2, containing a GARP DNA-binding domain, are likely partners of one or more GBFs . The results of yeast two-hybrid assays and in vitro binding assays indicated that GPRI1 can interact with the Pro-rich regions of GBF1 and GBF3 . GPRI2 interacted with the Pro-rich region of GBF1 . GPRI1 and GPRI2 transactivated transcription in yeast . In GPRI1 the region responsible for this activation was mapped in the N-terminal third of the protein . Transient assays showed that in Arabidopsis cells not only the N-terminal but also the C-terminal regions of GPRI1 can function as a separable activation domain . GPRI1 and GPRI2 may function in some promoters in concert with a GBF through interaction with its Pro-rich region to enhance the transcriptional level of the corresponding genes.

Plant Cell Physiol, 2002 Jan, 43(1), 1 - 11
Ped3p is a peroxisomal ATP-binding cassette transporter that might supply substrates for fatty acid beta-oxidation; Hayashi M et al.; Glyoxysomes, a group of specialized peroxisomes, are organelles that degrade fatty acids by the combination of fatty acid beta-oxidation and glyoxylate cycle . However, the mechanism underlying the transport of the fatty acids across the peroxisomal membrane is still obscure in higher plant cells . We identified and analyzed the PED3 gene and its gene product, Ped3p . The phenotype of the Arabidopsis ped3 mutant indicated that the mutation in the PED3 gene inhibits the activity of fatty acid beta-oxidation . Ped3p is a 149-kDa protein that exists in peroxisomal membranes . The amino acid sequence of Ped3p had a typical characteristic for "full-size" ATP-binding cassette (ABC) transporter consisting of two transmembrane regions and two ATP-binding regions . This protein was divided into two parts, that had 32% identical amino acid sequences . Each part showed a significant sequence similarity with peroxisomal "half" ABC transporters so far identified in mammals and yeast . Ped3p may contribute to the transport of fatty acids and their derivatives across the peroxisomal membrane.

Genome Res, 2002 Feb, 12(2), 255 - 71
Metallochaperones and metal-transporting ATPases: a comparative analysis of sequences and structures; Arnesano F et al.; A comparative structural genomic analysis of a new class of metal-trafficking proteins can provide insights into the intracellular chemistry of reactive cofactors such as copper and zinc . Starting from the sequences of the metallochaperone Atx1 and from the first soluble domain of the copper-transporting ATPase Ccc2, both from yeast, a search on the available genomes was performed using a homology criterion and a metal-binding motif x'-x"-C-x'''-x''''-C . By limiting ourselves to 20% identity with any of the proteins found, several soluble copper-transport proteins were identified, as well as soluble domains of membrane-bound ATPases . Structural models were calculated using high-resolution solution structures as templates, and the models were validated using statistical and energy criteria . Residue conservation and substitution have been interpreted and discussed in terms of structure-function relationship . The potential energy surfaces have been analyzed in terms of protein-protein interactions . We find that metallochaperones and their physiological partner ATPases from several phylogenetic kingdoms recognize one another, via an interplay of electrostatics, hydrogen bonding, and hydrophobic interactions, in a manner that precisely orients the metal-binding side chains for rapid metal transfer between otherwise tight binding sites . Finally, other putative metal-transport proteins are mentioned that have low homology and/or a different metal-binding consensus motif and that appear to use similar structures for recognition and transfer . This analysis highlights the wealth and the complexity of the field.

Curr Opin Chem Biol, 2002 Feb, 6(1), 92 - 6
One from column A and two from column B: the benefits of phage display in molecular-recognition studies; Rodi DJ et al.; Recent uses of phage-displayed combinatorial peptide and cDNA libraries have proven invaluable in mapping protein-protein interactions, protein-drug interactions, and the generation of 'molecular therapeutics' . This article reviews some of the findings of the past year and points out some of the pros and cons of phage display as compared with those of yeast two-hybrid screening.

Curr Opin Chem Biol, 2002 Feb, 6(1), 57 - 62
Two-hybrid arrays; Uetz P; The two-hybrid system is a genetic method for detecting protein-protein interactions . The assay can be applied to random libraries or arrays of colonies that express defined pairs of proteins . Arrays enable the testing of all possible protein pairs for interactions in a systematic fashion . The array format makes a large number of individual assays comparable and thus greatly simplifies the identification of false positives . Two-hybrid arrays have been used to study interactions among the proteins of yeast, hepatitis C virus, vaccinia virus, Drosophila, Caenorhabditis elegans, mouse and other species, and have already identified thousands of interactions.

Cardiovasc Res, 2002 Feb 1, 53(2), 423 - 30
Elevated hexokinase increases cardiac glycolysis in transgenic mice; Liang Q et al.; OBJECTIVE: Cardiac glucose metabolism is critical to normal and pathological function . The significance of the first committed metabolic step, glucose phosphorylation, has not been established . In this study a new transgenic model was developed in order to investigate the importance of this enzymatic step in cardiac glycolysis . METHODS: Transgenic mice were produced that overexpress yeast hexokinase B under the control of a cardiac specific promoter . Yeast hexokinase B is a high affinity enzyme that is not inhibited by glucose-6-phosphate . Hexokinase enzyme activity was measured by a modified radiometric procedure . Cardiac glucose metabolism and contractility were measured in the Langendorff mode . Cardiac glycogen content and glucose-6-phosphate independent glycogen synthase activity were also determined . RESULTS: In transgenic hearts hexokinase activity was significantly elevated and increased glucose metabolism, particularly in the presence of insulin and during cardiac reperfusion . However during ischemic perfusion the effect of the transgene on glycolysis was minimal . Under all conditions tested there was no effect of hexokinase on contractility . Glycogen content of transgenic hearts was elevated 2-fold and glucose-6-phosphate independent glycogen synthase was also increased . CONCLUSION: These results demonstrate that glucose phosphorylation is a key step in determining cardiac glucose metabolism under oxidative conditions.

Mol Cell Biochem, 2001 Nov, 227(1-2), 21 - 9
Functional specialization of CK2 isoforms and characterization of isoform-specific binding partners; Litchfield DW et al.; In mammals, protein kinase CK2 has two isozymic forms of its catalytic subunit, designated CK2alpha and CK2alpha' . CK2alpha and CK2alpha' exhibit extensive similarity within their catalytic domains but have completely unrelated C-terminal sequences . To systematically examine the cellular functions of each CK2 isoform in mammalian cells, we have generated human osteosarcoma U2-OS cell lines with the expression of active or inactive versions of each CK2 isoform under the control of an inducible promoter . Examination of these cell lines provides evidence for functional specialization of CK2 isoforms at the cellular level in mammals with indications that CK2alpha' is involved in the control of proliferation and/or cell survival . To understand the molecular basis for functional differences between CK2alpha and CK2alpha', we have undertaken studies to identify proteins that interact specifically with each isoform of CK2 and could contribute to the regulation of their independent functions . A novel pleckstrin-homology domain containing protein, designated CK2-interacting protein 1 (i.e . CKIP-1) was isolated using the yeast two hybrid system as a protein that interacts with CK2alpha but not CK2alpha' . When expressed in cells as a fusion with green fluorescent protein, CKIP-1 localizes to the cell membrane and to the nucleus . In this study, we present evidence from deletion analysis of CKIP-1 suggesting that a C-terminal region containing a putative leucine zipper has a role in regulating its nuclear localization . Collectively, our data supports a model whereby CKIP-1 is a non-enzymatic regulator of CK2alpha that regulates the cellular functions of CK2alpha by targeting or anchoring CK2alpha to specific cellular localization or by functioning as an adapter to integrate CK2alpha-mediated signaling events with components of other signal transduction pathways.

Plant Cell, 2002 Jan, 14(1), 33 - 46
SGR2, a phospholipase-like protein, and ZIG/SGR4, a SNARE, are involved in the shoot gravitropism of Arabidopsis; Kato T et al.; In higher plants, the shoot and the root generally show negative and positive gravitropism, respectively . To elucidate the molecular mechanisms involved in gravitropism, we have isolated many shoot gravitropism mutants in Arabidopsis . The sgr2 and zig/sgr4 mutants exhibited abnormal gravitropism in both inflorescence stems and hypocotyls . These genes probably are involved in the early step(s) of the gravitropic response . The sgr2 mutants also had misshapen seed and seedlings, whereas the stem of the zig/sgr4 mutants elongated in a zigzag fashion . The SGR2 gene encodes a novel protein that may be part of a gene family represented by bovine phosphatidic acid-preferring phospholipase A1 containing a putative transmembrane domain . This gene family has been reported only in eukaryotes . The ZIG gene was found to encode AtVTI11, a protein that is homologous with yeast VTI1 and is involved in vesicle transport . Our observations suggest that the two genes may be involved in a vacuolar membrane system that affects shoot gravitropism.

Plant Cell, 2002 Jan, 14(1), 17 - 32
Cytokinin growth responses in Arabidopsis involve the 26S proteasome subunit RPN12; Smalle J et al.; The 26S proteasome is an ATP-dependent eukaryotic protease responsible for degrading many important cell regulators, especially those conjugated with multiple ubiquitins . Bound on both ends of the 20S core protease is a multisubunit regulatory particle that plays a crucial role in substrate selection by an as yet unknown mechanism(s) . Here, we show that the RPN12 subunit of the Arabidopsis regulatory particle is involved in cytokinin responses . A T-DNA insertion mutant that affects RPN12a has a decreased rate of leaf formation, reduced root elongation, delayed skotomorphogenesis, and altered growth responses to exogenous cytokinins, suggesting that the mutant has decreased sensitivity to the hormone . The cytokinin-inducible genes CYCD3 and NIA1 are upregulated constitutively in rpn12a-1, indicating that feedback-inhibitory mechanisms also may be altered . rpn12a-1 seedlings also showed changes in auxin-induced growth responses, further illustrating the close interaction between auxin and cytokinin regulation . In yeast, RPN12 is necessary for the G1/S and G2/M transitions of the cell cycle, phases that have been shown to be under cytokinin control in plants . We propose that RPN12a is part of the Arabidopsis 26S proteasome that controls the stability of one or more of the factors involved in cytokinin regulation.

J Neurosci, 2002 Feb 1, 22(3), 803 - 14
Delphilin: a novel PDZ and formin homology domain-containing protein that synaptically colocalizes and interacts with glutamate receptor delta 2 subunit; Miyagi Y et al.; The glutamate receptor delta2 (GluRdelta2) subunit is selectively expressed in cerebellar Purkinje cells and plays an important role in cerebellar long-term depression, motor learning, motor coordination, and synapse development . We identified a novel GluRdelta2-interacting protein, named Delphilin, that contains a single PDZ domain and formin homology (FH) domains FH1 and FH2 plus coiled-coil structure . As far as we know, this is the first reported protein that contains both PDZ and FH domains . Yeast two-hybrid and surface plasmon resonance (SPR) analyses indicated that Delphilin interacts with the GluRdelta2 C terminus via its PDZ domain . This was also supported by coimmunoprecipitation experiments using a heterologous expression system in mammalian cells . Yeast cell and SPR analyses also demonstrated the possibility that the FH1 proline-rich region of Delphilin interacts with profilin, an actin-binding protein, and with the Src homology 3 domain of neuronal Src protein tyrosine kinase . In situ hybridization demonstrated the highest expression of Delphilin mRNA in Purkinje cells . Delphilin polypeptide was highly enriched in the synaptosomal membrane fraction of the cerebellum and coimmunoprecipitated with the GluRdelta2 subunit . The post-embedding immunogold technique demonstrated that Delphilin is selectively localized at the postsynaptic junction site of the parallel fiber-Purkinje cell synapse and colocalized with GluRdelta2 . Thus, Delphilin is a postsynaptic scaffolding protein at the parallel fiber-Purkinje cell synapse, where it may serve to link GluRdelta2 with actin cytoskeleton and various signaling molecules.

J Clin Microbiol, 2002 Feb, 40(2), 556 - 74
Identification of four distinct genotypes of Candida dubliniensis and detection of microevolution in vitro and in vivo; Gee SF et al.; The present study investigates further the population structure of Candida dubliniensis and its ability to exhibit microevolution . Using 98 isolates (including 80 oral isolates) from 94 patients in 15 countries, we confirmed the existence of two distinct populations within the species C . dubliniensis, designated Cd25 group I and Cd25 group II, respectively, on the basis of DNA fingerprints generated with the C . dubliniensis-specific probe Cd25 . The majority of Cd25 group I isolates (48 of 71, 67.6%) were from human immunodeficiency virus (HIV)-infected individuals, whereas the majority of Cd25 group II isolates (19 of 27, 70.4%) were from HIV-negative individuals (P < or = 0.001) . Nucleotide sequence analysis of the internal transcribed spacer (ITS) regions of the rRNA genes from 19 representative isolates revealed the presence of four separate genotypes . All of the Cd25 group I isolates tested belonged to genotype 1, while the Cd25 group II population was comprised of three distinct genotypes (genotypes 2 to 4), which corresponded to distinct clades within the Cd25 group II population . These findings were confirmed using genotype-specific PCR primers with 70 isolates . We also showed that C . dubliniensis can exhibit microevolution in vivo and in vitro as occurs in other yeast species . DNA fingerprinting using the C . dubliniensis probes Cd25, Cd24, and Cd1 and karyotype analysis of multiple oral isolates recovered from the same specimen from each of eight separate patients revealed microevolution in six of eight of the clonal populations . Similarly, sequential clonal isolates from various anatomical sites in two separate patients exhibited microevolution . Microevolution was also shown to occur when two clinical isolates susceptible to fluconazole were exposed to the drug in vitro . The epidemiological significance of the four C . dubliniensis genotypes and the ability of C . dubliniensis to undergo microevolution has yet to be established.

Trends Microbiol, 2001 Nov, 9(11), 541 - 6
Molecular mycology: a genetic toolbox for Histoplasma capsulatum; Magrini V et al.; Research in medical mycology has traditionally been a mix of exciting biology and frustrating genetics, although the excitement has been steadily increasing as genetic obstacles have been successfully overcome . Now, a variety of fungal pathogens can be studied using molecular techniques derived from classical bacterial and yeast genetics, but with selective and strategic adaptations . Histoplasma capsulatum is the best-studied of the primary pathogens known as 'dimorphic' fungi, and tailored molecular genetic strategies are beginning to reveal a repertoire of genes and gene products intimately associated with pathogenesis.

Biochim Biophys Acta, 2002 Jan 31, 1594(1), 40 - 53
The novel non-glycosylated invertase from Candida utilis (the properties and the conditions of production and purification); Belcarz A et al.; The Candida utilis yeast, which is cultivated in liquid media enriched with saccharose, synthesizes the well-known invertase of 300 kDa (EC 3.2.1.26) . This enzyme is present both intracellularly in the periplasmic space and extracellularly in the culture broth . However, it was determined that the same C . utilis strain cultured in certain conditions is simultaneously capable of producing another, still unknown form of invertase with a molecular mass of 60 kDa . The presence of the latter enzymatic form was detected in cells as well as in the liquid culture medium . Both invertase forms were purified using a three-step process (ion-exchange chromatography, affinity chromatography, and preparative column electrophoresis) and named, due to their different migration ratio in polyacrylamide gel electrophoresis, F-form (Fast; 60 kDa) and S-form (Slow; 300 kDa) . The F-form of invertase was found to be nonglycosylated as opposed to the well-known S-form of invertase from the same source . The physicochemical properties of the F-form of invertase (isoelectric point, substrate specificity, pH, and temperature optima) were determined and compared with those of the S-form of the enzyme.

Proc AMIA Symp . 2001;:110-4.
A metadata framework for interoperating heterogeneous genome data using XML; Cheung KH et al.; The rapid advances in the Human Genome Project and genomic technologies have produced massive amounts of data populated in a large number of network-accessible databases . These technological advances and the associated data can have a great impact on biomedicine and healthcare . To answer many of the biologically or medically important questions, researchers often need to integrate data from a number of independent but related genome databases . One common practice is to download data sets (text files) from various genome Web sites and process them by some local programs . One main problem with this approach is that these programs are written on a case-by-case basis because the data sets involved are heterogeneous in structure . To address this problem, we define metadata that maps these heterogeneously structured files into a common eXtensible Markup Language (XML) structure to facilitate data interoperation . We illustrate this approach by interoperating two sets of essential yeast genes that are stored in two yeast genome databases (MIPS and YPD).

J Nutr, 2002 Feb, 132(2), 292 - 7
Dietary selenite and azadeoxycytidine treatments affect dimethylhydrazine-induced aberrant crypt formation in rat colon and DNA methylation in HT-29 cells; Davis CD et al.; Several observations implicate a role for altered DNA methylation in cancer pathogenesis . The global level of DNA methylation is generally lower; however, DNA methyltransferase (Dnmt1) activity is usually higher in tumor cells than in normal cells . The purpose of this study was to investigate whether the Dnmt1 inhibitor, 5-aza-2'-deoxycytidine (aza-dC) would alter the effect of dietary selenium on the formation of aberrant crypts . Weanling rats (n = 60) were fed three concentrations of selenium (deficient, 0.1 and 2.0 mg/kg diet) in a Torula yeast-based diet . Half of the rats were injected weekly with aza-dC (1 mg/kg, subcutaneously) and half were injected with the vehicle control (PBS) . After 3.5 wk of consuming the experimental diets, the rats were given two injections of dimethylhydrazine (DMH; 25 mg/kg, intraperitoneally) . Rats fed the selenium-deficient diet and injected with PBS had significantly (P < 0.006) more aberrant crypts than rats fed 0.1 or 2.0 mg selenium/kg diet (244 +/- 21 vs . 165 +/- 9 and 132 +/- 14, respectively) . In contrast, when rats were injected with aza-dC, there was a significant (P < 0.0001) reduction in aberrant crypt formation and dietary selenium had no effect (62 +/- 8 vs . 77 +/- 13 vs . 54 +/- 8, in rats fed 0, 0.1 and 2.0 mg selenium/kg diet, respectively) . HT-29 cells cultured in the absence of selenium had significantly hypomethylated DNA but significantly more Dnmt1 protein expression than cells cultured in the presence of 1 or 2 micromol/L selenium . These results suggest that aza-dC treatment may protect selenium-deficient rats against carcinogen-induced aberrant crypt formation.

J Biol Chem, 2002 Apr 12, 277(15), 12846 - 53 Epub 2002 Jan 31.
The Oxa1 protein forms a homooligomeric complex and is an essential part of the mitochondrial export translocase in Neurospora crassa; Nargang FE et al.; The Oxa1 protein is a ubiquitous constituent of the inner membrane of mitochondria . Oxa1 was identified in yeast as a crucial component of the protein export machinery known as the OXA translocase, which facilitates the integration of proteins from the mitochondrial matrix into the inner membrane . We have identified the Neurospora crassa Oxa1 protein which shows a sequence identity of 22% to the yeast homologue . Despite the low level of identity, the function of the homologues is conserved as the N . crassa gene fully complemented a yeast null mutant . Genetic analysis revealed that Oxa1 is essential for viability in N . crassa . Cells propagated under conditions that severely reduce Oxa1 levels grew extremely slowly and were deficient in subunits of complex I and complex IV . Isolation of the Oxa1 complex from N . crassa mitochondria revealed a 170-180-kDa complex that contained exclusively Oxa1 . Since the Oxa1 monomer has a molecular weight of 43,000, our data suggest that the OXA translocase consists of a homooligomer most likely containing four Oxa1 subunits.

J Biol Chem, 2002 Apr 26, 277(17), 14844 - 52 Epub 2002 Jan 31.
Identification of novel point mutations in ERK2 that selectively disrupt binding to MEK1; Robinson FL et al.; Extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) are essential components of pathways through which signals received at membrane receptors are converted into specific changes in protein function and gene expression . As with other members of the mitogen-activated protein (MAP) kinase family, ERK1 and ERK2 are activated by phosphorylations catalyzed by dual-specificity protein kinases known as MAP/ERK kinases (MEKs) . MEKs exhibit stringent specificity for individual MAP kinases . Indeed, MEK1 and MEK2 are the only known activators of ERK1 and ERK2 . ERK2 small middle dotMEK1/2 complexes can be detected in vitro and in vivo . The biochemical nature of such complexes and their role in MAP kinase signaling are under investigation . This report describes the use of a yeast two-hybrid screen to identify point mutations in ERK2 that impair its interaction with MEK1/2, yet do not alter its interactions with other proteins . ERK2 residues identified in this screen are on the surface of the C-terminal domain of the kinase, either within or immediately preceding alpha-helix G, or within the MAP kinase insert . Some mutations identified in this manner impaired the two-hybrid interaction of ERK2 with both MEK1 and MEK2, whereas others had a predominant effect on the interaction with either MEK1 or MEK2 . Mutant ERK2 proteins displayed reduced activation in HEK293 cells following epidermal growth factor treatment, consistent with their impaired interaction with MEK1/2 . However, ERK2 proteins containing MEK-specific mutations retained kinase activity, and were similar to wild type ERK2 in their activation following overexpression of constitutively active MEK1 . Unlike wild type ERK2, proteins containing MEK-specific point mutations were constitutively localized in the nucleus, even in the presence of overexpressed MEK1 . These data suggest an essential role for the MAP kinase insert and residues within or just preceding alpha-helix G in the interaction of ERK2 with MEK1/2.

Hum Mol Genet, 2002 Feb 1, 11(3), 331 - 5
The human intronless melanocortin 4-receptor gene is NMD insensitive; Brocke KS et al.; Nonsense-mediated decay (NMD) is a phylogenetically widely conserved mechanism that contributes to the fidelity of gene expression . NMD inhibits the accumulation of nonsense- or frameshift-mutated mRNA and thus minimizes the synthesis of truncated proteins with potential dominant negative effects . Yeast and higher eukaryotes use somewhat diverse mechanisms to promote NMD and to discriminate between premature and physiological translation termination codons . NMD in yeast involves the binding of specific RNA-binding proteins to cis-acting exonic elements . In contrast, NMD of the intron-containing genes of higher eukaryotes is splicing-dependent . Here, we investigated the NMD sensitivity of nonsense-mutated transcripts of the naturally intronless human melanocortin 4-receptor (MC4-R) gene . Nonsense-mutated variants of MC4-R transcripts are stable and express truncated proteins that are detectable in the lysates of transfected cells . Thus, the naturally intronless MC4-R gene and probably many other intronless genes fail to be monitored by the NMD pathway.

Biotechnol Prog, 2002 Jan-Feb, 18(1), 43 - 50
Mechanical stability of immobilized biocatalysts (CLECs) in dilute agitated suspensions; Lee TS et al.; Cross-linked enzyme crystals (CLECs) are a novel form of immobilized biocatalyst designed for application in industrial biotransformation processes . In this work we have investigated the mechanical stability of agitated CLEC suspensions in relation to the design and scale-up of bioconversions carried out in stirred-tank reactors . By careful control of the crystallization conditions yeast alcohol dehydrogenase I (YADHI) microcrystals of different size were first prepared having either an hexagonal (approximately 12 microm) or rod-shaped (approximately 4.6 microm) morphology . These were then cross-linked with glutaraldehyde to form CLECs . The rate of breakage of the CLEC suspensions was subsequently measured in a rotating disk shear device (total volume, 11 mL) by monitoring the change in crystal size distribution with time . This device is designed to mimic the shear and energy dissipation rates found in a range of process scale equipment and may be used to study the mechanical stability of any immobilized biocatalyst preparation . Experiments were performed as a function of the speed and duration of disk rotation, CLEC concentration (0.26-2.5 mg.mL(-1)) and energy dissipation rate (2.2 x 10(3) to 6.8 x 10(5) W.kg(-1)) . No breakage of the rod-shaped CLECs was observed over the entire range of experimental conditions investigated . Breakage of the larger hexagonal-shaped CLECs did occur, however, at energy dissipation rates, epsilon(max), above 1.0 x 10(5) W.kg(-1), where the calculated length scale of turbulence was around 2.0 microm . Based on visual observation of the sheared CLEC suspensions and models of crystal breakage, it was concluded that breakage of the hexagonal-shaped CLECs occurred due to shear induced attrition . Measurement of the catalytic activity of both the hexagonal and rod-shaped CLECs showed no significant change in activity before and after shearing.

Oncogene, 2002 Jan 17, 21(3), 377 - 86
The hepatitis B virus X protein abrogates Bcl-2-mediated protection against Fas apoptosis in the liver; Terradillos O et al.; The role of the hepatitis B virus protein HBx in liver cell proliferation and apoptosis remains controversial . Using a transgenic mouse model, we have recently shown that HBx stimulates the apoptotic turnover of hepatocytes, independently of p53 . In this paper, we tested whether the proapoptotic function of HBx can interfere with Bcl-2 during hepatic apoptosis in vivo . HBx transgenic mice were crossed with PK-hBcl-2 mice that are protected against Fas killing by constitutive overexpression of Bcl-2 in hepatocytes . In a lethal challenge with Fas antibodies, HBx expressed at low levels restored sensitivity to Fas-mediated apoptosis and fulminant hepatic failure in mice overexpressing Bcl-2 . Furthermore, cytochrome c release from mitochondria and caspase 3 activation were restored to normal levels in HBx/Bcl-2 mice during transduction of the Fas signal . Thus, the proapoptotic activity of HBx overcomes or bypasses the inhibitory effect of Bcl-2 against Fas cytotoxicity . This effect was not apparently mediated through downregulation of the PK-hBcl-2 transgene or via delocalization of the Bcl-2 protein, and a direct interaction of HBx with Bcl-2, Bcl-X(L) or Bax could not be evidenced in yeast two-hybrid assays . We further show that apoptosis induced by ectopic expression of HBx is associated with mitochondrial membrane alterations and caspase 3 activation . Our data indicate that the dominant function of HBx upon Bcl-2-regulated control of apoptosis might play an important role in the pathogenesis of chronic hepatitis B.

J Biol Chem, 2002 Apr 5, 277(14), 12053 - 60 Epub 2002 Jan 30.
Insulin-like growth factor-binding protein 5 (IGFBP-5) interacts with a four and a half LIM protein 2 (FHL2); Amaar YG et al.; Recent studies using insulin-like growth factor I (IGF-I) knockout mice demonstrate that IGF-binding protein (IGFBP)-5, an important bone formation regulator, itself is a growth factor with cellular effects not dependent on IGFs . Because IGFBP-5 contains a nuclear localization sequence that mediates transport of IGFBP-5 into the nucleus, we propose that IGFBP-5 interacts with nuclear proteins to affect transcription of genes involved in bone formation . We therefore undertook studies to identify proteins that bind to IGFBP-5 using IGFBP-5 as bait in a yeast two-hybrid screen of a U2 human osteosarcoma cDNA library . Five related clones that interacted strongly with the bait corresponded to the FHL2 gene, which contains four and a half LIM domains . Co-immunoprecipitation studies with lysates from U2 cells overexpressing FHL2 and IGFBP-5 confirmed that interaction between IGFBP-5 and FHL2 occurs in whole cells . In vitro interaction studies revealed that purified FHL2 interacted with IGFBP-5 but not with IGFBP-3, -4, or -6 . Northern blot analysis showed that FHL2 was strongly expressed in human osteoblasts . Nuclear localization of both FHL2 and IGFBP-5 was evident from Western immunoblot analysis and immunofluorescence . The role of FHL2 as an intracellular mediator of the effects of IGFBP-5 and other osteoregulatory agents in osteoblasts will need to be verified in future studies.

J Biochem (Tokyo), 2002 Feb, 131(2), 171 - 3
Structure determination of the constitutive 20S proteasome from bovine liver at 2.75 A resolution; Unno M et al.; The crystal structure of the 20S proteasome from bovine liver was determined by the molecular replacement method using the structure of the 20S proteasome from the yeast Sacccharomyces cerevisiae . The initial phases were refined by density modification coupled with non-crystallographic symmetry averaging . The structural model was refined with the program CNS . The final R-factor and R(free) were 0.25 and 0.29, respectively . The constitutive proteasome without any contamination by the immunoproteasome was identified in the crystal structure.

Org Lett, 2002 Feb 7, 4(3), 415 - 8
Synthesis of chimeric 7alpha-substituted estradiol derivatives linked to cholesterol and cholesterylamine; Hussey SL et al.; We report the synthesis of 7alpha-substituted beta-estradiol derivatives bearing side chains terminated with cholesterol and 3beta-cholesterylamine . These chimeric compounds were designed to exhibit high affinity for estrogen receptors (ERs) and cellular plasma membranes to potentially enable regulated uptake of ERs by mammalian cells . Evaluation with recombinant yeast reporting compound-mediated ER dimerization revealed potencies similar to the antiestrogen ICI 182780 . Compounds that efficiently deliver dominant negative ERs into cells may provide novel therapeutics against breast cancers.

Mycoses, 2001 Dec, 44(11-12), 480 - 6
Phenotypic characterization of Microsporum canis isolated from cats and dogs; Maia ML et al.; To characterize strains of Microsporum canis that infect dogs and cats in Sao Paulo city, 30 isolates of this dermatophyte were tested for their ability to assimilate carbon and nitrogen sources, for proteinase and phospholipase secretion, for susceptibility to yeast killer toxins, and for susceptibility to the antifungals fluconazole, ketoconazole, itraconazole, 5-fluorocytosine and amphotericin B, in E test . All samples assimilated the nitrogen sources asparagine, ammonium sulphate, urea and sodium nitrate, as well as the carbon sources inulin, mannitol, trehalose, meso-erythritol, maltose, mannose, sorbitol, cellobiose, fructose and dextrin . Not all the samples assimilated adonitol, galactose, arabinose, rhamnose, raffinose, melibiose, ribose and sucrose, and none of them was capable of growing with dulcitol, lactose, or xylose as the only carbon source . Proteinase and phospholipase secretion was observed for most isolates . In the test of yeast killer toxin, 10 types could be identified, with four types exclusively observed in isolates from dogs and two types exclusively observed in isolates from cats . In the E test, all isolates were found to be resistant to the fluconazole and 5-fluorocytosine, while they were variably sensitive to amphotericin B, ketoconazole and itraconazole . When the data were submitted to the qualitative analysis in the matrix distance program FITOPAC, the similarity of the isolates could be assessed.

World J Gastroenterol, 1998 Apr, 4(2), 103 - 105
Inhibitory effects of two oligosaccharides on murine melanoma experimental liver metastasis; Liu YP et al.; AIM:To observe the effects of a chemically synthesized tetrose and a natural yeast mannan on experimental liver metastasis of mouse melanoma.METHODS: After treated with 4mg tetrose (tetrose group) or 4mg mannan (mannan group) for 30 minutes at 37&mgr;,0.5ml 1 10(6) B16-MBK melanoma cells were injected into the spleen of mice.Fifty-five days later, melanoma metastatic nodes on the surface of the liver and in other organs as well as mouse survival time were observed.RESULTS: Of the 6 mice in control (B16 cell+PBS) group, 4 died naturally within 55 days, and 2 were killed on the 55th day.All of the 6 mice had metastases in livers, the total number of the melanoma nodes on each liver surface ranged from 2 to 30, with the largest one merging into the whole liver . One mouse had a neoplasm in the remnant site of injection, and 3 had metastases in lungs.In contrast, of the 6 mice in tetrose group, only one died on the 50th day after injection, with 3 metastases in the liver, the largest being 10mm in diameter, the other 5 mice survived until being dissected on the 55th day after injection and had no liver metastasis,but 3 of them had neoplasms in their remnant sites of injection.In mannan group,all of the 6 mice survived and no metastasis was seen except for 2 liver nodes in one mouse with the largest diameter of 1mm.Neither tetrose nor mannan group had metastasis out of the liver, and the weight of liver in the two groups was significantly lower than those in the control group.CONCLUSION:Both tetrose and mannan had the effects of preventing melanoma cells from experimental metastasis to and out of the liver, and prolonging the survival time of the mouse.

Mol Endocrinol, 2002 Feb, 16(2), 287 - 300
Domain interactions between coregulator ARA(70) and the androgen receptor (AR); Zhou ZX et al.; The coregulator function of AR-associated protein 70 (ARA(70)) was investigated to further characterize its interaction with the AR . Using a yeast two-hybrid assay, androgen-dependent binding of ARA(70) deletion mutants to the AR ligand-binding domain (LBD) was strongest with ARA(70) amino acids 321-441 of the 614 amino acid ARA(70) protein . Mutations adjacent to or within an FxxLF motif in this 120-amino acid region abolished androgen-dependent binding to the AR-LBD both in yeast and in glutathione-S-transferase affinity matrix assays . Yeast one-hybrid assays revealed an intrinsic ARA(70) transcriptional activation domain within amino acids 296-441 . In yeast assays the ARA(70) domains for transcriptional activation and for binding to the AR-LBD were inhibited by the C-terminal region of ARA(70) . Full-length ARA(70) increased androgen-dependent AR transactivation in transient cotransfection assays using a mouse mammary tumor virus-luciferase reporter in CV1 cells . ARA(70) also increased constitutive transcriptional activity of an AR NH(2)-terminal-DNA binding domain fragment and bound this region in glutathione-S-transferase affinity matrix assays . Binding was independent of the ARA(70) FxxLF motif . The results identify an ARA(70) motif required for androgen-dependent interaction with the AR-LBD and demonstrate that ARA(70) can interact with the NH(2)-terminal and carboxyl-terminal regions of AR.

J Leukoc Biol, 2002 Feb, 71(2), 304 - 10
The microphthalmia transcription factor and the related helix-loop-helix zipper factors TFE-3 and TFE-C collaborate to activate the tartrate-resistant acid phosphatase promoter; Mansky KC et al.; The microphthalmia transcription factor (MITF) regulates different target genes in several distinct cell types, including osteoclasts . The role of the closely related factors TFE3 and TFEC in MITF action was studied . The TFE3 and TFEC proteins were expressed in osteoclast-like cells, and both could be immunoprecipitated in a complex with MITF . In transient transfection assays, TFE3 and TFEC could collaborate with MITF to superactivate the tartrate resistant acid phosphatase (TRAP) promoter, a target for MITF in osteoclasts . Although TFEC had been thought to act as a repressor, we could demonstrate that TFEC acted as a transactivator when fused to the gal4 DNA-binding domain in a yeast one-hybrid-type assay . Additionally, two mRNA isoforms of MITF, MITF-M and MITF-A, were detected in primary osteoclast-like cells by RT-PCR . In transient transfection assays, the MITF-A and MITF-M isoforms activated the promoter of the TRAP gene to the same extent, and both forms could collaborate equally well with TFE3 to activate the TRAP promoter . These results indicate that although different isoforms of MITF appear to be functionally similar, the TFE3 and TFEC proteins may collaborate with MITF to efficiently regulate expression of target genes in osteoclasts.

EMBO Rep, 2002 Feb, 3(2), 177 - 82 Epub 2002 Jan 29.
An intact NEDD8 pathway is required for Cullin-dependent ubiquitylation in mammalian cells; Ohh M et al.; Skp1-Cdc53/Cul1-F-box (SCF) complexes constitute a class of E3 ubiquitin ligases . Recently, a multiprotein complex containing pVHL, elongin C and Cul2 (VEC) was shown to structurally and functionally resemble SCF complexes . Cdc53 and the Cullins can become covalently linked to the ubiquitin-like molecule Rub1/NEDD8 . Inhibition of neddylation inhibits SCF function in vitro and in yeast and plants . Here we show that ongoing neddylation is likewise required for VEC function in vitro and for the degradation of SCF and VEC targets in mammalian cells . Thus, neddylation regulates the activity of two specific subclasses of mammalian ubiquitin ligases.

Curr Biol, 2002 Jan 22, 12(2), R71 - 3
Centrosome inheritance: birthright or the privilege of maturity?
Bornens M, Piel M.
Budding yeast cells exhibit a defined mode of centrosome inheritance--the 'old' spindle pole body always segregates into the bud . But it is the astral microtubule-cortex interaction which matters for controlling the asymmetric localization of Bfa1p/Bub2 at spindle pole bodies.

Curr Biol, 2002 Jan 22, 12(2), 85 - 94
SPLAYED, a novel SWI/SNF ATPase homolog, controls reproductive development in Arabidopsis; Wagner D et al.; BACKGROUND: The plant-specific transcriptional activator LEAFY (LFY) is a central regulator of the transition to reproductive development in Arabidopsis . LFY has a second, later role in the induction of floral homeotic gene expression . Available data suggests that, while LFY activity is controlled via interaction with tissue-specific coactivators, other mechanisms exist that regulate LFY activity, the identity of which are not known . RESULTS: We have identified a novel component in the temporal control of the switch from vegetative to reproductive development in Arabidopsis thaliana . The SPLAYED (SYD) gene product acts with LFY to regulate shoot apical meristem identity . SYD is also involved in the regulation of floral homeotic gene expression . In addition, mutations in SYD cause LFY-independent phenotypes that indicate that SYD is necessary for meristem maintenance during reproductive development and that SYD is required for proper carpel and ovule development . SYD encodes a presumptive Arabidopsis homolog of the yeast Snf2p ATPase, which is implicated in transcriptional control via chromatin remodeling . CONCLUSIONS: SYD acts as a LFY-dependent repressor of the meristem identity switch in the floral transition, most likely by altering the activity of the LFY transcription factor . That SYD regulates flowering in response to environmental stimuli suggests that the effect of environmental cues on plant development may be achieved in part by regulating transcription factor activity via alteration of the chromatin state.

Food Addit Contam, 2002 Jan, 19(1), 15 - 21
3-Monochloropropane-1,2-diol (3-MCPD) in food ingredients from UK food producers and ingredient suppliers; Hamlet CG et al.; A survey of 3-monochloropropane-1,2-diol (3-MCPD) in a range of food ingredients available in the UK is reported . The survey was conducted for the Food Standards Agency to assess the progress made by manufacturers in reducing levels of 3-MCPD in food ingredients in line with the UK Food Advisory Committee's recommendation, i.e . that 3-MCPD is undetectable (i.e . < 0.010 mg kg(-1) in foods and where technologically feasible, in food ingredients as well . Sixty-three samples of food ingredients available in the UK were analysed using a validated method of analysis with a limit of quantification of 0.010 mg kg(-1) . Samples included breadcrumbs, caramels, enzyme-hydrolysed vegetable proteins, gelatines, malt products (malt extracts, malt flours and other malt-based in gredients), meat extracts, modified starches, and yeast extracts . 3-MCPD was not quantified in 49 (78%) of the samples analysed . The remaining 14 samples (22%) contained levels of 3-MCPD between 0.014 and 0.488 mg kg(-1), the highest level being in a maize yellow dextrin . Malt-based ingredients accounted for the majority of samples containing 3-MCPD > 0.010 mg kg(-1), with nine of these 24 samples (38%) having quantifiable levels of 3-MCPD.

J Peripher Nerv Syst, 2001 Sep, 6(3), 111 - 29
Sciatic inflammatory neuritis (SIN): behavioral allodynia is paralleled by peri-sciatic proinflammatory cytokine and superoxide production; Gazda LS et al.; We have recently developed a model of sciatic inflammatory neuritis (SIN) to assess how immune activation near peripheral nerves influences somatosensory processing . Administration of zymosan (yeast cell walls) around a single sciatic nerve produces dose-dependent low-threshold mechanical allodynia without thermal hyperalgesia . Low (4 microg) doses produce both territorial and extraterritorial allodynia restricted to the injected hindleg . In contrast, higher (40 microg) doses produce territorial and extraterritorial allodynias of both hindlegs, an effect not accounted for by systemic spread of the zymosan . The aim of these experiments was to determine whether these behavioral allodynias were correlated with immunological and/or anatomical changes in or around the sciatic nerve . These experiments reveal that zymosan-induced bilateral allodynia was associated with the following: (a) increased release of both interleukin-1beta and tumor necrosis factor-alpha from peri-sciatic immune cells; (b) increased release of reactive oxygen species from perisciatic immune cells; (c) no change in circulating levels of proinflammatory cytokine; (d) no apparent zymosan-induced influx of immune cells into the sciatic nerve from the endoneurial blood vessels; (e) mild edema of the sciatic, which was predominantly restricted to superficial regions closest to the peri-sciatic immune cells; and (f) no anatomic evidence of changes in either the ipsilateral saphenous nerve or contralateral sciatic nerve that could account for the appearance of extraterritorial or contralateral ("mirror") allodynia, respectively . No reliable differences were found when the low-dose zymosan was compared with vehicle controls . Taken together, these data suggest that substances released by peri-sciatic immune cells may induce changes in the sciatic nerve, leading to the appearance of bilateral allodynia.

Indian J Med Res, 2001 Jun, 113, 214 - 20
In vitro susceptibility pattern of Sporothrix schenckii strains isolated from three centers in India; Ghosh A et al.; BACKGROUND & OBJECTIVES: With the availability of more number of antifungal agents in recent years, drugs other than saturated solution of potassium iodide (SSKI) are being increasingly used to treat sporotrichosis . It was therefore considered pertinent to evaluate in vitro antifungal susceptibility pattern of Sporothrix schenckii strains isolated at three centers in India against five commonly used antifungal agents . METHODS: Agar dilution method was used to evaluate 50 clinical isolates (25 from north, 17 from east and 8 from south India) both in its yeast and mycelial forms against amphotericin-B, 5-fluorocytosine, ketoconazole, fluconazole and itraconazole . RESULTS: No resistance was observed in the yeast form of S . schenckii against amphotericin B and azoles . However, 54 per cent strains in the yeast form were resistant to 5-fluorocytosine . None of the strains was susceptible to amphotericin B and ketoconazole, 56 and 10 per cent strains in the mycelial form were susceptible to itraconazole and fluconazole respectively . No significant difference was observed in the antifungal susceptibility pattern among the strains isolated from these three regions in India . INTERPRETATION & CONCLUSION: Clinical isolates of S . schenckii from three regions of India had a more or less uniform antifungal susceptibility pattern . Itraconazole had the best in vitro susceptibility results against the clinical isolates of S . schenckii and has the potential to replace SSKI.

Curr Microbiol, 2002 Feb, 44(2), 141 - 4
Production of linolenic acid by Mortierella isabellina grown on octadecanol; Xian M et al.; The production of linolenic acid in mycelial lipids reached 0.31 mg/ml of culture broth when Mortierella isabellina was cultivated in a medium consisting of 2% octadecanol, 1% yeast extract, and 25 mmol/L of Mg2+ at 23 degrees C for 5 days . Cultivation conditions were studied, and the results showed that (i) a suitable concentration of Mg2+ in the medium caused an increase in mycelial mass as well as linolenic acid production; (ii) when incubated at 23 degrees C, maximal linolenic acid productivity was reached, although a higher content of the acid in total fatty acids was found at the lower temperature; (iii) the effect of substrate concentration on linolenic acid yield showed that the latter increased with concentration of substrate, and maximal linolenic acid yield was obtained with concentrations of 2% octadecanol and 1% yeast extract.

J Cell Biol, 2002 Feb 4, 156(3), 481 - 94 Epub 2002 Jan 28.
Acyl-CoA oxidase is imported as a heteropentameric, cofactor-containing complex into peroxisomes of Yarrowia lipolytica; Titorenko VI et al.; Five isoforms of acyl-CoA oxidase (Aox), designated Aox1p to Aox5p, constitute a 443-kD heteropentameric complex containing one polypeptide chain of each isoform within the peroxisomal matrix of the yeast Yarrowia lipolytica . Assembly of the Aox complex occurs in the cytosol and precedes its import into peroxisomes . Peroxisomal targeting of the Aox complex is abolished in a mutant lacking the peroxin Pex5p, a component of the matrix protein targeting machinery . Import of the Aox complex into peroxisomes does not involve the cytosolic chaperone Pex20p, which mediates the oligomerization and import of peroxisomal thiolase . Aox2p and Aox3p play a pivotal role in the formation of the Aox complex in the cytosol and can substitute for one another in promoting assembly of the complex . In vitro, these subunits retard disassembly of the Aox complex and increase the efficiency of its reassembly . Neither Aox2p nor Aox3p is required for acquisition of the cofactor FAD by other components of the complex . We provide evidence that the Aox2p- and Aox3p-assisted assembly of the Aox complex in the cytosol is mandatory for its import into peroxisomes and that no component of the complex can penetrate the peroxisomal matrix as a monomer.

Gene, 2002 Jan 9, 282(1-2), 65 - 74
Effects of mouse strain, position of integration and tetracycline analogue on the tetracycline conditional system in transgenic mice; Robertson A et al.; The tetracycline conditional system is a very powerful method for achieving control of gene expression in transgenic mice, allowing one to turn expression both off and on in the same animal . We have used it to make a tissue-specific transgenic mouse model of Charcot-Marie-Tooth disease type 1A . This disease is most commonly caused by overexpression of peripheral myelin protein 22 (PMP22) in Schwann cells of the peripheral nervous system . Here we describe the effects of position of integration of the transgene, tetracycline analogue and mouse strain in this model . The small transgenes used to express tTA, the LacZ reporter and the pmp22 cDNA were all very dependent on the position of integration with few of the transgenic lines working successfully . In contrast, the single transgenic made with the 560 kb yeast artificial chromosome construct containing the tTA open reading frame worked well . Tetracycline was found to be cleared from mice relatively fast in comparison with doxycycline and is thus useful if one wants to switch on gene expression after extended periods of administration . Finally, the initial litters were on a mixed genetic background and the level of LacZ or pmp22 expression was very variable between mice . We found that expression became uniform between mice, and occurred in a higher proportion of cells, when the transgenes were crossed onto the CBA/Ca background in comparison with the C57BL/6J background.

Anal Biochem, 2002 Feb 15, 301(2), 243 - 54
Nonradioactive analysis of phosphatidylinositides and other anionic phospholipids by anion-exchange high-performance liquid chromatography with suppressed conductivity detection; Nasuhoglu C et al.; Phosphatidylinositol 4,5-biphosphate (PIP(2)) modulates the function of numerous ion transporters and channels, as well as cell signaling and cytoskeletal proteins . To study PIP(2) levels of cells without radiolabeling, we have developed a new method to quantify anionic phospholipid species . Phospholipids are extracted and deacylated to glycero-head groups, which are then separated by anion-exchange HPLC and detected by suppressed conductivity measurements . The major anionic head groups can be quantified in single runs with practical detection limits of about 100 pmol, and the D3 isoforms of phosphatidylinositol phosphate (PIP) and PIP(2) are detected as shoulder peaks . In HeLa, Hek 293 and COS cells, as well as intact heart, PIP(2) amounts to 0.5 to 1.5% of total anionic phospholipid (10 to 30 micromol/liter cell water or 0.15 to 0.45 nmol/mg protein) . In cell cultures, overexpression of Type I PIP5-kinase specifically increases PIP(2), whereas overexpression of Type II PI4-kinase can increase both PIP and PIP(2) . Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and the D3 isomers of PIP(2) are detected after treatment of cells with pervanadate; in yeast, overexpression of a phosphatidylinositol 3-kinase (VPS34) specifically increases phosphatidylinositol 3-phosphate (PI3P) . Using isolated cardiac membranes, lipid kinase and lipid phosphatase activities can be monitored with the same methods . Upon addition of ATP, PIP increases while PIP(2) remains low; exogenous PIP(2) is rapidly degraded to PIP and phosphatidylinositol (PI) . In summary, the HPLC methods described here can be used to probe multiple aspects of phosphatidylinositide (Ptide) metabolism without radiolabeling . (c)2002 Elsevier Science (USA).

J Cell Biochem, 2002, 84(3), 556 - 66
Interaction of the heart-specific LIM domain protein, FHL2, with DNA-binding nuclear protein, hNP220; Ng EK et al.; Using a yeast two-hybrid library screen, we have identified that the heart specific FHL2 protein, four-and-a-half LIM protein 2, interacted with human DNA-binding nuclear protein, hNP220 . Domain studies by the yeast two-hybrid interaction assay revealed that the second LIM domain together with the third and the fourth LIM domains of FHL2 were responsible to the binding with hNP220 . Using green fluorescent protein (GFP)-FHL2 and blue fluorescent protein (BFP)-hNP220 fusion proteins co-expressed in the same cell, we demonstrated a direct interaction between FHL2 and hNP220 in individual nucleus by two-fusion Fluorescence Resonance Energy Transfer (FRET) assay . Besides, Western blot analysis using affinity-purified anti-FHL2 antipeptide antibodies confirmed a 32-kDa protein of FHL2 in heart only . Virtually no expression of FHL2 protein was detected in brain, liver, lung, kidney, testis, skeletal muscle, and spleen . Moreover, the expression of FHL2 protein was also detectable in the human diseased heart tissues . Our results imply that FHL2 protein can shuttle between cytoplasm and nucleus and may act as a molecular adapter to form a multicomplex with hNP220 in the nucleus, thus we speculate that FHL2 may be particularly important for heart muscle differentiation and the maintenance of the heart phenotype .

J Cell Biochem, 2002, 84(3), 545 - 55
Necdin interacts with the ribonucleoprotein hnRNP U in the nuclear matrix; Taniura H et al.; Necdin is expressed predominantly in terminally differentiated neurons, and its ectopic expression suppresses cell proliferation . We screened a cDNA library from neurally differentiated embryonal carcinoma P19 cells for necdin-binding proteins by the yeast two-hybrid assay . One of the positive clones contained cDNA encoding a carboxyl-terminal portion of heterogeneous nuclear ribonucleoprotein U (hnRNP U), a nuclear matrix-associated protein that interacts with chromosomal DNA . We isolated cDNA encoding full-length mouse hnRNP U to analyze its physical and functional interactions with necdin . The necdin-binding site of hnRNP U was located near a carboxyl-terminal region that mediated the association between hnRNP U and the nuclear matrix . In postmitotic neurons, endogenously expressed necdin and hnRNP U were detected in the nuclear matrix and formed a stable complex . Ectopically expressed necdin was concentrated in the nucleoli, but coexpressed hnRNP U recruited necdin to the nucleoplasmic compartment of the nuclear matrix . Furthermore, under the same conditions necdin and hnRNP U cooperatively suppressed the colony formation of transfected SAOS-2 cells . These results suggest that necdin suppresses cell proliferation through its interaction with hnRNP U in the specific subnuclear structure .

J Biol Chem, 2002 Apr 12, 277(15), 12604 - 12 Epub 2002 Jan 25.
The basic helix-loop-helix factor, HAND2, functions as a transcriptional activator by binding to E-boxes as a heterodimer; Dai YS et al.; HAND2 (dHAND) is a basic helix-loop-helix (bHLH) transcription factor expressed in numerous tissues during development including the heart, limbs, and a subset of neural crest derivatives . Functional analysis has shown that HAND2 is involved in development of the branchial arches, heart, limb, vasculature, and nervous system . Although it is essential for development of numerous tissues, little is known about its mode of action . To this end, we have characterized HAND2 transcriptional regulatory mechanisms . Using mammalian one-hybrid analysis we show that HAND2 contains a strong transcriptional activation domain in the amino-terminal third of the protein . Like most tissue-restricted bHLH factors, HAND2 heterodimerizes with the broadly expressed bHLH factors, the E-proteins . We determined the consensus DNA binding site of HAND2 and show that HAND2 binds a subset of E-boxes as a heterodimer with E12 . Yeast two-hybrid screening of a neuroblastoma cDNA library for HAND2-interacting proteins selected HAND2 and numerous additional members of the E-protein family . Although HAND2 homodimer formation was confirmed by in vitro analysis, HAND2 fails to homodimerize in a mammalian two-hybrid assay but demonstrates robust HAND2/E12 interaction . We conclude that HAND2 functions as a transcription activator by binding a subset of E-boxes as a heterodimer with E-proteins.

J Biol Chem, 2002 Apr 12, 277(15), 12541 - 9 Epub 2002 Jan 25.
Ubc9 is a novel modulator of the induction properties of glucocorticoid receptors; Kaul S et al.; The EC(50) of agonists and the partial agonist activity of antagonists are crucial parameters for steroid hormone control of gene expression and endocrine therapies . These parameters have been shown to be modulated by a naturally occurring cis-acting element, called the glucocorticoid modulatory element (GME) that binds two proteins, GMEB-1 and -2 . We now present evidence that the GMEBs contact Ubc9, which is the mammalian homolog of a yeast E2 ubiquitin-conjugating enzyme . Ubc9 also binds to glucocorticoid receptors (GRs) . Ubc9 displays no intrinsic transactivation activity but modifies both the absolute amount of induced gene product and the fold induction by GR . With high concentrations of GR, added Ubc9 also reduces the EC(50) of agonists and increases the partial agonist activity of antagonists in a manner that is independent of the ability of Ubc9 to transfer SUMO-1 (small ubiquitin-like modifier-1) to proteins . This new activity of Ubc9 requires only the ligand binding domain of GR and part of the hinge region . Interestingly, Ubc9 modulation of full-length GR transcriptional properties can be seen in the absence of a GME . This, though, is consistent with the GME acting by increasing the local concentration of Ubc9, which then activates a previously unobserved target in the transcriptional machinery . With high concentrations of Ubc9 and GR, Ubc9 binding to GR appears to be sufficient to permit Ubc9 to act independently of the GME.

Microb Pathog, 2002 Feb, 32(2), 87 - 97
Transferrin independent serum inhibition of Blastomyces dermatitidis; Giles S et al.; Human serum has been reported to inhibit the growth of several fungal pathogens . Serum inhibitory activity has predominantly been associated with transferrin-mediated iron binding . We found that serum from several animal species (human, dog, mouse and fetal bovine) inhibited the growth of the dimorphic fungal pathogen, Blastomyces dermatitidisin vitro . In initial studies, we found no correlation between the total iron-binding capacity of various sera and their inhibitory activity against B . dermatitidis yeast . In addition, we were unable to abrogate the inhibitory activity of human serum against B . dermatitidis yeast by the addition of ferric chloride (10-200 microM) . We found that apo-transferrin had little or no inhibitory activity against B . dermatitidis yeast . Furthermore, serum from hypotransferrinemic mice (hpx/hpx) had inhibitory activity against B . dermatitidis yeast that was equivalent to that of normal mouse serum . These findings are consistent with our initial findings and suggest that serum inhibitory activity against B . dermatitidis yeast is transferrin independent . Although high concentrations (3.5-5 mg/ml) of lactoferrin did have inhibitory activity against B . dermatitidis yeast, these concentrations were orders of magnitude greater than those found in serum under normal physiological conditions, suggesting that lactoferrin was not likely to contribute to serum inhibitory activity against B . dermatitidis yeast . Our findings suggest that host iron-binding proteins may be relatively ineffective in innate host defense against infection with B . dermatitidis yeast .

Mycopathologia, 2001, 152(3), 113 - 23
Genetic diversity and transcriptional analysis of the bys1 gene from Blastomyces dermatitidis; Bono JL et al.; Blastomyces dermatitidis, a pathogenic fungal organism, is able to exist in two different morphologies, a multicellular mycelium or a unicellular yeast, according to temperature, 25 degrees C and 37 degrees C respectively . The switching between morphologies must be accompanied by a cascade of signaling events in which expression of genes responsible for the change of morphology is increased or decreased . bys1, a gene from B . dermatitidis isolate #58, is expressed at high levels in the unicellular yeast, but gradually diminishes as the temperature is lowered and the organism converts to the mycelial phase where there is no transcription of bys1 . We explored if bys1 homologs are found in other B . dermatitidis isolates and if the transcription of the homologs were regulated by temperature . bys1 was identified in all B . dermatitidis isolates tested and could be grouped into two classes by Southern blot, PCR, and DNA sequence . Although the bys1 transcripts of both classes were regulated by temperature, transcription rates varied between the three isolates tested.

Med Electron Microsc, 1999 Dec, 32(4), 221 - 225
Expression and localization of the LGN protein in the mouse brain with reference to its relationship with Galphai2; Satoh K et al.; It has been suggested that the LGN protein is associated with Galphai2 by the yeast two-hybrid system and in vitro pull-down assay . To determine the functions of LGN in the central nervous system, we examined the expression and localization of LGN in mouse brain by immunoblotting and immunofluorescence microscopy . By immunoblotting, almost similar amounts of LGN were detected in the olfactory bulb, cerebral cortex, hippocampus, and cerebellum of the adult mouse brain, and the levels of the postnatal LGN expression in the whole brain were fairly constant . Immunofluorescence microscopy showed that LGN is localized in nuclei of the neurons in the olfactory bulb, cerebral cortex, and hippocampus, but in both nuclei and cytoplasm of Purkinje cells in the cerebellum . On the other hand, Galphai2 was distributed throughout the neuronal elements except for the nuclei . Thus, LGN and Galphai2 were colocalized in the cytoplasm of Purkinje cells, but not in other neurons examined . These results suggest that LGN may be involved not only in the Galphai2-mediated signaling but also in other signaling pathways.

Hum Genet, 2002 Jan, 110(1), 64 - 7 Epub 2001 Nov 10.
Cytogenetic mapping of a novel locus for type II Waardenburg syndrome; Selicorni A et al.; An Italian family in which Waardenburg syndrome type II (WS2) segregates together with a der(8) chromosome from a (4p;8p) balanced translocation was studied . Cytogenetic analysis by painting and subtelomeric probe hybridization positioned the chromosome 8 breakpoint at p22-pter . Fluorescence in situ hybridization analysis with yeast artificial chromosomes from a contig spanning the 8p21-pter region refined the breakpoint in an interval of less than 170 kb between markers WI-3823 and D8S1819 . The only cloned gene for WS2 is that for microphtalmia (MITF) on chromosome 3p . In this family, MITF mutations were excluded by sequencing the whole coding region . The 8p23 region may represent a third locus for WS2 (WS2C).

Mol Genet Genomics, 2002 Jan, 266(5), 891 - 8 Epub 2001 Nov 29.
The matrix attachment regions (MARs) associated with the Heat Shock Cognate 80 gene ( HSC80) of tomato represent specific regulatory elements; Holmes-Davis R et al.; Matrix Attachment Regions (MARs) flank certain plant genes and appear in certain cases to be necessary for their proper regulation . For example, we previously demonstrated that the MARs and introns from the Heat Shock Cognate 80 gene of tomato (HSC80) are necessary for efficient expression of HSC80-based transgenes . MARs may exert their effect by anchoring the ends of a chromatin loop to the nuclear matrix, thereby establishing an independent chromatin domain . Alternatively, MARs may facilitate interactions between activating complexes and DNA . In the first case, MARs should enhance the expression of most genes, while in the latter case, their action might be gene-specific . We addressed this problem by testing whether the HSC80 MARs affected the regulation of an unrelated transgene . We constructed a chimeric transgene composed of the Arabidopsis ADENINE PHOSPHORIBOSYLTRANSFERASE (APT) promoter fused to the maize gene Lc, which encodes a regulator of anthocyanin synthesis, and compared the expression of Lc in Arabidopsis transparent testa glabra (ttg) mutants (which lack anthocyanin pigments) transformed with transgene constructs incorporating the MARs or control DNA fragments that do not bind to the nuclear matrix . Quantitative RT-PCR analysis was used to compare Lc expression in the different transgenic lines . Whether the APT-Lc transgene was flanked by the HSC80 MARs or a control fragment had no effect on expression, while the use of a different MAR, the ARS1 MAR from yeast, significantly decreased expression (P=0.03) . Comparison of single-copy and multicopy T-DNA insertions indicated that neither the HSC80 MARs nor the ARS1 MAR could protect the APT-Lc transgene from the negative effect of the integration of multiple copies . In conclusion, this work supports a model in which different regulatory elements within the HSC80 locus interact with the nuclear matrix to induce transcriptional competence.

J Biomed Sci, 2002 Jan-Feb, 9(1), 34 - 40
Angiogenesis inhibitors specific for methionine aminopeptidase 2 as drugs for malaria and leishmaniasis; Zhang P et al.; Methionine aminopeptidase 2 (MetAP2) is responsible for the hydrolysis of the initiator methionine molecule from the majority of newly synthesized proteins . We have cloned the MetAP2 gene from the malaria parasite Plasmodium falciparum (PfMetAP2; GenBank accession number AF348320) . The cloned PfMetAP2 has no intron, consists of 1,544 bp and encodes a protein of 354 amino acids with a molecular mass of 40,537 D and an overall base composition of 72.54% A + T . PfMetAP2 has 40% sequence identity with human MetAP2 and 45% identity with yeast MetAP2, and is located in chromosome 14 of P . falciparum . The three-dimensional structure of Pf MetAP2 has been modeled based on the crystal structure of human MetAP2, and several amino acid side chains protruding into the binding pocket that differ between the plasmodial and human enzyme have been identified . The specific MetAP2 inhibitors, fumagillin and TNP-470, potently blocked in vitro growth of P . falciparum and Leishmania donavani, with IC(50) values similar to the prototype drugs . Furthermore, in the case of P . falciparum, the chloroquine-resistant strains are equally susceptible to these two compounds .

Mol Biol Cell, 2002 Jan, 13(1), 158 - 68
Mammalian homolog of Drosophila tumor suppressor lethal (2) giant larvae interacts with basolateral exocytic machinery in Madin-Darby canine kidney cells; Musch A et al.; The Drosophila tumor suppressor protein lethal (2) giant larvae {l(2)gl} is involved in the establishment of epithelial cell polarity during development . Recently, a yeast homolog of the protein has been shown to interact with components of the post-Golgi exocytic machinery and to regulate a late step in protein secretion . Herein, we characterize a mammalian homolog of l(2)gl, called Mlgl, in the epithelial cell line Madin-Darby canine kidney (MDCK) . Consistent with a role in cell polarity, Mlgl redistributes from a cytoplasmic localization to the lateral membrane after contact-naive MDCK cells make cell-cell contacts and establish a polarized phenotype . Phosphorylation within a highly conserved region of Mlgl is required to restrict the protein to the lateral domain, because a recombinant phospho-mutant is distributed in a nonpolar manner . Membrane-bound Mlgl from MDCK cell lysates was coimmunoprecipitated with syntaxin 4, a component of the exocytic machinery at the basolateral membrane, but not with other plasma membrane soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins that are either absent from or not restricted to the basolateral membrane domain . These data suggest that Mlgl contributes to apico-basolateral polarity by regulating basolateral exocytosis.

J Biol Chem, 2002 Apr 5, 277(14), 11703 - 8 Epub 2002 Jan 24.
Insertional mutagenesis and immunochemical analysis of visual arrestin interaction with rhodopsin; Dinculescu A et al.; Visual arrestin inactivates the phototransduction cascade by specifically binding to light-activated phosphorylated rhodopsin . This study describes the combined use of insertional mutagenesis and immunochemical approaches to probe the structural determinants of arrestin function . Recombinant arrestins with insertions of a 10-amino acid c-Myc tag (EQKLISEEDL) were expressed in yeast and characterized . When the tag was placed on the C terminus after amino acid 399, between amino acids 99 and 100 or between residues 162 and 163, binding to rhodopsin was found to be very similar to that of wild-type arrestin . Two stable mutants with Myc insertions in the 68-78 loop were also generated . Binding to rhodopsin was markedly decreased for one (72myc73) and completely abolished for the other (77myc78) . Limited proteolysis assays using trypsin in the absence or presence of heparin were performed on all mutants and confirmed their overall conformational integrity . Rhodopsin binding to either 162myc163 or 72myc73 arrestins in solution was completely inhibited in the presence of less than a 2-fold molar excess of anti-Myc antibody relative to arrestin . In contrast, the antibody did not block the interaction of the 399myc or 99myc100 arrestins with rhodopsin . These results indicate that an interactive surface for rhodopsin is located on or near the concave region of the N-domain of arrestin.

Cancer Res, 2002 Jan 15, 62(2), 367 - 70
Functional evidence for a metastasis suppressor gene for rat prostate cancer within a 60-kilobase region on human chromosome 8p21-p12; Nihei N et al.; We recently demonstrated that the human chromosome 8p21-p12 region encodes a metastasis suppressor gene for rat prostate cancer . The presence of this region suppresses the metastatic ability of rat prostate cancer cells (N . Nihei et al., Genes Chromosomes Cancer, 17: 260-268, 1996) . To define further the region harboring the metastasis suppressor gene, a truncated human chromosome 8 containing this region was transferred into highly metastatic AT6.3 rat prostate cancer cells by microcell-mediated chromosome transfer . The region of human chromosome 8 retained in each microcell hybrid was determined by a PCR analysis with sequence-tagged site markers, and this analysis placed the metastasis suppressor gene in the interval between D8S2249 and D8S2244 on human chromosome 8p21-p12 . One of the metastasis-suppressed microcell hybrids was used for construction of representative yeast/bacterial artificial chromosome (YAC/BAC) library covering the candidate region using a transformation-associated recombination technology (N . Kouprina et al., Genomics, 53: 21-28, 1998) . The final contig corresponding to the candidate region was assembled by four YAC/BAC clones . Each clone was transfected into the AT6.3 cells, and the resultant transfectants were tested for their metastatic ability in athymic nude mice . Introduction of a 60-kb YAC/BAC clone resulted in significant suppression of the metastatic ability without suppression of the tumorigenicity . In contrast, other YAC/BAC clones in the contig had neither metastasis nor tumor suppressor activity . This demonstrates that the 60-kb fragment from the human chromosome 8p21-p12 region contains the metastasis suppressor gene for the AT6.3 cells . Frequent loss of heterozygosity of this region is detected in human prostate cancer, which suggests that our target metastasis suppressor gene may also play an important role in the progression of human prostate cancer.

J Nat Prod, 2002 Jan, 65(1), 11 - 5
Isolation and absolute configuration of ent-Halimane diterpenoids from Hymenaea courbaril from the Suriname rain forest; Abdel-Kader M et al.; Bioactivity-directed fractionation of a methanol extract of Hymenaea courbaril afforded the three new diterpenoids (13R)-13-hydroxy-1(10),14-ent-halimadien-18-oic acid (1), (2S,13R)-2,13-dihydroxy-1(10),14-ent-halimadien-18-oic acid (2), and (13R)-2-oxo-13-hydroxy-1(10),14-ent-halimadien-18-oic acid (3) . The configurations of these compounds were determined from X-ray crystallography of 1, circular dichroism of 2 and 3, and spectral studies of prepared derivatives . Compound 1 exhibited weak cytotoxicity toward the 1138 mutant yeast strain and the A2780 human ovarian cancer cell line.

Genes Chromosomes Cancer, 2002 Mar, 33(3), 235 - 42
Germline mutations of the BRCA1-associated ring domain (BARD1) gene in breast and breast/ovarian families negative for BRCA1 and BRCA2 alterations; Ghimenti C et al.; BARD1 (BRCA1-associated RING domain) was identified by yeast two-hybrid screening as a protein interacting with BRCA1 . Somatic and germline mutations of BARD1 have been detected in sporadic breast, ovarian, and endometrial cancers . The present study represents the first description of BARD1 germline mutations in hereditary breast and breast/ovarian cancer patients . We analyzed the BARD1 gene in 40 families with hereditary breast and breast/ovarian cancer, tested negative for BRCA1 and BRCA2 mutations . A mutational analysis by PCR-SSCP on the coding region and the exon-intron splice boundaries of the BARD1 gene yielded four different germline mutations . A group of 20 patients diagnosed with sporadic breast cancer below the age of 40 was also examined and only one germline mutation was found . A study of loss of heterozygosity at the BARD1 locus in neoplastic tissues from patients with BARD1 germline mutations was carried out . In all cases, we were unable to find any evidence for allelic deletions . The involvement of BARD1 mutations in the susceptibility to hereditary breast and breast/ovarian cancer is discussed.

Mol Carcinog, 2002 Jan, 33(1), 36 - 43
A p21(waf1)-independent pathway for inhibitory phosphorylation of cyclin-dependent kinase p34(cdc2) and concomitant G(2)/M arrest by the chemopreventive flavonoid apigenin; McVean M et al.; Apigenin, a nonmutagenic flavonoid, has been shown to inhibit ultraviolet light-induced skin tumorigenesis when topically applied to mouse skin . Our previous studies have shown that apigenin treatment of cultured mouse keratinocytes induces G(2)/M arrest accompanied by an increase in p53 protein stability and expression of p21(waf1) . In this study, we determined whether the G(2)/M arrest induced by apigenin was dependent upon the presence of the cyclin dependent kinase inhibitor p21(waf1) . We exposed WWT.8 (p21(waf1) wild-type) and WKO.16 (p21(waf1) null) mouse keratinocytes to various doses of apigenin for 24 h and observed G(2)/M arrest in both cell lines, thereby establishing that the apigenin-induced G(2)/M arrest was p21(waf1) independent . A 4-h treatment with apigenin induced increases in p53 protein level by sixfold and tenfold in the WWT.8 p21(waf1) wild-type cells and WKO.16 p21(waf1) null cells, respectively . After 24 h in WWT.8 cells, p21(waf1) protein also was induced in a dose-dependent manner, but it was not expressed in WKO.16 keratinocytes . We then measured the effect of apigenin treatment on the mammalian homologue of the yeast cdc2 gene (p34(cdc2)) cyclin-dependent kinase and cyclin B1 (cycB1), because these proteins complex to regulate G(2)/M progression . Apigenin treatment decreased the protein level of p34(cdc2), and p34(cdc2) kinase activity was inhibited in both p21(waf1)(+/+) and p21(waf1)(-/-) cell lines by approximately 40% . The inhibition of p34(cdc2) kinase activity by apigenin treatment correlated with increasing levels of p34(cdc2) phosphorylation at Tyr15, a site in the p34(cdc2) kinase that undergoes inhibitory phosphorylation by Wee1 kinase . Apigenin treatment also had no effect on the protein level or activity of the competing phosphatase, cdc25c, which dephosphorylates p34(cdc2) kinase at Tyr15 . Apigenin had little effect on the accumulation of cycB1 protein . These results supported the conclusion that G(2)/M arrest induced by apigenin was accompanied by inhibition of the p34(cdc2) cyclin-dependent kinase protein level and activity in a p21(waf1)-independent manner .

J Exp Bot, 2002 Feb, 53(367), 371 - 5
Interaction between Arabidopsis heat shock transcription factor 1 and 70 kDa heat shock proteins; Kim BH et al.; The activity of the Arabidopsis heat shock transcription factor (HSF) is repressed at normal conditions but activated by cellular stresses . Circumstantial evidence suggests that HSP70 may function as a negative feedback regulator of HSF activity . Here the interaction between HSF and HSP70 is reported using electrophoretic mobility shift and yeast two-hybrid assays . Subdomain mapping indicates an interaction of the activation domain and DNA-binding domain of HSF1 with HSP70.

J Cell Biol, 2002 Jan 21, 156(2), 361 - 76 Epub 2002 Jan 21.
Different splice variants of filamin-B affect myogenesis, subcellular distribution, and determine binding to integrin {beta} subunits; van der Flier A et al.; Integrins connect the extracellular matrix with the cell interior, and transduce signals through interactions of their cytoplasmic tails with cytoskeletal and signaling proteins . Using the yeast two-hybrid system, we isolated a novel splice variant (filamin-Bvar-1) of the filamentous actin cross-linking protein, filamin-B, that interacts with the cytoplasmic domain of the integrin beta1A and beta1D subunits . RT-PCR analysis showed weak, but wide, expression of filamin-Bvar-1 and a similar splice variant of filamin-A (filamin-Avar-1) in human tissues . Furthermore, alternative splice variants of filamin-B and filamin-C, from which the flexible hinge-1 region is deleted (DeltaH1), were induced during in vitro differentiation of C2C12 mouse myoblasts . We show that both filamin-Avar-1 and filamin-Bvar-1 bind more strongly than their wild-type isoforms to different integrin beta subunits . The mere presence of the high-affinity binding site for beta1A is not sufficient for targeting the filamin-Bvar-1 construct to focal contacts . Interestingly, the simultaneous deletion of the H1 region is required for the localization of filamin-B at the tips of actin stress fibers . When expressed in C2C12 cells, filamin-Bvar-1(DeltaH1) accelerates their differentiation into myotubes . Furthermore, filamin-B variants lacking the H1 region induce the formation of thinner myotubes than those in cells containing variants with this region . These findings suggest that specific combinations of filamin mRNA splicing events modulate the organization of the actin cytoskeleton and the binding affinity for integrins.

J Cell Biol, 2002 Jan 21, 156(2), 327 - 36 Epub 2002 Jan 21.
Assembly and function of AP-3 complexes in cells expressing mutant subunits; Peden AA et al.; The mouse mutants mocha and pearl are deficient in the AP-3 delta and beta3A subunits, respectively . We have used cells from these mice to investigate both the assembly of AP-3 complexes and AP-3 function . In mocha cells, the beta3 and mu3 subunits coassemble into a heterodimer, whereas the sigma3 subunit remains monomeric . In pearl cells, the delta and sigma3 subunits coassemble into a heterodimer, whereas mu3 gets destroyed . The yeast two hybrid system was used to confirm these interactions, and also to demonstrate that the A (ubiquitous) and B (neuronal-specific) isoforms of beta3 and mu3 can interact with each other . Pearl cell lines were generated that express beta3A, beta3B, a beta3Abeta2 chimera, two beta3A deletion mutants, and a beta3A point mutant lacking a functional clathrin binding site . All six constructs assembled into complexes and were recruited onto membranes . However, only beta3A, beta3B, and the point mutant gave full functional rescue, as assayed by LAMP-1 sorting . The beta3Abeta2 chimera and the beta3A short deletion mutant gave partial functional rescue, whereas the beta3A truncation mutant gave no functional rescue . These results indicate that the hinge and/or ear domains of beta3 are important for function, but the clathrin binding site is not needed.

Biophys J, 2002 Feb, 82(2), 996 - 1003
Orientation distributions for cytochrome c on polar and nonpolar interfaces by total internal reflection fluorescence; Tronin A et al.; The formation of chemisorbed monolayers of yeast cytochrome c on both uncharged polar and nonpolar soft surfaces of organic self-assembled monolayers (SAM) on solid inorganic substrates was followed in situ by polarized total internal reflection fluorescence . Two types of nonpolar surfaces and one type of uncharged polar surface were used . The first type of nonpolar surface contained only thiol endgroups, while the other was composed of a mixture of thiol and methyl endgroups . The uncharged polar surface was provided by the mixture of thiol and hydroxyl endgroups . The thiol endgroups were used to form a covalent disulfide bond with the unique surface-exposed cysteine residue 102 of the protein . The mean tilt angle of the protein's zinc-substituted porphyrin was found to be 41 degrees and 50 degrees for the adsorption onto the nonpolar and uncharged polar surfaces, respectively . The distribution widths for the pure thiol and the thiol/methyl and thiol/hydroxyl mixtures were 9 degrees, 1 degrees, and 18 degrees, respectively . The high degree of the orientational order and good stability achieved for the protein monolayer on the mixed thiol/methyl endgroup SAM makes this system very attractive for studies of both intramolecular and intermolecular electron transfer processes.

Genome Biol . 2002;3(1):RESEARCH0002 . Epub 2001 Dec 12.
Functional and phylogenetic analysis of the ubiquitylation system in Caenorhabditis elegans: ubiquitin-conjugating enzymes, ubiquitin-activating enzymes, and ubiquitin-like proteins; Jones D et al.; BACKGROUND: The eukaryotic ubiquitin-conjugation system sets the turnover rate of many proteins and includes activating enzymes (E1s), conjugating enzymes (UBCs/E2s), and ubiquitin-protein ligases (E3s), which are responsible for activation, covalent attachment and substrate recognition, respectively . There are also ubiquitin-like proteins with distinct functions, which require their own E1s and E2s for attachment . We describe the results of RNA interference (RNAi) experiments on the E1s, UBC/E2s and ubiquitin-like proteins in Caenorhabditis elegans . We also present a phylogenetic analysis of UBCs . RESULTS: The C . elegans genome encodes 20 UBCs and three ubiquitin E2 variant proteins . RNAi shows that only four UBCs are essential for embryogenesis: LET-70 (UBC-2), a functional homolog of yeast Ubc4/5p, UBC-9, an ortholog of yeast Ubc9p, which transfers the ubiquitin-like modifier SUMO, UBC-12, an ortholog of yeast Ubc12p, which transfers the ubiquitin-like modifier Rub1/Nedd8, and UBC-14, an ortholog of Drosophila Courtless . RNAi of ubc-20, an ortholog of yeast UBC1, results in a low frequency of arrested larval development . A phylogenetic analysis of C . elegans, Drosophila and human UBCs shows that this protein family can be divided into 18 groups, 13 of which include members from all three species . The activating enzymes and the ubiquitin-like proteins NED-8 and SUMO are required for embryogenesis . CONCLUSIONS: The number of UBC genes appears to increase with developmental complexity, and our results suggest functional overlap in many of these enzymes . The ubiquitin-like proteins NED-8 and SUMO and their corresponding activating enzymes are required for embryogenesis.

BMC Cell Biol . 2002;3(1):1 . Epub 2002 Jan 04.
MID1 and MID2 homo- and heterodimerise to tether the rapamycin-sensitive PP2A regulatory subunit, alpha 4, to microtubules: implications for the clinical variability of X-linked Opitz GBBB syndrome and other developmental disorders; Short KM et al.; BACKGROUND: Patients with Opitz GBBB syndrome present with a variable array of developmental defects including craniofacial, cardiac, and genital anomalies . Mutations in the X-linked MID1 gene, which encodes a microtubule-binding protein, have been found in approximately 50% of Opitz GBBB syndrome patients consistent with the genetically heterogeneous nature of the disorder . A protein highly related to MID1, called MID2, has also been described that similarly associates with microtubules . RESULTS: To identify protein partners of MID1 and MID2 we undertook two separate yeast two-hybrid screens . Using this system we identified Alpha 4, a regulatory subunit of PP2-type phosphatases and a key component of the rapamycin-sensitive signaling pathway, as a strong interactor of both proteins . Analysis of domain-specific deletions has shown that the B-boxes of both MID1 and MID2 mediate the interaction with Alpha 4, the first demonstration in an RBCC protein of a specific role for the B-box region . In addition, we show that the MID1/2 coiled-coil motifs mediate both homo- and hetero-dimerisation, and that dimerisation is a prerequisite for association of the MID-Alpha 4 complex with microtubules . CONCLUSIONS: Our findings not only implicate Alpha 4 in the pathogenesis of Opitz GBBB syndrome but also support our earlier hypothesis that MID2 is a modifier of the X-linked phenotype . Of further note is the observation that Alpha 4 maps to Xq13 within the region showing linkage to FG (Opitz-Kaveggia) syndrome . Overlap in the clinical features of FG and Opitz GBBB syndromes warrants investigation of Alpha 4 as a candidate for causing FG syndrome.

IUBMB Life, 2001 Jul, 52(1-2), 29 - 33
Redox regulation by thioredoxin and thioredoxin-binding proteins; Nishiyama A et al.; Recent works have shown the importance of reduction/oxidation (redox) regulation in various biological phenomena . Thioredoxin is a 12-kDa protein with redox-active dithiol in the active site -Cys-Gly-Pro-Cys- and constitutes a major thiol reducing system, the thioredoxin system . Thioredoxin plays multiple roles in cellular processes such as proliferation or apoptosis . It also promotes DNA binding of transcription factors such as NF-kappaB, AP-1, p53, and PEBP2 . Overexpression of thioredoxin suppresses the degradation of IkappaB and the transactivation of NF-kappaB, whereas overexpression of nuclear-targeted thioredoxin exhibits the enhancement of NF-kappaB-dependent transactivation . ASK1, a MAP kinase kinase kinase mediating the TNF-alpha signal has been identified as a thioredoxin binding protein . Thioredoxin shows an inhibitory effect on the TNF-alpha induced activation of ASK1 and p38 MAP kinase pathway . We identified p40phox as the thioredoxin binding protein-1 (TBP-1) and vitamin D3 up-regulated protein 1 (VDUP1) as the thioredoxin binding protein-2 (TBP-2) by yeast two-hybrid system . TBP-2/VDUP1 negatively regulates the expression and reducing activity of thioredoxin . Thioredoxin interacting proteins may be involved in thioredoxin-mediating redox regulation.

Ann N Y Acad Sci, 2001 Dec, 947, 398 - 402
Molecular mechanisms of morning onset of myocardial infarction; Maemura K et al.; We recently isolated a novel bHLH/PAS protein, CLIF (cycle like factor), by yeast two-hybrid screening of human umbilical endothelial cell cDNA library . CLIF is preferentially expressed in endothelial and neuronal cells . Because CLIF is expressed in vascular endothelial cells and forms a heterodimer with CLOCK, the key transcription factor controlling the circadian rhythm, we hypothesized that CLIF regulates the circadian oscillation of PAI-1 gene expression in endothelial cells . Northern blot analysis of mouse organs showed circadian oscillations of PAI-I mRNA levels . In addition, the clock-related genes also showed circadian oscillation in peripheral tissues . In endothelial cells, the heterodimer of CLIF and CLOCK upregulated the PAI-1 gene expression through E-box sites . Furthermore, Period and Cryptochrome, which are negative regulators in the feedback loop of the biological clock, inhibited PAI-1 promoter activation by the CLOCK:CLIF heterodimer . These results suggest that the peripheral tissues have their own biological clock and CLIF regulates the circadian oscillation of PAI-1 gene expression in endothelial cells . This study suggests a novel molecular mechanism of the morning onset of myocardial infarction . Here we review our recent work and literature.

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