Arthritis Rheum, 2000 Dec, 43(12), 2660 - 7 Regulatory effects of interleukin-4 and interleukin-10 on human neutrophil function ex vivo and on neutrophil influx in a rat model of arthritis; Bober LA et al.; OBJECTIVE: To assess the capacity of interleukin-4 (IL-4) and IL-10 to block polymorphonuclear neutrophil (PMN) activation in an ex vivo human model system, and to confirm their effect on neutrophil function in an animal model of arthritis . METHODS: The ex vivo phagocytic capacity of cytokine-activated human PMNs was assessed by use of assays for measuring the ingestion of heat-killed yeast and by subsequent hexose-monophosphate shunt activation using nitroblue tetrazolium reduction . The in vivo activity of IL-4 and IL-10 was measured using a rat adjuvant arthritis model in which the mycobacterial antigen concentration was titrated to modify disease intensity . RESULTS: IL-4 and IL-10 suppressed the ex vivo activation state of interferon-gamma- and tumor necrosis factor alpha-activated human neutrophils . In the rat adjuvant arthritis model, treatment with systemic murine IL-10 (mIL-10) effectively suppressed all disease parameters in rats that received the lower concentrations of mycobacteria, whereas systemic mIL-4 was effective against even the most severe disease . Both cytokines were effective in lowering the absolute PMN cell number recovered and the PMN activation state in the joint synovia . We also observed lower levels of the messenger RNA transcript for CINC protein (cytokine-induced neutrophil chemoattractant; a rat homolog for human IL-8) in the synovia . CONCLUSION: IL-10 is an effective antiarthritic agent and has a major effect on the presence and function of PMNs in the joint synovia when disease intensity is not severe . IL-4 has an inhibitory profile that is similar to that of IL-10, but is effective in modifying even the most severe disease . Both cytokines reduced the phagocytic activation of human PMNs in response to proinflammatory cytokines . These data demonstrate that IL-4 and IL-10 can exert powerful regulatory effects on neutrophil function that translate into a therapeutic response in a disease model of arthritis . Treatment with these cytokines alone or in combination may therefore be very useful in the management of patients with rheumatoid arthritis. Biochemistry, 2001 Jan 9, 40(1), 281 - 5 Effects of crowding by mono-, di-, and tetrasaccharides on cytochrome c-cytochrome c peroxidase binding: comparing experiment to theory; Morar AS et al.; In cells, protein-protein interactions occur in an environment that is crowded with other molecules, but in vitro studies are almost exclusively performed in dilute solution . To gain information about the effects of crowding on protein complex formation, we used isothermal titration calorimetry to measure the stoichiometry, the free energy change, and the enthalpy change for the binding of yeast iso-1-ferricytochrome c to yeast ferricytochrome c peroxidase in dilute solution and in solutions crowded with the sugars glucose, sucrose, and stachyose . The stoichiometry is 1:1 under all conditions . The sugars stabilize the complex, but by only 0.1-0.5 kcal.mol(-)(1), and the increased stability is not correlated with the change in enthalpy of complex formation . We then compared the measured stability changes to values obtained from several analyses that are currently used to predict crowding-induced changes in biomolecular equilibria . None of the analyses are completely successful by themselves, and the results suggest that a complete analysis must account for both excluded-volume and chemical interactions. Exp Cell Res, 2001 Jan 15, 262(2), 122 - 7 INCENP binds directly to tubulin and requires dynamic microtubules to target to the cleavage furrow; Wheatley SP et al.; Inner centromere protein (INCENP) is a chromosomal passenger protein with an essential role in mitosis . At the metaphase/anaphase transition, some INCENP transfers from the centromeres to the central spindle; the remainder then transfers to the equatorial cortex prior to cleavage furrow formation . The molecular associations dictating INCENP behavior during mitosis are currently unknown . Here we show that targeting INCENP to the cleavage plane requires dynamic microtubules, but not F-actin . When microtubules are eliminated, INCENP is dispersed across the entire cell cortex . Yeast two-hybrid and in vitro binding data demonstrate that INCENP binds directly to beta-tubulin via a conserved domain encompassing residues 48-85 . Furthermore, INCENP binds to microtubules polymerized from purified tubulin in vitro and appears to bundle microtubules when expressed in the interphase cytoplasm . These data indicate that INCENP is a microtubule-binding protein that targets to the equatorial cortex through interactions requiring microtubules . Nat Genet, 2001 Jan, 27(1), 121 - 4 Lipodystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin; Peterfy M et al.; Mice carrying mutations in the fatty liver dystrophy (fld) gene have features of human lipodystrophy, a genetically heterogeneous group of disorders characterized by loss of body fat, fatty liver, hypertriglyceridemia and insulin resistance . Through positional cloning, we have isolated the gene responsible and characterized two independent mutant alleles, fld and fld(2J) . The gene (Lpin1) encodes a novel nuclear protein which we have named lipin . Consistent with the observed reduction of adipose tissue mass in fld and fld(2J)mice, wild-type Lpin1 mRNA is expressed at high levels in adipose tissue and is induced during differentiation of 3T3-L1 pre-adipocytes . Our results indicate that lipin is required for normal adipose tissue development, and provide a candidate gene for human lipodystrophy . Lipin defines a novel family of nuclear proteins containing at least three members in mammalian species, and homologs in distantly related organisms from human to yeast. Nat Genet, 2001 Jan, 27(1), 89 - 93 A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy; Zhang K et al.; Stargardt-like macular dystrophy (STGD3, MIM 600110) and autosomal dominant macular dystrophy (adMD) are inherited forms of macular degeneration characterized by decreased visual acuity, macular atrophy and extensive fundus flecks . Genetic mapping data suggest that mutations in a single gene may be responsible for both conditions, already known to bear clinical resemblance . Here we limit the minimum genetic region for STGD3 and adMD to a 0.6-cM interval by recombination breakpoint mapping and identify a single 5-bp deletion within the protein-coding region of a new retinal photoreceptor-specific gene, ELOVL4, in all affected members of STGD3 and adMD families . Bioinformatic analysis of ELOVL4 revealed that it has homology to a group of yeast proteins that function in the biosynthesis of very long chain fatty acids . Our results are therefore the first to implicate the biosynthesis of fatty acids in the pathogenesis of inherited macular degeneration. J Ethnopharmacol, 2001 Jan, 74(1), 89 - 96 Screening antifungal activities of selected medicinal plants; Quiroga EN et al.; Plants synthesise a vast array of secondary metabolites that are gaining importance for their biotechnological applications . The antifungal activity of the ethanolic extracts of ten Argentinean plants used in native medicine is reported . Antifungal assays included radial growth inhibition, disk and well diffusion assays and growth inhibition by broth dilution tests . The chosen test fungi were yeasts, microfungi and wood-rot causing Basidiomycetes . Extracts of Larrea divaricata, Zuccagnia punctata and Larrea cuneifolia displayed remarkable activity in the assays against the majority of the test fungi . In addition to the former plants, Prosopanche americana also inhibited yeast growth. Immunol Lett, 2001 Jan 1, 75(2), 131 - 6 Ubiquitination of Lyn-kinase in rat basophilic leukemia RBL-2H3 cells; Bhattacharyya SP; Receptor-mediated signal transduction pathways of cells involved in allergy and inflammations are extremely significant . Lyn is a member of the Src family of non-receptor protein tyrosine kinases and is associated with a number of cell surface receptors, including the B-cell antigen receptor and immunoglobulin E receptor (FcepsilonRI) . Lyn is necessary for FcepsilonRI-mediated mast cell activation . To investigate how the level of Lyn is maintained in mast cell activation, it was studied whether Lyn binds to ubiquitin and is ubiquitinated for proteasomal degradation in cells . In the yeast two hybrid system, Lyn specifically interacted with ubiquitin in vivo . Furthermore, Lyn bound to ubiquitin-conjugated Sepharose beads in vitro and was efficiently competed by soluble ubiquitin . Pulse-chase experiments indicated intracellular degradation of Lyn was associated with the generation of a high molecular weight complex in the presence of proteasome-specific inhibitor, lactacystin . This high molecular weight complex cross-reacted with anti-Lyn and anti-ubiquitin demonstrating the ubiquitination Lyn . Overexpression of Lyn and ubiquitin in COS 7.2 cells also resulted in the ubiquitination of Lyn in the presence of lactacystin, supporting the ubiquitination of Lyn by a proteasome specific pathway. Curr Biol, 2000 Dec 14-28, 10(24), 1603 - 6 FIP-2, a coiled-coil protein, links Huntingtin to Rab8 and modulates cellular morphogenesis; Hattula K et al.; Huntington's disease is characterised by the death of cortical and striatal neurons, and is the result of an expanded polyglutamine tract in the Huntingtin protein {1} . Huntingtin is present on both endocytic and secretory membrane organelles but its function is unclear {2,3} . Rab GTPases regulate both of these transport pathways {4} . We have previously shown that Rab8 controls polarised membrane transport by modulating cell morphogenesis {5} . To understand Rab8-mediated processes, we searched for Rab8-interacting proteins by the yeast two-hybrid system . Here, we report that Huntingtin is linked to the Rab8 protein through FIP-2, a tumour necrosis factor-alpha (TNF-alpha)-inducible coiled-coil protein related to the NEMO protein {6,7} . The activated form of Rab8 interacted with the amino-terminal region of FIP-2, whereas dominant-negative Rab8 did not . Huntingtin bound to the carboxy-terminal region of FIP-2 . Coexpressed FIP-2 and Huntingtin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures, and FIP-2 promoted cell polarisation in a similar way to Rab8 . We propose a model in which Huntingtin, together with FIP-2 and Rab8, are part of a protein network that regulates membrane trafficking and cellular morphogenesis. Curr Biol, 2000 Dec 14-28, 10(24), 1547 - 56 Aberrant replication timing induces defective chromosome condensation in Drosophila ORC2 mutants; Loupart ML et al.; BACKGROUND: The accurate duplication and packaging of the genome is an absolute prerequisite to the segregation of chromosomes in mitosis . To understand the process of cell-cycle chromosome dynamics further, we have performed the first detailed characterization of a mutation affecting mitotic chromosome condensation in a metazoan . Our combined genetic and cytological approaches in Drosophila complement and extend existing work employing yeast genetics and Xenopus in vitro extract systems to characterize higher-order chromosome structure and function . RESULTS: Two alleles of the ORC2 gene were found to cause death late in larval development, with defects in cell-cycle progression (delays in S-phase entry and metaphase exit) and chromosome condensation in mitosis . During S-phase progression in wild-type cells, euchromatin replicates early and heterochromatin replicates late . Both alleles disrupted the normal pattern of chromosomal replication, with some euchromatic regions replicating even later than heterochromatin . Mitotic chromosomes were irregularly condensed, with the abnormally late replicating regions of euchromatin exhibiting the greatest problems in mitotic condensation . CONCLUSIONS: The results not only reveal novel functions for ORC2 in chromosome architecture in metazoans, they also suggest that the correct timing of DNA replication may be essential for the assembly of chromatin that is fully competent to undergo mitotic condensation. Cell, 2000 Dec 8, 103(6), 919 - 30 Pike . A nuclear gtpase that enhances PI3kinase activity and is regulated by protein 4.1N; Ye K et al.; While cytoplasmic PI3Kinase (PI3K) is well characterized, regulation of nuclear PI3K has been obscure . A novel protein, PIKE (PI3Kinase Enhancer), interacts with nuclear PI3K to stimulate its lipid kinase activity . PIKE encodes a 753 amino acid nuclear GTPase . Dominant-negative PIKE prevents the NGF enhancement of PI3K and upregulation of cyclin D1 . NGF treatment also leads to PIKE interactions with 4.1N, which has translocated to the nucleus, fitting with the initial identification of PIKE based on its binding 4.1N in a yeast two-hybrid screen . Overexpression of 4.1N abolishes PIKE effects on PI3K . Activation of nuclear PI3K by PIKE is inhibited by the NGF-stimulated 4.1N translocation to the nucleus . Thus, PIKE physiologically modulates the activation by NGF of nuclear PI3K. J Clin Microbiol, 2001 Jan, 39(1), 101 - 6 Isolation of an intron-containing partial sequence of the gene encoding dermatophyte actin (ACT) and detection of a fragment of the transcript by reverse transcription-nested PCR as a means of assessing the viability of dermatophytes in skin scales; Okeke CN et al.; An internal partial sequence of the gene encoding actin (ACT), 725 to 762 bp in length, was amplified by PCR from the genomic DNA extract of 12 species of dermatophytes and sequenced . An intron that is 56 to 93 bp in length was located along the ACT fragment of all of the dermatophytes at codon position 301 (-3) (a codon number followed by "-3" indicates that the intron directly follows the codon) with reference to the amino acid sequence of human alpha-smooth muscle actin . A primer pair that annealed to exon sequences flanking the ACT-associated intron produced a dermatophyte-specific 171-bp amplicon by reverse transcription-nested PCR (RT-PCR) of dermatophyte ACT mRNA . PCR primer pairs with antisense sequence based on the ACT intron sequence were species specific for dermatophytes, suggesting a potential for use in the identification of dermatophytes . The viability of dermatophytes in skin scales was subsequently assessed by the presence of ACT mRNA in total RNA extracted from a 48-h culture of scale samples in 250 microl of yeast carbon base broth . RT-nested PCR of dermatophyte-infected samples amplified an ACT fragment of the predicted size of 171 bp . The results of viability testing based on ACT mRNA detection by RT-nested PCR correlated with cultural isolation from skin scales . This method is a potential tool for rapidly assessing fungal viability in the therapeutic efficacy testing of antimycotics. Hum Mol Genet, 2001 Jan 1, 10(1), 39 - 45 Heterozygous HESX1 mutations associated with isolated congenital pituitary hypoplasia and septo-optic dysplasia; Thomas PQ et al.; We have previously shown that familial septo-optic dysplasia (SOD), a syndromic form of congenital hypopituitarism involving optic nerve hypoplasia and agenesis of midline brain structures, is associated with homozygosity for an inactivating mutation in the homeobox gene HESX1/Hesx1 in man and mouse . However, as most SOD/congenital hypopituitarism occurs sporadically, the possible contribution of HESX1 mutations to the aetiology of these cases is presently unclear . Interestingly, a small proportion of mice heterozygous for the Hesx1 null allele show a milder SOD phenocopy, implying that heterozygous mutations in human HESX1 could underlie some cases of congenital pituitary hypoplasia with or without midline defects . Accordingly, we have now scanned for HESX1 mutations in 228 patients with a broad spectrum of congenital pituitary defects, ranging in severity from isolated growth hormone deficiency to SOD with panhypopituitarism . Three different heterozygous missense mutations were detected in individuals with relatively mild pituitary hypoplasia or SOD, which display incomplete penetrance and variable phenotype amongst heterozygous family members . Gel shift analysis of the HESX1-S170L mutant protein, which is encoded by the C509T mutated allele, indicated that a significant reduction in relative DNA binding activity results from this mutation . Segregation analysis of a haplotype spanning 6.1 cM, which contains the HESX1 locus, indicated that only one HESX1 mutation was present in the families containing the C509T and A541G mutations . These results demonstrate that some sporadic cases of the more common mild forms of pituitary hypoplasia have a genetic basis, resulting from heterozygous mutation of the HESX1 gene. J Biol Chem, 2001 Feb 16, 276(7), 4539 - 42 Epub 2001 Jan 02. Deacetylase activity associates with topoisomerase II and is necessary for etoposide-induced apoptosis; Johnson CA et al.; DNA topoisomerase II (topo II) is a ubiquitous nuclear enzyme that is involved in DNA replication, transcription, chromosome segregation, and apoptosis . Here we show by immunoprecipitation, pull down with glutathione S-transferase fusion proteins, and yeast two-hybrid analysis that both topo IIalpha and -beta physically interact with the histone deacetylase HDAC1 . The in vitro DNA decatenation activity of recombinant topo IIalpha and -beta is inhibited by association with catalytically inactive, recombinant HDAC1 . We provide evidence for the in vivo significance of the topo II-HDAC1 association, showing that inhibition of HDAC activity with trichostatin A suppresses apoptosis induced by the topo II poison etoposide, but not by the topoisomerase I inhibitor camptothecin . We suggest that chromatin remodeling by an HDAC-containing complex facilitates both topo II-catalyzed DNA rearrangement and etoposide-induced DNA damage in vivo. J Cell Biochem, 2001, 80(3), 293 - 303 Protein-protein interaction of FHL3 with FHL2 and visualization of their interaction by green fluorescent proteins (GFP) two-fusion fluorescence resonance energy transfer (FRET); Li HY et al.; LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton . Human Four-and-a-half LIM-only protein 3 (FHL3) is a type of LIM-only protein that contains four tandemly repeated LIM motifs with an N-terminal single zinc finger (half LIM motif) . FHL3 expresses predominantly in human skeletal muscle . In this report, FHL3 was shown to be a novel interacting partner of FHL2 using the yeast two-hybrid assay . Furthermore, site-directed mutagenesis of FHL3 indicated that the LIM2 of FHL3 is the essential LIM domain for interaction with FHL2 . Green fluorescent protein (GFP) was used to tag FHL3 in order to study its distribution during myogenesis . Our result shows that FHL3 was localized in the focal adhesions and nucleus of the cells . FHL3 mainly stayed in the focal adhesion during myogenesis . Moreover, using site-directed mutagenesis, the LIM1 of FHL3 was identified as an essential LIM domain for its subcellular localization . Mutants of GFP have given rise to a novel technique, two-fusion fluorescence resonance energy transfer (FRET), in the determination of protein-protein interaction at particular subcellular locations of eukaryotic cells . To determine whether FHL2 and FHL3 can interact with one another and to locate the site of this interaction in a single intact mammalian cell, we fused FHL2 and FHL3 to different mutants of GFP and studied their interactions using FRET . BFP/GFP fusion constructs were cotransfected into muscle myoblast C2C12 to verify the colocalization and subcellular localization of FRET . We found that FHL2 and FHL3 were colocalized in the mitochondria of the C2C12 cells and FRET was observed by using an epi-fluorescent microscope equipped with an FRET specific filter set . Bioessays, 2001 Jan, 23(1), 77 - 85 The Ran-GTPase and cell-cycle control; Moore JD; RCC1, the chromatin-bound guanine-nucleotide exchange factor (GEF) for the small nuclear GTPase, Ran, is required for coordinating the onset of mitosis with S-phase completion in mammalian cells . Other defects in the Ran-GTPase network also result in disruption of cell-cycle processes such as DNA replication, exit from mitosis and, at least in budding yeast, accurate chromosome segregation . However, the Ran system is now best known for its pivotal role in nucleocytoplasmic transport, where RanGTP is used as a positional flag for the nucleus during interphase . Ran's effectors are the shuttling transport factors, importins and exportins, which facilitate the transit of cargoes between the nucleus and cytoplasm: RanGTP regulates their cargo-binding properties so that they can move their cargo in the correct direction . RanGTP also plays a separate role during mitosis, influencing microtubule polymerisation, possibly specifically in the vicinity of chromosomes . Most recently, Ran has been shown to be crucial for the regeneration of a nuclear envelope after exit from mitosis . So, can the problems with cell-cycle progression and control induced by perturbing the Ran-system be attributed to defects in these three processes? This article examines this issue, concentrating on vertebrate systems . BioEssays 23:77-85, 2001 . Plant J, 2000 Dec, 24(6), 775 - 84 Carboxyl-methylation of prenylated calmodulin CaM53 is required for efficient plasma membrane targeting of the protein; Rodriguez-Concepcion M et al.; Prenylation is necessary for association of the petunia calmodulin CaM53 with the plasma membrane . To determine whether post-prenylation processing of the protein was also required for plasma membrane targeting, we studied the subcellular localization of a GFP-labelled CaM53 reporter in yeast and plant cells . Blocking of carboxyl-methylation of prenylated proteins either by a specific inhibitor or in mutant yeast cells resulted in localization of green fluorescence to what appears to be the endomembrane system, in contrast with the plasma membrane localization observed in control cells . We show that a prenyl-cysteine methyltransferase (PCM) activity that carboxyl-methylates prenylated CaM53 also exists in plant cells, and that it is required for efficient plasma membrane targeting . We also report an Arabidopsis gene with homology to PCM and demonstrate that it encodes a protein with PCM activity that localizes to the endomembrane system of plant cells, similar to prenylated but unmethylated CaM53 . Together, our data suggest that, following prenylation, CaM53 is probably associated with the endomembrane system, where a PCM activity methylates the prenylated protein prior to targeting it to its final destination in the plasma membrane. Plant J, 2000 Dec, 24(6), 703 - 11 Arabidopsis ARR1 and ARR2 response regulators operate as transcriptional activators; Sakai H et al.; The genes coding for the response regulators ARR1 and ARR2 have previously been identified by in silico screening of an expression sequence tag database and subsequent cloning from both Arabidopsis cDNA and genomic libraries . Their structures, in which the N-terminal signal receiver domain is followed by the output domain, are characteristic of typical bacterial response regulators of the two-component regulatory systems that control responses to a variety of environmental stimuli . Here we present evidence that these response regulators actually work as transcription factors . ARR1 and ARR2 were localized in the nuclei of plant cells regardless of the presence or absence of their signal receiver domain . Their middle segments, which faintly resemble the mammalian oncogene product Myb, were capable of binding double-stranded DNA in a sequence-specific manner in vitro . Their C-terminal halves functioned as transactivation domains in plant cells when combined with the DNA-binding domain of yeast GAL4 . They thus possess all the essential components of a transcriptional activator . Both ARR1 and ARR2 promoted expression of a reporter gene in plant cells through their own target sequence . Truncation of their N-terminal signal receiver domain led to an increase in transactivation . An as yet unidentified phospho-relay signal may modulate the capability for transactivation and/or DNA binding through the signal receiver domain. J Biochem (Tokyo), 2001 Jan, 129(1), 43 - 9 Expression of chromatin remodeling factors during neural differentiation; Machida Y et al.; The yeast SWI/SNF complex is involved in remodeling of chromatin structure during transcriptional modulation . One of the key subunits of this complex, called SWI2/SNF2, has a DNA-dependent ATPase activity . Two different types of mammalian homolog of yeast SWI2/SNF2, called BRM and BRG1, were recently identified . They are closely similar in structure but have distinct functions . We investigated the expression of BRM and BRG1 during differentiation of neural precursor cells (NPCs) cultured in vitro . The expression of BRM was very low in NPCs and was induced to a high level during differentiation to neurons and astrocytes . In contrast, BRG1 was constantly expressed throughout differentiation . These phenomena were also observed in differentiation of P19 embryonal carcinoma cells to neural cells . Immunocytochemical analyses revealed that the expression of BRM started even in the undifferentiated nestin-positive cells . These results indicate that BRM may have an important role in neural cell differentiation. Proc Natl Acad Sci U S A, 2001 Jan 2, 98(1), 200 - 5 Blocking histone deacetylation in Arabidopsis induces pleiotropic effects on plant gene regulation and development; Tian L et al.; Histone acetylation and deacetylation play essential roles in eukaryotic gene regulation . Reversible modifications of core histones are catalyzed by two intrinsic enzymes, histone acetyltransferase and histone deacetylase (HD) . In general, histone deacetylation is related to transcriptional gene silencing, whereas acetylation correlates with gene activation . We produced transgenic plants expressing the antisense Arabidopsis HD (AtHD1) gene . AtHD1 is a homolog of human HD1 and RPD3 global transcriptional regulator in yeast . Expression of the antisense AtHD1 caused dramatic reduction in endogenous AtHD1 transcription, resulting in accumulation of acetylated histones, notably tetraacetylated H4 . Reduction in AtHD1 expression and AtHD1 production and changes in acetylation profiles were associated with various developmental abnormalities, including early senescence, ectopic expression of silenced genes, suppression of apical dominance, homeotic changes, heterochronic shift toward juvenility, flower defects, and male and female sterility . Some of the phenotypes could be attributed to ectopic expression of tissue-specific genes (e.g., SUPERMAN) in vegetative tissues . No changes in genomic DNA methylation were detected in the transgenic plants . These results suggest that AtHD1 is a global regulator, which controls gene expression during development through DNA-sequence independent or epigenetic mechanisms in plants . In addition to DNA methylation, histone modifications may be involved in a general regulatory mechanism responsible for plant plasticity and variation in nature. Mol Cell Biol, 2001 Jan, 21(2), 655 - 62 A novel 16-kilodalton cellular protein physically interacts with and antagonizes the functional activity of c-myc promoter-binding protein 1; Ghosh AK et al.; We initially identified c-myc promoter-binding protein 1 (MBP-1) from a human cervical carcinoma cell expression library which negatively regulates c-myc promoter activity . A recent study demonstrated that MBP-1 acts as a general transcriptional repressor (A . K . Ghosh, R . Steele, and R . B . Ray, Mol . Cell . Biol . 19:2880-2886, 1999) . In order to identify the cellular protein(s) interacting with MBP-1 for transcriptional regulation, a HeLa cell cDNA expression library was screened using a yeast two-hybrid system . An MBP-1-interacting cDNA encoding a polypeptide of 140 amino acid residues with an approximate molecular mass of 16 kDa was identified and named MBP-1 interacting protein-2A (MIP-2A) . MIP-2A has a sequence similarity with an unknown mRNA and SEDL . Mutations in the SEDL gene, located at human chromosome Xp22, has recently been implicated with an X-linked genetic disease, although the function of SEDL gene product was not determined (A . K . Gedeon et al., Nat . Genet . 22:400-404, 1999) . However, our results suggested the localization of MIP-2A at human chromosome 19 . The specificity of interaction between MBP-1 and MIP-2A was verified by an in vitro glutathione S-transferase pulldown experiment, a mammalian two-hybrid analysis, and in vivo coimmunoprecipitation assays . Further analysis revealed that the amino-terminal domain of MBP-1 (amino acids 1 to 95) interacts with MIP-2A . Immunofluorescent staining suggested colocalization of MIP-2A and MBP-1 primarily in the perinuclear membrane of cells . Functional analysis demonstrated that MIP-2A relieves MBP-1 mediated transcriptional repression on c-myc promoter . Additionally, MIP-2A antagonizes cell growth regulatory role of MBP-1 . Taken together, these results suggest the functional interaction of MIP-2A and MBP-1 in cell growth regulation. Mol Cell Biol, 2001 Jan, 21(2), 614 - 23 Prodos is a conserved transcriptional regulator that interacts with dTAF(II)16 in Drosophila melanogaster; Hernandez-Hernandez A et al.; The transcription factor TFIID is a multiprotein complex that includes the TATA box binding protein (TBP) and a number of associated factors, TAF(II) . Prodos (PDS) is a conserved protein that exhibits a histone fold domain (HFD) . In yeast two-hybrid tests using PDS as bait, we cloned the Drosophila TAF(II), dTAF(II)16, as a specific PDS target . dTAF(II)16 is closely related to human TAF(II)30 and to another recently discovered Drosophila TAF, dTAF(II)24 . PDS and dTAF(II)24 do not interact, however, thus establishing a functional difference between these dTAFs . The PDS-dTAF(II)16 interaction is mediated by the HFD motif in PDS and the N terminus in dTAF(II)16, as indicated by yeast two-hybrid assays with protein fragments . Luciferase-reported transcription tests in transfected cells show that PDS or an HFD-containing fragment activates transcription only with the help of dTAF(II)16 and TBP . Consistent with this, the eye phenotype of flies expressing a sev-Ras1 construct is modulated by PDS and dTAF(II)16 in a gene dosage-dependent manner . Finally, we show that PDS function is required for cell viability in somatic mosaics . These findings indicate that PDS is a novel transcriptional coactivator that associates with a member of the general transcription factor TFIID. J Am Soc Nephrol, 2001 Jan, 12(1), 107 - 13 Human adolescent nephronophthisis: gene locus synteny with polycystic kidney disease in pcy mice; Omran H et al.; In a large Venezuelan kindred, a new type of nephronophthisis was recently identified: Adolescent nephronophthisis (NPH3) is a late-onset recessive renal cystic disorder of the nephronophthisis/medullary cystic group of diseases causing end-stage renal disease at a median age of 19 yr . With the use of a homozygosity mapping strategy, the gene (NPHP3) was previously localized to chromosome 3q22 within a critical interval of 2.4 cM . In the current study, the NPHP3 genetic region was cloned and seven genes, eight expressed sequence-tagged sites, and seven microsatellites were physically localized within the critical disease interval . By human-mouse synteny analysis based on expressed genes, synteny between the human NPHP3 locus on chromosome 3q and the pcy locus on mouse chromosome 9 was clearly demonstrated, thus providing the first evidence of synteny between a human and a spontaneous murine renal cystic disease . By fluorescence in situ hybridization the chromosomal assignment of NPHP3 to chromosome 3q21-q22 was refined . Renal pathology in NPH3 was found to consist of tubular basement membranes changes, tubular atrophy and dilation, and sclerosing tubulointerstitial nephropathy . This pathology clearly resembled findings observed in the recessive pcy mouse model of late-onset polycystic kidney disease . In analogy to pcy, renal cyst development at the corticomedullary junction was found to be an early sign of the disease . Through cloning of the NPH3 critical region and mapping of expressed genes, synteny between human NPH3 and murine pcy was established, thus generating the hypothesis that both diseases are caused by recessive mutations of homologous genes. Dev Biol, 2001 Jan 1, 229(1), 250 - 61 Cytoplasmic occurrence of the Chk1/Cdc25 pathway and regulation of Chk1 in Xenopus oocytes; Oe T et al.; Chk1, a nuclear DNA damage/replication G2 checkpoint kinase, phosphorylates Cdc25 and causes its nuclear exclusion in yeast and mammalian cells, thereby arresting the cell at the G2 phase until DNA repair/replication is completed . Chk1 is also involved, at least in part, in the natural G2 arrest of immature Xenopus oocytes, but it is unknown how Chk1 inhibits Cdc25 function and undergoes regulation during oocyte maturation . By using enucleated oocytes, we show here that Chk1 inhibits Cdc25 function in the cytoplasm of G2-arrested oocytes and that Cdc25 is activated exclusively in the cytoplasm of maturing oocytes . Moreover, we show that Chk1 activity is not appreciably altered during maturation, being maintained at basal levels, and that C-terminal truncation mutants of Chk1 have very high kinase activities, strong abilities to inhibit maturation, and altered subcellular localization in oocytes . These results, together with other results, suggest that the Chk1/Cdc25 pathway is involved cytoplasmically in G2 arrest of Xenopus oocytes, but moderately and independent of the G2 checkpoint, and that the C-terminal region of Chk1 negatively regulates its kinase activity and also determines its subcellular localization . Based on these results, we discuss the possibility that Chk1 (with the basal activity) may function as an ordinary regulator of Cdc25 in oocytes (and in other cell types) and that Chk1 might be hyperactivated in response to the G2 checkpoint via its dramatic conformational change . Am J Hum Genet, 2001 Feb, 68(2), 313 - 24 Epub 2000 Dec 20. Wild-type huntingtin reduces the cellular toxicity of mutant huntingtin in vivo; Leavitt BR et al.; We have developed yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 or YAC72) human huntingtin (htt), in a developmental- and tissue-specific manner, that is identical to endogenous htt . YAC72 mice develop selective degeneration of medium spiny projection neurons in the lateral striatum, similar to what is observed in Huntington disease . Mutant human htt expressed by YAC transgenes can compensate for the absence of endogenous htt and can rescue the embryonic lethality that characterizes mice homozygous for targeted disruption of the endogenous Hdh gene (-/-) . YAC72 mice lacking endogenous htt (YAC72 -/-) manifest a novel phenotype characterized by infertility, testicular atrophy, aspermia, and massive apoptotic cell death in the testes . The testicular cell death in YAC72 -/- mice can be markedly reduced by increasing endogenous htt levels . YAC72 mice with equivalent levels of both wild-type and mutant htt (YAC72 +/+) breed normally and have no evidence of increased testicular cell death . Similar findings are seen in YAC46 -/- mice compared with YAC46 +/+ mice, in which wild-type htt can completely counteract the proapoptotic effects of mutant htt . YAC18 -/- mice display no evidence of increased cellular apoptosis, even in the complete absence of endogenous htt, demonstrating that the massive cellular apoptosis observed in YAC46 -/- mice and YAC72 -/- mice is polyglutamine-mediated toxicity from the mutant transgene . These data provide the first direct in vivo evidence of a role for wild-type htt in decreasing the cellular toxicity of mutant htt. J Biol Chem, 2001 Mar 30, 276(13), 10153 - 60 Epub 2000 Dec 21. The human VPAC1 receptor: three-dimensional model and mutagenesis of the N-terminal domain; Lins L et al.; The human VPAC(1) receptor for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide belongs to the class II family of G-protein-coupled receptors with seven transmembrane segments . Like for all class II receptors, the extracellular N-terminal domain of the human VPAC(1) receptor plays a predominant role in peptide ligand recognition . To determine the three-dimensional structure of this N-terminal domain (residues 1-144), the Protein Data Bank (PDB) was screened for a homologous protein . A subdomain of yeast lipase B was found to have 27% sequence identity and 50% sequence homology with the N-terminal domain (8) of the VPAC(1) receptor together with a good alignment of the hydrophobic clusters . A model of the N-terminal domain of VPAC(1) receptor was thus constructed by homology . It indicated the presence of a putative signal sequence in the N-terminal extremity . Moreover, residues (Glu(36), Trp(67), Asp(68), Trp(73), and Gly(109)) which were shown to be crucial for VIP binding are gathered around a groove that is essentially negatively charged . New putatively important residues for VIP binding were suggested from the model analysis . Site-directed mutagenesis and stable transfection of mutants in CHO cells indicated that Pro(74), Pro(87), Phe(90), and Trp(110) are indeed important for VIP binding and activation of adenylyl cyclase activation . Combination of molecular modeling and directed mutagenesis provided the first partial three-dimensional structure of a VIP-binding domain, constituted of an electronegative groove with an outspanning tryptophan shell at one end, in the N-terminal extracellular region of the human VPAC(1) receptor. Bioorg Med Chem Lett, 2000 Dec 18, 10(24), 2735 - 9 Novel antifungals based on 4-substituted imidazole: solid-phase synthesis of substituted aryl sulfonamides towards optimization of in vitro activity; Saha AK et al.; The in vitro activity of novel 4-substituted imidazole antifungals was optimized by solid-phase chemistry and parallel synthesis . Potent yeast-selective as well as broad-spectrum antifungal compounds (32 and 20) were discovered. J Biol Chem, 2001 Mar 23, 276(12), 9375 - 82 Epub 2000 Dec 19. Topogenesis of peroxisomal membrane protein requires a short, positively charged intervening-loop sequence and flanking hydrophobic segments . study using human membrane protein PMP34; Honsho M et al.; Human 34-kDa peroxisomal membrane protein (PMP34) consisting of 307 amino acids was previously identified as an ortholog of, or a similar protein (with 27% identity) to the, 423-amino acid-long PMP47 of the yeast Candida boidinii . We investigated membrane topogenesis of PMP34 with six putative transmembrane segments, as a model peroxisomal membrane protein . PMP34 was characterized as an integral membrane protein of peroxisomes . Transmembrane topology of PMP34 was determined by differential permeabilization and immunofluorescent staining of HeLa cells ectopically expressing PMP34 as well as of Chinese hamster ovary-K1 expressing epitope-tagged PMP34 . As opposed to PMP47, PMP34 was found to expose its N- and C-terminal parts to the cytosol . Various deletion variants of PMP34 and their fusion proteins with green fluorescent protein were expressed in Chinese hamster ovary-K1 and were verified with respect to intracellular localization . The loop region between transmembrane segments 4 and 5 was required for the peroxisome-targeting activity, in which Ala substitution for basic residues abrogated the activity . Three hydrophobic transmembrane segments linked in a flanking region of the basic loop were essential for integration of PMP34 to peroxisome membranes . Therefore, it is evident that the intervening basic loop plus three transmembrane segments of PMP34 function as a peroxisomal targeting and topogenic signal. Results Probl Cell Differ, 2001, 32, 95 - 101 Two conformations of G-actin related to two conformations of F-actin; Egelman EH et al.; In summary, a number of different conformational states of F-actin have been described by several different laboratories . Crystal structures have revealed that an opening of the nucleotide-binding cleft, produced by a large rotation of subdomain 2, can occur in G-actin . We have shown that two crystal states of beta-actin, in an open and closed form, can provide a very good model for the conformational difference in F-actin between yeast the wild-type and a V159N mutant . This suggests that some of the dynamics associated with G-actin may provide insights into dynamic processes within the F-actin filament. Mamm Genome, 2000 Dec, 11(12), 1087 - 92 Abnormal growth plate function in pigs carrying a dominant mutation in type X collagen; Nielsen VH et al.; We have identified a naturally occurring, dominant mutation that causes dwarfism in domestic pigs (Sus scrofa) . With a positional candidate gene approach, the dwarf phenotype was shown to be a result of a single amino acid change, G590R, in the alpha1 (X) chain of type X collagen . Type X collagen is a homotrimer of alpha1(X) chains encoded by the COL10A1 gene, which is expressed in hypertrophic chondrocytes during the process of endochondral ossification . An amino acid substitution at the equivalent position in human type X collagen, G595E, has previously been shown to cause Schmid metaphyseal chondrodysplasia (SMCD), which is a relatively mild skeletal disorder associated with dwarfism and growth plate abnormality . Consistent with the clinical phenotype of SMCD patients, radiological and histological examination of the dwarf pigs revealed metaphyseal chondrodysplasia in the long bones . Yeast-based, two-hybrid protein interaction studies and in vitro assembly experiments demonstrated that the amino acid substitution interfered with the ability of the mutated collagen molecules to engage in trimerization . This work establishes that the chondrodysplastic dwarf pigs by genetic, biochemical, radiological and histological criteria provide a valid animal model of SMCD. Mamm Genome, 2000 Dec, 11(12), 1053 - 7 Genomic organization and mapping of mouse CDV (carnitine deficiency-associated gene expressed in ventricle)-1 and its related CDV-1R gene; Higashi M et al.; We have previously reported that CDV (carnitine deficiency-associated gene expressed in ventricle)-1 was a downregulated gene in the hypertrophied ventricle of carnitine-deficient juvenile visceral steatosis mice and that the related gene (CDV-1R) showed no tissue specificity and no sensitivity to carnitine deficiency . In the present paper, the CDV-1/1R gene was isolated from a mouse genomic BAC library, and the genomic structure was characterized . We found that the CDV-1/1R gene consisted of at least 19 exons and encompassed approximately 48 kb . The splice sites conformed to the GT-AG rule, and the CDV-1R mRNA containing 19 exons was processed . CDV-1 mRNA containing 5 exons was constructed from the 3' half of CDV-1R . The first exon of CDV-1 consisted of the 3' side (116 bp) of intron 14 and exon 15 (87 bp) of CDV-1R . The presumed promoter sequence for CDV-1 located in the intron 14 of CDV-1R contained the common TATA box and consensus binding sites for various transcription factors (Nkx-2.5, Spl, C/EBP, SRF, YY1, and CREB), which seem to play roles in the heart-specific expression and carnitine deficiency-associated suppression of CDV-1 . In the upstream region of the CDV-1 promoter, we found two VNTRs, 13 repeats of GATA1, and 16 copies of STRE involved in yeast stress response . The CDV-1/1R gene was located close to DSMIT68 on mouse Chromosome (Chr) 5, corresponding to human Chr 12q24 . All these data revealed that two mRNA species, CDV-1 and CDV-1R, are expressed tissue-specifically by using promoters peculiar to each transcript in a single gene. Nature, 2000 Dec 14, 408(6814), 881 - 4 Metal-ion coordination by U6 small nuclear RNA contributes to catalysis in the spliceosome; Yean SL et al.; Introns are removed from nuclear messenger RNA precursors through two sequential phospho-transesterification reactions in a dynamic RNA-protein complex called the spliceosome . But whether splicing is catalysed by small nuclear RNAs in the spliceosome is unresolved . As the spliceosome is a metalloenzyme, it is important to determine whether snRNAs coordinate catalytic metals . Here we show that yeast U6 snRNA coordinates a metal ion that is required for the catalytic activity of the spliceosome . With Mg2+, U6 snRNA with a sulphur substitution for the pro-Rp or pro-Sp non-bridging phosphoryl oxygen of nucleotide U80 reconstitutes a fully assembled yet catalytically inactive spliceosome . Adding a thiophilic ion such as Mn2+ allows the first transesterification reaction to occur in the U6/sU80(Sp)- but not the U6/sU80(Rp)-reconstituted spliceosome . Mg2+ competitively inhibits the Mn2+-rescued reaction, indicating that the metal-binding site at U6/U80 exists in the wild-type spliceosome and that the site changes its metal requirement for activity in the Sp spliceosome . Thus, U6 snRNA contributes to pre-messenger RNA splicing through metal-ion coordination, which is consistent with RNA catalysis by the spliceosome. Cell Struct Funct, 2000 Aug, 25(4), 205 - 6 New steps toward the nucleocytoplasmic traffic of macromolecules; Yoneda Y; The nucleocytoplasmic transport of functional molecules is mediated bidirectionally through the nuclear pore complex (NPC), which spans the double membranes of the nuclear envelope . It has recently been shown that signaling between the nucleus and the cytoplasm plays a key role in coordinating the cellular processes such as the cell cycle and cell differentiation (Yoneda, 2000) . As the result of recent extensive analysis, significant progress has been made in our understanding of the fundamental mechanism of nuclear transport of proteins and RNAs and numerous transport factors have now been identified . In this special issue of review articles, we focus on our rapid growing knowledge of nucleocytoplasmic transport, especially the translocation of proteins through the NPC and mRNA export, and review this exciting field from various points of view including cell biology, structural biology and yeast genetics. Biosci Biotechnol Biochem, 2000 Oct, 64(10), 2276 - 9 New scheme of the biosynthesis of formononetin involving 2,7,4'-trihydroxyisoflavanone but not daidzein as the methyl acceptor; Akashi T et al.; Glycyrrhiza echinata cell-free extract produced isoformononetin by the 7-O-transmethylation of daidzein from S-adenosyl-L-methionine (SAM) . When the yeast microsome expressing 2-hydroxyisoflavanone synthase was mixed with the cell-free extract and incubated with liquiritigenin and SAM, formononetin emerged . Furthermore, the cell-free extract yielded formononetin on incubation with 2,7,4'-trihydroxyisoflavanone and SAM . We propose a novel pathway of formononetin biosynthesis involving 2,7,4'-trihydroxyisoflavanone as the methyl acceptor. J Commun Dis, 2000 Mar, 32(1), 17 - 21 Response to hepatitis B vaccination in high risk population; Prakash C et al.; Hepatitis B vaccine is well established as very efficacious, but immune response to the vaccine is highly individual specific . A study involving fifty vaccinees was undertaken at the Hepatitis Laboratory, National Institute of Communicable Disease, Delhi . One ml (20 microgram) of Engerix B vaccine (recombinant yeast derived vaccine) was administered in the standard three dose schedule (0, 1 and 6 months) . The sero-conversion of the vaccinees was 24%, 66%, 76% and 78% at 1 month, 6 months, 7 months, and 12 months respectively . There was no seroconversion in 22% of the vaccinees . Sero-conversion was assessed using Macro ELISA test (Ausab, Abbott Labs) for Anti HBs reactivity. Hum Genet, 2000 Oct, 107(4), 376 - 84 Components of the human spindle checkpoint control mechanism localize specifically to the active centromere on dicentric chromosomes; Saffery R et al.; The spindle checkpoint control mechanism functions to ensure faithful chromosome segregation by delaying cell division until all chromosomes are correctly oriented on the mitotic spindle . Initially identified in budding yeast, several mammalian spindle checkpoint-associated proteins have recently been identified and partially characterized . These proteins associate with all active human centromeres, including neocentromeres, in the early stages of mitosis prior to the commencement of anaphase . We have examined the status of proteins associated with the checkpoint protein complex (BUB1, BUBR1, BUB3, MAD2), the anaphase-promoting complex (Tsg24, p55CDC), and other proteins associated with mitotic checkpoint control (ERK1, 3F3/2 epitope, hZW10), on a human dicentric chromosome . Each of these proteins was found to specifically associate with only the active centromere, suggesting that only active centromeres participate in the spindle checkpoint . This finding complements previous studies on multicentric chromosomes demonstrating specific association of structural and motor-related centromere proteins with active centromeres, and suggests that centromere inactivation is accompanied by loss of all functionally important centromere proteins. Mol Gen Genet, 2000 Nov, 264(4), 411 - 8 The MAK-V protein kinase regulates endocytosis in mouse; Korobko IV et al.; We report the cloning of a mouse cDNA encoding the MAK-V protein kinase, with a putative specificity for serine/threonine residues . The mak-v gene is transcribed in adult brain and in the mouse embryo from at least 7.5 dpc . Using the yeast two-hybrid system, we showed that MAK-V interacts with Rabaptin-5, a protein which plays an important role in endocytosis . Functional studies of the MAK-V protein suggest that it regulates endocytosis . We also constructed a human mak-v cDNA and localized the human mak-v gene at 21q22.11 . Its chromosomal location suggests that mak-v could be involved in disorders of the nervous system, development or in malignancies. J Neural Transm Suppl, 2000, (58), 1 - 17 Huntington disease: new insights on the role of huntingtin cleavage; Wellington CL et al.; Huntington Disease (HD) results from polyglutamine expansion within the N-terminus of huntingtin . We have produced yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) human huntingtin in a developmentally appropriate and tissue-specific manner identical to the pattern of expression of endogenous huntingtin . YAC46 and YAC72 mice show early electrophysiological abnormalities indicating neuronal cytoplasmic dysfunction prior to developing nuclear inclusions or neurodegeneration . YAC72 mice display a hyperkinetic movement disorder by 7 months of age, and have evidence for selective and specific degeneration of medium spiny neurons in the lateral striatum by 12 months of age . A key molecular feature of pathology of these YAC72 mice is cleavage of huntingtin in the cytoplasm following by translocation of the resulting huntingtin N-terminal fragments into the nucleus of striatal neurons . Increasing nuclear localization of huntingtin N-terminal fragments within medium spiny neurons of the striatum occurs concomitantly with the onset of selective neurodegeneration . Because huntingtin is a caspase substrate and truncated huntingtin fragments are toxic in vitro, inhibiting caspase cleavage of huntingtin may be of potential therapeutic benefit in HD . We show that caspase inhibitors eliminate huntingtin cleavage in cells and protects them from an apoptotic stress . We also identify caspase-6 and caspase-3 cleavage sites in huntingtin and demonstrate that neuronal and non-neuronal cells expressing a caspase-resistant huntingtin with an expanded polyglutamine tract are less susceptible to apoptosis and aggregate formation . These results suggest that caspase cleavage of huntingtin may be a crucial step in aggregate formation and neurotoxicity in HD. J Biol Chem, 2001 Mar 23, 276(12), 9303 - 7 Epub 2000 Dec 13. The neuronal adaptor protein X11alpha interacts with the copper chaperone for SOD1 and regulates SOD1 activity; McLoughlin DM et al.; The neuronal adaptor protein X11alpha participates in the formation of multiprotein complexes and intracellular trafficking . It contains a series of discrete protein-protein interaction domains including two contiguous C-terminal PDZ domains . We used the yeast two-hybrid system to screen for proteins that interact with the PDZ domains of human X11alpha, and we isolated a clone encoding domains II and III of the copper chaperone for Cu,Zn-superoxide dismutase-1 (CCS) . The X11alpha/CCS interaction was confirmed in coimmunoprecipitation studies plus glutathione S-transferase fusion protein pull-down assays and was shown to be mediated via PDZ2 of X11alpha and a sequence within the carboxyl terminus of domain III of CCS . CCS delivers the copper cofactor to the antioxidant superoxide dismutase-1 (SOD1) enzyme and is required for its activity . Overexpression of X11alpha inhibited SOD1 activity in transfected Chinese hamster ovary cells which suggests that X11alpha binding to CCS is inhibitory to SOD1 activation . X11alpha also interacts with another copper-binding protein found in neurons, the Alzheimer's disease amyloid precursor protein . Thus, X11alpha may participate in copper homeostasis within neurons. Tumour Biol, 2001 Mar-Apr, 22(2), 59 - 66 Rare somatic p53 mutation identified in breast cancer: a case report; Smardova J et al.; Most p53 mutations occur in the central part of the p53 gene that codes for the DNA-binding domain . Missense mutations are prevalent . However, 10-25% of all mutations occur outside exons 5-8 and include a prevalence of frameshift, nonsense and splice site mutations . Functional analysis of p53 transactivation ability in yeast (FASAY) was used to screen for p53 mutations in tumors and a mutant p53 protein retaining partial activity was identified . We characterized this somatic p53 mutation in codon 337: transition C-->T, changing codon CGC to TGC and causing substitution of arginine for cysteine in exon 10, which codes for the tetramerization domain of p53 . We detected high accumulation of this mutant p53 protein within the tumor tissue and found that it cannot be immunoprecipitated by either a wild-type p53-specific antibody (PAb1620) or by a mutant p53-specific antibody (PAb240) . We confirmed the somatic origin of the mutation by analysis of p53 status in peripheral leukocytes . Genes Dev, 2000 Dec 15, 14(24), 3126 - 39 Mutation of a Drosophila gamma tubulin ring complex subunit encoded by discs degenerate-4 differentially disrupts centrosomal protein localization; Barbosa V et al.; We have cloned the Drosophila gene discs degenerate-4 (dd4) and find that it encodes a component of the gamma-tubulin ring complex (gammaTuRC) homologous to Spc98 of budding yeast . This provides the first opportunity to study decreased function of a member of the gamma-tubulin ring complex, other than gamma-tubulin itself, in a metazoan cell . gamma-tubulin is no longer at the centrosomes but is dispersed throughout dd4 cells and yet bipolar metaphase spindles do form, although these have a dramatically decreased density of microtubules . Centrosomin (CNN) remains in broad discrete bodies but only at the focused poles of such spindles, whereas Asp (abnormal spindle protein) is always present at the presumptive minus ends of microtubules, whether or not they are focused . This is consistent with the proposed role of Asp in coordinating the nucleation of mitotic microtubule organizing centers . The centrosome associated protein CP190 is partially lost from the spindle poles in dd4 cells supporting a weak interaction with gamma-tubulin, and the displaced protein accumulates in the vicinity of chromosomes . Electron microscopy indicates not only that the poles of dd4 cells have irregular amounts of pericentriolar material, but also that they can have abnormal centrioles . In six dd4 cells subjected to serial sectioning centrioles were missing from one of the two poles . This suggests that in addition to its role in nucleating cytoplasmic and spindle microtubules, the gammaTuRC is also essential to the structure of centrioles and the separation of centrosomes. Curr Atheroscler Rep, 2001 Jan, 3(1), 93 - 6 Herbs and atherosclerosis; Heber D; It is now widely accepted that atherosclerosis is a complex multicellular process involving oxidation of cholesterol and the intracellular accumulation of oxidized cholesterol . This accumulation causes a cascade of inflammatory processes, resulting in an unstable atherosclerotic plaque that ultimately bursts, causing myocardial infarction . Botanical dietary supplements (herbs) can ameliorate this process and prevent cardiovascular disease at many steps in the process . Many herbs have antioxidant activity and can reduce low-density lipoprotein oxidation . Some phytosterols found in botanicals can inhibit cholesterol absorption . After a brief review of herbs being promoted for achieving and maintaining healthy cholesterol levels, the evidence and future prospects for Chinese red yeast rice, the main component of dietary supplements with HMG-CoA reductase inhibiting activity, are discussed in detail . Initial phase II clinical trials are highly encouraging . This herb is likely to be able to directly impact the process of atherosclerosis, but large-scale clinical trials are needed to assess the public health potential of this herbal supplement. Plant J, 2000 Dec, 24(5), 637 - 44 Studies on the topology of the protein import channel in relation to the plant mitochondrial processing peptidase integrated into the cytochrome bc1 complex; Dessi P et al.; The mitochondrial processing peptidase (MPP) specifically cleaves N-terminal targeting signals from hundreds of nuclear-encoded, matrix-targeted precursor proteins . In contrast to yeast and mammals, the plant MPP is an integral component of the respiratory cytochrome bc1 complex . The topology of the protein import channel in relation to MPP/bc1 in plants was studied using chimeric precursors containing truncated cytochrome b2 (cyt b2) proteins of 55-167 residues in length, fused to dihydrofolate reductase (DHFR) . The DHFR domain could be tightly folded by methotrexate (MTX), generating translocation intermediates trapped in the import channel with only the cyt b2 pre-sequence/mature domain protruding into the matrix . Spinach and soybean mitochondria imported and processed unfolded precursors . MTX-folded intermediates were not processed in spinach but the longest (1-167) MTX-folded cyt b2-DHFR construct was processed in soybean, while yeast mitochondria successfully processed even shorter MTX-folded constructs . The MTX-folded precursors were cleaved with high efficiency by purified spinach MPP/bc1 complex . We interpret these results as indicating that the protein import channel is located distantly from the MPP/bc1 complex in plants, and that there is no link between protein translocation and protein processing. Plant J, 2000 Dec, 24(5), 583 - 9 Functional characterization of the EMCV IRES in plants; Urwin P et al.; The translation of eukaryotic messenger RNA is typically dependent upon the presence of an m7GpppN cap structure at the 5' end of the transcript . However, several animal viruses, including the Picorna viruses, have been shown to exhibit cap-independent translation through the presence of an internal ribosome entry site or IRES . This IRES-mediated cap-independent internal translation initiation has been exploited to generate bicistronic transcripts that function in animal cells . Recently IRES elements have also been identified in a small number of vertebrate, insect and yeast cellular messenger RNAs although no such sequences have been identified in endogenous plant genes and there are no reports of animal virus derived IRES activity in plant cells . Here we have constructed a bicistronic gene containing both green fluorescent protein and luciferase open-reading frames separated by the encephalomyocarditis IRES element under the control of the CaMV 35S promoter . Northern analysis reveals expression of the bicistronic transcript and in vivo imaging of GFP and luciferase activities demonstrates the functional presence of both proteins . Western blot analysis confirms the independent translation of both reporter proteins . These data suggest that insertion of the encephalomyocarditis virus (EMCV) IRES element between two open-reading frames of a plant bicistronic transcript can mediate translation of the second open-reading frame . This activity is more apparent in the leaves, than in the roots, of transgenic seedlings carrying the bicistronic reporter gene construct. Mol Microbiol, 2000 Dec, 38(5), 1034 - 47 The abaA homologue of Penicillium marneffei participates in two developmental programmes: conidiation and dimorphic growth; Borneman AR et al.; Penicillium marneffei is the only known species of its genus that is dimorphic . At 25 degrees C, P . marneffei exhibits true filamentous growth and undergoes asexual development producing spores borne on complex structures called conidiophores . At 37 degrees C, P . marneffei undergoes a dimorphic transition to produce uninucleate yeast cells that divide by fission . We have cloned a homologue of the Aspergillus nidulans abaA gene encoding an ATTS/TEA DNA-binding domain transcriptional regulator and shown that it is involved in both these developmental programs . Targeted deletion of abaA blocks asexual development at 25 degrees C before spore production, resulting in aberrant conidiophores with reiterated terminal cells . At 37 degrees C, the abaA deletion strain fails to switch correctly from multinucleate filamentous to uninucleate yeast cells . Both the transitional hyphal cells, which produce the yeast cells, and the yeast cells themselves contain multiple nuclei . Expression of the abaA gene is activated during both conidiation and the hyphal-yeast switch . Interestingly, the abaA gene of the filamentous monomorphic fungus A . nidulans can complement both conidiation and dimorphic switching defects in the P . marneffei abaA mutant . In addition, ectopic overexpression of abaA results in anucleate yeast cells and multinucleate vegetative filamentous cells . These data suggest that abaA regulates cell cycle events and morphogenesis in two distinct developmental programmes. Jpn J Cancer Res, 2000 Dec, 91(12), 1211 - 21 Isolation of differentiated squamous and undifferentiated spindle carcinoma cell lines with differing metastatic potential from a 4-nitroquinoline N-Oxide-induced tongue carcinoma in a F344 rat; Takeuchi S et al.; One differentiated squamous cell carcinoma (SCC) cell line (RSC3-E2) and two undifferentiated tumor cell lines (RSC3-LM and RSC3-E2R) with different metastatic potential were established from a 4-nitroquinoline N-oxide (4NQO)-induced differentiated SCC in F344 rat tongue . The RSC3-E2 subline was isolated from a parental cell line (RSC3-P) by single cell cloning in vitro, whereas the RSC3-LM subline was isolated from a lung metastatic focus after subcutaneous (s.c.) injection of RSC3-P cells . The RSC3-E2R cell line was isolated from a lung metastatic focus following s.c . injection of RSC3-E2 cells after X-irradiation in vitro . The RSC3-E2 cell line is keratin-positive and grows as a keratinizing tumor in nude mice, whereas RSC3-LM and RSC3-E2R cells are keratin-negative, vimentin-positive and form undifferentiated tumors . When s.c . injected into nude mice, the RSC3-E2 cell line proved to be non-metastatic, while the RSC3-LM cell line was metastatic by both hematogenous and lymphogenous routes, and the RSC3-E2R cell line was metastatic only hematogenously . In vitro relative growth rates and in vitro invasion activity of these cell lines were in the order RSC3-LM > RSC3-E2R > RSC3-E2 . Chromosome analysis revealed two peaks with modal chromosome numbers of 83 and 78 for RSC3-P cells and single peaks at 83, 78 and 56 for RSC3-LM, RSC3-E2 and RSC3-E2R cell lines, respectively . Common structural abnormalities on chromosome 11 were shared by all cell lines . Mutation analysis of the p53 gene using a yeast functional assay demonstrated RSC3-LM cell line to have a point mutation at codon 269, whereas RSC3-E2 and RSC3-E2R had double mutations at codons 106 and 170 on each allele . These results suggest that the two undifferentiated RSC3-LM and RSC3-E2R tumor cell lines with different metastatic potential were generated from differentiated SCC cells via different genetic pathways as a consequence of tumor progression in vivo and in vitro, respectively . These cell lines should provide a useful model for understanding mechanisms of hematogenous and lymphogenous metastasis, as well as tumor progression of oral SCCs. Insect Mol Biol, 2000 Dec, 9(6), 591 - 604 Sequence, secondary structure and phylogenetic analyses of the ribosomal internal transcribed spacer 2 (ITS2) in the Timarcha leaf beetles (Coleoptera: Chrysomelidae); Gomez-Zurita J et al.; Internal transcribed spacer 2 (ITS2) sequences of the nuclear rDNA in forty-seven specimens (thirty-four species) of the leaf beetle genus Timarcha have been studied . Timarcha ITS2 (523 bp on average) share some sequence features with other Chrysomeloidea relatives (Chrysolina, Diabrotica and Bruchus) but have no clear similarity with any other arthropod ITS2 sequences . Interspecific divergences are in the range 0.002-0.166, and 0.124-0.206 in the comparisons between subgenera . No evidence of intragenomic divergent ITS2 sequences has been found . Secondary structures are concordant with the four-domain model proposed for vertebrates and yeast, but differs from those proposed for dipterans . Phylogenetic analysis of the ITS2 data confirms the results of a previous study based on mitochondrial sequences, as the basality of the Metallotimarcha subgenus and the absence of phylogenetic support for the Timarchostoma subgenus. Genes Cells, 2000 Nov, 5(11), 913 - 927 Extracellular matrix tenascin-X in combination with vascular endothelial growth factor B enhances endothelial cell proliferation; Ikuta T et al.; BACKGROUND: An extracellular matrix tenascin-X (TNX) is highly expressed in muscular tissues, especially heart and skeletal muscle, and is also prominent around blood vessels . The precise in vivo role of TNX remains to be elucidated . To identify proteins that interact with TNX in the extracellular environment, we searched for TNX-binding proteins using a yeast two-hybrid system . RESULTS: We used mouse TNX-specific fibronectin type III repeats (mTNX/FNIII13-25) as a bait for the screening . We found that vascular endothelial growth factor B (VEGF-B) binds to mTNX/FNIII13-25 . This interaction was confirmed by pull-down assays and co-immunoprecipitation assays . The full-length mTNX, as well as mTNX/FNIII13-25, interacted with both alternative splice isoforms VEGF-B186 and VEGF-B167 . Furthermore, the full-length mTNX also bound to VEGF-A . The minimal region of TNX that interacts with VEGF-B was mapped to the FNIII repeats (FNIII13-25) but not to the other characteristic domains of TNX . The TNX-binding site of VEGF-B was located in the N-terminal 115-amino acid region . mTNX/FNIII13-25 did not prevent the interaction of VEGF-B with VEGFR-1 (VEGF receptor 1), and VEGF-B could simultaneously bind to both mTNX/FNIII13-25 and VEGFR-1 . A conditioned medium from transfected 293T cells coexpressing full-length TNX and VEGF-B could promote DNA synthesis in bovine endothelial cells in which VEGFR-1 were expressed . VEGFR-1 phosphorylation triggered by VEGF-B186 were increased in cells plated with mTNX/FNIII13-25 or full-length mTNX, compared with cells plated with VEGF-B186 alone . CONCLUSION: TNX interacts with VEGF-B and enhances the ability of VEGF-B to stimulate cell proliferation . This enhanced mitogenecity is caused by increased signals mediated by the VEGFR-1 receptor . This finding suggests a role for TNX in the regulation of the development of blood vessels such as vasculogenesis and angiogenesis. Eur J Neurosci, 2000 Dec, 12(12), 4215 - 21 Interaction of the C-terminal tail region of the metabotropic glutamate receptor 7 with the protein kinase C substrate PICK1; El Far O et al.; Group III metabotropic glutamate receptors (mGluRs) are highly enriched in the presynaptic terminals of glutamatergic synapses where they mediate feedback inhibition of neurotransmitter release . Here, we used the yeast two-hybrid system to identify a direct interaction of the C-terminal tail region of mGluR7 with the rat homologue of the protein kinase C substrate PICK1 . This interaction is specifically mediated by the very C-terminal amino acids of the receptor and can be reconstituted in human embryonic kidney 293 cells by transfection of full-length mGluR7 and PICK1 cDNAs . Quantitative beta-galactosidase assays revealed that among the different group III mGluRs, mGluR7 is the major PICK1 binding partner although other subfamily members can also interact with PICK1 . These data indicate that PDZ domain-containing proteins might contribute to the presynaptic localization of group III mGluRs. J Cell Biol, 2000 Dec 11, 151(6), 1141 - 54 Nischarin, a novel protein that interacts with the integrin alpha5 subunit and inhibits cell migration; Alahari SK et al.; Integrins have been implicated in key cellular functions, including cytoskeletal organization, motility, growth, survival, and control of gene expression . The plethora of integrin alpha and beta subunits suggests that individual integrins have unique biological roles, implying specific molecular connections between integrins and intracellular signaling or regulatory pathways . Here, we have used a yeast two-hybrid screen to identify a novel protein, termed Nischarin, that binds preferentially to the cytoplasmic domain of the integrin alpha5 subunit, inhibits cell motility, and alters actin filament organization . Nischarin is primarily a cytosolic protein, but clearly associates with alpha5beta1, as demonstrated by coimmunoprecipitation . Overexpression of Nischarin markedly reduces alpha5beta1-dependent cell migration in several cell types . Rat embryo fibroblasts transfected with Nischarin constructs have "basket-like" networks of peripheral actin filaments, rather than typical stress fibers . These observations suggest that Nischarin might affect signaling to the cytoskeleton regulated by Rho-family GTPases . In support of this, Nischarin expression reverses the effect of Rac on lamellipodia formation and selectively inhibits Rac-mediated activation of the c-fos promoter . Thus, Nischarin may play a negative role in cell migration by antagonizing the actions of Rac on cytoskeletal organization and cell movement. Radiat Res, 2001 Jan, 155(1 Pt 2), 181 - 187 Expression of the protein product of the PCPH proto-oncogene in human tumor cell lines; Rouzaut A et al.; Expression of the Protein Product of the PCPH Proto-oncogene in Human Tumor Cell Lines . Exposure of Syrian hamster embryo fibroblasts to chemical carcinogens resulted in the oncogenic activation of the PCPH proto-oncogene by induction of a single base-pair deletion that generated a truncated PCPH oncoprotein (mutated PCPH) . Recently, we isolated and characterized the cDNA for the human PCPH proto-oncogene and determined that in humans PCPH is a single-copy gene located in chromosome 14 (14q24.3) . Pilot mRNA expression studies indicated that PCPH was expressed in the majority of normal organs tested, particularly in liver and kidney, but it appeared to be expressed either at low levels or not at all in tumor cells or cell lines derived from the high-expressing tissues . We have generated an antiserum against bacterial recombinant Syrian hamster PCPH . This antiserum recognizes both the normal and truncated, oncogenic Syrian hamster PCPH proteins and cross-reacts with the yeast, mouse, rat and human homologue proteins . Using this antibody, we have performed a study of PCPH expression in a larger sample of human neoplastic cell lines, including some derived from breast, nervous system, colon, lung and pancreas tumors . Results confirmed the frequent lack of PCPH expression in malignant cells and identified several immunoreactive forms of PCPH being differentially expressed in cells of diverse tissue origins. Proc Natl Acad Sci U S A, 2000 Dec 19, 97(26), 14784 - 8 Completing the heterotrimer: isolation and characterization of an Arabidopsis thaliana G protein gamma-subunit cDNA; Mason MG et al.; Heterotrimeric G proteins consist of three subunits (alpha, beta, and gamma) . alpha- and beta- subunits have been previously cloned in plants, but the gamma-subunit has remained elusive . To isolate the gamma-subunit of a plant heterotrimeric G protein an Arabidopsis thaliana yeast two-hybrid library was screened by using a tobacco G-beta-subunit as the bait protein . One positive clone (AGG1) was isolated several times; it displays significant homology to the conserved domains of mammalian gamma-subunits . The predicted AGG1 protein sequence contains all of the typical characteristics of mammalian gamma-subunits such as small size (98 amino acids, 10.8 kDa), presence of a C-terminal CAAX box to direct isoprenyl modification, and an N-terminal alpha-helix region capable of forming a coiled-coil interaction with the beta-subunit . Northern and Southern analyses showed that AGG1 is a single-copy gene in Arabidopsis with a similar expression pattern to the Arabidopsis beta-subunit, AGB1 {Weiss, C . A., Garnaat, C . W., Mukai, K., Hu, Y . & Ma, H . (1994) Proc . Natl . Acad . Sci . USA 91, 9554-9558} . By using the yeast two-hybrid system, we show that AGG1 strongly interacts with tobacco and Arabidopsis beta-subunits . The in vivo results have been confirmed by using in vitro methods to prove the interaction between AGG1 and the Arabidopsis beta-subunit . As previously observed in mammalian systems, both the coiled-coil domain and the WD repeat regions of the beta-subunit are essential for AGG1 interaction . Also in agreement with previous observations, the removal of the N-terminal alpha-helix of the AGG1 greatly reduces but does not completely block the interaction. Physiol Genomics, 2000 Dec 18, 4(2), 109 - 126 Analysis of molecular profile data using generative and discriminative methods; Moler EJ et al.; A modular framework is proposed for modeling and understanding the relationships between molecular profile data and other domain knowledge using a combination of generative (here, graphical models) and discriminative {Support Vector Machines (SVMs)} methods . As illustration, naive Bayes models, simple graphical models, and SVMs were applied to published transcription profile data for 1,988 genes in 62 colon adenocarcinoma tissue specimens labeled as tumor or nontumor . These unsupervised and supervised learning methods identified three classes or subtypes of specimens, assigned tumor or nontumor labels to new specimens and detected six potentially mislabeled specimens . The probability parameters of the three classes were utilized to develop a novel gene relevance, ranking, and selection method . SVMs trained to discriminate nontumor from tumor specimens using only the 50-200 top-ranked genes had the same or better generalization performance than the full repertoire of 1,988 genes . Approximately 90 marker genes were pinpointed for use in understanding the basic biology of colon adenocarcinoma, defining targets for therapeutic intervention and developing diagnostic tools . These potential markers highlight the importance of tissue biology in the etiology of cancer . Comparative analysis of molecular profile data is proposed as a mechanism for predicting the physiological function of genes in instances when comparative sequence analysis proves uninformative, such as with human and yeast translationally controlled tumour protein . Graphical models and SVMs hold promise as the foundations for developing decision support systems for diagnosis, prognosis, and monitoring as well as inferring biological networks. FEBS Lett, 2000 Dec 15, 486(3), 305 - 9 Regulation of Wee1 kinase in response to protein synthesis inhibition; Suda M et al.; To investigate the mechanism coupling growth (protein synthesis) with cell division, we examined the relationship between the tyrosine kinase Wee1 that inhibits Cdc2-Cdc13 mitosis-inducing kinase by phosphorylating it, and protein synthesis inhibition in fission yeast . The wee1-50 mutant showed supersensitivity to protein synthesis inhibitor, cycloheximide . Wee1 was essential for the G(2) delay upon a partial inhibition of protein synthesis . Indeed, the protein synthesis inhibition caused an increase in the Wee1 protein by the Sty1/Spc1 MAPK-dependent transcriptional and the Sty1/Spc1 MAPK-independent post-transcriptional regulations . Further, the results indicated that the post-transcriptional regulation is important for the G(2) delay. J Virol, 2001 Jan, 75(1), 384 - 95 An Epstein-Barr virus protein interacts with Notch; Kusano S et al.; The Epstein-Barr virus (EBV) BamHI A mRNAs were originally identified in cDNA libraries from nasopharyngeal carcinoma, where they are expressed at high levels . The RNAs are differentially spliced to form several open reading frames and also contain the BARF0 open reading frame at the 3' end . One cDNA, RK-BARF0, included a potential endoplasmic reticulum-targeting signal peptide sequence . The RK-BARF0 protein is shown here to interact with the Notch4 ligand binding domain, using yeast two-hybrid screening, coimmunoprecipitation, and confocal microscopy . This interaction induces translocation of a portion of the full-length unprocessed Notch4 to the nucleus by using the Notch nuclear localization signal . These effects of RK-BARF0 on Notch intracellular location indicate that EBV possibly modulates Notch signaling . Unprocessed Notch4 was also detected in immunoprecipitated complexes from EBV-infected cells by using a rabbit antiserum raised against a BARF0-specific peptide . This finding provides additional evidence for expression of RK-BARF0 and its interaction with Notch during EBV infection . In EBV-infected, EBNA2-negative cells, RK-BARF0 induced the expression of EBV latent membrane protein 1 (LMP1), and this induction was dependent on the RK-BARF0/Notch interaction domain . The activation of LMP1 expression by RK-BARF0 may be responsible for expression of LMP1 in EBV latent infections in the absence of EBNA2. J Virol, 2001 Jan, 75(1), 242 - 50 Viral DNA synthesis defects in assembly-competent Rous sarcoma virus CA mutants; Cairns TM et al.; The major structural protein of the retroviral core (CA) contains a conserved sequence motif shared with the CA-like proteins of distantly related transposable elements . The function of this major region of homology (MHR) has not been defined, in part due to the baffling array of phenotypes in mutants of several viruses and the yeast TY3 . This report describes new mutations in the CA protein of Rous sarcoma virus (RSV) that were designed to test whether these different phenotypes might indicate distinct functional subdomains in the MHR . A comparison of 25 substitutions at 10 positions in the RSV conserved motif argues against this possibility . Most of the replacements destroyed virus infectivity, although either of two lethal phenotypes was obtained depending on the residue introduced . At most of the positions, one or more replacements (generally the more conservative substitutions) caused a severe replication defect without having any obvious effects on virus assembly, budding, Gag-Pol and genome incorporation, or protein processing . The mutant particles exhibited a defect in endogenous viral DNA synthesis and showed increased sensitivity of the core proteins to detergent, indicating that the mutations interfere with the formation and/or activity of the virion core . The distribution of these mutations across the MHR, with no evidence of clustering, suggests that the entire region is important for a critical postbudding function . In contrast, a second class of lethal substitutions (those that destroyed virus assembly and release) consists of alterations that are expected to cause severe effects on protein structure by disruption either of the hydrophobic core of the CA carboxyl-terminal domain or of the hydrogen bond network that stabilizes the domain . We suggest that this duality of phenotypes is consistent with a role for the MHR in the maturation process that links the two parts of the life cycle. J Virol, 2001 Jan, 75(1), 205 - 14 Mutations that affect dimer formation and helicase activity of the hepatitis C virus helicase; Khu YL et al.; Interaction between viral proteins is necessary for viral replication and viral particle assembly . We used the yeast two-hybrid assay to identify interactions among all the mature proteins of the hepatitis C virus . The interaction between NS3 and NS3 was one of the strongest viral protein-protein interactions detected . The minimal region required for this interaction was mapped to a specific subdomain of 174 amino acids in the N terminus of the helicase region . Random mutations in the minimal region were generated by PCR, and mutants that failed to interact with a wild-type minimal fragment were isolated using the yeast two-hybrid assay as a screen . Three of these mutations resulted in a reduction or a loss of interaction between helicases . Analytical gel filtration showed that in the presence of an oligonucleotide, wild-type helicases form dimers whereas the mutants remain mostly monomeric . All three mutants were partially or almost inactive when assayed for helicase activity in vitro . Mixing a mutant helicase (Y267S) with wild-type helicase did not dramatically affect helicase activity . These data indicate that dimerization of the helicase is important for helicase activity . The mutations that reduce self-association of the helicase may define the key residues involved in NS3-NS3 dimerization. J Virol, 2001 Jan, 75(1), 151 - 60 Gps2, a protein partner for human papillomavirus E6 proteins; Degenhardt YY et al.; We have used the yeast two-hybrid system to screen a cDNA library prepared from normal human epidermal keratinocytes and identified protein partners for human papilloma virus (HPV) E6 proteins . A clone that encoded Gps2 interacted with E6 proteins from HPVs of high and low oncogenic risk . The specificity of these reactions was verified and the regions of E6 that were required for interaction were mapped . Steady-state and pulse-chase analyses of cells cotransfected with DNAs expressing E6 from either HPV6 or HPV18 and Gps2 demonstrated that the E6 proteins induced the degradation of Gps2 in vivo but not in vitro . Gps2 exhibited transcriptional activation activity, and high-risk E6 suppressed this activity. J Virol, 2001 Jan, 75(1), 26 - 35 Dominance of virus over host factors in cross-species activation of human cytomegalovirus early gene expression; Garcia-Ramirez JJ et al.; Human cytomegalovirus (HCMV) exhibits a highly restricted host range . In this study, we sought to examine the relative significance of host and viral factors in activating early gene expression of the HCMV UL54 (DNA polymerase) promoter in murine cells . Appropriate activation of the UL54 promoter at early times is essential for viral DNA replication . To study how the HCMV UL54 promoter is activated in murine cells, a transgenesis system based on yeast artificial chromosomes (YACs) was established for HCMV . A 178-kb YAC, containing a subgenomic fragment of HCMV encompassing the majority of the unique long (UL) region, was constructed by homologous recombination in yeast . This HCMV YAC backbone is defective for viral growth and lacks the major immediate-early (IE) gene region, thus permitting the analysis of essential cis-acting sequences when complemented in trans . To quantitatively measure the level of gene expression, we generated HCMV YACs containing a luciferase reporter gene inserted downstream of either the UL54 promoter or, as a control for late gene expression, the UL86 promoter, which directs expression of the major capsid protein . To determine the early gene activation pathway, point mutations were introduced into the inverted repeat 1 (IR1) element of the UL54 promoter of the HCMV YAC . In the transgenesis experiments, HCMV YACs and derivatives generated in yeast were introduced into NIH 3T3 murine cells by polyethylene glycol-mediated fusion . We found that infection of YAC, but not plasmid, transgenic lines with HCMV was sufficient to fully recapitulate the UL54 expression program at early times of infection, indicating the importance of remote regulatory elements in influencing regulation of the UL54 promoter . Moreover, YACs containing a mutant IR1 in the UL54 promoter led to reduced ( approximately 30-fold) reporter gene expression levels, indicating that HCMV major IE gene activation of the UL54 promoter is fully permissive in murine cells . In comparison with HCMV, infection of YAC transgenic NIH 3T3 lines with murine cytomegalovirus (MCMV) resulted in lower (more than one order of magnitude) efficiency in activating UL54 early gene expression . MCMV is therefore not able to fully activate HCMV early gene expression, indicating the significance of virus over host determinants in the cross-species activation of key early gene promoters . Finally, these studies show that YAC transgenesis can be a useful tool in functional analysis of viral proteins and control of gene expression for large viral genomes. FEMS Immunol Med Microbiol, 2000 Dec, 29(4), 241 - 5 Amino acid- or protein-dependent growth of Trichophyton mentagrophytes and Trichophyton rubrum; Takasuka T; Culture conditions were examined for Trichophyton mentagrophytes and Trichophyton rubrum, which are major pathogens involved in dermatophytosis . They grew well in Sabouraud's dextrose broth or RPMI 1640 . Growth in phosphate-buffered yeast nitrogen base supplemented with glucose was very slow, although growth improved significantly with the addition of amino acids or proteins to the medium . The fungi could also grow using human nail fragments as the only source of nutrition . Examination of proteases by substrate gel electrophoresis indicated that distinct sets of proteases are secreted from the dermatophytes in two different media, Sabouraud's dextrose broth and nail fragments . A protease inhibitor, phenylmethanesulfonyl fluoride, inhibited the growth of the fungi on nail fragments, but it did not inhibit their growth in Sabouraud's dextrose broth. Plant Mol Biol, 2000 Sep, 44(2), 167 - 76 Functional analysis of a RPD3 histone deacetylase homologue in Arabidopsis thaliana; Wu K et al.; Histone acetylation is modulated through the action of histone acetyltransferase and deacetylase, which play key roles in the regulation of eukaryotic gene expression . We have screened the expressed sequence tag database with the yeast histone deacetylase RPD3 sequence and identified two Arabidopsis homologues, AtRPD3A and AtRPD3B . The deduced amino acid sequences of AtRPD3A and AtRPD3B show high overall homology (55% identity) to each other . AtRPD3A encodes a putative protein of 502 amino acids with 49% identity to the yeast RPD3 . AtRPD3B encodes a putative protein of 471 amino acids and shares 55% amino acid identity with the yeast RPD3 . Northern analysis indicated that AtRPD3A was highly expressed in the leaves, stems, flowers and young siliques of Arabidopsis plants, whereas the AtRPD3B transcript was not detected in these organs . An AtRPD3A fusion protein repressed transcription when directed to a promoter driving a reporter gene, indicating a role for AtRPD3A protein in gene repression . Arabidopsis plants were transformed with a gene construct comprising a truncated AtRPD3A cDNA in the antisense orientation driven by a strong constitutive promoter, -394tCUP . Antisense expression of AtRPD3A resulted in decreased endogenous AtRPD3A transcript and delayed flowering in transgenic Arabidopsis plants, suggesting that the transition from the vegetative to reproductive phase of development could be affected by histone acetylation . Our study demonstrates the important role of histone deacetylases in plant growth and development. Plant Mol Biol, 2000 Sep, 44(2), 129 - 40 Plant lipoxygenase 2 is a translation initiation factor-4E-binding protein; Freire MA et al.; The eukaryotic initiation factor 4E (eIF4E) emerged recently as a target for different types of regulation affecting translation . In animal and yeast cells, eIF4E-binding proteins modulate the availability of eIF4E . A search for plant eIF4E-binding proteins from Arcabictopsis thaliana using the yeast genetic interaction system identified a clone encoding a lipoxygenase type 2 (AtLOX2) . In vitro and in vivo biochemical assays confirm an interaction between AtLOX2 and plant eIF4E(iso) factor . A two-hybrid assay revealed that AtLOX2 is also able to interact with both wheat initiation factors 4E and 4E(iso) . Deletion analysis maps the region of AtLOX2 involved in interaction with AteIF(iso)4E between amino acids 175 and 232 . A sequence related to the conserved motif present in several eIF4E-binding proteins was found in this region . Furthermore, the wheat p86 subunit, a component of the plant translation eIF(iso)4F complex, was found to interfere with the AteIF(iso)4E-AtLOX2 interaction suggesting that p86 and AtLOX2 compete for the same site on eIF(iso)4E . These results may reflect a link between eIF4Es factors mediating translational control with LOX2 activity, which is probably conserved throughout the plant kingdom. J Biol Chem, 2001 Mar 16, 276(11), 8014 - 20 Epub 2000 Dec 14. GDP dissociation inhibitor domain II required for Rab GTPase recycling; Gilbert PM et al.; Rab GTPases are localized to distinct subsets of organelles within the cell, where they regulate SNARE-mediated membrane trafficking between organelles . One factor required for Rab localization and function is Rab GDP dissociation inhibitor (GDI), which is proposed to recycle Rab after vesicle fusion by extracting Rab from the membrane and loading Rab onto newly formed transport intermediates . GDI is composed of two domains; Rab binding is mediated by Domain I, and the function of Domain II is not known . In this study, Domain II of yeast GDI, encoded by the essential GDI1/SEC19 gene, was targeted in a genetic screen to obtain mutants that might lend insight into the function of this domain . In one gdi1 mutant, the cytosolic pools of all Rabs tested were depleted, and Rab accumulated on membranes, suggesting that this mutant Gdi1 protein has a general defect in extraction of Rab from membranes . In a second gdi1 mutant, the endosomal/vacuolar Rabs Vps21/Ypt51p and Ypt7p accumulated in the cytosol bound to Gdi1p, but localization of Ypt1p and Sec4p were not significantly affected . Using an in vitro assay which reconstitutes Gdi1p-mediated membrane loading of Rab, this mutant Gdi1p was found to be defective in loading of Vps21p but not Ypt1p . Loading of Vps21p by loading-defective Gdi1p was restored when acceptor membranes prepared from a deletion strain lacking Vps21p were used . These results suggest that membrane-associated Rab may regulate recruitment of GDI-Rab from the cytosol, possibly by regulating a GDI-Rab receptor . We conclude that Domain II of Gdi1p is essential for Rab loading and Rab extraction, and confirm that each of these activities is required for Gdi1p function in vivo. Plant Physiol, 2000 Dec, 124(4), 1558 - 69 The Arabidopsis genome . An abundance of soluble N-ethylmaleimide-sensitive factor adaptor protein receptors; Sanderfoot AA et al.; Many factors have been characterized as essential for vesicle trafficking, including a number of proteins commonly referred to as soluble N-ethylmaleimide-sensitive factor adaptor protein receptor (SNARE) components . The Arabidopsis genome contains a remarkable number of SNAREs . In general, the vesicle fusion machinery appears highly conserved . However, whereas some classes of yeast and mammalian genes appear to be lacking in Arabidopsis, this small plant genome has gene families not found in other eukaryotes . Very little is known about the precise function of plant SNAREs . By contrast, the intracellular localization of and interactions between a large number of plant SNAREs have been determined, and these data are discussed in light of the phylogenetic analysis. Oncol Rep, 2001 Jan-Feb, 8(1), 39 - 42 Mutation analysis of hBUB1, human mitotic checkpoint gene in multiple carcinomas; Mimori K et al.; hBUB1 is a human homolog of yeast mitotic check point gene that plays an important role in chromosome segregation . Recently mutations of hBUB1 were reported in colorectal cancer cell lines, indicating that inactivation of this gene could be directly involved in aneuploidy in human carcinoma cells . To obtain information of the magnitude of hBUB1 inactivation in multiple carcinomas, we examined mutations in 59 multiple carcinoma cell lines showing single base alteration, however, there was no mutation of hBUB1 with amino acid change in these carcinomas . There were four silent mutations at codon 93, codon 735, codon 430 and codon 98 in KYSE190, TE8 esophageal carcinoma cells, KATOIII gastric carcinoma cells and 697 B cell leukemia cells, respectively . Two candidates of mutation were identified in TE3 esophageal carcinoma cells and 697 B cell leukemia cell line at codon 9 and codon 285, respectively . This result suggests that the inactivation of hBUB1 may be very rare in human carcinomas, or restricted to certain cell lines of colorectal carcinomas. J Paediatr Child Health, 2000 Dec, 36(6), 609 - 10 Hansenula anomala infection in a neonate; Wong AR et al.; We present an unusual neonatal fungal infection, Hansenula anomala in a very low birthweight infant who underwent abdominal surgery for an omphalocele . Despite treatment with adequate doses of amphotericin B, the yeast continued to grow from the blood culture, and was only eradicated with the use of oral ketoconazole. Curr Biol, 2000 Nov 30, 10(23), R860 - 2 Microtubule dynamics: the view from the tip; Sawin KE; Recent studies have suggested that proteins found at the tips of microtubules in vertebrate cells may play an important role in intracellular membrane transport processes . Evidence from fission yeast indicates that such proteins can also regulate microtubule dynamics. Curr Biol, 2000 Nov 30, 10(23), 1535 - 8 Interaction of the Arabidopsis polycomb group proteins FIE and MEA mediates their common phenotypes; Spillane C et al.; Genes of the FERTILISATION INDEPENDENT SEED (FIS) class regulate cell proliferation during reproductive development in Arabidopsis {1-5} . The FIS genes FERTILISATION INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA) encode homologs of animal Polycomb group (Pc-G) proteins, transcriptional regulators that modify chromatin structure and are thought to form multimeric complexes {3-11} . To test whether similarities in fis mutant phenotypes reflect interactions between their protein products, we characterised FIE RNA and protein localisation in vivo, and FIE protein interactions in yeast and in vitro . Expression of FIE mRNA overlaps with that of MEA during embryo sac and seed development and is unaffected in mea mutants . Results from the yeast two-hybrid system and an in vitro pull-down assay indicate that MEA and FIE proteins interact . The relevance of this interaction in vivo is supported by the finding that FIE and MEA co-localise in the nucleus in transfected plant cells . Interaction of MEA and FIE is mediated by the amino-terminal region of MEA . Despite sequence divergence in this domain, MEA can interact with its corresponding animal partner Extrasexcombs (ESC) in the yeast two-hybrid system . We conclude that FIE and MEA act together as part of a multimeric complex and that this accounts for the similarities in mutant phenotypes . We propose that an ancient mechanism for chromatin modification has been independently recruited to different developmental processes in the two kingdoms. Cell, 2000 Nov 22, 103(5), 733 - 43 Transcription factor dosage affects changes in higher order chromatin structure associated with activation of a heterochromatic gene; Lundgren M et al.; The mechanisms of transcriptional activation in heterochromatin were investigated by using FISH to directly visualize changes in chromatin organization during activation of a heterochromatic lambda5 transgene . A DNase I hypersensitive site was shown to relocate the transgene to the outside of the pericentromeric heterochromatin complex in the absence of transcription . Activation of transcription, which is dependent on the transcription factor EBF, occurs in a stochastic manner that resembles telomeric silencing in yeast, with the transcribed gene remaining closely associated with the heterochromatin complex . Reducing the dosage of EBF results in a reduced frequency of localization of the transgene to the outside of the heterochromatin complex and lower levels of transcription . These data provide evidence that transcription factors can initiate changes in higher order chromatin structure during the earliest stages of gene activation. J Mol Biol, 2001 Jan 5, 305(1), 61 - 9 Electron microscopic analysis supports a dual role for the mitochondrial telomere-binding protein of Candida parapsilosis; Tomaska L et al.; Linear mitochondrial genomes exist in several yeast species which are closely related to yeast that harbor circular mitochondrial genomes . Several lines of evidence suggest that the conversion from one form to another occurred accidentally through a relatively simple mechanism . Previously, we (L.T . & J.N.) reported the identification of the first mitochondrial telomere-binding protein (mtTBP) that specifically binds a sequence derived from the extreme end of Candida parapsilosis linear mtDNA, and sequence analysis of the corresponding nuclear gene MTP1 revealed that mtTBP shares homology with several bacterial and mitochondrial single-stranded (ss) DNA-binding (SSB) proteins . In this study, the DNA-binding properties of mtTBP in vitro and in vivo were analyzed by electron microscopy (EM) . When M13 ssDNA was used as a substrate, mtTBP exhibited similar DNA binding characteristics as human mitochondrial SSB: mtTBP formed protein globules along the DNA substrate, and the bound proteins were randomly distributed, indicating that the binding of mtTBP to M13 ssDNA is not highly cooperative . EM analysis demonstrated that mtTBP is able to recognize the 5' single-stranded telomeric overhangs in their natural context . Using isopycnic centrifugation of mitochondrial lysates of C . papsilosis we show that mtTBP is a structural part of mitochondrial nucleoids of C . parapsilosis and is predominantly bound to the mitochondrial telomeres . These data support a dual role of mtTBP in mitochondria of C . parapsilosis, serving both as a typical mitochondrial SSB and as a specific component of the mitochondrial telomeric chromatin . Brain Res Mol Brain Res, 2000 Dec 8, 84(1-2), 150 - 7 Molecular cloning and expression analysis of human glycogen synthase kinase-3 alpha promoter; Lee KF et al.; Human glycogen synthase kinase-3 alpha (GSK-3 alpha) is a serine/threonine kinase that phosphorylates a variety of cytoplasmic and nuclear proteins . It also phosphorylates components of the neuronal cytoskeleton including tau and neurofilament heavy chain . Hyperphosphorylated tau is found in neurofibrillary tangles, a hallmark of Alzheimer's disease and aberrant phosphorylation of neurofilament heavy chain is observed in motor neuron disease . Alterations in GSK-3 alpha activity may therefore contribute to the disease process in these disorders . As a first step to understand the transcriptional regulation of GSK-3 alpha, a 2-kb (p-1751/+243) DNA fragment upstream of the GSK-3 alpha initiation codon was obtained from a YAC clone and characterised . Using primer extension assays, a putative transcriptional start site was located to a G nucleotide 244 bp upstream of the ATG codon . Several transcription factor-binding sites were identified on the promoter region, but no TATA-like element was located close to the start site . Deletion mutants of the 2-kb DNA fragment were generated and fused to a promoterless chloramphenicol acetyltransferase (CAT) gene . Transfection study in a neuroblastoma cell line revealed the 1-kb (p-719/+243) fragment carried strong promoter activity, while the 2-kb construct that contains an Alu-like sequence was only 50% active. FEBS Lett, 2000 Dec 8, 486(2), 99 - 102 Disruption of SMN function by ectopic expression of the human SMN gene in Drosophila; Miguel-Aliaga I et al.; Spinal muscular atrophy is a neurodegenerative disorder caused by mutations or deletions in the survival motor neuron (SMN) gene . We have cloned the Drosophila ortholog of SMN (DmSMN) and disrupted its function by ectopically expressing human SMN . This leads to pupal lethality caused by a dominant-negative effect, whereby human SMN may bind endogenous DmSMN resulting in non-functional DmSMN/human SMN hetero-complexes . Ectopic expression of truncated versions of DmSMN and yeast two-hybrid analysis show that the C-terminus of SMN is necessary and sufficient to replicate this effect . We have therefore generated a system which can be utilized to carry out suppressor and high-throughput screens, and provided in vivo evidence for the importance of SMN oligomerization for SMN function at the level of an organism as a whole. Mol Cell Biol, 2001 Jan, 21(1), 343 - 53 CIA, a novel estrogen receptor coactivator with a bifunctional nuclear receptor interacting determinant; Sauve F et al.; Coregulators for nuclear receptors (NR) are factors that either enhance or repress their transcriptional activity . Both coactivators and corepressors have been shown to use similar but functionally distinct NR interacting determinants containing the core motifs LxxLL and PhixxPhiPhi, respectively . These interactions occur through a hydrophobic cleft located on the surface of the ligand-binding domain (LBD) of the NR and are regulated by ligand-dependent activation function 2 (AF-2) . In an effort to identify novel coregulators that function independently of AF-2, we used the LBD of the orphan receptor RVR (which lacks AF-2) as bait in a yeast two-hybrid screen . This strategy led to the cloning of a nuclear protein referred to as CIA (coactivator independent of AF-2 function) that possesses both repressor and activator functions . Strikingly, we observed that CIA not only interacts with RVR and Rev-ErbAalpha in a ligand-independent manner but can also form complexes with estrogen receptor alpha (ERalpha) and ERbeta in vitro and enhances ERalpha transcriptional activity in the presence of estradiol (E(2)) . CIA-ERalpha interactions were found to be independent of AF-2 and enhanced by the antiestrogens EM-652 and ICI 182,780 but not by 4-hydroxytamoxifen and raloxifene . We further demonstrate that CIA-ERalpha interactions require the presence within CIA of a novel bifunctional NR recognition determinant containing overlapping LxxLL and PhixxPhiPhi motifs . The identification and functional characterization of CIA suggest that hormone binding can create a functional coactivator interaction interface in the absence of AF-2. Mol Cell Biol, 2001 Jan, 21(1), 185 - 8 Requirement of DNA polymerase eta for error-free bypass of UV-induced CC and TC photoproducts; Yu SL et al.; The yeast RAD30-encoded DNA polymerase eta (Poleta) bypasses a cis-syn thymine-thymine dimer efficiently and accurately . Human DNA polymerase eta functions similarly in the bypass of this lesion, and mutations in human Poleta result in the cancer prone syndrome, the variant form of xeroderma pigmentosum . UV light, however, also elicits the formation of cis-syn cyclobutane dimers and (6-4) photoproducts at 5'-CC-3' and 5'-TC-3' sites, and in both yeast and human DNA, UV-induced mutations occur primarily by 3' C to T transitions . Genetic studies presented here reveal a role for yeast Poleta in the error-free bypass of cyclobutane dimers and (6-4) photoproducts formed at CC and TC sites . Thus, by preventing UV mutagenesis at a wide spectrum of dipyrimidine sites, Poleta plays a pivotal role in minimizing the incidence of sunlight-induced skin cancers in humans. J Biol Chem, 2001 Mar 16, 276(11), 7782 - 90 Epub 2000 Dec 11. Properties of a native cation channel activated by Ca2+ store depletion in vascular smooth muscle cells; Trepakova ES et al.; Depletion of intracellular Ca(2+) stores activates capacitative Ca(2+) influx in smooth muscle cells, but the native store-operated channels that mediate such influx remain unidentified . Recently we demonstrated that calcium influx factor produced by yeast and human platelets with depleted Ca(2+) stores activates small conductance cation channels in excised membrane patches from vascular smooth muscle cells (SMC) . Here we characterize these channels in intact cells and present evidence that they belong to the class of store-operated channels, which are activated upon passive depletion of Ca(2+) stores . Application of thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca(2+) ATPase, to individual SMC activated single 3-pS cation channels in cell-attached membrane patches . Channels remained active when inside-out membrane patches were excised from the cells . Excision of membrane patches from resting SMC did not by itself activate the channels . Loading SMC with BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), which slowly depletes Ca(2+) stores without a rise in intracellular Ca(2+), activated the same 3-pS channels in cell-attached membrane patches as well as whole cell nonselective cation currents in SMC . TG- and BAPTA-activated 3-pS channels were cation-selective but poorly discriminated among Ca(2+), Sr(2+), Ba(2+), Na(+), K(+), and Cs(+) . Open channel probability did not change at negative membrane potentials but increased significantly at high positive potentials . Activation of 3-pS channels did not depend on intracellular Ca(2+) concentration . Neither TG nor a variety of second messengers (including Ca(2+), InsP3, InsP4, GTPgammaS, cyclic AMP, cyclic GMP, ATP, and ADP) activated 3-pS channels in inside-out membrane patches . Thus, 3-pS nonselective cation channels are present and activated by TG or BAPTA-induced depletion of intracellular Ca(2+) stores in intact SMC . These native store-operated cation channels can account for capacitative Ca(2+) influx in SMC and can play an important role in regulation of vascular tone. J Biol Chem, 2001 Mar 16, 276(11), 8104 - 10 Epub 2000 Dec 11. Amphiphysin 1 binds the cyclin-dependent kinase (cdk) 5 regulatory subunit p35 and is phosphorylated by cdk5 and cdc2; Floyd SR et al.; Amphiphysin 1 is a phosphoprotein expressed at high levels in neurons, where it participates in synaptic vesicle endocytosis and neurite outgrowth . It is a substrate for cyclin-dependent kinase (cdk) 5, a member of the cyclin-dependent protein kinase family, which has been functionally linked to neuronal migration and neurite outgrowth via its action on the actin cytoskeleton . The yeast homologue of amphiphysin, Rvs167, functions in endocytosis and actin dynamics, is phosphorylated by the cdk5 homologue Pho85, and binds the Pho85 regulatory subunit Pcl2 . We show here that amphiphysin 1 interacts with the cdk5-activating subunit p35 and that this interaction is mediated by the conserved NH2-terminal region of amphiphysin . Amphiphysin 1 colocalizes with p35 in the growth cones of neurons and at actin-rich peripheral lamellipodia in transfected fibroblasts . Amphiphysin is phosphorylated by cdk5 in a region including serines 272, 276, and 285 . Amphiphysin 1 is also phosphorylated by the cdc2/cyclin B kinase complex in the same region and undergoes mitotic phosphorylation in dividing cells . These data indicate that phosphorylation by members of the cyclin-dependent kinase family is a conserved property of amphiphysin and suggest that this phosphorylation may play an important physiological role both in mitosis and in differentiated cells. J Biol Chem, 2001 Mar 23, 276(12), 9206 - 13 Epub 2000 Nov 30. Interaction of the type IIa Na/Pi cotransporter with PDZ proteins; Gisler SM et al.; The type IIa Na(+)-dependent inorganic phosphate (Na/P(i)) cotransporter is localized in the apical membrane of proximal tubular cells and is regulated by an endocytotic pathway . Because molecular processes such as apical sorting, internalization, or subsequent degradation might be assisted by associated proteins, a yeast two-hybrid screen against the C-terminal, cytosolic tail of type IIa cotransporter was designed . Most of the potential proteins found belonged to proteins with multiple PDZ modules and were either identical/related to PDZK1 or identical to NHERF-1 . Yeast trap truncation assays confined the peptide-protein association to the C-terminal amino acid residues TRL of type IIa cotransporter and to single PDZ domains of each identified protein, respectively . The specificity of these interactions were confirmed in yeast by testing other apical localized transmembraneous proteins . Moreover, the type IIa protein was recovered in vitro by glutathione S-transferase-fused PDZ proteins from isolated renal brush border membranes or from type IIa-expressing oocytes . Further, these PDZ proteins are immunohistochemically detected either in the microvilli or in the subapical compartment of proximal tubular cells . Our results suggest that the type IIa Na/P(i) cotransporter interacts with various PDZ proteins that might be responsible for the apical sorting, parathyroid hormone controlled endocytosis or the lysosomal sorting of internalized type IIa cotransporter. J Biol Chem, 2001 Mar 23, 276(12), 8705 - 12 Epub 2000 Nov 29. Uncoupling protein 2, in vivo distribution, induction upon oxidative stress, and evidence for translational regulation; Pecqueur C et al.; Uncoupling protein 2 (UCP2) belongs to the mitochondrial anion carrier family and partially uncouples respiration from ATP synthesis when expressed in recombinant yeast mitochondria . We generated a highly sensitive polyclonal antibody against human UCP2 . Its reactivity toward mitochondrial proteins was compared between wild type and ucp2(-/-) mice, leading to non-ambiguous identification of UCP2 . We detected UCP2 in spleen, lung, stomach, and white adipose tissue . No UCP2 was detected in heart, skeletal muscle, liver, and brown adipose tissue . The level of UCP2 in spleen mitochondria is less than 1% of the level of UCP1 in brown adipose tissue mitochondria . Starvation and LPS treatments increase UCP2 level up to 12 times in lung and stomach, which supports the hypothesis that UCP2 responds to oxidative stress situations . Stimulation of the UCP2 expression occurs without any change in UCP2 mRNA levels . This is explained by translational regulation of the UCP2 mRNA . We have shown that an upstream open reading frame located in exon two of the ucp2 gene strongly inhibits the expression of the protein . This further level of regulation of the ucp2 gene provides a mechanism by which expression can be strongly and rapidly induced under stress conditions. Biochem Biophys Res Commun, 2000 Dec 9, 279(1), 6 - 10 Interaction of Daxx, a Fas binding protein, with sentrin and Ubc9; Ryu SW et al.; Sentrin is a ubiquitin-like protein that can covalently modify cellular proteins, and is a Fas binding protein that protects cells against anti-Fas induced cell death . However, the mechanism by which sentrin exerts its effect upon Fas-mediated apoptosis is not well known . Thus, this study examined the interaction of sentrin with Daxx . Sentrin interacted with Daxx but not with FADD when analyzed by yeast two-hybrid assay . In vitro translated Daxx bound to GST-sentrin fusion protein . FLAG-sentrin fusion protein was also coimmunoprecipitated with Daxx in BOSC23 cells . Also, Daxx interacted with Ubc9, an essential protein as a key conjugating enzyme . Amino acids 625-740 of Daxx, known as Fas binding region, was also m