Yeasts

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 mapped as sentrin and Ubc9 binding region . Colocalization of Fas, sentrin, and Ubc9 binding regions suggests the importance of that region upon the regulation of Daxx . Our data also demonstrated that sentrin could homooligomerize by protein-protein interaction .

Dev Neurosci, 2000 Sep-Dec, 22(5-6), 481 - 7
Diet, lipids and brain development; Salvati S et al.; Brain development is a sequential anatomical process characterised by specific well-defined stages of growth and maturation . One of the fundamental and necessary events in the normal development of the central nervous system in vertebrates is the formation of a myelin sheath . It is becoming more evident that this process is influenced by dietary lipids . A number of findings have indicated that the administration of a diet deficient in essential fatty acids during development causes hypomyelination in the rat brain . Our studies have shown that lipids can also play a role in accelerating myelinogenesis in the brain of rats whose mothers had been fed, during pregnancy and lactation, a lipid fraction extracted from yeast grown on n-alkanes . Further studies have shown that accelerated myelinogenesis is connected to a precocious appearance of behavioural reflexes . Thus, the use of particular lipids in human nutrition must be carefully screened for possible effects on brain development .

Brain Dev, 2000 Dec, 22(8), 465 - 8
Friedreich's ataxia and iron metabolism; Gordon N; The possible causes of abnormal iron metabolism in patients with Friedreich's ataxia are considered . Reduced expression of a frataxin homologue in yeast is associated with mitochondrial iron accumulation at the expense of cytosolic iron, and the same phenomenon can be demonstrated in these patients . A decrease in cytosolic iron causes the expression of a high-affinity iron-uptake protein, and therefore Friedreich's ataxia can be considered to be a disease of abnormal intracellular iron distribution . Friedreich's ataxia is of autosomal recessive inheritance, and the gene associated with it has been mapped to chromosome 9 . This encodes the protein frataxin which regulates mitochondrial iron transport . The commonest mutation causing this disorder is an expanded GAA repeat in the gene for this protein . Different point mutations may account for some of the variations in the phenotypic features that are often found, and these variations are discussed . These findings have raised therapeutic possibilities in a condition for which previously there was no specific treatment . There are intracellular enzymes which are very sensitive to injury by oxygen-free radicals . Treatment has therefore been tried with ibebenone which acts as a free-radical scavenger, with some evidence of improvement . Iron chelating agents, such as deferoxamine, have also been given, but the finding of normal serum iron and ferritin casts doubt on the rationale of this . However the finding that the accumulation of iron in the mitochondria of the cells in patients with this form of ataxia will cause oxidative stress and cell death, gives hope for more effective treatment in the future, possibly with gene therapy.

J Nutr, 2000 Dec, 130(12), 2903 - 9
Dietary selenium and arsenic affect DNA methylation in vitro in Caco-2 cells and in vivo in rat liver and colon; Davis CD et al.; Selenium is an essential trace element for human health, and it has received considerable attention for its possible role as an anticarcinogenic agent . The purpose of the present study was to determine whether changes in the amount and the chemical form of selenium would affect DNA methylation and whether this effect would be modified by arsenic . Caco-2 cells, a human colon cancer cell line, were exposed to 0, 1 or 2 micromol supplemental selenite/L and 0, 1 or 2 micromol supplemental arsenite/L for 7 d . DNA isolated from Caco-2 cells not treated with selenite was significantly (P: < 0 . 0001) hypomethylated compared with that from cells treated with 1 or 2 micromol selenite/L . DNA isolated from Caco-2 cells not treated with arsenite was significantly (P: < 0.0001) hypomethylated compared with DNA isolated from cells treated with 1 or 2 micromol arsenite/L . In addition, methylation of the p53 promoter region of Caco-2 cells decreased when cells were cultured in the absence of selenite and in the absence of arsenite . Sixty weanling male Fischer 344 rats were fed a torula yeast-based diet supplemented with 0, 0.1 or 2 mg selenium/kg diet as either selenite or selenomethionine in the presence or absence of 5 mg arsenic/kg diet as arsenite for 6 wk . Similar to the results with Caco-2 cells, rats fed selenium-deficient diets had significantly (P: < 0.0001) hypomethylated liver and colon DNA compared with rats fed 0.1 or 2.0 microg selenium/g diets as either selenite or selenomethionine . Thus, alterations in DNA methylation may be a potential mechanism, whereby deficient dietary selenium increases liver and colon tumorigenesis.

Blood, 2000 Dec 15, 96(13), 4236 - 45
Human CLP36, a PDZ-domain and LIM-domain protein, binds to alpha-actinin-1 and associates with actin filaments and stress fibers in activated platelets and endothelial cells; Bauer K et al.; A 38-kd protein that associates with F-actin structures in activated platelets and endothelial cells was purified, cloned, and characterized . The protein contains an N-terminal PDZ motif, a large intervening sequence, and a C-terminal LIM domain and was identified as the human homolog of rat CLP36 . The study showed that CLP36 associates with actin filaments and stress fibers that are formed during shape change and spreading of platelets and during migration and contraction of endothelial cells . CLP36 binds to alpha-actinin-1 as shown by coimmunoprecipitation, pull-down experiments, yeast 2-hybrid analysis, and blot overlay assays and colocalizes with alpha-actinin-1 along endothelial actin stress fibers . In contrast to alpha-actinin-1, CLP36 was absent from focal adhesions in both activated platelets and endothelial cells . The N-terminal part of CLP36 containing the PDZ domain and the intervening region, but not the LIM domain, targeted enhanced green fluorescent protein fusion proteins to stress fibers in endothelial cells . Yeast 2-hybrid analysis demonstrated that the intervening sequence, but not the PDZ or the LIM domain of CLP36, binds to the spectrinlike repeats 2 and 3 of alpha-actinin-1 . The study further shows that CLP36 binds to alpha-actinin in resting platelets and translocates as a CLP36/alpha-actinin complex to the newly formed actin cytoskeleton in activated platelets . The results indicate that CLP36 binds via alpha-actinin-1 to actin filaments and stress fibers in activated human platelets and endothelial cells . The study suggests that CLP36 may direct alpha-actinin-1 to specific actin structures and at this position might modulate the function of alpha-actinin-1 . (Blood . 2000;96:4236-4245)

J Biol Chem, 2001 Mar 9, 276(10), 7069 - 78 Epub 2000 Dec 07.
Hrs interacts with sorting nexin 1 and regulates degradation of epidermal growth factor receptor; Chin LS et al.; Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a mammalian homologue of yeast vacuolar protein sorting (Vps) protein Vps27p; however, the role of Hrs in lysosomal trafficking is unclear . Here, we report that Hrs interacts with sorting nexin 1 (SNX1), a recently identified mammalian homologue of yeast Vps5p that recognizes the lysosomal targeting code of epidermal growth factor receptor (EGFR) and participates in lysosomal trafficking of the receptor . Biochemical analyses demonstrate that Hrs and SNX1 are ubiquitous proteins that exist in both cytosolic and membrane-associated pools, and that the association of Hrs and SNX occurs on cellular membranes but not in the cytosol . Furthermore, endogenous SNX1 and Hrs form a approximately 550-kDa complex that excludes EGFR . Immunofluorescence and subcellular fractionation studies show that Hrs and SNX1 colocalize on early endosomes . By using deletion analysis, we have mapped the binding domains of Hrs and SNX1 that mediate their association . Overexpression of Hrs or its SNX1-binding domain inhibits ligand-induced degradation of EGFR, but does not affect either constitutive or ligand-induced receptor-mediated endocytosis . These results suggest that Hrs may regulate lysosomal trafficking through its interaction with SNX1.

J Biol Chem, 2001 Mar 2, 276(9), 6177 - 84 Epub 2000 Nov 10.
Three novel components of the human exosome; Brouwer R et al.; The yeast exosome is a complex of 3' --> 5' exoribonucleases . Sequence analysis identified putative human homologues for exosome components, although several were found only as expressed sequence tags . Here we report the cloning of full-length cDNAs, which encode putative human homologues of the Rrp40p, Rrp41p, and Rrp46p components of the exosome . Recombinant proteins were expressed and used to raise rabbit antisera . In Western blotting experiments, these decorated HeLa cell proteins of the predicted sizes . All three human proteins were enriched in the HeLa cells nucleus and nucleolus, but were also clearly detected in the cytoplasm . Size exclusion chromatography revealed that hRrp40p, hRrp41p, and hRrp46p were present in a large complex . This cofractionated with the human homologues of other exosome components, hRrp4p and PM/Scl-100 . Anti-PM/Scl-positive patient sera coimmunoprecipitated hRrp40p, hRrp41p, and hRrp46p demonstrating their physical association . The immunoprecipitated complex exhibited 3' --> 5' exoribonuclease activity in vitro . hRrp41p was expressed in yeast and shown to suppress the lethality of genetic depletion of yeast Rrp41p . We conclude that hRrp40p, hRrp41p, and hRrp46p represent novel components of the human exosome complex.

J Biol Chem, 2001 Apr 13, 276(15), 11949 - 55 Epub 2000 Nov 22.
Immunocytochemical localization and crystal structure of human frequenin (neuronal calcium sensor 1); Bourne Y et al.; Frequenin, a member of a large family of myristoyl-switch calcium-binding proteins, functions as a calcium-ion sensor to modulate synaptic activity and secretion . We show that human frequenin colocalizes with ARF1 GTPase in COS-7 cells and occurs in similar cellular compartments as the phosphatidylinositol-4-OH kinase PI4Kbeta, the mammalian homolog of the yeast kinase PIK1 . In addition, the crystal structure of unmyristoylated, calcium-bound human frequenin has been determined and refined to 1.9 A resolution . The overall fold of frequenin resembles those of neurocalcin and the photoreceptor, recoverin, of the same family, with two pairs of calcium-binding EF hands and three bound calcium ions . Despite the similarities, however, frequenin displays significant structural differences . A large conformational shift of the C-terminal region creates a wide hydrophobic crevice at the surface of frequenin . This crevice, which is unique to frequenin and distinct from the myristoyl-binding box of recoverin, may accommodate a yet unknown protein ligand.

FEBS Lett, 2000 Dec 1, 486(1), 73 - 8
Characterization of wheat DP, a heterodimerization partner of the plant E2F transcription factor which stimulates E2F-DNA binding; Ramirez-Parra E et al.; Recent studies suggest that the G1/S transition in plants depends on the activity of E2F transcription factors . In animal cells, E2Fs interact with DP proteins, whose identification in plants has been elusive, so far . Here we show that although an E2F-containing DNA-binding activity can be detected in plant cell extracts, purified E2F protein binds poorly to DNA . In a yeast two-hybrid screening, using wheat E2F as a bait, we have isolated a cDNA clone encoding a wheat DP (TmDP) protein . TmDP is expressed ubiquitously and exhibits a domain organization similar to animal DPs . Contrary to the specificity observed for the plant RBR/E2F interaction, human and plant E2F and DP proteins can interact in a heterologous manner . Purified TmDP protein stimulates E2F-DNA complex formation.

Int Microbiol, 1998 Jun, 1(2), 149 - 58
The fungus Ustilago maydis, from the aztec cuisine to the research laboratory; Ruiz-Herrera J et al.; Ustilago maydis is a plant pathogen fungus responsible for corn smut . It has a complex life cycle . In its saprophitic stage, it grows as haploid yeast cells, while in the invasive stage it grows as a mycelium formed by diploid cells . Thus, a correlation exists between genetic ploidy, pathogenicity and morphogenesis . Dimorphism can be modulated in vitro by changing environmental parameters such as pH . Studies with auxotrophic mutants have shown that polyamines play a central role in regulating dimorphism . Molecular biology approaches are being employed for the analysis of fundamental aspects of the biology of this fungus, such as mating type regulation, dimorphism or cell wall biogenesis.

Int Microbiol, 1998 Jun, 1(2), 123 - 30
Yarrowia lipolytica: a model organism for protein secretion studies; Beckerich JM et al.; This paper reviews the advantages of the yeast Yarrowia lipolytica as a tool in the study of protein secretion . Work has been focused on the early steps leading the polypeptide, from the cytoplasmic ribosomes where it is synthesized, to the lumen of the endoplasmic reticulum . Using a thermosensitive allele of the 7SL RNA, the first in vivo evidence for a co-translational translocation was shown . Genetic screens allowed the identification of several new components of the translocation apparatus: Sls1p, an ER lumenal component involved in both translocation and lumenal transit; Tsr1p, involved in SRP-ribosome targeting; Tsr3p . Major translocation partners were also identified by reverse genetics (Sec61p, Sec62p, Kar2p, Srp54p, Sec65p).

J Comput Biol, 2000, 7(3-4), 429 - 47
NOTUNG: a program for dating gene duplications and optimizing gene family trees; Chen K et al.; Large scale gene duplication is a major force driving the evolution of genetic functional innovation . Whole genome duplications are widely believed to have played an important role in the evolution of the maize, yeast, and vertebrate genomes . The use of evolutionary trees to analyze the history of gene duplication and estimate duplication times provides a powerful tool for studying this process . Many studies in the molecular evolution literature have used this approach on small data sets, using analyses performed by hand . The rapid growth of genetic sequence data will soon allow similar studies on a genomic scale, but such studies will be limited unless the analysis can be automated . Even existing data sets admit alternative hypotheses that would be too tedious to consider without automation . In this paper, we describe a program called NOTUNG that facilitates large scale analysis, using both rooted and unrooted trees . When tested on trees analyzed in the literature, NOTUNG consistently yielded results that agree with the assessments in the original publications . Thus, NOTUNG provides a basic building block for inferring duplication dates from gene trees automatically and can also be used as an exploratory analysis tool for evaluating alternative hypotheses.

Cell, 2000 Nov 10, 103(4), 583 - 94
Kinesin-dependent axonal transport is mediated by the sunday driver (SYD) protein; Bowman AB et al.; A broadly conserved membrane-associated protein required for the functional interaction of kinesin-I with axonal cargo was identified . Mutations in sunday driver (syd) and the axonal transport motor kinesin-I cause similar phenotypes in Drosophila, including aberrant accumulations of axonal cargoes . GFP-tagged mammalian SYD localizes to tubulovesicular structures that costain for kinesin-I and a marker of the secretory pathway . Coimmunoprecipitation analysis indicates that mouse SYD forms a complex with kinesin-I in vivo . Yeast two-hybrid analysis and in vitro interaction studies reveal that SYD directly binds kinesin-I via the tetratricopeptide repeat (TPR) domain of kinesin light chain (KLC) with K(d) congruent with 200 nM . We propose that SYD mediates the axonal transport of at least one class of vesicles by interacting directly with KLC.

Mol Cell, 2000 Nov, 6(5), 1183 - 93
Crystal structure of eukaryotic DNA ligase-adenylate illuminates the mechanism of nick sensing and strand joining; Odell M et al.; Chlorella virus DNA ligase is the smallest eukaryotic ATP-dependent ligase known; it has an intrinsic nick-sensing function and suffices for yeast cell growth . Here, we report the 2.0 A crystal structure of the covalent ligase-AMP reaction intermediate . The conformation of the adenosine nucleoside and contacts between the enzyme and the ribose sugar have undergone a significant change compared to complexes of T7 ligase with ATP or mRNA capping enzyme with GTP . The conformational switch allows the 3' OH of AMP to coordinate directly the 5' PO(4) of the nick . The structure explains why nick sensing is restricted to adenylated ligase and why the 5' phosphate is required for DNA binding . We identify a metal binding site on ligase-adenylate and propose a mechanism of nick recognition and catalysis supported by mutational data.

Mol Cell, 2000 Nov, 6(5), 1099 - 108
Regulated translation initiation controls stress-induced gene expression in mammalian cells; Harding HP et al.; Protein kinases that phosphorylate the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) are activated in stressed cells and negatively regulate protein synthesis . Phenotypic analysis of targeted mutations in murine cells reveals a novel role for eIF2alpha kinases in regulating gene expression in the unfolded protein response (UPR) and in amino acid starved cells . When activated by their cognate upstream stress signals, the mammalian eIF2 kinases PERK and GCN2 repress translation of most mRNAs but selectively increase translation of Activating Transcription Factor 4 (ATF4), resulting in the induction of the downstream gene CHOP (GADD153) . This is the first example of a mammalian signaling pathway homologous to the well studied yeast general control response in which eIF2alpha phosphorylation activates genes involved in amino acid biosynthesis . Mammalian cells thus utilize an ancient pathway to regulate gene expression in response to diverse stress signals.

Mol Cell, 2000 Nov, 6(5), 989 - 98
Chromosome synapsis defects and sexually dimorphic meiotic progression in mice lacking Spo11; Baudat F et al.; Spo11, a protein first identified in yeast, is thought to generate the chromosome breaks that initiate meiotic recombination . We now report that disruption of mouse Spo11 leads to severe gonadal abnormalities from defective meiosis . Spermatocytes suffer apoptotic death during early prophase; oocytes reach the diplotene/dictyate stage in nearly normal numbers, but most die soon after birth . Consistent with a conserved function in initiating meiotic recombination, Dmc1/Rad51 focus formation is abolished . Spo11(-/-) meiocytes also display homologous chromosome synapsis defects, similar to fungi but distinct from flies and nematodes . We propose that recombination initiation precedes and is required for normal synapsis in mammals . Our results also support the view that mammalian checkpoint responses to meiotic recombination and/or synapsis defects are sexually dimorphic.

Mutat Res, 2001 Jan 5, 461(4), 325 - 38
Sequence variation in the human uracil-DNA glycosylase (UNG) gene; Kvaloy K et al.; Spontaneous deamination of cytosine results in a premutagenic G:U mismatch that may result in a GC-->AT transition during replication . The human UNG-gene encodes the major uracil-DNA glycosylase (UDG or UNG) which releases uracil from DNA, thus, initiating base excision repair to restore the correct DNA sequence . Bacterial and yeast mutants lacking the homologous UDG exhibit elevated spontaneous mutation frequencies . Hence, mutations in the human UNG gene could presumably result in a mutator phenotype . We screened all seven exons including exon-intron boundaries, both promoters, and one intron of the UNG gene and identified considerable sequence variation in cell lines derived from normal fibroblasts and tumour tissue . None of the sequence variants was accompanied by significantly reduced UDG activity . In the UNG gene from 62 sources, we identified 12 different variant alleles, with allele frequencies ranging from 0.01 to 0.23 . We identified one variant allele per 3.8kb in non-coding regions, but none in the coding region of the gene . In promoter B we identified four different variants . A substitution within an AP2 element was observed in tumour cell lines only and had an allele frequency of 0.10 . Introduction of this substitution into chimaeric promoter-luciferase constructs affected transcription from the promoter . UDG-activity varied little in fibroblasts, but widely between tumour cell lines . This variation did not however correlate with the presence of any of the variant alleles . In conclusion, mutations affecting the function of human UNG gene are seemingly infrequent in human tumour cell lines.

J Clin Invest, 2000 Dec, 106(11), 1381 - 9
Mutation of the WI-1 gene yields an attenuated blastomyces dermatitidis strain that induces host resistance; Wuthrich M et al.; Systemic fungal infections are becoming more common and difficult to treat, and vaccine prevention is not available . Pulmonary infection with the dimorphic fungus Blastomyces dermatitidis often progresses and requires treatment to prevent fatality . We recently created a recombinant strain of the fungus lacking the WI-1 adhesin and pathogenicity . We show here that administration of viable yeast of this attenuated strain vaccinates against lethal pulmonary experimental infection due to isogenic and nonisogenic strains from diverse geographic regions . To our knowledge, this is the first example of a recombinant attenuated vaccine against fungi . The vaccine induces delayed-type hypersensitivity and polarized type 1 cytokine responses, which are linked with resistance . A cell-wall/membrane (CW/M) antigen from the vaccine strain also induces polarized and protective immune responses . Lymph node cells and CD4(+) T-cell lines raised with CW/M antigen transfer protective immunity when they release type 1 cytokine IFN-gamma, but not when they release IL-4, and neutralization of IFN-gamma confirmed its role in vivo . Thus, by mutating a pathogenetic locus in a dimorphic fungus, we have created an attenuated vaccine strain and have begun to elucidate fungal and host elements requisite for vaccine immunity.

Biochem J, 2000 Dec 15, 352 Pt 3, 795 - 800
Interaction between DNA-damage protein GADD34 and a new member of the Hsp40 family of heat shock proteins that is induced by a DNA-damaging reagent; Hasegawa T et al.; GADD34 is one of a subset of proteins induced after DNA damage or cell growth arrest . To examine the function of GADD34, we used the yeast two-hybrid system to clone the protein that interacts with murine GADD34 . As bait we used the product of the partial GADD34 cDNA, including the regions rich in proline, glutamic acid, serine and threonine (PEST) and gamma(1)34.5 regions . A cDNA clone, named GAHSP40, which is a mouse DnaJ family protein with a high similarity to human HLJ1 was cloned . The interaction between GADD34 and GAHSP40 in cultured cells was confirmed by a co-immunoprecipitation experiment and in NIH 3T3 cells by two-hybrid analysis in vivo . For binding of the two proteins, the gamma(1)34.5-similar region of GADD34 was necessary; however, the PEST region was also involved and the C-terminus of GAHSP40, but not the J-domain, was important . GAHSP40 was detected in all mouse tissues examined, but a different transcript was found in the testis . Both GADD34 mRNA and GAHSP40 mRNA were significantly elevated by treatment with methyl methanesulphonate, although the time courses were different . In addition, both GAHSP40 and GADD34 mRNA were induced by heat shock.

Genome Biol . 2000;1(1):REVIEWS104 . Epub 2000 Jun 09.
Membrane traffic between genomes; Armstrong J; Proteins of the Rab and SNARE families target vesicles to their intracellular destinations . A comparison of these families from the budding yeast, fission yeast, nematode and fruitfly genomes has implications for the organization of membrane traffic in different organisms.

Genome Biol . 2000;1(1):RESEARCH001 . Epub 2000 Apr 27.
Are plant formins integral membrane proteins?
Cvrckova F.
BACKGROUND: The formin family of proteins has been implicated in signaling pathways of cellular morphogenesis in both animals and fungi; in the latter case, at least, they participate in communication between the actin cytoskeleton and the cell surface . Nevertheless, they appear to be cytoplasmic or nuclear proteins, and it is not clear whether they communicate with the plasma membrane, and if so, how . Because nothing is known about formin function in plants, I performed a systematic search for putative Arabidopsis thaliana formin homologs . RESULTS: I found eight putative formin-coding genes in the publicly available part of the Arabidopsis genome sequence and analyzed their predicted protein sequences . Surprisingly, some of them lack parts of the conserved formin-homology 2 (FH2) domain and the majority of them seem to have signal sequences and putative transmembrane segments that are not found in yeast or animals formins . CONCLUSIONS: Plant formins define a distinct subfamily . The presence in most Arabidopsis formins of sequence motifs typical or transmembrane proteins suggests a mechanism of membrane attachment that may be specific to plant formins, and indicates an unexpected evolutionary flexibility of the conserved formin domain.

Life Sci, 2000 Oct 13, 67(21), 2631 - 8
Lagenin, a novel ribosome-inactivating protein with ribonucleolytic activity from bottle gourd (Lagenaria siceraria) seeds; Wang HX et al.; The seeds of Lagenaria siceraria (Family Cucurbitaceae) were extracted with water and the extract was lyophilized . The lyophilized extract was chromatographed on a DEAE-cellulose column in 10 mM Tris-HCl buffer (pH 7.2) . The unadsorbed fraction was applied to an Affi-gel Blue gel column previously equilibrated with the same buffer . After removal of unadsorbed materials, the adsorbed proteins were eluted with 1.5 M NaCl in the Tris-HCl buffer . After dialysis the adsorbed fraction was loaded on a CM-Sepharose CL-6B column which had been equilibrated with and was eluted with the same buffer . After elution of unadsorbed proteins, the column was eluted with a gradient of 0-1 M NaCl in 10 mM Tris-HCl buffer (pH 7.2) . The fraction eluting at about 0.55 M NaCl, which represented pure ribosome inactivating protein (RIP), inhibited cell-free translation in a rabbit reticulocyte system with an IC50 of 0.21 nM and exerted ribonuclease activity on yeast tRNA with an activity of 45 U/mg . The RIP was designated lagenin . It possessed a molecular weight of 20 kDa, smaller than the range of 26-32 kDa reported for other RIPs . The N-terminal sequence of lagenin exhibited a lesser extent of similarity to those of other Cucurbitaceae RIPs, characterized by a deletion of the first three amino acid residues and a replacement of the 4th (Phe), 17th (Phe), 18th (Ile) and 22nd (Arg) residues which are invariant in other RIPs.

Biochem Cell Biol, 2000, 78(5), 613 - 28
Neurons from stem cells: implications for understanding nervous system development and repair; Mansergh FC et al.; Neurodegenerative diseases cost the economies of the developed world billions of dollars per annum . Given ageing population profiles and the increasing extent of this problem, there has been a surge of interest in neural stem cells and in neural differentiation protocols that yield neural cells for therapeutic transplantation . Due to the oncogenic potential of stem cells a better characterisation of neural differentiation, including the identification of new neurotrophic factors, is required . Stem cell cultures undergoing synchronous in vitro neural differentiation provide a valuable resource for gene discovery . Novel tools such as microarrays promise to yield information regarding gene expression in stem cells . With the completion of the yeast, C . elegans, Drosophila, human, and mouse genome projects, the functional characterisation of genes using genetic and bioinformatic tools will aid in the identification of important regulators of neural differentiation.

Mol Biol Cell, 2000 Dec, 11(12), 4359 - 68
Dual requirement for rho and protein kinase C in direct activation of phospholipase D1 through G protein-coupled receptor signaling; Du G et al.; G protein-coupled and tyrosine kinase receptor activation of phospholipase D1 (PLD1) play key roles in agonist-stimulated cellular responses such as regulated exocytosis, actin stress fiber formation, and alterations in cell morphology and motility . Protein Kinase C, ADP-ribosylation factor (ARF), and Rho family members activate PLD1 in vitro; however, the actions of the stimulators on PLD1 in vivo have been proposed to take place through indirect pathways . We have used the yeast split-hybrid system to generate PLD1 alleles that fail to bind to or to be activated by RhoA but that retain wild-type responses to ARF and PKC . These alleles then were employed in combination with alleles unresponsive to PKC or to both stimulators to examine the activation of PLD1 by G protein-coupled receptors . Our results demonstrate that direct stimulation of PLD1 in vivo by RhoA (and by PKC) is critical for significant PLD1 activation but that PLD1 subcellular localization and regulated phosphorylation occur independently of these stimulatory pathways.

Mol Biol Cell, 2000 Dec, 11(12), 4173 - 87
Identification of ribonucleotide reductase protein R1 as an activator of microtubule nucleation in Xenopus egg mitotic extracts; Takada S et al.; Microtubule nucleation on the centrosome and the fungal equivalent, the spindle pole body (SPB), is activated at the onset of mitosis . We previously reported that mitotic extracts prepared from Xenopus unfertilized eggs convert the interphase SPB of fission yeast into a competent state for microtubule nucleation . In this study, we have purified an 85-kDa SPB activator from the extracts and identified it as the ribonucleotide reductase large subunit R1 . We further confirmed that recombinant mouse R1 protein was also effective for SPB activation . On the other hand, another essential subunit of ribonucleotide reductase, R2 protein, was not required for SPB activation . SPB activation by R1 protein was suppressed in the presence of anti-R1 antibodies or a partial oligopeptide of R1; the oligopeptide also inhibited aster formation on Xenopus sperm centrosomes . In accordance, R1 was detected in animal centrosomes by immunofluorescence and immunoblotting with anti-R1 antibodies . In addition, recombinant mouse R1 protein bound to gamma- and alpha/beta-tubulin in vitro . These results suggest that R1 is a bifunctional protein that acts on both ribonucleotide reduction and centrosome/SPB activation.

J Biol Chem, 2001 Feb 23, 276(8), 5643 - 9 Epub 2000 Nov 30.
Characterization of histatin 5 with respect to amphipathicity, hydrophobicity, and effects on cell and mitochondrial membrane integrity excludes a candidacidal mechanism of pore formation; Helmerhorst EJ et al.; Histatin 5 is a 24-residue peptide from human saliva with antifungal properties . We recently demonstrated that histatin 5 translocates across the yeast membrane and targets to the mitochondria, suggesting an unusual antifungal mechanism (Helmerhorst, E . J., Breeuwer, P., van't Hof, W., Walgreen-Weterings, E., Oomen, L . C . J . M., Veerman, E . C . I., Nieuw Amerongen, A . V., and Abee, T . (1999) J . Biol . Chem . 274, 7286-7291) . The present study used specifically designed synthetic analogs of histatin 5 to elucidate the role of peptide amphipathicity, hydrophobicity, and the propensity to adopt alpha-helical structures in relation to membrane permeabilization and fungicidal activity . Studies included circular dichroism measurements, evaluation of the effects on the cytoplasmic transmembrane potential and on the respiration of isolated mitochondria, and analysis of the peptide hydrophobicity/amphipathicity relationship (Eisenberg, D . (1984) Annu . Rev . Biochem . 53, 595-623) . The 14-residue synthetic peptides used were dh-5, comprising the functional domain of histatin 5, and dhvar1 and dhvar4, both designed to maximize amphipathic characteristics . The results obtained show that the amphipathic analogs exhibited a high fungicidal activity, a high propensity to form an alpha-helix, dissipated the cytoplasmic transmembrane potential, and uncoupled the respiration of isolated mitochondria, similar to the pore-forming peptide PGLa (Peptide with N-terminal Glycine and C-terminal Leucine-amide) . In contrast, histatin 5 and dh-5 showed fewer or none of these features . The difference in these functional characteristics between histatin 5 and dh-5 on the one hand and dhvar1, dhvar4, and PGLa on the other hand correlated well with their predicted affinity for membranes based on hydrophobicity/amphipathicity analysis . These data indicate that the salivary protein histatin 5 exerts its antifungal function through a mechanism other than pore formation.

DNA Cell Biol, 2000 Nov, 19(11), 679 - 88
Genomic organization, expression, and chromosome localization of a third aurora-related kinase gene, Aie1; Hu HM et al.; We previously reported two novel testis-specific serine/threonine kinases, Aie1 (mouse) and AIE2 (human), that share high amino acid identities with the kinase domains of fly aurora and yeast Ipl1 . Here, we report the entire intron-exon organization of the Aie1 gene and analyze the expression patterns of Aie1 mRNA during testis development . The mouse Aie1 gene spans approximately 14 kb and contains seven exons . The sequences of the exon-intron boundaries of the Aie1 gene conform to the consensus sequences (GT/AG) of the splicing donor and acceptor sites of most eukaryotic genes . Comparative genomic sequencing revealed that the gene structure is highly conserved between mouse Aie1 and human AIE2 . However, much less homology was found in the sequence outside the kinase-coding domains . The Aie1 locus was mapped to mouse chromosome 7A2-A3 by fluorescent in situ hybridization . Northern blot analysis indicates that Aie1 mRNA likely is expressed at a low level on day 14 and reaches its plateau on day 21 in the developing postnatal testis . RNA in situ hybridization indicated that the expression of the Aie1 transcript was restricted to meiotically active germ cells, with the highest levels detected in spermatocytes at the late pachytene stage . These findings suggest that Aie1 plays a role in spermatogenesis.

Cancer Lett, 2000 Nov 10, 160(1), 59 - 66
Aminohexanoic hydroxamate is a potent inducer of the differentiation of mouse neuroblastoma cells; Lu J et al.; Deoxyhypusine synthase is the key enzyme for modifying a lysine residue to hypusine in the cellular protein eukaryotic initiation factor 5A (eIF-5A) . Deletion of the deoxyhypusine synthase or the eIF-5A gene in yeast produces lethal phenotype . Inhibition of deoxyhypusine synthase by 1-guanidino-7-aminoheptane (GC7) suppresses tumor cell growth . Hypusine formation represents one of the most specific polyamine-dependent biochemical reactions . In view of the importance of polyamines in growth regulation and cancer biology, deoxyhypusine synthase has been considered to be a good target for chemotherapeutic drug design . Using GC7 as a prototype we have synthesized and tested three classes of diamine analogs, namely, guanidino-, pyrimidino-, and hydroxamate derivatives, as potential inhibitors for deoxyhypusine synthase . Our study shows that (i) among all the compounds tested, GC7 remained to be the most potent inhibitor for deoxyhypusine synthase; (ii) N,N'-bispyrimidino-1, 9-diaminononane, although a poor inhibitor of deoxyhypusine synthase, was a potent growth inhibitor; and (iii) one of the hydroxamate derivatives, 6-aminohexanoic hydroxamate (HC6), prominently induced the differentiation of mouse neuroblastoma cells at sub-millimolar concentrations . Interestingly, other hydroxamates with different chain length were not nearly as effective as HC6 in inducing neuroblastoma cell differentiation . The effect of HC6 was also unique in that it could induce neurite outgrowth and the expression of neuron-specific genes such as synapsin I and MAP-2 in neuroblastoma cells in the absence of other promoting agents such as cAMP . The effect of HC6 on neuroblastoma cell differentiation was comparable with, or better than that of N(6),O(2)'-dibutyryl cAMP (Bt(2)cAMP), a standard reagent commonly used for inducing the differentiation of mouse and human neuroblastoma cells in culture.

Allergy, 2000 Nov, 55(11), 1056 - 8
The relevance of skin prick tests for Pityrosporum ovale in patients with head and neck dermatitis; Devos SA et al.; BACKGROUND: Patients with atopic dermatitis often develop immunoglobulin E antibodies against the yeast Pityrosporum ovale . This organism may produce positive skin prick reactions in a higher rate in patients with atopic dermatitis of the head, scalp, and neck region . METHODS: We investigated whether positive prick tests to P . ovale were associated with a specific localization in the head and neck region . A total of 589 consecutive patients were prick tested with a P . ovale extract from ALK Abello . RESULTS: Thirty-seven patients (6.3%) showed a positive reaction to the P . ovale extract . We could not find significant differences in the localizations of the dermatitis and the pattern of reaction to the tested intracutaneous allergens between the 37 patients positive to P . ovale and the control group of 55 subjects with negative reactions . In a subgroup, we found an elevated level of P . ovale-specific IgE in 100.0% of the patients with head and neck dermatitis, compared with 13.6% in the atopic dermatitis patients with lesions in any other localization . CONCLUSIONS: Our results clearly show that P . ovale-specific IgE is strongly related to the head and neck localization of atopic dermatitis, but RAST seems more sensitive than a prick test with the extract we used.

Parasitol Res, 2000 Nov, 86(11), 916 - 22
Molecular characterization of the histone H2A gene from the parasite Trypanosoma rangeli; Puerta C et al.; The sequence, genomic organization, and transcription of the gene encoding the H2A histone protein of the protozoan parasite Trypanosoma rangeli is described in this paper . The locus encoding the T . rangeli H2A protein is formed by at least 11 gene units measuring 790 nucleotides in length, organized in tandem, and located in a single chromosome of approximately 1.9 Mb . The gene units actively transcribe only one size class of mRNA measuring 0.7 kb in length . The T . rangeli H2A protein contains in the amino-terminal the AGLXFPV motif, which is conserved in a broad range of H2A proteins, and the RSAK motif, which is implicated in repression of the histone's basal transcription in yeast . The carboxyl-terminal of the protein contains a two-lysine residue described as the ubiquitin binding site and the histidine residue implicated in DNA binding.

Dev Immunol, 2000, 7(2-4), 51 - 65
Cell polarization: a comparative cell biology and immunological view; Vicente-Manzanares M et al.; Cell polarization and the establishment of functionally specialized domains play a pivotal role in many cellular processes such as vectorial transport of molecules, cell division and differentiation, directional movement of the cells in a chemotactic gradient and activation of the immune response . Cell polarization is a complex phenomenon, in which the interplay among cell cytoskeletal components, extra- and intracellular signals and organelle and membrane reorganization is crucial to achieve a correct cell shape change . The intracellular machinery needed for cell polarization has been elucidated in several well-established models, including yeast, epithelial, neuronal and germ-line cells . Cells of the immune system also polarize in response to extracellular cues, but many of the intracellular signals that control cell polarization and the role of genes with a well-defined function in other polarization processes are still unknown . In this review, recent advances in the study of leukocyte polarization are examined highlighting the similarities and differences with other models of cell polarization . The extracellular signals which direct cell polarization, the signal transduction pathways involved as well as the role of cell polarization in the development of the immune response are discussed.

Biochem Biophys Res Commun, 2000 Nov 30, 278(3), 671 - 8
HD-PTP: A novel protein tyrosine phosphatase gene on human chromosome 3p21.3; Toyooka S et al.; A human cDNA encoding a novel protein tyrosine phosphatase has been isolated . The phosphatase has unique features in its domain structure: a "Zn-hand" domain containing several SH3-binding motifs, a tyrosine phosphatase domain, a C-terminal PEST motif, and an N-terminal domain similar to yeast BRO1, an apoptosis-related mammalian AIP1 and to a RHO-binding protein, Rhophilin . The gene is located at chromosome 3p21.3, an area frequently deleted in many types of cancer, especially within the functionally defined narrow region . The gene may be a human homolog of the rat PTP-TD14 gene reported by others, which can suppress H-ras-mediated transformation . We identified a hemizygous missense mutation in a lung cancer cell line . Thus, the phosphatase gene may be a candidate for one of the tumor suppressor genes located on 3p21.3 .

Proc Natl Acad Sci U S A, 2000 Dec 5, 97(25), 13871 - 6
A PPxY motif within the VP40 protein of Ebola virus interacts physically and functionally with a ubiquitin ligase: implications for filovirus budding; Harty RN et al.; VP40, the putative matrix protein of both Ebola and Marburg viruses, possesses a conserved proline-rich motif (PY motif) at its N terminus . We demonstrate that the VP40 protein can mediate its own release from mammalian cells, and that the PY motif is important for this self-exocytosis (budding) function . In addition, we used Western-ligand blotting to demonstrate that the PY motif of VP40 can mediate interactions with specific cellular proteins that have type I WW-domains, including the mammalian ubiquitin ligase, Nedd4 . Single point mutations that disrupted the PY motif of VP40 abolished the PY/WW-domain interactions . Significantly, the full-length VP40 protein was shown to interact both physically and functionally with full-length Rsp5, a ubiquitin ligase of yeast and homolog of Nedd4 . The VP40 protein was multiubiquitinated by Rsp5 in a PY-dependent manner in an in vitro ubiquitination assay . These data demonstrate that the VP40 protein of Ebola virus possesses a PY motif that is functionally similar to those described previously for Gag and M proteins of specific retroviruses and rhabdoviruses, respectively . Last, these studies imply that VP40 likely plays an important role in filovirus budding, and that budding of retroviruses, rhabdoviruses, and filoviruses may proceed via analogous mechanisms.

J Am Soc Nephrol, 2000 Dec, 11(12), 2167 - 78
Characterization of ANKRA, a novel ankyrin repeat protein that interacts with the cytoplasmic domain of megalin; Rader K et al.; Ankyrin-repeat family A protein (ANKRA) is a novel protein that interacts directly and specifically with the cytoplasmic tail of megalin in the yeast two-hybrid system and glutathione-S-transferase pull-down assays . ANKRA has three ankyrin repeats and shows 61% overall homology to regulatory factor X, ankyrin repeat-containing protein . Mapping studies show that the three ankyrin repeats and C-terminus of ANKRA are required for binding to a unique juxtamembrane, 19-amino acid sequence on the megalin tail . Point mutational analysis reveals that a proline-rich motif (PXXPXXP) within this region is the site of ANKRA binding . ANKRA interacts with megalin but not with low-density lipoprotein receptor related protein, in keeping with the fact that the sequence of the megalin tail is unique . By cell fractionation, ANKRA is found both in the cytosol and associated with membranes enriched in megalin in L2 cells and proximal tubule cells . By immunofluorescence, ANKRA is concentrated near megalin along the plasma membrane of L2 cells and in the kidney cortex is expressed in glomerular and proximal tubule epithelia which also express megalin . These observations suggest that ANKRA may play a unique role in megalin's function as a clearance receptor in the kidney and L2 cells . In addition, ANKRA may have other partners because northern blot analysis reveals that ANKRA is more broadly expressed than megalin, and by immunofluorescence ANKRA is also expressed in connecting tubule cells and principal cells of collecting ducts.

Plant Mol Biol, 2000 Sep, 44(1), 61 - 71
An Arabidopsis gene induced by wounding functionally homologous to flavoprotein oxidoreductases; Costa CL et al.; The regulation of genes in response to wounding is mediated in part by the octadecanoids 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA) and its methyl ester methyl jasmonate (MeJA) . We identified, by differential display, an Arabidopsis gene (OPR3) induced after wounding . OPR3 is homologous to members of the flavin mononucleotide (FMN) binding proteins, including the old yellow enzyme (OYE) from yeast and 12-oxophytodienoate-10,11-reductase (OPR) from Arabidopsis . Transcripts of OPR3 rapidly accumulated in leaves after wounding and MeJA treatment, but they were detected in various tissues of unwounded plants at relatively low levels . Expression of the OPR3 gene was significantly reduced in wounded leaves of the coil mutant, indicating partial dependence on jasmonate perception for full induction of the gene . The recombinant protein of OPR3 cross-reacted with an antiserum raised against the OYE protein, and showed oxidation of beta-NADPH when OPDA or 15-deoxy-delta(12,14) prostaglandin J2 (PGJ2), an analogue of OPDA, was used as substrate . Beta-NADPH oxidation was not observed when MeJA, which lacks the double bond in the ketone ring, was used as substrate . The recombinant OPR3 protein also showed beta-NADPH oxidation activity in the presence of cyclohexenone, but not cyclohexanone, suggesting that the enzyme has specificity to cleavage of olefinic bonds in cyclic enones . The results show that the OPR3 gene product represents a new OPR of Arabidopsis induced after wounding.

Mol Cell Biol, 2000 Dec, 20(24), 9212 - 24
Vav3 mediates receptor protein tyrosine kinase signaling, regulates GTPase activity, modulates cell morphology, and induces cell transformation; Zeng L et al.; A recently reported new member of the Vav family proteins, Vav3 has been identified as a Ros receptor protein tyrosine kinase (RPTK) interacting protein by yeast two-hybrid screening . Northern analysis shows that Vav3 has a broad tissue expression profile that is distinct from those of Vav and Vav2 . Two species of Vav3 transcripts, 3.4 and 5.4 kb, were detected with a differential expression pattern in various tissues . Transient expression of Vav in 293T and NIH 3T3 cells demonstrated that ligand stimulation of several RPTKs (epidermal growth factor receptor {EGFR}, Ros, insulin receptor {IR}, and insulin-like growth factor I receptor {IGFR}) led to tyrosine phosphorylation of Vav3 and its association with the receptors as well as their downstream signaling molecules, including Shc, Grb2, phospholipase C (PLC-gamma), and phosphatidylinositol 3 kinase . In vitro binding assays using glutathione S-transferase-fusion polypeptides containing the GTPase-binding domains of Rok-alpha, Pak, or Ack revealed that overexpression of Vav3 in NIH 3T3 cells resulted in the activation of Rac-1 and Cdc42 whereas a deletion mutant lacking the N-terminal calponin homology and acidic region domains activated RhoA and Rac-1 but lost the ability to activate Cdc42 . Vav3 induced marked membrane ruffles and microspikes in NIH 3T3 cells, while the N-terminal truncation mutants of Vav3 significantly enhanced membrane ruffle formation but had a reduced ability to induce microspikes . Activation of IR further enhanced the ability of Vav3 to induce membrane ruffles, but IGFR activation specifically promoted Vav3-mediated microspike formation . N-terminal truncation of Vav3 activated its transforming potential, as measured by focus-formation assays . We conclude that Vav3 mediates RPTK signaling and regulates GTPase activity, its native and mutant forms are able to modulate cell morphology, and it has the potential to induce cell transformation.

J Biol Chem, 2001 Mar 16, 276(11), 7727 - 33 Epub 2000 Nov 28.
Human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine 5'-monophosphate cyclohydrolase . A bifunctional protein requiring dimerization for transformylase activity but not for cyclohydrolase activity; Vergis JM et al.; The bifunctional enzyme aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is responsible for catalysis of the last two steps in the de novo purine pathway . Gel filtration studies performed on human enzyme suggested that this enzyme is monomeric in solution . However, cross-linking studies performed on both yeast and avian ATIC indicated that this enzyme might be dimeric . To determine the oligomeric state of this protein in solution, we carried out sedimentation equilibrium analysis of ATIC over a broad concentration range . We find that ATIC participates in a monomer/dimer equilibrium with a dissociation constant of 240 +/- 50 nM at 4 degrees C . To determine whether the presence of substrates affects the monomer/dimer equilibrium, further ultracentrifugation studies were performed . These showed that the equilibrium is only significantly shifted in the presence of both AICAR and a folate analog, resulting in a 10-fold reduction in the dissociation constant . The enzyme concentration dependence on each of the catalytic activities was studied in steady state kinetic experiments . These indicated that the transformylase activity requires dimerization whereas the cyclohydrolase activity only slightly prefers the dimeric form over the monomeric form.

Int J Cancer, 2000 Dec 15, 88(6), 938 - 42
Single nucleotide polymorphism analyses of the human proliferating cell nuclear antigen (pCNA) and flap endonuclease (FEN1) genes; Ma X et al.; The products of proliferating cell nuclear antigen (PCNA) and flap endonuclease (FEN1) genes are multifunctional proteins that are involved in DNA replication and damage repair . Yeast models suggest association of mutant forms of PCNA and FEN1 with genomic instability . In our study, we have determined the single nucleotide polymorphisms in human PCNA and FEN1 genes . We sequenced the coding region and adjacent noncoding region of both the PCNA and FEN1 genes in 120 alleles (60 individuals) . In the PCNA gene, we detected 9 sequence variants with Hardy-Weinberg allele frequency ranging from 0.008 to 0.088 . No polymorphism was detected in the FEN1 gene . The sequence variants in the PCNA gene included 7 intronic single nucleotide polymorphisms (SNP) and 2 synonymous exonic SNPs . All the intronic SNPs were located in introns 1 and 4, which contain several regulatory elements involved in the control of PCNA gene expression . Six of the 7 intronic SNPs showed complete linkage disequilibrium . We confirmed this allelic linkage disequilibrium by allele-specific PCR sequencing . We genotyped 117 additional individuals belonging to 3 population subgroups using the PCR-RFLP method . Finally, to see if the detected polymorphisms are associated with any cancer type, we genotyped cases with melanomas (37 cases), breast cancers (118 cases) and lung cancers (100 cases) . We did not find statistical difference in frequency of polymorphism in any cancer type compared with healthy controls, although in breast cancer the frequency was low . Our results suggest that the coding regions of the PCNA and FEN1 genes are highly conserved when compared with other DNA repair genes . The potential of some of the detected intronic polymorphisms to effect regulation of the PCNA gene expression remains to be determined .

Biopolymers, 2001 Feb, 58(2), 138 - 51
Solution structure of native proteins with irregular folds from Raman optical activity; Smyth E et al.; Raman optical activity (ROA) spectra have been measured for the proteins hen phosvitin, yeast invertase, bovine alpha-casein, soybean Bowman-Birk protease inhibitor, and rabbit Cd(7)-metallothionein, all of which have irregular folds in the native state . The results show that ROA is able to distinguish between two types of disorder . Specifically, invertase, alpha-casein, the Bowman-Birk inhibitor, and metallothionein appear to possess a "static" type of disorder similar to that in disordered states of poly(L-lysine) and poly(L-glutamic acid); whereas phosvitin appears to possess a more "dynamic" type of disorder similar to that in reduced (unfolded) lysozyme and ribonuclease A and also in molten globule protein states . In the delimiting cases, static disorder corresponds to that found in loops and turns within native proteins with well-defined tertiary folds that contain sequences of residues with fixed but nonrepetitive phi,psi angles; and dynamic disorder corresponds to that envisaged for the model random coil in which there is a distribution of Ramachandran phi,psi angles for each amino acid residue, giving rise to an ensemble of interconverting conformers . In both cases there is a propensity for the phi,psi angles to correspond to the alpha, beta and poly(L-proline) II (PPII) regions of the Ramachandran surface, as in native proteins with well-defined tertiary folds . Our results suggest that, with the exception of invertase and metallothionein, an important conformational element present in the polypeptide and protein states supporting the static type of disorder is that of the PPII helix . Long sequences of relatively unconstrained PPII helix, as in alpha-casein, may impart a plastic (rheomorphic) character to the structure .

Annu Rev Genet, 2000, 34, 255 - 295
Protein and lipid requirements for endocytosis; D'Hondt K et al.; Genetic and biochemical studies in yeast and animal cells have led to the identification of many components required for endocytosis . In this review, we summarize our understanding of the endocytic machinery with an emphasis on the proteins regulating the internalization step of endocytosis and endosome fusion . Even though the overall endocytic machinery appears to be conserved between yeast and animals, clear differences exist . We also discuss the roles of phosphoinositides, sterols, and sphingolipid precursors in endocytosis, because in addition to proteins, these lipids have emerged as important determinants in the spatial and most likely temporal specificity of endocytic membrane trafficking events.

J Biol Chem, 2001 Mar 2, 276(9), 6720 - 6 Epub 2000 Nov 22.
Eukaryotic translation initiation factor 5 functions as a GTPase-activating protein; Das S et al.; Eukaryotic translation initiation factor 5 (eIF5) forms a complex with eIF2 by interacting with the beta subunit of eIF2 . This interaction is essential for eIF5-promoted hydrolysis of GTP bound to the 40 S initiation complex . In this work, we show that, in addition to the eIF2 beta-binding region at the C terminus of eIF5, the N-terminal region of eIF5 is also required for eIF5-dependent GTP hydrolysis . Like other GTPase-activating proteins, eIF5 contains an invariant arginine residue (Arg-15) at its N terminus that is essential for its function . Mutation of this arginine residue to alanine or even to conservative lysine caused a severe defect in the ability of eIF5 to promote GTP hydrolysis from the 40 S initiation complex, although the ability of these mutant proteins to bind to eIF2 beta remained unchanged . These mutants were also defective in overall protein synthesis as well as in their ability to support cell growth of a Delta TIF5 yeast strain . Additionally, alanine substitution mutagenesis of eIF5 defined Lys-33 and Lys-55 as also critical for eIF5 function in vitro and in vivo . The implications of these results in relation to other well characterized GAPs are discussed and provide additional evidence that eIF5 functions as a GTPase-activating protein.

DNA Seq, 2000, 11(3-4), 353 - 61
A sequence-ready map for human chromosome 12q15-21; Lee SG et al.; Construction of sequence-ready clone map is an essential step toward sequencing the human genome . We chose a region that is frequently amplified in liposarcoma between D12S350 and D12S106 in chromosome 12q15-21 to build a PAC/BAC clone contig map . This region was spanned by 4 YACs and contained 30 STS on the YAC and radiation hybrid (RH) framework maps, providing an average STS spacing of 160 kb if each YAC is approximately 1.2 Mb in size . To convert a STS-based YAC map to a STS-based contig map of bacterial clones, 22 non-polymorphic STS markers were used as probes to screen the high density gridded arrays of PAC and BAC clones by filter hybridizations, followed by assembly of clones into contigs by marker content . Contigs have been extended and joined by direct end sequencing of appropriate clones, generating new STSs and rescreening the library as necessary . Using these approaches, we have constructed 5 contigs covering the region with the largest single contig being 1.4 Mb and a final size estimation of 3.6 Mb . The map is comprised of 17 YACs, 187 PACs, 160 BACs, and 17 cosmids; onto this, 6 polymorphic, 97 non-polymorphic, 24 ESTs, and 4 gene-based markers are now placed in a unique order, providing an average resolution of approximately 28 kb . Of a total of 131 markers, 97 were developed in the present study . The sequence-ready map should provide a framework to generate complete DNA sequence and ultimately gene map of this segment of chromosome 12.

J Clin Virol, 2000 Oct, 19(1-2), 75 - 8
Immunization against human papillomavirus infection and associated neoplasia; Osen W et al.; BACKGROUND: Chimeric virus like particles (CVLPs) constructed by fusing human papillomavirus type 16 (HPV16) E7 sequences into the C-terminus of the viral L1 gene constitute the first generation of preventive and therapeutic HPV vaccines . Even though vaccination with DNA is highly efficient in the induction of a cytotoxic T-cell (CTL) response utilization of a DNA vaccine in the HPV context, it has been hampered by concern for the oncogenic potential of the E6 and E7 proteins encoded by the viral oncogenes . OBJECTIVE: To consider the use and impact of E7 DNA for immunization . EXPERIMENTAL: In addition to hemagglutination inhibition, a versatile assay to measure neutralization of yeast cell-derived pseudovirions carrying a green fluorescence reporter gene has now been developed . Mice immunized with the HPV16 CVLPs generate E7-specific CTLs, which kill E7 expressing or E7 peptide loaded RMA-cells, protect against tumor formation by syngeneic HPV transformed cells and also induce regression of already established tumors . Since generation of CTL response is achieved by presentation of epitopes as short peptides together with appropriate MHC class I molecules, complete proteins are not required . Instead a shuffled E7 protein has now been used successfully for generating CTL responses comparable to the CVLP responses in mice . CONCLUSIONS: Our preliminary results suggest that immunization with E7 shuffled DNA yields a response directed against the authentic E7 protein . Furthermore, booster immunization with E7 shuffled DNA would avoid inhibition by neutralizing antibodies, however, further studies are needed to guarantee that the shuffled E7 protein lacks oncogenic activity.

Mech Dev, 2000 Dec, 99(1-2), 191 - 4
Expression of TAFII70 RNA and protein during oogenesis and development of the amphibian Pleurodeles waltl; Giani L et al.; TAFs are thought to play an essential role in eukaryotic RNA polymerase II transcription by mediating the expression of distinct subsets of genes . TAFII60/70 was studied in yeast, Drosophila and humans: in the present work, we analyzed the homologue PwTAFII70 in Pleurodeles . The gene is expressed in ovarian oocytes and throughout development, and the level of expression decreases in late embryos . The transcripts are localized in the animal hemisphere of the fertilized eggs and in the animal blastomeres of embryos at cleavage; later PwTAFII70 mRNA is expressed in the neural plate and folds . TAFII70 protein, which is present in fertilized eggs and throughout development, progressively shows a lower level of expression starting from the neurula stage.

Mol Cell, 2000 Oct, 6(4), 791 - 802
The late flowering phenotype of fwa mutants is caused by gain-of-function epigenetic alleles of a homeodomain gene; Soppe WJ et al.; The transition to flowering in Arabidopsis thaliana is delayed in fwa mutant plants . FWA was identified by loss-of-function mutations in normally flowering revertants of the fwa mutant, and it encodes a homeodomain-containing transcription factor . The DNA sequence of wild-type and fwa mutant alleles was identical in the genomic region of FWA . Furthermore, the FWA gene is ectopically expressed in fwa mutants and silenced in mature wild-type plants . This silencing is associated with extensive methylation of two direct repeats in the 5' region of the gene . The late flowering phenotype, ectopic FWA expression, and hypomethylation of the repeats were also induced in the ddm1 hypomethylated background . Mechanisms for establishment and maintenance of the epigenetic mark on FWA are discussed.

Science, 2000 Nov 24, 290(5496), 1574 - 7
Beta-arrestin 2: a receptor-regulated MAPK scaffold for the activation of JNK3; McDonald PH et al.; beta-Arrestins, originally discovered in the context of heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) desensitization, also function in internalization and signaling of these receptors . We identified c-Jun amino-terminal kinase 3 (JNK3) as a binding partner of beta-arrestin 2 using a yeast two-hybrid screen and by coimmunoprecipitation from mouse brain extracts or cotransfected COS-7 cells . The upstream JNK activators apoptosis signal-regulating kinase 1 (ASK1) and mitogen-activated protein kinase (MAPK) kinase 4 were also found in complex with beta-arrestin 2 . Cellular transfection of beta-arrestin 2 caused cytosolic retention of JNK3 and enhanced JNK3 phosphorylation stimulated by ASK1 . Moreover, stimulation of the angiotensin II type 1A receptor activated JNK3 and triggered the colocalization of beta-arrestin 2 and active JNK3 to intracellular vesicles . Thus, beta-arrestin 2 acts as a scaffold protein, which brings the spatial distribution and activity of this MAPK module under the control of a GPCR.

J Mol Biol, 2000 Dec 1, 304(3), 385 - 95
Structural homology between DNA binding sites of DNA polymerase beta and DNA topoisomerase II; Mizushina Y et al.; Unsaturated long-chain fatty acids selectively bind to the DNA binding sites of DNA polymerase beta and DNA topoisomerase II, and inhibit their activities, although the amino acid sequences of these enzymes are markedly different from each other . Computer modeling analysis revealed that the fatty acid interaction interface in both enzymes has a group of four amino acid residues in common, forming a pocket which binds to the fatty acid molecule . The four amino acid residues were Thr596, His735, Leu741 and Lys983 for yeast DNA topoisomerase II, corresponding to Thr79, His51, Leu11 and Lys35 for rat DNA polymerase beta . Using three-dimensional structure model analysis, we determined the spatial positioning of specific amino acid residues binding to the fatty acids in DNA topoisomerase II, and subsequently obtained supplementary information to build the structural model .

Plant Cell, 2000 Nov, 12(11), 2247 - 58
SIMKK, a mitogen-activated protein kinase (MAPK) kinase, is a specific activator of the salt stress-induced MAPK, SIMK; Kiegerl S et al.; In eukaryotes, mitogen-activated protein kinases (MAPKs) play key roles in the transmission of external signals, such as mitogens, hormones, and different stresses . MAPKs are activated by MAPK kinases through phosphorylation of MAPKs at both the threonine and tyrosine residues of the conserved TXY activation motif . In plants, several MAPKs are involved in signaling of hormones, stresses, cell cycle, and developmental cues . Recently, we showed that salt stress-induced MAPK (SIMK) is activated when alfalfa cells are exposed to hyperosmotic conditions . Here, we report the isolation and characterization of the alfalfa MAPK kinase SIMKK (SIMK kinase) . SIMKK encodes an active protein kinase that interacts specifically with SIMK, but not with three other MAPKs, in the yeast two-hybrid system . Recombinant SIMKK specifically activates SIMK by phosphorylating both the threonine and tyrosine residues in the activation loop of SIMK . SIMKK contains a putative MAPK docking site at the N terminus that is conserved in mammalian MAPK kinases, transcription factors, and phosphatases . Removal of the MAPK docking site of SIMKK partially compromises but does not completely abolish interaction with SIMK, suggesting that other domains of SIMKK also are involved in MAPK binding . In transient expression assays, SIMKK specifically activates SIMK but not two other MAPKs . Moreover, SIMKK enhances the salt-induced activation of SIMK . These data suggest that the salt-induced activation of SIMK is mediated by the dual-specificity protein kinase SIMKK.

Plant Cell, 2000 Nov, 12(11), 2219 - 36
In situ localization and in vitro induction of plant COPI-coated vesicles; Pimpl P et al.; Coat protein (COP)-coated vesicles have been shown to mediate protein transport through early steps of the secretory pathway in yeast and mammalian cells . Here, we attempt to elucidate their role in vesicular trafficking of plant cells, using a combined biochemical and ultrastructural approach . Immunogold labeling of cryosections revealed that COPI proteins are localized to microvesicles surrounding or budding from the Golgi apparatus . COPI-coated buds primarily reside on the cis-face of the Golgi stack . In addition, COPI and Arf1p show predominant labeling of the cis-Golgi stack, gradually diminishing toward the trans-Golgi stack . In vitro COPI-coated vesicle induction experiments demonstrated that Arf1p as well as coatomer could be recruited from cauliflower cytosol onto mixed endoplasmic reticulum (ER)/Golgi membranes . Binding of Arf1p and coatomer is inhibited by brefeldin A, underlining the specificity of the recruitment mechanism . In vitro vesicle budding was confirmed by identification of COPI-coated vesicles through immunogold negative staining in a fraction purified from isopycnic sucrose gradient centrifugation . Similar in vitro induction experiments with tobacco ER/Golgi membranes prepared from transgenic plants overproducing barley alpha-amylase-HDEL yielded a COPI-coated vesicle fraction that contained alpha-amylase as well as calreticulin.

J Virol, 2000 Dec, 74(24), 11811 - 24
A human nuclear shuttling protein that interacts with human immunodeficiency virus type 1 matrix is packaged into virions; Gupta K et al.; Active nuclear import of the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC) is essential for the productive infection of nondividing cells . Nuclear import of the PIC is mediated by the HIV-1 matrix protein, which also plays several critical roles during viral entry and possibly during virion production facilitating the export of Pr55(Gag) and genomic RNA . Using a yeast two-hybrid screen, we identified a novel human virion-associated matrix-interacting protein (VAN) that is highly conserved in vertebrates and expressed in most human tissues . Its expression is upregulated upon activation of CD4(+) T cells . VAN is efficiently incorporated into HIV-1 virions and, like matrix, shuttles between the nucleus and cytoplasm . Furthermore, overexpression of VAN significantly inhibits HIV-1 replication in tissue culture . We propose that VAN regulates matrix nuclear localization and, by extension, both nuclear import of the PIC and export of Pr55(Gag) and viral genomic RNA during virion production . Our data suggest that this regulatory mechanism reflects a more global process for regulation of nucleocytoplasmic transport.

Genes Dev, 2000 Nov 15, 14(22), 2906 - 17
Drosophila homologs of transcriptional mediator complex subunits are required for adult cell and segment identity specification; Boube M et al.; The origins of specificity in gene expression are a central concern in understanding developmental control . Mediator protein complexes regulate transcriptional initiation, acting as modular adaptors linking specific transcription factors to core RNA polymerase II . Here, we identified the Drosophila homologs of 23 human mediator genes and mutations of two, dTRAP240 and of dTRAP80 (the putative fly homolog of yeast SRB4) . Clonal analysis indicates a general role for dTRAP80 necessary for cell viability . The dTRAP240 gene is also essential, but cells lacking its function are viable and proliferate normally . Clones reveal localized developmental activities including a sex comb cell identity function . This contrasts with the ubiquitous nuclear accumulation of dTRAP240 protein in imaginal discs . Synergistic genetic interactions support shared developmental cell and segment identity functions of dTRAP240 and dTRAP80, potentially within a common complex . Further, they identify the homeotic Sex combs reduced product, required for the same cell/tissue identities, as a functional partner of these mediator proteins.

Plant Mol Biol, 2000 Aug, 43(5-6), 705 - 18
Stressing the role of MAP kinases in mitogenic stimulation; Bogre L et al.; In yeast and animal cells, distinct subfamilies of mitogen-activated protein kinases (MAPKs) have evolved for transmitting different types of signals, such as the extracellular signal-regulated kinase (ERK) for mitogenic stimuli and differentiation, p38 and JUN kinase (JNK) for stress factors . Based on sequence analysis, the presently known plant MAPKs are most similar to ERKs, even though compelling evidence implies a role in various forms of biotic and abiotic stress responses . However, knowledge of their involvement in controlling proliferation is just emerging . A subgroup of the plant MAPKs, containing the alfalfa MMK3 and tobacco NTF6, are only active in mitotic cells and their localisation to the cell plate suggests a role in cytokinesis . An upstream regulator of MAPKs, the tobacco NPK1, appears to be also activated during mitosis . NPK1 might be associated and regulated by a microtubule motor protein . The localisation of NPK1 to the cell plate and its mitosis-specific activation suggest that together with NTF6 it could constitute a mitotic MAPK signalling module in tobacco . NPK1 appears to have a second role in repression of auxin-induced gene expression . MAPKs might also be involved in signalling within the meristems as suggested by the recruitement of a small G-protein to the CLAVATA 1 receptor-like protein kinase upon activation . In animal and yeast cells some of the small G-proteins relay signals from receptors to MAPK pathways.

Plant Mol Biol, 2000 Aug, 43(5-6), 677 - 90
Factors controlling cyclin B expression; Ito M; Cyclins control the transition between the phases of the eukaryotic cell cycle as regulatory subunits of the cyclin-dependent kinases (CDKs) . Phase-specific activation of the CDK is in part regulated by phase-specific expression of their cyclin component . In most eukaryotic cells including higher plant, B-type cyclin genes are expressed specifically at G2/M phase during the cell cycle . Promoters from yeast, plant and animal B-type cyclin genes are all activated in a cell cycle-regulated manner . In yeast, a transcription factor, Mcm1, in cooperation with an uncloned factor SFF, regulates the cell cycle-dependent promoter activation of mitotic B-type cyclin genes, CLB1 and CLB2 . Activity of the human cyclin B1 promoter is regulated by a complex mechanism involving multiple cis-acting elements, none of which are sufficient for G2/M-specific promoter activation . In contrast, plants employ a simple mechanism for cell cycle-regulated promoter activation of B-type cyclin genes . Plant B-type cyclin gene promoters contain a common cis-acting element, called the MSA element, which is necessary and sufficient for the phase-specific promoter activation . MSA-like sequences are also found in the promoters of G2/M-specific genes encoding kinesin-like proteins, suggesting that a defined set of G2/M-specific genes are co-regulated by a common MSA-mediated mechanism in plants . Thus, the molecular mechanisms regulating B-type cyclin gene expression are evolutionarily divergent, and the MSA-mediated mechanism seems to be specific to plants . The consensus sequence of the MSA element resembles the binding sites of animal Myb transcription factors . A set of our data suggest the possibility that plant Myb may have unexpected roles in G2/M by inducing B-type cyclin genes, together with other cell cycle-related genes in plants.

Plant Mol Biol, 2000 Aug, 43(5-6), 607 - 20
CDK-related protein kinases in plants; Joubes J et al.; Cyclin-dependent kinases (CDK) form a conserved superfamily of eukaryotic serine-threonine protein kinases, which require binding to a cyclin protein for activity . CDK are involved in different aspects of cell biology and notably in cell cycle regulation . The comparison of nearly 50 plant CDK-related cDNAs with a selected set of their animal and yeast counterparts reveals five classes of these genes in plants . These are described here with respect to their phylogenetic, structural and functional properties . A plant-wide nomenclature of CDK-related genes is proposed, using a system similar to that of the plant cyclin genes . The most numerous class, CDKA, includes genes coding for CDK with the PSTAIRE canonical motif . CDKB makes up a class of plant-specific CDK divided into two groups: CDKB1 and CDKB2 . CDKC, CDKD and CDKE form less numerous classes . The CDKD class includes the plant orthologues of metazoan CDK7, which correspond to the CDK-activating kinase (CAK) . At present, no functional information is available in plants for CDKC and CDKE.

Plant Mol Biol, 2000 Aug, 43(5-6), 595 - 605
Multiple cyclin-dependent kinase complexes and phosphatases control G2/M progression in alfalfa cells; Meszaros T et al.; Reversible phosphorylation of proteins by kinases and phosphatases plays a key regulatory role in several eukaryotic cellular functions including the control of the division cycle . Increasing numbers of sequence and biochemical data show the involvement of cyclin-dependent kinases (CDKs) and cyclins in regulation of the cell cycle progression in higher plants . The complexity represented by different types of CDKs and cyclins in a single species such as alfalfa, indicates that multicomponent regulatory pathways control G2/M transition . A set of cdc2-related genes (cdc2Ms A, B, D and F) was expressed in G2 and M cells . Phosphorylation assays also revealed that at least three kinase complexes (Cdc2Ms A/B, D and F) were successively active in G2/M cells after synchronization . Interaction between alfalfa mitotic cyclin (Medsa;CycB2;1) and a kinase partner has been reported previously . The present yeast two-hybrid analyses showed differential interaction between defined D-type cyclins and Cdc2Ms kinases functioning in G2/M phases . Localization of Cdc2Ms F kinase to the preprophase band (PPB), the perinuclear ring in early prophase, the mitotic spindle and the phragmoplast indicated a pivotal role for this kinase in mitotic plant cells . So far limited research efforts have been devoted to the functions of phosphatases in the control of plant cell division . A homologue of dual phosphatase, cdc25, has not been cloned yet from alfalfa; however tyrosine phosphorylation was indicated in the case of Cdc2Ms A kinase and the p(13suc1)-bound kinase activity was increased by treatment of this complex with recombinant Drosophila Cdc25 . The potential role of serine/threonine phosphatases can be concluded from inhibitor studies based on okadaic acid or endothall . Endothall elevated the kinase activity of p(13suc1)-bound fractions in G2-phase alfalfa cells . These biochemical data are in accordance with observed cytological abnormalities . The present overview with selected original data outlines a conclusion that emphasizes the complexity of G2/M regulatory events in flowering plants.

J Biomol Struct Dyn, 2000 Oct, 18(2), 311 - 23
Computer simulations of glycolytic enzyme interactions with F-actin; Ouporov IV et al.; Muscle actin and fructose-1,6-bisphosphate aldolase (aldolase) were chemically crosslinked to produce an 80 kDa product representing one subunit of aldolase linked to one subunit of actin . Hydroxylamine digestion of the crosslinked product resulted in two 40.5 kDa fragments, one that was aldolase linked to the 12 N-terminal residues of actin . Brownian dynamics simulations of muscle aldolase and GAPDH with F-actin (muscle, yeast, and various mutants) estimated the association free energy . Mutations of residues 1-4 of muscle actin to Ala individually or two in combination of the first four residues reduced the estimated binding free energy . Simulations showed that muscle aldolase binds with the same affinity to the yeast actin as to the double mutated muscle actin; these mutations make the N-terminal of muscle actin identical to yeast, supporting the conclusion that the actin N-terminus participates in binding . Because the depth of free energy wells for yeast and the double mutants is less than for native rabbit actin, the simulations support experimental findings that muscle aldolase and GAPDH have a higher affinity for muscle actin than for yeast actin . Furthermore, Brownian dynamics revealed that the lower affinity of yeast actin for aldolase and GAPDH compared to muscle actin, was directly related to the acidic residues at the N-terminus of actin.

Z Gastroenterol, 2000 Oct, 38(10), 821 - 5
Fungal colonization of the esophagus in alcoholic liver disease; Peter Z et al.; OBJECTIVES: Little is known about the fungal colonization of the esophagus . Since alcoholic liver disease (ALD) patients are prone to fungal esophagitis, we have investigated the esophageal fungal colonization of this patient group . METHODS: One hundred consecutive ALD patients were enrolled in this prospective study . 22 patients with dyspeptic symptoms acted as controls . After taking an oropharyngeal swab, patients underwent an upper gastrointestinal endoscopy and surface material was obtained from the esophagus for direct smears and culture . RESULTS: In the ALD group pseudohyphae were found in 21.5% and yeast forms in 6.4% of the direct smears . The culture was positive in 40.8% of the ALD patients, the isolated strains were: 30 C . albicans, 2 C . kefyr, 2 C . krusei, 1 C . zeylanoides and in 3 cases the species could not be identified . 41.9% of ALD patients and 13.6% of control patients (p = 0.013) had fungi in their esophagus . Significantly more ALD patients had fungal esophagitis than in the control group (19.3% vs . 0%, p = 0.021), the rate of fungal colonization was also higher, but the difference was not significant (22.5% vs . 13.6%) . A significantly higher rate of fungal esophagitis and esophageal colonization was found in patients with fungi in their oropharyngeal swabs (p = 0.00001) . CONCLUSIONS: Fungal colonization of the esophagus is frequent in ALD patients . Its presence might have clinical significance in the case of liver transplantation.

Curr Opin Genet Dev, 2000 Dec, 10(6), 612 - 6
The Drosophila genome; Celniker SE; The past year has been a spectacular one for Drosophila research . The sequencing and annotation of the Drosophila melanogaster genome has allowed a comprehensive analysis of the first three eukaryotes to be sequenced-yeast, worm and fly-including an analysis of the fly's influences as a model for the study of human disease . This year has also seen the initiation of a full-length cDNA sequencing project and the first analysis of Drosophila development using high-density DNA microarrays containing several thousand Drosophila genes . For the first time homologous recombination has been demonstrated in flies and targeted gene disruptions may not be far off.

Mutat Res, 2000 Nov 30, 456(1-2), 45 - 57
Werner's syndrome cell lines are hypersensitive to camptothecin-induced chromosomal damage; Pichierri P et al.; Werner's syndrome (WS) is a recessive human genetic disorder associated with an elevated incidence of many types of cancer . The WS gene product, WRNp, belongs to the RecQ family of DNA helicases and is required for the maintenance of genomic stability in human cells . A possible interaction between helicases and topoisomerases that could co-operate in many aspects of DNA metabolism such as progression of the replication forks, recombination and repair has been recently suggested . In addition, sgs1 gene product in yeast, homologous to WS gene, has been shown to physically interact with topoisomerase types I and II . Earlier data from our laboratory suggested that WRN helicase might play a role in a G2 recombinational pathway of double strand breaks (DSBs) repair, co-operating with topoisomerase II . In this work, the effect of the topoisomerase I inhibitor camptothecin in WS cells has been investigated at the chromosomal level.The data from the present work suggest that the inhibition of topoisomerase I activity by camptothecin results in a higher induction of chromosomal damage in WS cell lines in the G2-phase and in the S-phase of the cell cycle compared to normal cells, perhaps associated with the defects in DNA replication synthesis.

Proc Natl Acad Sci U S A, 2000 Dec 5, 97(25), 13967 - 72
The GABAB receptor interacts directly with the related transcription factors CREB2 and ATFx; White JH et al.; gamma-Aminobutyric acid type B (GABA(B)) receptors mediate the metabotropic actions of the inhibitory neurotransmitter GABA . These seven-transmembrane receptors are known to signal primarily through activation of G proteins to modulate the action of ion channels or second messengers . The functional GABA(B) receptor is made up of a heterodimer consisting of two subunits, GABA(B)-R1 and GABA(B)-R2, which interact via coiled-coil domains in their C-terminal tails . By using a yeast two-hybrid approach, we have identified direct interactions between the C-terminal tails of GABA(B)-R1 and GABA(B)-R2 with two related transcription factors, CREB2 (ATF4) and ATFx . In primary neuronal cultures as well in recombinant Chinese hamster ovary cells expressing GABA(B) receptors, CREB2 is localized within the cytoplasm as well as the nucleus . Activation of the GABA(B) receptor by the specific agonist baclofen leads to a marked translocation and accumulation of CREB2 from the cytoplasm into the nucleus . We demonstrate that receptor stimulation results in activation of transcription from a CREB2 responsive reporter gene . Such a signaling mechanism is unique among Family C G protein-coupled receptors and, in the case of the GABA(B) receptor and CREB2, may play a role in long-term changes in the nervous system.

Proc Natl Acad Sci U S A, 2000 Dec 5, 97(25), 13991 - 6
Mutants of Arabidopsis defective in a sequence-specific mRNA degradation pathway; Johnson MA et al.; One of the ways a cell can rapidly and tightly regulate gene expression is to target specific mRNAs for rapid decay . A number of mRNA instability sequences that mediate rapid mRNA decay have been identified, particularly from multicellular eukaryotes, but pinpointing the cellular components that play critical roles in sequence-specific decay in vivo has been more difficult . In contrast, general pathways of mRNA degradation in yeast have been well established through the analysis of mutants affecting the general mRNA decay machinery . Strategies to isolate mutants in sequence-specific mRNA decay pathways, although extremely limited so far, have the potential to be just as powerful . In the study reported here, a selection in transgenic plants allowed the isolation of rare mutants of Arabidopsis thaliana that elevate the abundance of mRNAs that contain the plant mRNA instability sequence called DST (downstream element) . This instability sequence is highly conserved in unstable small auxin up RNA (SAUR) transcripts . Genetic analysis of two dst mutants isolated via this selection showed that they are incompletely dominant and represent two independent loci . In addition to affecting DST-containing transgene mRNAs, mutations at both loci increased the abundance of the endogenous DST-containing SAUR-AC1 mRNA, but not controls lacking DST sequences . That these phenotypes are caused by deficiencies in DST-mediated mRNA decay was supported by mRNA stability measurements in transgenic plants . Isolation of the dst mutants provides a means to study sequence-specific mRNA degradation in vivo and establishes a method to isolate similar mutants from other organisms.

Genomics, 2000 Nov 15, 70(1), 113 - 22
Expression and genetic analysis of XIAP-associated factor 1 (XAF1) in cancer cell lines; Fong WG et al.; X-linked inhibitor of apoptosis protein (XIAP) is a potent modulator of programmed cell death . XIAP specifically binds and inhibits the function of caspase-3, -7, and -9, key effector proteases of apoptosis . We recently isolated, by yeast two-hybrid screening, a novel 34-kDa zinc finger protein, XIAP-associated factor 1 (XAF1) . Both the caspase inhibiting and the anti-apoptotic abilities of XIAP were found to be blocked by overexpressed XAF1 . Here, we report the isolation and characterization of the human XAF1 gene . The xaf1 gene consists of seven exons spanning 18 kb . Fluorescence in situ hybridization analysis localized the xaf1 locus at 17p13.2, telomeric to the p53 gene . The xaf1 locus was further refined to YAC 746C10, approximately 3 cM distal to TP53 . Microsatellite analysis of the xaf1 locus using the NCI 60 cell line panel revealed significantly decreased heterozygosity at all three polymorphic markers tested, suggesting that allelic loss of the xaf1 gene is prevalent in cancer cell lines . Examination of the same NCI cell line panel for xaf1 RNA expression demonstrated that cancer cell lines exhibited very low levels of mRNA relative to normal human liver . In contrast, XIAP mRNA levels were relatively high in the majority of cancer cell lines tested . We propose that a high level of XIAP to XAF1 expression in cancer cells may provide a survival advantage through the relative increase of XIAP anti-apoptotic function .

J Nat Prod, 2000 Nov, 63(11), 1461 - 4
Bioactive saponins from Swartzia schomburgkii from the suriname rainforest; Abdel-Kader MS et al.; Bioassay-guided fractionation of the MeOH extract of Swartzia schomburgkii using the engineered yeast strains 1138, 1140, and 1353 as the bioassay tool resulted in the isolation of five active (2, 4-7) and three inactive (1, 3, 8) saponins . Saponins 4 and 6 are previously unreported . The structures of all of the saponins were established based on 1D and 2D NMR spectral analysis, on acid and alkaline hydrolysis followed by TLC and GC-MS, and by comparison with literature data for known compounds . Three of the isolated compounds (4-6) showed weak cytotoxicity against the M-109 cell line.

J Physiol Paris, 2000 May-Aug, 94(3-4), 193 - 8
Identification of proteins interacting with the rat somatostatin receptor subtype 2; Kreienkamp HJ et al.; Using the yeast two hybrid system we have identified a novel protein termed somatostatin receptor interacting protein (SSTRIP) from human brain which interacts with the rat somatostatin receptor subtype 2 . Interaction with the receptor C-terminus is mediated by a PSD-95/discs large/ZO-1 (PDZ) domain which exhibits high similarity to the PDZ domain of cortactin binding protein 1 (CortBP1) . SSTRIP and CortBP1 define a novel family of multidomain proteins containing ankyrin repeats, SH3- and SH3 binding regions and a sterile alpha motif (SAM domain) in addition to the PDZ domain . Both SSTRIP and CortBP1 can be co-immunoprecipitated with the somatostatin receptor when co-expressed in HEK cells . Interestingly, co-localization of SSTR2 and CortBP1 at the plasma membrane is increased when SSTR2 is stimulated by agonists.

J Clin Invest, 2000 Nov, 106(10), 1239 - 49
Interaction between complement receptor gC1qR and hepatitis C virus core protein inhibits T-lymphocyte proliferation; Kittlesen DJ et al.; Hepatitis C virus (HCV) is an important human pathogen that is remarkably efficient at establishing persistent infection . The HCV core protein is the first protein expressed during the early phase of HCV infection . Our previous work demonstrated that the HCV core protein suppresses host immune responses, including anti-viral cytotoxic T-lymphocyte responses in a murine model . To investigate the mechanism of HCV core-mediated immunosuppression, we searched for host proteins capable of associating with the core protein using a yeast two-hybrid system . Using the core protein as bait, we screened a human T cell-enriched expression library and identified a gene encoding the gC1q receptor (gC1qR) . C1q is a ligand of gC1qR and is involved in the early host defense against infection . Like C1q, HCV core can inhibit T-cell proliferative responses in vitro . This core-induced anti-T-cell proliferation is reversed by addition of anti-gC1qR Ab in a T-cell proliferation assay . Furthermore, biochemical analysis of the interaction between core and gC1qR indicates that HCV core binds the region spanning amino acids 188 to 259 of gC1qR, a site distinct from the binding region of C1q . The inhibition of T-cell responsiveness by HCV core may have important implications for HCV persistence in humans.

Biochem J, 2000 Dec 1, 352 Pt 2, 443 - 8
Association of FHIT (fragile histidine triad), a candidate tumour suppressor gene, with the ubiquitin-conjugating enzyme hUBC9; Shi Y et al.; FHIT (fragile histidine triad), a candidate tumour suppressor gene, has recently been identified at chromosomal region 3p14.2, and deletions of the gene have been reported in many types of human cancer . However, the biological function of the Fhit protein has not been fully characterized yet . Using the yeast two-hybrid screen to search for proteins that interact with Fhit in vivo, we identified a protein that is specifically associated with Fhit . This association was confirmed in both immunoprecipitation and glutathione S-transferase pull-down assays . The sequence of the protein is identical with that of human ubiquitin-conjugating enzyme 9 (hUBC9) . The last 21 amino acids at the C-terminus of hUBC9 appear to be unimportant for its biological activity, since an hUBC9 mutant harbouring a deletion of these amino acids could still restore normal growth of yeast containing a temperature-sensitive mutation in the homologue UBC9 gene . Mutational analysis indicated that hUBC9 was associated with the C-terminal portion of Fhit . Neither a single amino acid substitution at codon 96 (His-->Asn) nor triple amino acid substitutions (His-->Asn) at a histidine triad (codons 94, 96 and 98) affected the association, whereas Fhit triphosphate (diadenosine 5',5"'-P(1),P(3)-triphosphate) hydrolase activity has been reported to be eliminated by either type of mutation, suggesting that the interaction between Fhit and hUBC9 is independent of Fhit enzymic activity . Given that yeast UBC9 is involved in the degradation of S- and M-phase cyclins, Fhit may be involved in cell cycle control through its interaction with hUBC9.

J Biol Chem, 2001 Feb 23, 276(8), 5814 - 20 Epub 2000 Nov 20.
Functional interaction of human Cdc37 with the androgen receptor but not with the glucocorticoid receptor; Rao J et al.; Cdc37 is a molecular chaperone closely associated with the folding of protein kinases . Results from studies using a yeast model system showed that it was also important for activation of the human androgen receptor (AR) . Based on results from the yeast model system (Fliss, A . E., Fang, Y., Boschelli, F., and Caplan, A . J . (1997) Mol . Biol . Cell 8, 2501-2509), we initiated studies to address whether AR and Cdc37 interact with each other in animal cell systems . Our results show that Cdc37 binds to AR but not to glucocorticoid receptors (GR) synthesized in rabbit reticulocyte lysates . This binding occurs via the ligand-binding domain of the AR in a manner that is partially dependent on Hsp90 and the presence of hormone . Further studies using the yeast system showed that Cdc37 is not interchangeable with Hsp90, suggesting that it functions at a distinct step in the activation pathway . Expression of a dominant negative form of Cdc37 in animal cells down-regulates full-length AR but has very little effect on an AR truncation lacking the ligand-binding domain or full-length GR . These results reveal differences in the mechanisms by which AR and GR become active transcription factors and strengthen the notion that Cdc37 has a wider range of polypeptide clients than was realized previously.

J Biol Chem, 2001 Feb 16, 276(7), 5074 - 84 Epub 2000 Nov 20.
Identification and characterization of SNX15, a novel sorting nexin involved in protein trafficking; Phillips SA et al.; Sorting nexins are a family of phox homology domain containing proteins that are homologous to yeast proteins involved in protein trafficking . We have identified a novel 342-amino acid residue sorting nexin, SNX15, and a 252-amino acid splice variant, SNX15A . Unlike many sorting nexins, a SNX15 ortholog has not been identified in yeast or Caenorhabditis elegans . By Northern blot analysis, SNX15 mRNA is widely expressed . Although predicted to be a soluble protein, both endogenous and overexpressed SNX15 are found on membranes and in the cytosol . The phox homology domain of SNX15 is required for its membrane association and for association with the platelet-derived growth factor receptor . We did not detect association of SNX15 with receptors for epidermal growth factor or insulin . However, overexpression of SNX15 led to a decrease in the processing of insulin and hepatocyte growth factor receptors to their mature subunits . Immunofluorescence studies showed that SNX15 overexpression resulted in mislocalization of furin, the endoprotease responsible for cleavage of insulin and hepatocyte growth factor receptors . Based on our data and the existing findings with yeast orthologs of other sorting nexins, we propose that overexpression of SNX15 disrupts the normal trafficking of proteins from the plasma membrane to recycling endosomes or the trans-Golgi network.

J Biol Chem, 2001 Jan 26, 276(4), 2325 - 8 Epub 2000 Nov 20.
A novel glucokinase regulator in pancreatic beta cells: precursor of propionyl-CoA carboxylase beta subunit interacts with glucokinase and augments its activity; Shiraishi A et al.; A glucokinase regulatory protein has been reported to exist in the liver, which suppresses enzyme activity in a complex with fructose 6-phosphate, whereas no corresponding protein has been found in pancreatic beta cells . To search for such a protein in pancreatic beta cells, we screened for a cDNA library of the HIT-T15 cell line with the cDNA of glucokinase from rat islet by the yeast two hybrid system . We detected a cDNA encoding the precursor of propionyl-CoA carboxylase beta subunit (pbetaPCCase), and glutathione S-transferase pull-down assay illustrated that pbetaPCCase interacted with recombinant rat islet glucokinase and with glucokinase in rat liver and islet extracts . Functional analysis indicated that pbetaPCCase decreased the K(m) value of recombinant islet glucokinase for glucose by 18% and increased V(max) value by 23% . We concluded that pbetaPCCase might be a novel activator of glucokinase in pancreatic beta cells.

J Exp Med, 2000 Nov 20, 192(10), 1479 - 90
Hematopoietic expression of HOXB4 is regulated in normal and leukemic stem cells through transcriptional activation of the HOXB4 promoter by upstream stimulating factor (USF)-1 and USF-2; Giannola DM et al.; The homeobox genes encode a family of transcription factors that regulate development and postnatal tissue homeostasis . Since HOXB4 plays a key role in regulating the balance between hematopoietic stem cell renewal and differentiation, we studied the molecular regulation of HOXB4 expression in human hematopoietic stem cells . HOXB4 expression in K562 cells is regulated at the level of transcription, and transient transfection defines primary HOXB4 regulatory sequences within a 99-bp 5' promoter . Culture of highly purified human CD34(+) bone marrow cells in thrombopoietin/Flt-3 ligand/stem cell factor induced HOXB4 3-10-fold, whereas culture in granulocyte/macrophage colony-stimulating factor, only increased HOXB4/luciferase expression 20-50% . Mutations within the HOXB4 promoter identified a potential E box binding site (HOX response element {HXRE}-2) as the most critical regulatory sequence, and yeast one hybrid assays evaluating bone marrow and K562 libraries for HXRE-2 interaction identified upstream stimulating factor (USF)-2 and micropthalmia transcription factor (MITF) . Electrophoretic mobility shift assay with K562 extracts confirmed that these proteins, along with USF-1, bind to the HOXB4 promoter in vitro . Cotransfection assays in both K562 and CD34(+) cells showed that USF-1 and USF-2, but not MITF, induce the HOXB4 promoter in response to signals stimulating stem cell self-renewal, through activation of the mitogen-activated protein kinase pathway . Thus hematopoietic expression of the human HOXB4 gene is regulated by the binding of USF-1 and USF-2, and this process may be favored by cytokines promoting stem cell self-renewal versus differentiation.

Neurogenetics, 2000 Sep, 3(1), 41 - 4
CLN-encoded proteins do not interact with each other; Zhong NA et al.; The lysosomal storage of lipofuscins is the common pathological feature that characterizes the infantile, late-infantile, juvenile (Batten's disease), and Finnish-variant neuronal ceroid lipofuscinosis (INCL, LINCL, JNCL and FNCL), which are due to mutations in the genes CLN1, CLN2, CLN3, and CLN5, respectively . The CLN1 and CLN2 genes encode lysosomal enzymes, but the CLN3 and CLN5 genes encode membrane-spanning proteins . Why deficiencies of lysosomal enzymes and membrane-spanning proteins produce similar clinical phenotypes and pathological changes is still unanswered . We hypothesize that CLN-encoded proteins may comprise a functional pathogenic pathway, in which protein associations may play important roles . To test this hypothesis, we studied protein-protein interactions among the CLN1-, CLN2-, and CLN3-encoded proteins using a yeast two-hybrid system . Our results provided no evidence that CLN-encoded proteins interact with each other . This suggests there may be unidentified components in NCL pathogenesis.

Rapid Commun Mass Spectrom, 2000, 14(21), 2070 - 3
Improved matrix-assisted laser desorption/ionization mass spectrometric analysis of tryptic hydrolysates of proteins following guanidination of lysine-containing peptides; Brancia FL et al.; Analysis of tryptic digests of proteins using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry commonly results in superior detection of arginine-containing peptides compared with lysine-containing counterparts . The effect is attributable in part to the greater stability of the arginine-containing peptide ions associated with the sequestration of the single ionizing proton on the arginine side-chain . Reaction of peptides with O-methylisourea resulted in conversion of lysine to homoarginine residues with consequent improved detection during MALDI-MS . Analysis of the underivatized tryptic digest of the yeast protein, enolase, revealed peptides representing 20% of the protein; the corresponding figure after derivatization was 46%.

Mol Gen Genet, 2000 Oct, 264(3), 257 - 67
Molecular and expression analysis of a LIM protein gene family from flowering plants; Eliasson A et al.; LIM-domain proteins participate in important cellular processes in eukaryotes, including gene transcription and actin cytoskeleton organization . They are predominantly found in animals, but have also been identified in yeast and plants . Following the characterization ofa LIM-domain protein in sunflower pollen, we carried out an extensive search for these proteins in flowering plants . We have isolated and studied cDNAs and/or genomic sequences for two novel LIM-domain proteins from sunflower, three from tobacco, and one from Arabidopsis . The plant proteins are structurally related to the cytoskeleton-associated CRP class of LIM proteins in animals, but show several distinctive features, including a second, atypical, LIM domain . We have performed comparative expression studies of these genes, as well as of one other gene from tobacco and two additional Arabidopsis genes whose sequences are available from databases . These studies, carried out by RT-PCR in the presence of gene-specific primers, showed that, in sunflower and tobacco, pollen grains and sporophytic tissues express different sets of LIM proteins . With the exception of one Arabidopsis gene--which has two introns--all the genes analyzed contain four introns at conserved positions, indicating that the ancestral gene from which the various copies evolved in higher plants allready had this split structure.

Curr Biol, 2000 Nov 2, 10(21), 1329 - 38
Cut8, essential for anaphase, controls localization of 26S proteasome, facilitating destruction of cyclin and Cut2; Tatebe H et al.; BACKGROUND: Anaphase-promoting complex (APC)/cyclosome and 26S proteasome are respectively required for polyubiquitination and degradation of mitotic cyclin and anaphase inhibitor Cut2 (Pds1/securin) . In fission yeast, mutant cells defective in cyclosome and proteasome fail to complete mitosis and have hypercondensed chromosomes and a short spindle . A similar phenotype is seen in a temperature-sensitive strain cut8-563 at 36 degrees C, but the molecular basis for Cut8 function is little understood . RESULTS: At high temperature, the level of Cut8 greatly increases and it becomes essential to the progression of anaphase . In cut8 mutants, chromosome mis-segregation and aberrant spindle dynamics occur, but cytokinesis takes place with normal timing, leading to the cut phenotype . This is due to the fact that destruction of mitotic cyclin and Cut2 in the nucleus is dramatically delayed, though polyubiquitination of Cdc13 occurs in cut8 mutant . Cut8 is localized chiefly to the nucleus and nuclear periphery, a distribution highly similar to that of 26S proteasome . In cut8 mutant, however, 26S proteasome becomes mostly cytoplasmic, showing that Cut8 is needed for its proper localization . CONCLUSION: Cut8 is a novel evolutionarily conserved heat-inducible regulator . It facilitates anaphase-promoting proteolysis by recruiting 26S proteasome to a functionally efficient nuclear location.

Curr Biol, 2000 Nov 2, 10(21), 1319 - 28
Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype; Uren AG et al.; BACKGROUND: Survivin is a mammalian protein that carries a motif typical of the inhibitor of apoptosis (IAP)proteins, first identified in baculoviruses . Although baculoviral IAP proteins regulate cell death, the yeast Survivin homolog Bir1 is involved in cell division . To determine the function of Survivin in mammals, we analyzed the pattern of localization of Survivin protein during the cell cycle, and deleted its gene by homologous recombination in mice . RESULTS: In human cells, Survivin appeared first on centromeres bound to a novel para-polar axis during prophase/metaphase, relocated to the spindle midzone during anaphase/telophase, and disappeared at the end of telophase . In the mouse, Survivin was required for mitosis during development . Null embryos showed disrupted microtubule formation, became polyploid, and failed to survive beyond 4.5days post coitum . This phenotype, and the cell-cycle localization of Survivin, resembled closely those of INCENP . Because the yeast homolog of INCENP, Sli15, regulates the Aurora kinase homolog Ipl1p, and the yeast Survivin homolog Bir1 binds to Ndc10p, a substrate of Ipl1p, yeast Survivin, INCENP and Aurora homologs function in concert during cell division . CONCLUSIONS: In vertebrates, Survivin and INCENP have related roles in mitosis, coordinating events such as microtubule organization, cleavage-furrow formation and cytokinesis . Like their yeast homologs Bir1 and Sli15, they may also act together with the Aurora kinase.

Mol Cell Neurosci, 2000 Nov, 16(5), 620 - 30
The human preprotachykinin-A gene promoter has been highly conserved and can drive human-like marker gene expression in the adult mouse CNS; MacKenzie A et al.; Toward an understanding of the mechanisms controlling Preprotachykinin-A (PPTA) transcription, we introduced a 380-kb human yeast artificial chromosome containing the PPTA gene tagged with the beta-galactosidase gene into transgenic mice . This resulted in a pattern of LacZ expression in the central nervous system (CNS) remarkably similar to that reported for PPTA mRNA in the rat . However, the human gene drove expression in areas of the mouse CNS not associated with strong PPTA expression in rodents but which have been shown to express PPTA in the human . This study clearly demonstrates the high degree of conservation of the mechanisms involved in PPTA transcription that has occurred throughout 100 million of divergent human and rodent evolution . This study also defines the maximum linear extent of the human PPT-A promoter . We believe these findings constitute the removal of a significant obstacle in studying the transcriptional regulation of the human PPTA gene in vivo .

J Biol Chem, 2001 Feb 16, 276(7), 5331 - 8 Epub 2000 Nov 17.
Dlxin-1, a novel protein that binds Dlx5 and regulates its transcriptional function; Masuda Y et al.; Dlx5, a member of the Dlx family of homeodomain proteins, plays a critical role in bone development and fracture healing . To understand the molecular mechanism underlying the transcriptional regulation by Dlx5, we performed yeast two-hybrid screening and isolated a novel protein, Dlxin-1, that binds Dlx5 and regulates its transcriptional function . Dlxin-1 cDNA encodes a 775-amino acid protein that has a partial homology with necdin at the C terminus and 25 repeats of hexapeptides (WQXPXX) in the middle region . Dlxin-1 mRNA is expressed in various adult tissues, but not the spleen, and also in osteoblastic and chondrogenic cell lines . During embryogenesis, a strong signal for Dlxin-1 mRNA was found in cell layers surrounding cartilaginous elements in bone rudiment during digit formation . Dlxin-1 binds not only Dlx5 but also Dlx7 and Msx2 and forms homomultimers in vivo . Transfection and reporter gene assays indicate that Dlxin-1 activates the transcriptional function of Dlx5 . Therefore, Dlxin-1 may act as a regulator of the function of Dlx family members in bone formation.

J Biol Chem, 2001 Feb 23, 276(8), 5836 - 40 Epub 2000 Nov 17.
Arginine-rich peptides . An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery; Futaki S et al.; A basic peptide derived from human immunodeficiency virus (HIV)-1 Tat protein (positions 48-60) has been reported to have the ability to translocate through the cell membranes and accumulate in the nucleus, the characteristics of which are utilized for the delivery of exogenous proteins into cells . Based on the fluorescence microscopic observations of mouse macrophage RAW264.7 cells, we found that various arginine-rich peptides have a translocation activity very similar to Tat-(48-60) . These included such peptides as the d-amino acid- and arginine-substituted Tat-(48-60), the RNA-binding peptides derived from virus proteins, such as HIV-1 Rev, and flock house virus coat proteins, and the DNA binding segments of leucine zipper proteins, such as cancer-related proteins c-Fos and c-Jun, and the yeast transcription factor GCN4 . These segments have no specific primary and secondary structures in common except that they have several arginine residues in the sequences . Moreover, these peptides were able to be internalized even at 4 degrees C . These results strongly suggested the possible existence of a common internalization mechanism ubiquitous to arginine-rich peptides, which is not explained by a typical endocytosis . Using (Arg)(n) (n = 4-16) peptides, we also demonstrated that there would be an optimal number of arginine residues (n approximately 8) for the efficient translocation.

Eur J Biochem, 2000 Dec, 267(23), 6832 - 40
Purification and properties of hydroquinone hydroxylase, a FAD-dependent monooxygenase involved in the catabolism of 4-hydroxybenzoate in Candida parapsilosis CBS604; Eppink MH et al.; The ascomycetous yeast Candida parapsilosis CBS604 catabolizes 4-hydroxybenzoate through the initial formation of hydroquinone (1, 4-dihydroxybenzene) . High levels of hydroquinone hydroxylase activity are induced when the yeast is grown on either 4-hydroxybenzoate, 2,4-dihydroxybenzoate, 1,3-dihydroxybenzene or 1, 4-dihydroxybenzene as the sole carbon source . The monooxygenase constitutes up to 5% of the total amount of protein and is purified to apparent homogeneity in three chromatographic steps . Hydroquinone hydroxylase from C . parapsilosis is a homodimer of about 150 kDa with each 76-kDa subunit containing a tightly noncovalently bound FAD . The flavin prosthetic group is quantitatively resolved from the protein at neutral pH in the presence of chaotropic salts . The apoenzyme is dimeric and readily reconstituted with FAD . Hydroquinone hydroxylase from C . parapsilosis catalyzes the ortho-hydroxylation of a wide range of monocyclic phenols with the stoichiometric consumption of NADPH and oxygen . With most aromatic substrates, no uncoupling of hydroxylation occurs . Hydroxylation of monofluorinated phenols is highly regiospecific with a preference for C6 hydroxylation . Binding of phenol highly stimulates the rate of flavin reduction by NADPH . At pH 7.6, 25 degrees C, this step does not limit the rate of overall catalysis . During purification, hydroquinone hydroxylase is susceptible towards limited proteolysis . Proteolytic cleavage does not influence the enzyme dimeric nature but results in relatively stable protein fragments of 55, 43, 35 and 22 kDa . N-Terminal peptide sequence analysis revealed the presence of two nick sites and showed that hydroquinone hydroxylase from C . parapsilosis is structurally related to phenol hydroxylase from Trichosporon cutaneum . The implications of these findings for the catalytic mechanism of hydroquinone hydroxylase are discussed.

Science, 2000 Nov 17, 290(5495), 1368 - 72
Intracellular parasitism by Histoplasma capsulatum: fungal virulence and calcium dependence; Sebghati TS et al.; Histoplasma capsulatum is an effective intracellular parasite of macrophages and causes the most prevalent fungal respiratory disease in the United States . A "dimorphic" fungus, H . capsulatum exists as a saprophytic mold in soil and converts to the parasitic yeast form after inhalation . Only the yeasts secrete a calcium-binding protein (CBP) and can grow in calcium-limiting conditions . To probe the relation between calcium limitation and intracellular parasitism, we designed a strategy to disrupt CBP1 in H . capsulatum using a telomeric linear plasmid and a two-step genetic selection . The resultingcbp1 yeasts no longer grew when deprived of calcium, and they were also unable to destroy macrophages in vitro or proliferate in a mouse model of pulmonary infection.

J Cell Sci, 2000 Dec, 113 Pt 24, 4523 - 31
Human Cdc5, a regulator of mitotic entry, can act as a site-specific DNA binding protein; Lei XH et al.; G(2)/M progression requires coordinated expression of many gene products, but little is known about the transcriptional regulators involved . We recently identified human Cdc5, a positive regulator of G(2)/M in mammalian cells . We also demonstrated the presence of a latent activation domain in its carboxyl terminus, suggesting that human Cdc5 regulates G(2)/M through transcriptional activation . Despite the presence of a DNA binding domain, studies by others have failed to identify a preferential binding site for Cdc5 family members . In addition, Cdc5 recently has been associated with the splicesome in several organisms, suggesting that it may not act through DNA binding . We now report the identification of a 12 bp sequence to which human Cdc5 binds specifically and with high affinity through its amino terminus . We show that this DNA-protein interaction is capable of activating transcription . We also used a selection system in yeast to identify human genomic fragments that interact with human Cdc5 . Several of these contained sequences similar to the binding site . We demonstrate that these bind human Cdc5 with similar specificity and affinity . These experiments provide the first evidence that Cdc5 family members can act as site-specific DNA binding proteins, and that human Cdc5 may interact with specific, low abundance sequences in the human genome . This raises the possibility that Cdc5 proteins may participate in more than one process necessary for regulated cell division.

J Cell Sci, 2000 Dec, 113 Pt 24, 4391 - 7
Recent structural insights into transcription preinitiation complexes; Nogales E; Our understanding of the elaborate mechanism of gene transcription initiation in eukaryotes has been widened by recent structural information on some of the key components of the complex preinitiation transcriptional machinery . The high-resolution structures of both bacterial and eukaryotic polymerases are technical landmarks of great biological significance that have given us the first molecular insight into the mechanism of this large enzyme . While new atomic structures of different domains of general transcription factors, such as the double bromodomain of TAF250, have become available by means of X-ray crystallography and NMR studies, more global pictures of multisubunit transcription complexes, such as TFIID, TFIIH or the yeast mediator, have now been obtained by electron microscopy and image-reconstruction techniques . A combination of methodologies may prove essential for a complete structural description of the initial steps in the expression of eukaryotic genes.

Cell, 2000 Oct 27, 103(3), 457 - 66
A common core RNP structure shared between the small nucleoar box C/D RNPs and the spliceosomal U4 snRNP; Watkins NJ et al.; The box C/D snoRNAs function in directing 2'-O-methylation and/or as chaperones in the processing of ribosomal RNA . We show here that Snu13p (15.5 kD in human), a component of the U4/U6.U5 tri-snRNP, is also associated with the box C/D snoRNAs . Indeed, genetic depletion of Snu13p in yeast leads to a major defect in RNA metabolism . The box C/D motif can be folded into a stem-internal loop-stem structure, almost identical to the 15.5 kD binding site in the U4 snRNA . Consistent with this, the box C/D motif binds Snu13p/ 15.5 kD in vitro . The similarities in structure and function observed between the U4 snRNP (chaperone for U6) and the box C/D snoRNPs raises the interesting possibility that these particles may have evolved from a common ancestral RNP.

Cell, 2000 Oct 27, 103(3), 399 - 410
Two distinct pathways remove mammalian cohesin from chromosome arms in prophase and from centromeres in anaphase; Waizenegger IC et al.; In yeast, anaphase depends on cohesin cleavage . How anaphase is controlled in vertebrates is unknown because their cohesins dissociate from chromosomes before anaphase . We show that residual amounts of the cohesin SCC1 remain associated with human centromeres until the onset of anaphase when a similarly small amount of SCC1 is cleaved . In Xenopus extracts, SCC1 cleavage depends on the anaphase-promoting complex and separin . Separin immunoprecipitates are sufficient to cleave SCC1, indicating that separin is associated with a protease activity . Separin activation coincides with securin destruction and partial separin cleavage, suggesting that several mechanisms regulate separin activity . We propose that in vertebrates, a cleavage-independent pathway removes cohesin from chromosome arms during prophase, whereas a separin-dependent pathway cleaves centromeric cohesin at the metaphase-anaphase transition.

Nature, 2000 Nov 2, 408(6808), 115 - 20
Structural basis for the activation of 20S proteasomes by 11S regulators; Whitby FG et al.; Most of the non-lysosomal proteolysis that occurs in eukaryotic cells is performed by a nonspecific and abundant barrel-shaped complex called the 20S proteasome . Substrates access the active sites, which are sequestered in an internal chamber, by traversing a narrow opening (alpha-annulus) that is blocked in the unliganded 20S proteasome by amino-terminal sequences of alpha-subunits . Peptide products probably exit the 20S proteasome through the same opening . 11S regulators (also called PA26 (ref . 4), PA28 (ref . 5) and REG) are heptamers that stimulate 20S proteasome peptidase activity in vitro and may facilitate product release in vivo . Here we report the co-crystal structure of yeast 20S proteasome with the 11S regulator from Trypanosoma brucei (PA26) . PA26 carboxy-terminal tails provide binding affinity by inserting into pockets on the 20S proteasome, and PA26 activation loops induce conformational changes in alpha-subunits that open the gate separating the proteasome interior from the intracellular environment . The reduction in processivity expected for an open conformation of the exit gate may explain the role of 11S regulators in the production of ligands for major histocompatibility complex class I molecules.

AIDS Res Hum Retroviruses, 2000 Nov 1, 16(16), 1633 - 8
Pleiotropic effects of HTLV type 1 Tax protein on cellular metabolism: mitotic checkpoint abrogation and NF-kappaB activation; Iha H et al.; Tax protein expressed by human T cell leukemia virus type 1 (HTLV-1) is a strong trans-activator of its own LTR promoter; it also affects the function of multiple cellular genes involved in cell cycle control and transcription . One way in which Tax exerts its pleiotropic effects is through protein-protein interaction with cellular cofactors . By using yeast two-hybrid technology, we have isolated several cellular proteins that bind to Tax . Two of these are MAD1, a mitotic checkpoint control protein, and TXBP151, a suppressor of tumor necrosis factor alpha-induced apoptosis . Here we discuss findings describing the role of MAD1 in exit of cells from mitosis and TXBP151 in NF-kappaB activation.

Gene, 2000 Oct 31, 257(2), 223 - 32
The murine DSCR1-like (Down syndrome candidate region 1) gene family: conserved synteny with the human orthologous genes; Strippoli P et al.; A recently recognized gene family, conserved from yeast to humans, includes Down syndrome candidate region 1 gene (DSCR1), Adapt78 (recognized as the hamster ortholog of the DSCR1 isoform 4), ZAKI-4 (renamed DSCR1-like 1, DSCR1L1) and DSCR1L2 (a novel gene on human chromosome 1), along with yeast and C . elegans single members (Strippoli P., Lenzi L., Petrini M., Carinci P., Zannotti M., 2000 . A new gene family including DSCR1 (Down Syndrome Candidate Region 1) and ZAKI-4: characterization from yeast to human and identification of DSCR1-like 2, a novel human member . Genomics 64, 252-263) . The proposed family labels were a putative single-strand nucleic acid binding domain similar to the RNA recognition motif, and a unique, highly-conserved serine-proline motif . We have used a bioinformatics-driven molecular biology approach to characterize the murine members of DSCR1-like gene family . Systematic expressed-sequence-tags (EST) database search and reverse-transcription polymerase chain rection (RT-PCR) product sequencing allowed identification of the murine DSCR1, DSCR1L1 and DSCR1L2 . The sequences of the respective protein products are of 198, 197 and 241 amino acids, respectively, and are very similar to the corresponding human proteins . The very broad expression pattern of the murine DSCR1 genes is similar to that of the human genes . Using a radiation hybrid panel, we mapped the murine DSCR1-like family members . The murine DSCR1 ortholog is located on the chromosome 16, in a region corresponding to that on human chromosome 21 just upstream of the Down syndrome candidate region . DSCR1L1 and DSCR1L2 murine genes are also located in chromosomal segments of chromosome 17 and 4, respectively, exactly corresponding to those containing the respective human homologs on chromosomes 6 and 1 . Description of the mouse orthologs for DSCR1-like genes will allow knockout mice to be obtained for specific family members.

Plant Physiol, 2000 Nov, 124(3), 979 - 90
Modular domain structure of Arabidopsis COP1 . Reconstitution of activity by fragment complementation and mutational analysis of a nuclear localization signal in planta; Stacey MG et al.; The Arabidopsis COP1 protein functions as a developmental regulator, in part by repressing photomorphogenesis in darkness . Using complementation of a cop1 loss-of-function allele with transgenes expressing fusions of cop1 mutant proteins and beta-glucuronidase, it was confirmed that COP1 consists of two modules, an amino terminal module conferring a basal function during development and a carboxyl terminal module conferring repression of photomorphogenesis . The amino-terminal zinc-binding domain of COP1 was indispensable for COP1 function . In contrast, the debilitating effects of site-directed mutations in the single nuclear localization signal of COP1 were partially compensated by high-level transgene expression . The carboxyl-terminal module of COP1, though unable to substantially ameliorate a cop1 loss-of-function allele on its own, was sufficient for conferring a light-quality-dependent hyperetiolation phenotype in the presence of wild-type COP1 . Moreover, partial COP1 activity could be reconstituted in vivo from two non-covalently linked, complementary polypeptides that represent the two functional modules of COP1 . Evidence is presented for efficient association of the two sub-fragments of the split COP1 protein in Arabidopsis and in a yeast two-hybrid assay.

EMBO J, 2000 Nov 15, 19(22), 6020 - 9
Vezatin, a novel transmembrane protein, bridges myosin VIIA to the cadherin-catenins complex; Kussel-Andermann P et al.; Defects in myosin VIIA are responsible for deafness in the human and mouse . The role of this unconventional myosin in the sensory hair cells of the inner ear is not yet understood . Here we show that the C-terminal FERM domain of myosin VIIA binds to a novel transmembrane protein, vezatin, which we identified by a yeast two-hybrid screen . Vezatin is a ubiquitous protein of adherens cell-cell junctions, where it interacts with both myosin VIIA and the cadherin-catenins complex . Its recruitment to adherens junctions implicates the C-terminal region of alpha-catenin . Taken together, these data suggest that myosin VIIA, anchored by vezatin to the cadherin-catenins complex, creates a tension force between adherens junctions and the actin cytoskeleton that is expected to strengthen cell-cell adhesion . In the inner ear sensory hair cells vezatin is, in addition, concentrated at another membrane-membrane interaction site, namely at the fibrillar links interconnecting the bases of adjacent stereocilia . In myosin VIIA-defective mutants, inactivity of the vezatin-myosin VIIA complex at both sites could account for splaying out of the hair cell stereocilia.

Proc AMIA Symp . 2000;:141-5.
Graphically-enabled integration of bioinformatics tools allowing parallel execution; Cheung KH et al.; Rapid analysis of large amounts of genomic data is of great biological as well as medical interest . This type of analysis will greatly benefit from the ability to rapidly assemble a set of related analysis programs and to exploit the power of parallel computing . TurboGenomics, which is a software package currently in its alpha-testing phase, allows integration of heterogeneous software components to be done graphically . In addition, the tool is capable of making the integrated components run in parallel . To demonstrate these abilities, we use the tool to develop a Web-based application that allows integrated access to a set of large-scale sequence data analysis programs used by a transposon-insertion based yeast genome project . We also contrast the differences in building such an application with and without using the TurboGenomics software.

J Biol Chem, 2001 Mar 2, 276(9), 6695 - 702 Epub 2000 Nov 14.
Core LXXLL motif sequences in CREB-binding protein, SRC1, and RIP140 define affinity and selectivity for steroid and retinoid receptors; Heery DM et al.; An alpha-helical motif containing the sequence LXXLL is required for the ligand-dependent binding of transcriptional co-activators to nuclear receptors . By using a peptide inhibition assay, we have defined the minimal "core" LXXLL motif as an 8-amino acid sequence spanning positions -2 to +6 relative to the primary conserved leucine residue . In yeast two-hybrid assays, core LXXLL motif sequences derived from steroid receptor co-activator (SRC1), the 140-kDa receptor interacting protein (RIP140), and CREB-binding protein (CBP) displayed differences in selectivity and affinity for nuclear receptor ligand binding domains . Although core LXXLL motifs from SRC1 and RIP140 mediated strong interactions with steroid and retinoid receptors, three LXXLL motifs present in the global co-activator CBP were found to have very weak affinity for these proteins . Core motifs with high affinity for steroid and retinoid receptors were generally found to contain a hydrophobic residue at position -1 relative to the first conserved leucine and a nonhydrophobic residue at position +2 . Our results indicate that variant residues in LXXLL core motifs influence the affinity and selectivity of co-activators for nuclear receptors.

J Biol Chem, 2001 Feb 23, 276(8), 5900 - 7 Epub 2000 Nov 14.
Diacylglycerol kinase zeta in hypothalamus interacts with long form leptin receptor . Relation to dietary fat and body weight regulation; Liu Z et al.; Leptin and its long form receptor, Ob-Rb, in hypothalamic nuclei play a key role in regulating energy balance . The mutation of Ob-Rb into one of its natural variants, Ob-Ra, results in severe obesity in rodents . We demonstrate here that diacylglycerol kinase zeta (DGKzeta) interacts, via its ankyrin repeats, with the cytoplasmic portion of Ob-Rb in yeast two-hybrid systems, in protein precipitation experiments in vitro and in vivo . It does not interact, however, with the short form, Ob-Ra, which mediates the entry of leptin into the brain . Furthermore, we show by in situ hybridization that DGKzeta is expressed in neurons of hypothalamic nuclei known to synthesize Ob-Rb and to participate in energy homeostasis . The mutant ob-/ob- and db-/db- mice exhibit increased hypothalamic DGKzeta mRNA level compared with their wild-type controls, suggesting a role for the leptin/OB-Rb system in regulating DGKzeta expression . Further experiments show that hypothalamic DGKzeta mRNA level is stimulated by the consumption of a high-fat diet . In addition, DGKzeta mRNA is statistically significantly lower in rats and inbred mice that become obese on a high-fat diet compared with their lean counterparts . In fact, it is strongly, negatively correlated with both body fat and circulating levels of leptin . Taken together, our evidence suggests that DGKzeta constitutes a downstream component of the leptin signaling pathway and that reduced hypothalamic DGKzeta mRNA, and possibly activity, is associated with obesity.

Am J Med Genet, 2000 Nov 13, 95(2), 178 - 81
Mapping of X chromosome inversion breakpoints {inv(X)(q11q28)} associated with FG syndrome: a second FG locus {FGS2}?
Briault S, Villard L, Rogner U, Coy J, Odent S, Lucas J, Passage E, Zhu D, Shrimpton A, Pembrey M, Till M, Guichet A, Dessay S, Fontes M, Poustka A, Moraine C.
FG syndrome is an X-linked condition comprising mental retardation, congenital hypotonia, macrocephaly, distinctive facial changes, and constipation or anal malformations . In a linkage analysis, we mapped a major FG syndrome locus {FGS1} to Xq13, between loci DXS135 and DXS1066 . The same data, however, clearly demonstrated genetic heterogeneity . Recently, we studied a French family in which an inversion {inv(X)(q12q28)} segregates with clinical symptoms of FG syndrome . This suggests that one of the breakpoints corresponds to a second FG syndrome locus {FGS2} . We report the results of fluorescence in situ hybridization analysis performed in this family using YACs and cosmids encompassing the Xq11q12 and Xq28 regions . Two YACs, one positive for the DXS1 locus at Xq11.2 and one positive for the color vision pigment genes and G6PD loci at Xq28, were found to cross the breakpoints, respectively . We postulate that a gene might be disrupted by one of the breakpoints .

Oncogene, 2000 Nov 2, 19(46), 5291 - 7
PCNA interacts with hHus1/hRad9 in response to DNA damage and replication inhibition; Komatsu K et al.; The hHus1 and several hRad proteins are involved in the control of DNA integrity checkpoints, although the mechanisms underlying these processes are unknown . Using a yeast two-hybrid system to detect protein-protein interactions, we found that human proliferating cell nuclear antigen (PCNA), a protein known to function in both DNA replication and repair, interacts with the human checkpoint-related protein Hus1 (hHus1) . In human skin fibroblast cells, exposure to ionizing radiation of hydroxyurea triggers translocation of hHus1 from the cytosol to the nucleus, where it associates with PCNA as well as another checkpoint protein, hRad9 . This nuclear translocation and the complex formation or hHus1 with PCNA and hRad9 correlate closely with changes in cell cycle distribution in response to radiation exposure . These results suggest that this multi-protein complex may be important for coordinating cell-cycle progression, DNA replication and repair of damaged DNA.

J Cell Biol, 2000 Nov 13, 151(4), 749 - 62
Characterization of vertebrate cohesin complexes and their regulation in prophase; Sumara I et al.; In eukaryotes, sister chromatids remain connected from the time of their synthesis until they are separated in anaphase . This cohesion depends on a complex of proteins called cohesins . In budding yeast, the anaphase-promoting complex (APC) pathway initiates anaphase by removing cohesins from chromosomes . In vertebrates, cohesins dissociate from chromosomes already in prophase . To study their mitotic regulation we have purified two 14S cohesin complexes from human cells . Both complexes contain SMC1, SMC3, SCC1, and either one of the yeast Scc3p orthologs SA1 and SA2 . SA1 is also a subunit of 14S cohesin in Xenopus . These complexes interact with PDS5, a protein whose fungal orthologs have been implicated in chromosome cohesion, condensation, and recombination . The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase . Reconstitution of this process in mitotic Xenopus extracts shows that cohesin dissociation does neither depend on cyclin B proteolysis nor on the presence of the APC . Cohesins can also dissociate from chromatin in the absence of cyclin-dependent kinase 1 activity . These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.

Mol Endocrinol, 2000 Nov, 14(11), 1709 - 24
FIF {fibroblast growth factor-2 (FGF-2)-interacting-factor}, a nuclear putatively antiapoptotic factor, interacts specifically with FGF-2; Van den Berghe L et al.; Numerous evidence indicates that some of the activities of fibroblast growth factor 2 (FGF-2) depend on an intracrine mode of action . Recently, we showed that three high molecular mass (HMM) nuclear forms of FGF-2 are part of a 320-kDa protein complex while the cytoplasmic AUG-initiated form is included in a 130-kDa complex . Consequently, the characterization of FGF endogenous targets has become crucial to allow the elucidation of their endogenous activities . Through the screening of GAL4-based yeast two-hybrid expression libraries, we have isolated a gene encoding a nuclear protein of 55 kDa, FIF (FGF-2-interacting-factor), which interacts specifically with FGF-2 but not with FGF-1, FGF-3, or FGF-6 . In this system, FIF interacts equally well with the NH2-extended 24-kDa FGF form as with the 18-kDa form, indicating that the FIF-binding motif is located in the last 155 amino acids of FGF-2 . Nevertheless, coimmunoprecipitation experiments showed an exclusive association with HMM FGF-2 . The predicted protein contains a canonical leucine zipper domain and three overlapping hydrophobic heptad repeats . The region spanning these repeats is, together with a region located in the N-terminal part of the FIF protein, implicated in the binding to FGF-2 . In contrast to the full-length FIF protein, several deletion constructs were able to transactivate a lac-Z reporter gene . Furthermore, the COOH-terminal part, but not the full-length FIF protein, has previously been shown to exhibit antiapoptotic properties . Thus we discuss the possibility that these activities could reflect a physiological function of FIF through its interaction with FGF-2.

Nahrung, 2000 Oct, 44(5), 344 - 8
Legumes-whey weaning food: storage studies; el-Adawy TA et al.; Legume-whey weaning foods formulas were stored for 6 months in aluminum foil coated by polyester at room temperature (22-25 degrees C) and refrigerator (4 degrees C) . Peroxide values and free fatty acids values of weaning food formulas reached to its maximum after 3 and 2 months, respectively, and after that continuously decreased during storage . Thio-barbituric acid values of all samples increased gradually throughout the storage period at different condition of storage (room temperature and refrigerator) . The protein in-vitro digestibility of all samples was not affected during storage . Protein solubility index in distilled water, 5% NaCl and 5% sucrose decreased with different obvious rates, but bulk density of weaning food formula decreased within a very narrow ranges during storage period . Total bacterial counts as well as yeast and molds decreased gradually throughout the storage period at different conditions of storage, and cold stored samples became free from molds and yeast after 6 months of storage.

Mol Cell Biol, 2000 Dec, 20(23), 9009 - 17
Mice with an increased glucocorticoid receptor gene dosage show enhanced resistance to stress and endotoxic shock; Reichardt HM et al.; Targeted mutagenesis of the glucocorticoid receptor has revealed an essential function for survival and the regulation of multiple physiological processes . To investigate the effects of an increased gene dosage of the receptor, we have generated transgenic mice carrying two additional copies of the glucocorticoid receptor gene by using a yeast artificial chromosome . Interestingly, overexpression of the glucocorticoid receptor alters the basal regulation of the hypothalamo-pituitary-adrenal axis, resulting in reduced expression of corticotropin-releasing hormone and adrenocorticotrope hormone and a fourfold reduction in the level of circulating glucocorticoids . In addition, primary thymocytes obtained from transgenic mice show an enhanced sensitivity to glucocorticoid-induced apoptosis . Finally, analysis of these mice under challenge conditions revealed that expression of the glucocorticoid receptor above wild-type levels leads to a weaker response to restraint stress and a strongly increased resistance to lipopolysaccharide-induced endotoxic shock . These results underscore the importance of tight regulation of glucocorticoid receptor expression for the control of physiological and pathological processes . Furthermore, they may explain differences in the susceptibility of humans to inflammatory diseases and stress, depending on individual prenatal and postnatal experiences known to influence the expression of the glucocorticoid receptor.

Mol Cell Biol, 2000 Dec, 20(23), 8996 - 9008
TAP (NXF1) belongs to a multigene family of putative RNA export factors with a conserved modular architecture; Herold A et al.; Vertebrate TAP (also called NXF1) and its yeast orthologue, Mex67p, have been implicated in the export of mRNAs from the nucleus . The TAP protein includes a noncanonical RNP-type RNA binding domain, four leucine-rich repeats, an NTF2-like domain that allows heterodimerization with p15 (also called NXT1), and a ubiquitin-associated domain that mediates the interaction with nucleoporins . Here we show that TAP belongs to an evolutionarily conserved family of proteins that has more than one member in higher eukaryotes . Not only the overall domain organization but also residues important for p15 and nucleoporin interaction are conserved in most family members . We characterize two of four human TAP homologues and show that one of them, NXF2, binds RNA, localizes to the nuclear envelope, and exhibits RNA export activity . NXF3, which does not bind RNA or localize to the nuclear rim, has no RNA export activity . Database searches revealed that although only one p15 (nxt) gene is present in the Drosophila melanogaster and Caenorhabditis elegans genomes, there is at least one additional p15 homologue (p15-2 {also called NXT2}) encoded by the human genome . Both human p15 homologues bind TAP, NXF2, and NXF3 . Together, our results indicate that the TAP-p15 mRNA export pathway has diversified in higher eukaryotes compared to yeast, perhaps reflecting a greater substrate complexity.

J Biol Chem, 2001 Feb 23, 276(8), 5720 - 5 Epub 2000 Nov 09.
Inhibition of RNA polymerase III elongation by a T10 peptide nucleic acid; Dieci G et al.; The terminator elements of eukaryotic class III genes strongly contribute to overall transcription efficiency by allowing fast RNA polymerase III (pol III) recycling . Being constituted by a run of thymidine residues on the coding strand (a poly(dA) tract on the transcribed strand), pol III terminators are expected to form highly stable triple-helix complexes with oligothymine peptide nucleic acids (PNAs) . We analyzed the effect of a T10 PNA on in vitro transcription of three yeast class III genes (coding for two different tRNAs and the U6 small nuclear RNA) having termination signals of at least ten T residues . At nanomolar concentrations, the PNA almost completely inhibited transcription of supercoiled, but not linearized, templates in a sequence-specific manner . The total RNA output of the first transcription cycle was not affected by PNA concentrations strongly inhibiting multiple round transcription . Thus, an impairment of pol III recycling fully accounts for the observed inhibition . As revealed by the size and the state (free or transcription complex-associated) of the RNAs produced in PNA-inhibited reactions, pol III is "roadblocked" by the DNA-PNA adduct before reaching the terminator region . On different templates, the distance between the active site and the leading edge of the arrested polymerase ranged from 10 to 20 base pairs . Given their ability to efficiently block pol III elongation, oligothymine PNAs lend themselves as potential cell growth inhibitors interfering with eukaryotic class III gene transcription.

J Biol Chem, 2001 Feb 16, 276(7), 5059 - 67 Epub 2000 Nov 09.
Human chromatid cohesin component hRad21 is phosphorylated in M phase and associated with metaphase centromeres; Hoque MT et al.; Sister chromatids duplicated in S phase are connected with each other during G(2) and M phase until the onset of anaphase . This chromatid cohesion is essential for correct segregation of genetic material to daughter cells . Recently, understanding of the molecular mechanisms governing chromatid cohesion in yeast has been greatly advanced, whereas these processes in mammalian cells remain unclear . We report here biochemical and cytological analyses of human Rad21, a homologue of the yeast cohesin subunit, Scc1p/Mcd1p . hRad21 is a nuclear phosphorylated protein . Its abundance does not change during the cell cycle, and it becomes hyperyphosphorylated in M phase . Most hRad21 is not associated with chromatin when the nuclear envelope breakdown takes place in prophase . However, a detailed analysis of the spread chromosomes indicated that hRad21 remains associated with prometaphase-like chromosomes along their entire lengths . The mitotic chromatin-bound hRad21 becomes dissociated in a highly regulated manner because hRad21 remains specifically at the centromeres but disappears from the arm regions on metaphase-like chromosomes . Interestingly, hRad21 at the metaphase centromeres appears to be present at the inner pairing domain where the two sister chromatids are supposed to be in intimate contact . These results suggest that hRad21 has a critical role in chromatid cohesion in human mitotic cells.

J Biol Chem, 2001 Feb 16, 276(7), 5093 - 100 Epub 2000 Nov 09.
MEKK2 associates with the adapter protein Lad/RIBP and regulates the MEK5-BMK1/ERK5 pathway; Sun W et al.; MEKK2 and MEKK3 are two closely related mitogen-activated protein kinase (MAPK) kinase kinases . The kinase domains of MEKK2 and MEKK3 are nearly identical, although their N-terminal regulatory domains are significantly divergent . By yeast two-hybrid library screening, we have identified MEK5, the MAPK kinase in the big mitogen-activated protein kinase 1 (BMK1)/ERK5 pathway, as a binding partner for MEKK2 . MEKK2 expression stimulates BMK1/ERK5 activity, the downstream substrate for MEK5 . Compared with MEKK3, MEKK2 activated BMK1/ERK5 to a greater extent, which might correlate with a higher affinity MEKK2-MEK5 interaction . A dominant negative form of MEK5 blocked the activation of BMK1/ERK5 by MEKK2, whereas activation of c-Jun N-terminal kinase (JNK) was unaffected, showing that MEK5 is a specific downstream effector of MEKK2 in the BMK1/ERK5 pathway . Activation of BMK1/ERK5 by epidermal growth factor and H2O2 in Cos7 and HEK293 cells was completely blocked by a kinase-inactive MEKK3 (MEKK3kin(-)), whereas MEKK2kin(-) had no effect . However, in D10 T cells, expression of MEKK2kin(-) but not MEKK3kin(-) inhibited BMK1/ERK5 activity . Two-hybrid screening also identified Lck-associated adapter/Rlk- and Itk-binding protein (Lad/RIBP), a T cell adapter protein, as a binding partner for MEKK2 . MEKK2 and Lad/RIBP colocalize at the T cell contact site with antigen-loaded presenting cells, demonstrating cotranslocation of MEKK2 and Lad/RIBP during T cell activation . MEKK3 neither binds Lad/RIBP nor is recruited to the T cell contact with antigen presenting cell . MEKK2 and MEKK3 are differentially associated with signaling from specific upstream receptor systems, whereas both activate the MEK5-BMK1/ERK5 pathway.

RNA, 2000 Oct, 6(10), 1347 - 55
The crystal structure of HIV reverse-transcription primer tRNA(Lys,3) shows a canonical anticodon loop; Benas P et al.; We have solved to 3.3 A resolution the crystal structure of the HIV reverse-transcription primer tRNA(Lys,3) . The overall structure is exactly comparable to the well-known L-shape structure first revealed by yeast tRNA(Phe) . In particular, it unambiguously shows a canonical anticodon loop . This contradicts previous results in short RNA fragment studies and leads us to conclude that neither frameshifting specificities of tRNA(Lys) nor tRNA(Lys,3) primer selection by HIV are due to a specific three-dimensional anticodon structure . Comparison of our structure with the results of an NMR study on a hairpin representing a nonmodified anticodon stem-loop makes plausible the conclusion that chemical modifications of the wobble base U34 to 5-methoxycarbonyl-methyl-2-thiouridine and of A37 to 2-methylthio-N-6-threonylcarbamoyl-adenosine would be responsible for a canonical 7-nt anticodon-loop structure, whereas the unmodified form would result in a noncanonical UUU short triloop . The hexagonal crystal packing is remarkable and shows tight dimers of tRNAs forming a right-handed double superhelix . Within the dimers, the tRNAs are associated head-to-tail such that the CCA end of one tRNA interacts with the anticodon of the symmetry-related tRNA . This provides us with a partial view of a codon-anticodon interaction and gives insights into the positioning of residue 37, and of its posttranscriptional modifications, relative to the first base of the codon.

Life Sci, 2000 Sep 8, 67(16), 2025 - 32
First demonstration of lactoribonuclease, a ribonuclease from bovine milk with similarity to bovine pancreatic ribonuclease; Ye XY et al.; The isolation of a ribonuclease designated lactoribonuclease, with a molecular weight and an N-terminal amino acid sequence identical to those of bovine pancreatic ribonuclease, was first reported from bovine milk . After removal of globulin from acid whey by precipitation with 1.8 M (NH4)2SO4, (NH4)2SO4 was added to attain a concentration of 3.6 M . Adsorption on the ion exchanger CM-Sepharose and subsequently on Mono S by fast protein liquid chromatography yielded pure lactoribonuclease . The enzyme, like pancreatic ribonuclease, was most active at pH 7.5 with yeast transfer RNA (tRNA) as substrate . Lactoribonuclease and pancreatic ribonuclease showed a strong preference for poly(C) over poly(U) . However, pancreatic ribonuclease did so with a higher specific activity, suggesting that the two ribonucleases are not identical . No inhibitory effect was shown by either lactoribonuclease or pancreatic ribonuclease toward poly (A) and poly (G) . The effect of lactoribonuclease and pancreatic ribonuclease on tRNA increased with the concentration of tRNA . Lactoribonuclease inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of 3.5 nM while the corresponding IC50 for pancreatic ribonuclease was 0.09 nM.

Carbohydr Res, 2000 Sep 22, 328(3), 331 - 41
Structural characterization of beta-D-(1 --> 3, 1 --> 6)-linked glucans using NMR spectroscopy; Kim YT et al.; Nondestructive structural analysis of a series of beta-D-(1 --> 3, 1 --> 6)-linked glucans (laminaran, curdlan, yeast glucan, scleroglucan, etc.) was performed using two-dimensional NMR spectroscopy . The relative ratios of H-1 at different AGUs provided the information about DPn and DB . The alpha-, and beta-anomeric protons on reducing terminals were assigned at 5.02 to approximately 5.03 ppm (J 3.6 to approximately 3.7 Hz), and 4.42 to approximately 4.43 ppm (J 7.6 to approximately 7.9 Hz), respectively, whereas the H-1 protons of internal AGUs and beta-(1 --> 6)-branched AGUs appeared at 4.56 to approximately 4.59 ppm (J 7.6 to approximately 7.8 Hz), and 4.26 to approximately 4.28 ppm (J 7.6 to approximately 10.6 Hz), respectively, in a mixed solvent of 6:1 Me2SO-d6-D2O at 80 degrees C . In the solvent, the OH peaks were eliminated from the spectra allowing the H-1 protons to appear clearly . In addition, the nonreducing terminal H-1 and H-1 at the AGU next to reducing terminal could be assigned at 4.45 to approximately 4.46 ppm (J 7.8 to approximately 7.9 Hz), and 4.51 to approximately 4.53 ppm (J 7.8 Hz), respectively . The DPn of the laminaran was 33 (polydispersity 1.12) and the DB was 0.07 . The number of glucosyl units in the side chain of laminaran is more than one . The DPn and DB of the water-insoluble yeast glucan were 228 and 0.003, respectively . However the DPn of water soluble yeast glucan phosphate and curdlan was changed upon the number of freeze-drying processes and the content of water in the mixed solvent, respectively . And the DB of those were calculated as 0.02 and 0, respectively . The DB of scleroglucan was precisely calculated as 0.33, compared with the previously reported data . The H-1s at different AGUs of the various beta-D-(1 --> 3, 1 --> 6)-linked glucans having different DB can be exactly assigned by their chemical shifts in the mixed solvent system . This NMR analysis can be effectively used to determine the DP and DB of polysaccharides in a simple and non-destructive manner.

Biochem Biophys Res Commun, 2000 Nov 11, 278(1), 167 - 74
Characterization of the CIN85 adaptor protein and identification of components involved in CIN85 complexes; Watanabe S et al.; CIN85 is an 85-kDa adaptor protein whose functions in signaling pathways are presently unknown . Using the yeast two-hybrid screen, the B cell linker protein (BLNK) was identified as a binding partner of CIN85 . Coimmunoprecipitation experiments using mammalian cells revealed that CIN85 directly bound to BLNK through its SH3 domains . Immunostaining analysis showed that CIN85 and BLNK were colocalized in the cytoplasm . These results indicate a potential role of CIN85 in the B cell receptor-mediated signaling pathway . It was also found that Crk-I, Crk-II, p130(Cas), p85-PI3K, Grb2, and Sos1 were components of CIN85 complexes . CIN85 interacted with itself through its coiled-coil region, resulting in formation of a tetramer . Both the coiled-coil region and SH3 domains of CIN85 were responsible for its subcellular localization . Our data suggest that CIN85 may serve for regulation of various signaling events through formation of its diverse complexes.

Biochem Biophys Res Commun, 2000 Nov 11, 278(1), 38 - 43
Suppressor of cytokine signaling (SOCS)-3 protein interacts with the insulin-like growth factor-I receptor; Dey BR et al.; SOCS proteins are a class of proteins that are negative regulators of cytokine receptor signaling via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway . In a yeast two-hybrid screen of a human fetal brain library, we have previously identified SOCS-2 as a binding partner of the activated IGF-I receptor (IGFIR) . To test whether or not SOCS-3 also binds to the IGFIR, we cloned human SOCS-3 by reverse transcription-polymerase chain reaction from human skeletal muscle mRNA . SOCS-3 mRNA was expressed in many human fetal and adult tissues and in some human cancer cell lines (Hela, A549 pulmonary adenocarcinoma and G361 human melanoma) . We found that human SOCS-3 protein interacts directly with the cytoplasmic domains of the activated IGFIR and the insulin receptor (IR) in the yeast two-hybrid assay . In GST-SOCS-3 pull-down experiments using IGFIR from mammalian cells and in immunoprecipitation experiments in which IGFIR and FLAG-SOCS-3 were transiently expressed in human embryonic kidney 293 cells, we found that SOCS-3 interacts constitutively with IGFIR in vitro and in intact cells . Unlike SOCS-2, hSOCS-3 was phosphorylated on tyrosines in response to IGF-I addition to 293 cells . We conclude that SOCS-3 binds to the IGFIR and may be a direct substrate for the receptor tyrosine kinase .

Dev Biol, 2000 Nov 15, 227(2), 324 - 42
The murine gene, Traube, is essential for the growth of preimplantation embryos; Thomas T et al.; Little is known about the genetic control of preimplantation development . We have isolated, characterized, and mutated a previously undescribed mouse gene, Traube (Trb), essential for preimplantation development . Similar protein coding sequences are found in rats, humans, and yeast . The TRB protein contained two amino-terminal acidic domains, a leucine zipper, and three putative nuclear localization signals . The Trb gene was expressed at low levels ubiquitously early in development and became restricted to the liver and the central nervous system from E11.5 onward . Myc-tagged TRB protein was localized to the nucleus, and in a large proportion of the cells to the nucleoli . The Trb mutant embryos halted in development at the compacted morula stage at E2.5 . At E3.5 they started to decompact and a day later they disintegrated and died . The observed defect was cell autonomous, as mutant cells failed to participate in the formation of chimeric embryos . The Trb mutant embryos showed a 50% reduction of the total cell number . The mutant embryos exhibited a paucity of ribosomes, polyribosomes, and rough endoplasmic reticulum . This paucity of ribosomes together with the localization of TRB to the nucleoli, the site of ribosome synthesis, suggests that TRB is involved in the synthesis of ribosomes .

Genome Inform Ser Workshop Genome Inform, 1999, 10, 73 - 82
A Greedy Algorithm for Minimizing the Number of Primers in Multiple PCR Experiments; Doi K et al.; The selection of a suitable set of primers is very important for polymerase chain reaction (PCR) experiments . Most existing algorithms for primer selection are concerned with producing a primer pair for each DNA sequence . However, when all the DNA sequences of the target objects are already known, like the approximately 6,000 yeast ORFs, we may want to design a small set of primers to PCR amplify all the targets, which can then be resolved electrophoretically in a series of experiments . This would be quite useful, because decreasing the number of primers greatly reduces the cost of an experiment . This paper extends the problem of primer selection for a single experiment presented in Doi and Imai (Genome Informatics, 8:43-52, 1997) to primer selection for multiple PCR experiments, and proposes algorithms for the extended problem . The algorithms design primer sets one at a time . We extend the greedy algorithm for one PCR experiment in (Genome Informatics, 8:43-52, 1997) by handling amplified segments in DNA sequences that have been identified by primer pairs already selected and by changing the priorities in the greedy algorithm . This algorithm is applied to real yeast data . The number of primers equaled 85% of the number of identified DNA sequences, which represented more than 90% of all the target DNA sequences . This is 42% the number of primers needed for multiplex PCR . Furthermore, the length of each primer is less than half the length of multiplex PCR primers so the cost of producing the primers is reduced to 20% of the cost in the multiplex PCR case.

Genome Inform Ser Workshop Genome Inform, 1997, 8, 43 - 52
Greedy Algorithms for Finding a Small Set of Primers Satisfying Cover and Length Resolution Conditions in PCR Experiments; Doi K et al.; Selecting a good collection of primers is very important for polymerase chain reaction (PCR) experiments . Most existing algorithms for primer selection are concerned with computing a primer pair for each DNA sequence . In generalizing the arbitrarily primed PCR, etc., to the case that all DNA sequences of target objects are already known, like about 6000 ORFs of yeast, we may design a small set of primers so that all the targets are PCR amplified and resolved electrophoretically in a series of experiments . This is quite useful because deceasing the number of primers greatly reduces the cost of experiments . Pearson et al . (ISMB 1995: 285-291, 1995; Discrete Appl . Math . 71: 231-246, 1996) consider finding a minimum set of primers covering all given DNA sequences, but their method does not meet necessary biological conditions such as primer amplification and electrophoresis resolution . In this paper, based on the modeling and computational complexity analysis by Doi, we propose algorithms for this primer selection problem . These algorithms do not necessarily minimize the number of primers, but, since basic versions of these problems are shown to be computationally intractable, especially even for approximability with the length resolution condition, this is inevitable . In the algorithms, the amplification condition by a primer pair and the length resolution condition by electrophoresis are incorporated . These algorithms are based on the theoretically well-founded greedy algorithm for the set cover in computer science . Preliminary computational results are presented to show the validity of this approach . The number of computed primers is much less than a half of the number of targets, and hence is less than one forth of the number needed in the multiplex PCR.

J Biol Chem, 2001 Mar 2, 276(9), 6656 - 65 Epub 2000 Nov 08.
Pescadillo, a novel cell cycle regulatory protein abnormally expressed in malignant cells; Kinoshita Y et al.; Using a culture model of glial tumorigenesis, we identified a novel gene that was up-regulated in malignant mouse astrocytes following the loss of p53 . The gene represents the murine homologue of pescadillo, an uncharacterized gene that is essential for embryonic development in zebrafish . Pescadillo is a strongly conserved gene containing unique structural motifs such as a BRCA1 C-terminal domain, clusters of acidic amino acids and consensus motifs for post-translational modification by SUMO-1 . Pescadillo displayed a distinct spatial and temporal pattern of gene expression during brain development, being detected in neural progenitor cells and postmitotic neurons . Although it is not expressed in differentiated astrocytes in vivo, the pescadillo protein is dramatically elevated in malignant human astrocytomas . Yeast strains harboring temperature-sensitive mutations in the pescadillo gene were arrested in either G(1) or G(2) when grown in nonpermissive conditions, demonstrating that pescadillo is an essential gene in yeast and is required for cell cycle progression . Consistent with the latter finding, DNA synthesis was only observed in mammalian cells expressing the pescadillo protein . These results suggest that pescadillo plays a crucial role in cell proliferation and may be necessary for oncogenic transformation and tumor progression.

J Biol Chem, 2001 Feb 9, 276(6), 4298 - 303 Epub 2000 Nov 08.
Multiple C-terminal motifs of the 46-kDa mannose 6-phosphate receptor tail contribute to efficient binding of medium chains of AP-2 and AP-3; Storch S et al.; The interaction of adaptor protein (AP) complexes with signal structures in the cytoplasmic domains of membrane proteins is required for intracellular sorting . Tyrosine- or dileucine-based motifs have been reported to bind to medium chain subunits (mu) of AP-1, AP-2, or AP-3 . In the present study, we have examined the interaction of the entire 67-amino acid cytoplasmic domain of the 46-kDa mannose 6-phosphate receptor (MPR46-CT) containing tyrosine- as well as dileucine-based motifs with mu2 and mu3A chains using the yeast two-hybrid system . Both mu2 and mu3A bind specifically to the MPR46-CT . In contrast, mu3A fails to bind to the cytoplasmic domain of the 300-kDa mannose 6-phosphate receptor . Mutational analysis of the MPR46-CT revealed that the tyrosine-based motif and distal sequences rich in acidic amino acid residues are sufficient for effective binding to mu2 . However, the dileucine motif was found to be one part of a consecutive complex C-terminal structure comprising tyrosine and dileucine motifs as well as clusters of acidic residues necessary for efficient binding of mu3A . Alanine substitution of 2 or 4 acidic amino acid residues of this cluster reduces the binding to mu3A much more than to mu2 . The data suggest that the MPR46 is capable of interacting with different AP complexes using multiple partially overlapping sorting signals, which might depend on posttranslational modifications or subcellular localization of the receptor.

Drug Chem Toxicol, 2000 Nov, 23(4), 575 - 601
Role of prolactin in chloro-S-triazine rat mammary tumorigenesis; O'Connor JC et al.; Chloro-S-triazine herbicides {cyanazine (CZ), atrazine (AZ), simazine (SZ)} increase mammary tumors in Crl:CD BR rats but not in F-344 rats or in mice . A nongenotoxic mechanism was investigated since the chloro-S-triazines are negative in short-term tests for genotoxicity . An in vivo battery was used to assess the chloro-S-triazines for estrogenic activity or for their ability to increase prolactin (PRL) levels, both of which play important roles in enhancing mammary gland tumorigenesis in rodents . Ovariectomized (OVX) female rats were treated with AZ, CZ, SZ, or three CZ metabolites for 4 days via intraperitoneal injection . The pattern of responses between the chloro-S-triazines and four controls (estradiol, estriol, haloperidol, reserpine) was compared . For the 6 end-points examined, the responses from rats treated with AZ, CZ, SZ, and the metabolites of CZ most closely matched the responses from the reserpine-treated rats (a PRL rather than estrogenic mechanism) . In addition, AZ, CZ, and SZ were tested in several other in vitro models (estrogen/biogenic amine receptor competition assays and a yeast-expressed human estrogen receptor transcription assay) as well as an in vivo 24 h time-course experiment to characterize the CZ-induced increases in PRL levels . AZ, CZ, and SZ are not estrogen receptor (ER) activating compounds based on yeast transactivation and receptor competition data . CZ and AZ demonstrated marginal competition (at mM levels) to the D and alpha2 adrenergic receptors . Ligands to the D2 receptor, but not the alpha2 adrenergic receptor, are known to induce mammary tumors . CZ was also found to produce elevated PRL levels in a time-course similar to that seen with reserpine and haloperidol . Overall, the pattern of responses obtained with the chloro-S-triazines most closely matched the responses observed for reserpine . Taken together, these data suggest chloro-S-triazine-induced mammary tumors in rats are mediated through a PRL mechanism, which is thought to be of low relevance to humans.

Mol Biochem Parasitol, 2000 Oct, 110(2), 373 - 90
Identification and functional characterization of a member of the PUR-alpha family from Schistosoma mansoni; Fantappie MR et al.; Schistosoma mansoni p14 gene encodes an eggshell precursor that is expressed only in vitelline cells of mature female worms in response to a male stimulus . The upstream region of the p14 gene contains several potential cis-acting regulatory sequences . We used the upstream region of the p14 gene as bait in a yeast-one-hybrid screen of a S . mansoni cDNA library to identify interacting proteins . We report the identification and characterization of a cDNA (S . mansoni PUR-alpha (SmPUR-alpha)) encoding a protein homologous to single-stranded DNA transcription activator PUR-alpha, that binds to the p14 upstream region and activates transcription of the HIS3 reporter gene in yeast . SmPUR-alpha has a predicted molecular mass of 30 kDa and shares an overall homology of 63% with mammalian PUR-alpha . The DNA binding domain of SmPUR-alpha is highly conserved . We show by gel shift assays that GST-SmPUR-alpha binds to oligonucleotides comprising the p14 upstream region . SmPUR-alpha binds preferentially to single-stranded DNA and also binds RNA . Unlike the mammalian homologue, SmPUR-alpha exhibits little specificity for the PUR element GGn, but shows strong preference for a sequence containing alternating pyrimidines . Our data support that SmPUR-alpha is a single-copy gene and through reverse transcriptase-polymerase chain reaction and in situ hybridization, we show that SmPUR-alpha is constitutively transcribed in many cell types and thus likely plays a role as a general transcription activator in schistosomes.

J Neurosci Res, 2000 Nov 15, 62(4), 491 - 502
Interaction between two isoforms of the NF2 tumor suppressor protein, merlin, and between merlin and ezrin, suggests modulation of ERM proteins by merlin; Meng JJ et al.; The product of the neurofibromatosis type II (NF2) tumor suppressor gene, merlin, is closely related to the ezrin-radixin-moesin (ERM) family, a group of proteins believed to link the cytoskeleton to the plasma membrane . Mutation in the NF2 locus is associated with Schwann cell tumors (schwannomas) . The two predominant merlin isoforms, I and II, differ only in the carboxy-terminal 16 residues and only isoform I is anti-proliferative . Merlin lacks an actin-binding domain conserved among ezrin, radixin and moesin . Because merlin, ezrin and moesin are co-expressed in Schwann cells, and all homodimerize, we have examined whether merlin and ezrin dimerize with one another . We found by immunoprecipitation and yeast two-hybrid assays that both merlin isoforms interact with ezrin . The interaction occurs in a head-to-tail orientation, with the amino-terminal half of one protein interacting with the carboxy-terminal half of the other . The two merlin isoforms behave differently in their interaction with ezrin . Isoform I binds only ezrin whose carboxy-terminus is exposed, whereas isoform II binds ezrin regardless of whether ezrin is in the open or closed conformation . The heterodimerization of merlin is a much stronger interaction than the interaction between either merlin isoform and ezrin, and can inhibit merlin-ezrin binding . This suggests that, in vivo, merlin dimerization could regulate merlin-ERM protein interaction, and could thus indirectly regulate other interactions involving ERM proteins .

Proc Natl Acad Sci U S A, 2000 Nov 7, 97(23), 12463 - 8
The x-ray structure of D-amino acid oxidase at very high resolution identifies the chemical mechanism of flavin-dependent substrate dehydrogenation; Umhau S et al.; Flavin is one of the most versatile redox cofactors in nature and is used by many enzymes to perform a multitude of chemical reactions . d-Amino acid oxidase (DAAO), a member of the flavoprotein oxidase family, is regarded as a key enzyme for the understanding of the mechanism underlying flavin catalysis . The very high-resolution structures of yeast DAAO complexed with d-alanine, d-trifluoroalanine, and l-lactate (1.20, 1.47, and 1.72 A) provide strong evidence for hydride transfer as the mechanism of dehydrogenation . This is inconsistent with the alternative carbanion mechanism originally favored for this type of enzymatic reaction . The step of hydride transfer can proceed without involvement of amino acid functional groups . These structures, together with results from site-directed mutagenesis, point to orbital orientation/steering as the major factor in catalysis . A diatomic species, proposed to be a peroxide, is found at the active center and on the Re-side of the flavin . These results are of general relevance for the mechanisms of flavoproteins and lead to the proposal of a common dehydrogenation mechanism for oxidases and dehydrogenases.

J Virol, 2000 Dec, 74(23), 11322 - 8
Interaction between herpes simplex virus type 1 IE63 protein and cellular protein p32; Bryant HE et al.; The herpes simplex virus type 1 (HSV-1) immediate-early gene IE63 (ICP27), the only HSV-1 regulatory gene with a homologue in every mammalian and avian herpesvirus sequenced so far, is a multifunctional protein which regulates transcriptional and posttranscriptional processes . One of its posttranscriptional effects is the inhibition of splicing of viral and cellular transcripts . We previously identified heterogeneous nuclear ribonucleoprotein (hnRNP) K and casein kinase 2 (CK2) as two protein partners of IE63 (H . Bryant et al., J . Biol . Chem . 274:28991-28998, 1999) . Here, using a yeast two-hybrid assay, we identify another partner of IE63, the cellular protein p32 . Confirmation of this interaction was provided by coimmunoprecipitation from virus-infected cells and recombinant p32 binding assays . A p32-hnRNP K-CK2 complex, which required IE63 to form, was isolated from HSV-1-infected cells, and coimmunoprecipitating p32 was phosphorylated by CK2 . Expression of IE63 altered the cytoplasmic distribution of p32, with some now colocalizing with IE63 in the nuclei of infected and transfected cells . As p32 copurifies with splicing factors and can inhibit splicing, we propose that IE63 together with p32, possibly with other IE63 partner proteins, acts to disrupt or regulate pre-mRNA splicing . As well as contributing to host cell shutoff, this effect could facilitate splicing-independent nuclear export of viral transcripts.

J Virol, 2000 Dec, 74(23), 11270 - 7
Human T-lymphotropic virus type 1 p30(II) functions as a transcription factor and differentially modulates CREB-responsive promoters; Zhang W et al.; Human T-lymphotropic virus type 1 (HTLV-1), a complex retrovirus, causes adult T-cell lymphoma/leukemia and is linked to a variety of immune-mediated disorders . The roles of proteins encoded in the pX open reading frame (ORF) II gene region in HTLV-1 replication or in mediating virus-associated diseases remain to be defined . A nucleus-localizing 30-kDa protein, p30(II), encoded within pX ORF II has limited homology with the POU family of transcription factors . Recently, we reported that selected mutations in pX ORF II diminish the ability of HTLV-1 to maintain high viral loads in infected rabbits . Herein we have tested the transcriptional ability of p30(II) in mammalian cells by using yeast Gal4 fusion protein vectors and transfection of luciferase reporter genes driven by CREB-responsive promoters . p30(II) as a Gal4 DNA-binding domain (DBD) fusion protein transactivates Gal4-driven luciferase reporter gene activity up to 25-fold in 293 and HeLa-tat cells . We confirmed nuclear localization of p30(II) and demonstrate dose-dependent binding of p30(II)-Gal4(DBD) to Gal4 DNA-binding sites . The transcriptional activity of p30(II)-Gal4(DBD) was independent of TATA box flanking sequences, as shown by using two different Gal4 reporter systems . Studies of selected p30(II) mutants indicated that domains that mediate transcription are restricted to a central core region of the protein between amino acids 62 and 220 . Transfection of a p30(II)-expressing plasmid repressed cellular CRE-driven reporter gene activity, with or without Tax expression . In contrast, p30(II) at lower concentrations enhanced HTLV-1 long terminal repeat-driven reporter gene activity independent of Tax expression . These data are the first to demonstrate a transcriptional function for p30(II) and suggest a mechanism by which this nuclear protein may influence HTLV-1 replication or cellular gene expression in vivo.

J Virol, 2000 Dec, 74(23), 11027 - 39
The carboxy-terminal fragment of nucleolin interacts with the nucleocapsid domain of retroviral gag proteins and inhibits virion assembly; Bacharach E et al.; A yeast two-hybrid screen for cellular proteins that interact with the murine leukemia virus (MuLV) Gag protein resulted in the identification of nucleolin, a host protein known to function in ribosome assembly . The interacting fusions contained the carboxy-terminal 212 amino acids of nucleolin {Nuc(212)} . The nucleocapsid (NC) portion of Gag was necessary and sufficient to mediate the binding to Nuc(212) . The interaction of Gag with Nuc(212) could be demonstrated in vitro and was manifested in vivo by the NC-dependent incorporation of Nuc(212) inside MuLV virions . Overexpression of Nuc(212), but not full-length nucleolin, potently and specifically blocked MuLV virion assembly and/or release . A mutant of MuLV, selected to specifically disrupt the binding to Nuc(212), was found to be severely defective for virion assembly . This mutant harbors a single point mutation in capsid (CA) adjacent to the CA-NC junction, suggesting a role for this region in Moloney MuLV assembly . These experiments demonstrate that selection for proteins that bind assembly domain(s) can yield potent inhibitors of virion assembly . These experiments also raise the possibility that a nucleolin-Gag interaction may be involved in virion assembly.

Genes Dev, 2000 Nov 1, 14(21), 2712 - 24
Regulation of cellular growth by the Drosophila target of rapamycin dTOR; Zhang H et al.; The TOR protein kinases (TOR1 and TOR2 in yeast; mTOR/FRAP/RAFT1 in mammals) promote cellular proliferation in response to nutrients and growth factors, but their role in development is poorly understood . Here, we show that the Drosophila TOR homolog dTOR is required cell autonomously for normal growth and proliferation during larval development, and for increases in cellular growth caused by activation of the phosphoinositide 3-kinase (PI3K) signaling pathway . As in mammalian cells, the kinase activity of dTOR is required for growth factor-dependent phosphorylation of p70 S6 kinase (p70(S6K)) in vitro, and we demonstrate that overexpression of p70(S6K) in vivo can rescue dTOR mutant animals to viability . Loss of dTOR also results in cellular phenotypes characteristic of amino acid deprivation, including reduced nucleolar size, lipid vesicle aggregation in the larval fat body, and a cell type-specific pattern of cell cycle arrest that can be bypassed by overexpression of the S-phase regulator cyclin E . Our results suggest that dTOR regulates growth during animal development by coupling growth factor signaling to nutrient availability.

J Cell Sci, 2000 Dec, 113 Pt 23, 4263 - 73
Dynein light chain interacts with NRF-1 and EWG, structurally and functionally related transcription factors from humans and drosophila; Herzig RP et al.; Nuclear respiratory factor-1 is a transcriptional activator that has been implicated in the nuclear control of respiratory chain expression . Yeast two-hybrid screens were performed to identify proteins that physically interact with nuclear respiratory factor-1 . Saturation screening of both mouse embryo and mouse testis libraries yielded 14 independent clones, all of which represented two different isoforms of dynein light chain . In addition to using the two-hybrid method, the specificity of the nuclear respiratory factor-1/dynein light chain interaction was established by chemical crosslinking of the purified native proteins and by co-immunoprecipitation of nuclear respiratory factor-1 and dynein light chain from mammalian cells . Both two-hybrid and chemical crosslinking assays demonstrated that binding of dynein light chain required the first 26 amino acids of nuclear respiratory factor-1 . Although dynein light chain is associated with dynein, a cytoplasmic motor molecule, immunolocalizations showed substantial nuclear staining using several different anti-dynein light chain antibodies . Moreover, fluorescence overlays of confocal images established that nuclear respiratory factor-1 and dynein light chain displayed a very similar nuclear staining pattern . The significance of the nuclear respiratory factor-1/dynein light chain interaction was investigated further by determining whether a similar interaction was conserved between dynein light chain and the erect wing gene product of Drosophila, a protein related to nuclear respiratory factor-1 through its DNA binding domain . Here, we establish that the erect wing gene product can bind and trans-activate transcription through authentic nuclear respiratory factor-1 binding sites . Moreover, the erect wing gene product, like nuclear respiratory factor-1, interacted specifically with dynein light chain both in vitro and in transfected cells . Thus, the interaction with dynein light chain is conserved between transcription factors that are structurally and functionally similar between humans and Drosophila.

J Cell Sci, 2000 Dec, 113 Pt 23, 4253 - 61
The genomic silencing of position-effect variegation in Drosophila melanogaster: interaction between the heterochromatin-associated proteins Su(var)3-7 and HP1; Delattre M et al.; Position-effect variegation results from mosaic silencing by chromosomal rearrangements juxtaposing euchromatin genes next to pericentric heterochromatin . An increase in the amounts of the heterochromatin-associated Su(var)3-7 and HP1 proteins augments silencing . Using the yeast two-hybrid protein interaction trap system, we have isolated HP1 using Su(var)3-7 as a bait . We have then delimited three binding sites on Su(var)3-7 for HP1 . On HP1, the C-terminal moiety, including the chromo shadow domain, is required for interaction . In vivo, both proteins co-localise not only in heterochromatin, but also in a limited set of sites in euchromatin and at telomeres . When delocalised to the sites bound by the protein Polycomb in euchromatin, HP1 recruits Su(var)3-7 . Finally, and in contrast with euchromatin genes, a decrease in the amounts of both proteins enhances variegation of the light gene, one of the few genetic loci mapped within pericentric heterochromatin . This body of data supports a direct link between Su(var)3-7 and HP1 in the genomic silencing of position-effect variegation.

Plant J, 2000 Nov, 24(3), 397 - 411
Transient expression and heat-stress-induced co-aggregation of endogenous and heterologous small heat-stress proteins in tobacco protoplasts; Kirschner M et al.; Heat-stress granules (HSG) are highly ordered, cytoplasmic chaperone complexes found in all heat-stressed plant cells . We have developed an experimental system involving expression of cytosolic class I and class II small heat-stress proteins (Hsps) of pea, Arabidopsis and tomato in tobacco protoplasts to study the structural prerequisites for the assembly of HSG or HSG-like complexes . Class I and class II small Hsps formed class-specific dodecamers of 210-280 kDa, which, upon heat stress, were incorporated into HSG complexes . Interestingly, class II dodecamers alone could form HSG-like complexes (auto-aggregation), whereas class I dodecamers could do so only in the presence of class II proteins (recruitment) . By analysing C-terminal deletion forms of Hsp17 class II, we obtained evidence that the intact C-terminus is critical for the oligomerization state, for the heat-stress-induced auto-aggregation and for recruitment of class I proteins . The class-specific formation of dimers as a prerequisite for oligomerization was analysed by the yeast two-hybrid system . In the presence of the endogenous (tobacco) set of heat-stress-induced proteins, all heterologous class I and class II proteins were incorporated into HSG complexes, whose ultrastructure was different from that of complexes formed by class I and class II proteins alone . Although other, more distantly related, members of the Hsp20 family, i.e . the plastidic pea Hsp21, the Drosophila Hsp23 and the mouse Hsp25, were well expressed in tobacco protoplasts and formed homo-oligomers of 200-700 kDa, none of them could be recruited to HSG complexes.

Plant J, 2000 Nov, 24(3), 305 - 16
A potato alpha-glucosidase gene encodes a glycoprotein-processing alpha-glucosidase II-like activity . Demonstration of enzyme activity and effects of down-regulation in transgenic plants; Taylor MA et al.; In order to elucidate more fully the function of a potato gene (MAL1) encoding alpha-glucosidase activity, transgenic plants in which MAL1 expression was down-regulated were generated using antisense technology . In transgenic lines severely down-regulated in the expression of MAL1, total alpha-glucosidase activity was not decreased in leaves and tubers, and the contents of starch, glucose, fructose and sucrose remained unchanged in tubers . Phylogenetic analysis indicated that the MAL1 gene product was more similar to the glycoprotein-processing alpha-glucosidase II of mammalian and yeast origin than to other plant alpha-glucosidases . Using {14C-Glc}-labelled Glc2Man9GlcNAc2 as a substrate, it was demonstrated that glucosidase II activity was markedly down-regulated in microsomes isolated from tubers of four independent antisense lines studied in detail, strongly suggesting that MAL1 encodes glucosidase II activity . In field trials (but not in the glasshouse), MAL1 down-regulation produced an extremely stunted phenotype - the leaves were curled and tuber yield was decreased by 90% compared to control values . Microscopic analysis of leaves revealed significant differences between the antisense and control samples . Plants with down-regulated glucosidase II activity showed a greater degree of plasmolysis, and an increase in the size of mesophyll intracellular spaces . Analysis of cell walls also indicated changes in structure as a result of MAL1 down-regulation . In leaves from four antisense lines, the steady-state transcript level corresponding to the endoplasmic reticulum chaperone, BiP, was enhanced . This is diagnostic of stress in the endoplasmic reticulum.

Lett Appl Microbiol, 2000 Nov, 31(5), 400 - 3
A single PCR-restriction endonuclease analysis for rapid identification of Malassezia species; Guillot J et al.; AIMS: The present study describes a system based on PCR and restriction endonuclease analysis (REA) to distinguish the seven currently recognized Malassezia species . METHODS AND RESULTS: Fifty-five representative yeast isolates were examined . A single primer pair was designed to amplify the large subunit ribosomal RNA (LSU rRNA) gene of the seven Malassezia species, and identification was achieved by digestion of the PCR products with three restriction endonucleases: BanI, HaeII and MspI . A specific restriction endonuclease analysis pattern was determined for each species investigated . Moreover, PCR-REA allowed the detection and characterization of mixtures of several Malassezia species . CONCLUSION: PCR-REA of only the LSU rRNA gene is a reliable and rapid method to distinguish all Malassezia species . SIGNIFICANCE AND IMPACT OF THE STUDY: PCR-REA represents a considerable saving in time over currently available identification procedures . This method should be evaluated on clinical material directly.

J Exp Zool, 2000 Oct 15, 288(3), 193 - 204
p68, a DEAD-box RNA helicase, is expressed in chordate embryo neural and mesodermal tissues; Seufert DW et al.; The p68 DEAD-box RNA helicases have been identified in diverse organisms, including yeast, invertebrates, and mammals . DEAD-box RNA helicases are thought to unwind duplexed RNAs, and the p68 family may participate in initiating nucleolar assembly . Recent evidence also suggests that they are developmentally regulated in chordate embryos . bobcat, a newly described member of this gene family, has been found in eggs and developing embryos of the ascidian urochordate, Molgula oculata . Antisense RNA experiments have implicated this gene in establishing basic chordate features, including the notochord and neural tube in ascidians (Swalla et al . 1999) . We have isolated p68 homologs from chick and Xenopus in order to investigate their possible role in vertebrate development . We show that embryonic expression of p68 in chick, frog, and ascidian embryos is high in the developing brain and spinal cord as well as in the sensory vesicles . In frog embryos, p68 expression also marks the streams of migrating cranial neural crest cells throughout neural tube development and in tailbud stages, but neural crest expression is faint in chick embryos . Ascidian embryos also show mesodermal p68 expression during gastrulation and neurulation, and we document some p68 mesodermal expression in both chick and frog . Thus, as shown in these studies, p68 is expressed in early neural development and in various mesodermal tissues in a variety of chordate embryos, including chick, frog, and ascidian . Further functional experiments will be necessary to understand the role(s) p68 may play in vertebrate development.

Curr Biol, 2000 Oct 19, 10(20), 1295 - 8
Alternative splicing of dystrobrevin regulates the stoichiometry of syntrophin binding to the dystrophin protein complex; Newey SE et al.; Dystrophin coordinates the assembly of a complex of structural and signalling proteins that is required for normal muscle function . A key component of the dystrophin-associated protein complex (DPC) is alpha-dystrobrevin, a dystrophin-related and -associated protein whose absence results in muscular dystrophy and neuromuscular junction defects {1,2} . The current model of the DPC predicts that dystrophin and dystrobrevin each bind a single syntrophin molecule {3} . The syntrophins are PDZ-domain-containing proteins that facilitate the recruitment of signalling proteins such as nNOS (neuronal nitric oxide synthase) to the DPC {4} . Here we show, using yeast two-hybrid analysis and biochemical binding studies, that alpha-dystrobrevin in fact contains two independent syntrophin-binding sites in tandem . The previously undescribed binding site is situated within an alternatively spliced exon of alpha-dystrobrevin, termed the variable region-3 (vr3) sequence, which is specifically expressed in skeletal and cardiac muscle {5,6} . Analysis of the syntrophin-binding region of dystrobrevin reveals a tandem pair of predicted alpha helices with significant sequence similarity . These alpha helices, each termed a syntrophin-binding motif, are also highly conserved in dystrophin and utrophin . Together these data show that there are four potential syntrophin-binding sites per dystrophin complex in skeletal muscle: two on dystrobrevin and two on dystrophin or utrophin . Furthermore, alternative splicing of dystrobrevin provides a mechanism for regulating the stoichiometry of syntrophin association with the DPC . This is likely to have important consequences for the recruitment of specific signalling molecules to the DPC and ultimately for its function.

J Biol Chem, 2000 Dec 29, 275(52), 40910 - 5
Characterization of a novel trans-activation domain of BRCA1 that functions in concert with the BRCA1 C-terminal (BRCT) domain; Hu YF et al.; Mutations in the breast cancer susceptibility gene, BRCA1, account for a significant proportion of hereditary breast and ovarian cancers . The BRCA1 C-terminal (BRCT) domain, which can activate transcription when fused to a heterologous DNA binding domain, is required for BRCA1 function in suppression of tumorigenesis . Here, we provide evidence for a new activation domain in BRCA1 that lies adjacent to the BRCT domain . We name the two domains AD1 and AD2, respectively . Like AD2, the newly discovered AD1 can act independently as an activation domain in both yeast and human cells . However, unlike AD2, AD1 activity in mammalian cells is cell type context-dependent . Furthermore, combination of these two domains in mammalian cells can result in a robust synergy in transcriptional activation . A highly conserved coiled-coil motif in AD1 is required for the cooperative transcription activation . Interestingly, the functional cooperativity between AD1 and AD2 is absent in certain breast and ovarian cancer cell lines, although each domain can still activate transcription . Therefore, the differential and cooperative actions of the two activation modules may contribute to the heterogeneous risk of BRCA1 mutations in different tissues.

Genes Chromosomes Cancer, 2000 Dec, 29(4), 356 - 62
Expression and mutational analyses of the human MAD2L1 gene in breast cancer cells; Percy MJ et al.; Breast cancer is a heterogeneous disorder in which most tumors display some degree of aneuploidy, especially those at later stages of the disease . Aneuploidy and associated chromosome instability may be important in the progression of mammary tumorigenesis . Aneuploidy is prevented during normal cell division in part through regulation of a mitotic spindle checkpoint where mitotic arrest prevents segregation of misaligned chromosomes into daughter cells at anaphase . Mitotic arrest genes, including the MAD family, which was originally characterized in yeast, help regulate normal function of the mitotic spindle checkpoint . Decreased expression of the human gene MAD2L1 was previously reported in a breast cancer cell line exhibiting chromosome instability and aneuploidy . To explore further the potential role of MAD2L1 in breast cancer, we analyzed MAD2L1 gene expression in 13 minimally to grossly aneuploid human breast cancer cell lines and found significant differences of expression in three lines . Sequence analysis of MAD2L1 cDNA in these as well as nine additional aneuploid breast cancer and five immortalized normal human mammary epithelial cell lines revealed one heterozygous frameshift (572 del A) mutation in a cancer cell line that demonstrated a high level of transcript expression . In addition, two 3'UTR sequence variants were noted in breast cancer cell lines . The 572 del A mutation creates a truncated MAD2 protein product . Further functional studies in primary breast tumors are therefore warranted to determine the potential role MAD2L1 may play in breast cancer .

Biotechnol Bioeng, 2000 Dec 20, 70(6), 638 - 46
Stability and activity of alcohol dehydrogenases in W/O-microemulsions: enantioselective reduction including cofactor regeneration; Orlich B et al.; Microemulsions provide an interesting alternative to classical methods for the conversion of less water-soluble substrates by alcohol dehydrogenase, but until now stability and activity were too low for economically useful processes . The activity and stability of the enzymes are dependent on the microemulsion composition, mostly the water and the surfactant concentration . Therefore, it is necessary to know the exact phase behavior of a given microemulsion reaction system and the corresponding enzyme behavior therein . Because of their economic and ecologic suitability polyethoxylated fatty alcohols were investigated concerning their phase behavior and their compatibility with enzymes in ternary mixtures . The phase behavior of Marlipal O13-60 (C13EO6 in industrial quality)/cyclohexane/water and its effect on the activity and stability of alcohol dehydrogenase from Yeast (YADH) and horse liver (HLADH) and the carbonyl reductase from Candida parapsilosis (CPCR) is presented in this study . Beside the macroscopic phase behavior of the reaction system, the viscosity of the system indicates structural changes of aggregates in the microemulsion . The changes of the enzyme activities with the composition are discussed on the basis of transitions from reverse micelles to swollen reverse micelles and finally, the transition to the phase separation . The formate dehydrogenase from Candida boidinii was used for the NADH-regeneration during reduction reactions . While the formate dehydrogenase did not show any kinetic effect on the microemulsion composition, the other enzymes show significant changes of activity and stability varying the water or surfactant concentration of the microemulsion . Under certain conditions, stability could be maintained with HLADH for several weeks . Successful experiments with semi-batch processes including cofactor regeneration and product separation were performed.

Mol Cell Endocrinol, 2000 Oct 25, 168(1-2), 21 - 9
Apparent coactivation due to interference of expression constructs with nuclear receptor expression; Hofman K et al.; Transient cotransfection in COS-7 cells, a standard approach to demonstrate coactivation, was used to study the coactivation properties of NuRIP183, a new nuclear receptor interacting protein of 183 kDa, isolated by a yeast two-hybrid screening . Transfection with a NuRIP183 expression construct strongly increased the ligand-dependent response of reporter constructs for several nuclear receptors when compared to transfection with the empty expression vector . A more detailed study, however, revealed major changes in the expression level of the nuclear receptors in cotransfection experiments, indicating that the observed changes in receptor activity were not due to coactivation but to differences in receptor concentration caused by interference from the cotransfected expression constructs with the expression of the receptor . Such interference, which is inversely related to the length of the insert, was observed, not only in COS-7 cells but also in CV-1 and MCF-7 cells, using different transfection techniques (FuGENE-6 and calcium phosphate) and different expression vectors (pSG5, pcDNA1.1 and pIRESneo) . These data cast some doubt on coactivation of nuclear receptors based on similar cotransfection experiments without measurement of receptor concentration . Moreover, it is recommended to limit the amounts of (co)transfected expression plasmid and to avoid the use of empty expression plasmid as a control . Finally, one should be aware of similar misleading results in other experimental set-ups based on cotransfection.

Cancer Genet Cytogenet, 2000 Sep, 121(2), 133 - 8
Reciprocal translocation (3;5)(q26;q22) and possible BCHE gene involvement in an unusual myelogenous disorder with both myeloproliferative and dysplastic features; Fureder W et al.; We report on a 77-year-old male patient who presented with an unusual myelogenous disorder exhibiting both myeloproliferative and dysplastic features . The patient suffered from leukocytosis, eosinophilia, basophilia, transfusion dependent anemia, and rapidly progressing thrombocytopenia . Classical chromosome analysis and fluorescence in situ hybridization (FISH) revealed a reciprocal t(3;5)(q26;q22) . Using yeast artificial chromosome (YAC) probes, the breakpoint on chromosome 3 was localized to the butyrylcholinesterase (BCHE) gene (3q26.1-q26.2) . This gene has recently been implicated in the regulation of myeloid cells . Whether the BCHE gene was also involved in the deregulation of myelopoiesis, causing the unusual clinical picture in this case, remains unknown.

Cancer Genet Cytogenet, 2000 Sep, 121(2), 128 - 32
Unusual breakpoint distribution of 8p abnormalities in T-prolymphocytic leukemia: a study with YACS mapping to 8p11-p12; Sorour A et al.; Chromosome 8 abnormalities are seen in 80% of patients with T-cell prolymphocytic leukemia (T-PLL) . The abnormalities described are idic(8)(p11),t(8;8)(p11;q12),+8, and 8p+ with the involvement of 8p . To localize 8p11-p12 breakpoints in T-PLL, metaphases from seven cases were karyotyped . Those with idic(8)(p11) and add(8)(p11) were probed with a panel of contiguous YACs derived from 8p11-p12 using fluorescence in situ hybridization (FISH) . Analysis of FISH results showed that 8p11-p12 breakpoints cluster into two regions . The first region is telomeric to YAC 899e2, which contains the fibroblast growth factor receptor-1 gene (FGFR1) and appears to cluster within a 1.5-MB YAC 807a2 . The second region is more centromeric with breakpoints on either side of YAC 806e9, flanked by YAC 940f10 distally and YAC 910d7 proximally, the latter containing the MOZ gene . These findings showed that a segment of 8p was still present in the isodicentric, but the pattern of clustering does not seem to correspond to a breakpoint affecting a single gene . The clustering regions are likely to be hot spots for recombination and result in idic(8)(p11) and 8p+ . These changes point to the pathogenesis of T-PLL involving deletion of a gene sequence on 8p and/or gain of a copy of 8q.

Cancer Genet Cytogenet, 2000 Sep, 121(2), 117 - 23
Establishment of a human malignant fibrous histiocytoma cell line, COMA . Characterization By conventional cytogenetics, comparative genomic hybridization, and multiplex fluorescence In situ hybridization; Mairal A et al.; The human COMA cell line has been established from a storiform pleomorphic malignant fibrous histiocytoma (MFH) . As expected for this tumor type, a very complex karyotype was observed after R-banding analysis . An extensive analysis by 24-color painting, comparative genomic hybridization (CGH), and fluorescence in situ hybridization (FISH) was performed . Twelve complex marker chromosomes recurrently observed were clearly identified; among them, three were systematically present in all analyzed metaphases . Amplifications detected by CGH were refined by FISH with probes specific for various candidate loci . A significant aneuploidy and numerous micronuclei were observed, which could be related to the anomalies of centriole numbers detected in a proportion of cells . Such an analysis, performed on a series of MFH cell lines, would allow the delineation of the genomic alterations specific for the oncogenesis or progression of this complex tumor type or both.

Genetics, 2000 Nov, 156(3), 1349 - 62
GL3 encodes a bHLH protein that regulates trichome development in arabidopsis through interaction with GL1 and TTG1; Payne CT et al.; Arabidopsis trichome development and differentiation is a well-studied model for plant cell-fate determination and morphogenesis . Mutations in TRANSPARENT TESTA GLABRA1 (TTG1) result in several pleiotropic defects including an almost complete lack of trichomes . The complex phenotype caused by ttg1 mutations is suppressed by ectopic expression of the maize anthocyanin regulator R . Here it is demonstrated that the Arabidopsis trichome development locus GLABRA3 (GL3) encodes an R homolog . GL3 and GLABRA1 (GL1) interact when overexpressed together in plants . Yeast two-hybrid assays indicate that GL3 participates in physical interactions with GL1, TTG1, and itself, but that GL1 and TTG1 do not interact . These data suggest a reiterated combinatorial model for the differential regulation of such diverse developmental pathways as trichome cell-fate determination, root hair spacing, and anthocyanin secondary metabolism.

Genetics, 2000 Nov, 156(3), 1157 - 67
Two genes become one: the genes encoding heterochromatin protein Su(var)3-9 and translation initiation factor subunit eIF-2gamma are joined to a dicistronic unit in holometabolic insects; Krauss V et al.; The Drosophila suppressor of position-effect variegation Su(var)3-9 encodes a heterochromatin-associated protein that is evolutionarily conserved . In contrast to its yeast and mammalian orthologs, the Drosophila Su(var)3-9 gene is fused with the locus encoding the gamma subunit of translation initiation factor eIF2 . Synthesis of the two unrelated proteins is resolved by alternative splicing . A similar dicistronic Su(var)3-9/eIF-2gamma transcription unit was found in Clytus arietis, Leptinotarsa decemlineata, and Scoliopterix libatrix, representing two different orders of holometabolic insects (Coleoptera and Lepidoptera) . In all these species the N terminus of the eIF-2gamma, which is encoded by the first two exons, is fused to SU(VAR)3-9 . In contrast to Drosophila melanogaster, RT-PCR analysis in the two coleopteran and the lepidopteran species demonstrated the usage of a nonconserved splice donor site located within the 3' end of the SU(VAR)3-9 ORF, resulting in removal of the Su(var)3-9-specific stop codon from the mRNA and complete in-frame fusion of the SU(VAR)3-9 and eIF-2gamma ORFs . In the centipede Lithobius forficatus eIF-2gamma and Su(var)3-9 are unconnected . Conservation of the dicistronic Su(var)3-9/eIF-2gamma transcription unit in the studied insects indicates its origin before radiation of holometabolic insects and represents a useful tool for molecular phylogenetic analysis in arthropods.

Genetics, 2000 Nov, 156(3), 1025 - 33
Self-compatible B mutants in coprinus with altered pheromone-receptor specificities; Olesnicky NS et al.; A successful mating in the mushroom Coprinus cinereus brings together a compatible complement of pheromones and G-protein-coupled receptors encoded by multiallelic genes at the B mating-type locus . Rare B gene mutations lead to constitutive activation of B-regulated development without the need for mating . Here we characterize a mutation that arose in the B6 locus and show that it generates a mutant receptor with a single amino acid substitution (R96H) at the intracellular end of transmembrane domain III . Using a heterologous yeast assay and synthetic pheromones we show that the mutation does not make the receptor constitutively active but permits it to respond inappropriately to a normally incompatible pheromone encoded within the same B6 locus . Parallel experiments carried out in Coprinus showed that a F67W substitution in this same pheromone enabled it to activate the normally incompatible wild-type receptor . Together, our experiments show that a single amino acid replacement in either pheromone or receptor can deregulate the specificity of ligand-receptor recognition and confer a self-compatible B phenotype . In addition, we use the yeast assay to demonstrate that different receptors and pheromones found at a single B locus belong to discrete subfamilies within which receptor activation cannot normally occur.

J Biol Chem, 2001 Feb 2, 276(5), 3550 - 4 Epub 2000 Nov 03.
Molecular mechanism of hypoxia-inducible factor 1alpha -p300 interaction . A leucine-rich interface regulated by a single cysteine; Gu J et al.; Hypoxia-inducible factor 1alpha (HIF1alpha) plays a pivotal role in embryogenesis, angiogenesis, and tumorigenesis . HIF1alpha-mediated transcription requires the coactivator p300, at least in part, through interaction with the cysteine- and histidine-rich 1 domain of p300 . To understand the molecular basis of this interaction, we have developed a random mutagenesis screen in yeast approach for efficient identification of residues that are functionally critical for protein interactions . As a result, four residues (Leu-795, Cys-800, Leu-818, and Leu-822) in the C-terminal activation domain of HIF1alpha have been identified as crucial for HIF1 transactivation in mammalian systems . Moreover, data from residue substitution experiments indicate the stringent necessity of leucine and hydrophobic cysteine for C-terminal activation domain function . Likewise, Leu-344, Leu-345, Cys-388, and Cys-393 in the cysteine- and histidine-rich 1 domain of p300 have also been shown to be essential for the functional interaction . We propose that hypoxia-induced HIF1alpha-p300 interaction relies upon a leucine-rich hydrophobic interface that is regulated by the hydrophilic and hydrophobic sulfhydryls of HIF1alpha Cys-800.

J Anim Sci, 2000 Nov, 78(11), 2950 - 6
Chromium supplementation does not influence glucose metabolism or insulin action in response to cold exposure in mature sheep; Sano H et al.; An isotope dilution method using {U-(13)C} glucose infusion and a glucose clamp approach were applied to determine the effects of supplemental Cr and cold exposure on blood glucose turnover rate and tissue responsiveness and sensitivity to insulin in eight sheep . The daily profiles of blood metabolites and hormones were also determined . The sheep consumed diets containing either 0 or 1 mg of Cr/kg from a high-Cr yeast and were exposed from a thermoneutral environment (20 degrees C) to a cold environment (0 to 4 degrees C) for 9 d . The experiment used a crossover design . Body weight was lost (P = 0.02) during cold exposure, regardless of Cr supplementation . Blood glucose turnover rate and the maximal glucose infusion rate did not differ between diets, but both were higher (P = 0.0004 and P = 0.0001, respectively) during cold exposure than in the thermoneutral environment . The plasma insulin concentration at half-maximal glucose infusion rate changed with neither diet nor environment . Plasma concentrations of glucose and NEFA increased (P < 0.05) during cold exposure for both diets . In sheep, Cr supplementation, 1 mg/kg of diet as high-Cr yeast, has little influence on blood glucose metabolism and insulin action, whereas cold exposure enhances both without further modification by Cr supplementation.

Mamm Genome, 2000 Nov, 11(11), 1024 - 9
Genome scan identifies a locus affecting gamma-globin level in human beta-cluster YAC transgenic mice; Lin SD et al.; Genetic factors affecting postnatal gamma-globin expression--a major modifier of the severity of both beta-thalassemia and sickle cell anemia--have been difficult to study . This is especially so in mice, an organism lacking a globin gene with an expression pattern equivalent to that of human gamma-globin . To model the human beta-cluster in mice, with the goal of screening for loci affecting human gamma-globin expression in vivo, we introduced a human beta-globin cluster YAC transgene into the genome of FVB/N mice . The beta-cluster contained a Greek hereditary persistence of fetal hemoglobin (HPFH) gamma allele, resulting in postnatal expression of human gamma-globin in transgenic mice . The level of human gamma-globin for various F1 hybrids derived from crosses between the FVB/N transgenics and other inbred mouse strains was assessed . The gamma-globin level of the (C3HeB/FeJ x FVB/N)F1 transgenic mice was noted to be significantly elevated . To map genes affecting postnatal y-globin expression, we performed a 20-centiMorgan (cM) genome scan of a (C3HeB/FeJ x FVB/N)F1 transgenics x FVB/N backcross, followed by high-resolution marker analysis of promising loci . From this analysis we mapped a locus within an 18-cM interval of mouse Chromosome (Chr) 1 (LOD = 4.3) that contributes 10.9% of variation in gamma-globin level . Combining transgenic modeling of the human beta-globin gene cluster with quantitative trait analysis, we have identified and mapped a murine locus that impacts on human gamma-globin level in vivo.

Mamm Genome, 2000 Nov, 11(11), 967 - 71
Localization of the mouse kidney disease (kd) gene to a YAC/BAC contig on Chromosome 10; Dell KM et al.; Mice that are homozygous for the kidney disease (kd) gene on Chromosome (Chr) 10 spontaneously develop a progressive and fatal interstitial nephritis . The disease phenotype is similar to that of the human disease, juvenile nephronophthisis . Using a backcross and intercross breeding strategy and analysis of over 900 resultant progeny, this genetic locus has now been mapped to a minimal co-segregating region of approximately two megabases between D10Mit 193 and D10Mit 38 . The location assigned to kd by this study is over 3 cM from the current Mouse Genome Database location . The entire interval has been cloned in yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) clones . Recombinant analysis has permitted assignment of 13 Mit microsatellite markers to positions near or within the region . Two new markers have been identified by using single-strand conformation polymorphism (SSCP) analysis of sequenced BAC ends . Several BAC end sequences align with human BAC clones from Chr 6q2 that contain NR2E1 . Snx3, and Ros1 . Three murine genes, CD24a, fyn, and ColX reported to map in or near the kd region as defined by this study have been evaluated . Though not definitely excluded, they appear to be unlikely candidates.

J Cell Biol, 2000 Oct 30, 151(3), 601 - 12
Rabenosyn-5, a novel Rab5 effector, is complexed with hVPS45 and recruited to endosomes through a FYVE finger domain; Nielsen E et al.; Rab5 regulates endocytic membrane traffic by specifically recruiting cytosolic effector proteins to their site of action on early endosomal membranes . We have characterized a new Rab5 effector complex involved in endosomal fusion events . This complex includes a novel protein, Rabenosyn-5, which, like the previously characterized Rab5 effector early endosome antigen 1 (EEA1), contains an FYVE finger domain and is recruited in a phosphatidylinositol-3-kinase-dependent fashion to early endosomes . Rabenosyn-5 is complexed to the Sec1-like protein hVPS45 . hVPS45 does not interact directly with Rab5, therefore Rabenosyn-5 serves as a molecular link between hVPS45 and the Rab5 GTPase . This property suggests that Rabenosyn-5 is a closer mammalian functional homologue of yeast Vac1p than EEA1 . Furthermore, although both EEA1 and Rabenosyn-5 are required for early endosomal fusion, only overexpression of Rabenosyn-5 inhibits cathepsin D processing, suggesting that the two proteins play distinct roles in endosomal trafficking . We propose that Rab5-dependent formation of membrane domains enriched in phosphatidylinositol-3-phosphate has evolved as a mechanism for the recruitment of multiple effector proteins to mammalian early endosomes, and that these domains are multifunctional, depending on the differing activities of the effector proteins recruited.

Biochem J, 2000 Nov 15, 352 Pt 1, 165 - 73
Identification of Rab6 as an N-ethylmaleimide-sensitive fusion protein-binding protein; Han SY et al.; In this study we show the interaction of N-ethylmaleimide-sensitive fusion protein (NSF) with a small GTP-binding protein, Rab6 . NSF is an ATPase involved in the vesicular transport within eukaryotic cells . Using the yeast two-hybrid system, we have isolated new NSF-binding proteins from the rat lung cDNA library . One of them was Rab6, which is involved in the vesicular transport within the Golgi and trans-Golgi network as a Ras-like GTPase . We demonstrated that the N-terminal domain of NSF interacted with the C-terminal domain of Rab6, and these proteins were co-immunoprecipitated from the rat brain extract . This interaction was maintained preferentially in the presence of hydrolysable ATP . Recombinant NSF-His(6) can also bind to C-terminal Rab6-glutathione S-transferase under the conditions to allow the ATP hydrolysis . Surprisingly, Rab6 stimulates the ATPase activity of NSF by approx . 2-fold as does alpha-soluble NSF attachment protein receptor . Anti-Rab6 polyclonal antibodies significantly inhibited the Rab6-stimulated ATPase activity of NSF . Furthermore, we found that Rab3 and Rab4 can also associate with NSF and stimulate its ATPase activity . Taken together, we propose a model in which Rab can form an ATP hydrolysis-regulated complex with NSF, and function as a signalling molecule to deliver the signal of vesicle fusion through the interaction with NSF.

Virology, 2000 Nov 10, 277(1), 184 - 92
Transcription of Epstein-Barr virus-encoded nuclear antigen 1 promoter Qp is repressed by transforming growth factor-beta via Smad4 binding element in human BL cells; Liang CL et al.; In Epstein-Barr virus (EBV)-infected BL cells, the oncogenic EBV-encoded nuclear antigen 1 (EBNA 1) gene is directed from the latent promoter Qp . Yeast one-hybrid screen analysis using the -50 to -37 sequence of Qp as the bait was carried out to identify transcriptional factors that may control Qp activity . Results showed that Smad4 binds the -50 to -37 sequence of Qp, indicating that this promoter is potentially regulated by TGF-beta . The association of Smad4 with Qp was further confirmed by supershift of EMSA complexes using Smad4-specific antibody . The transfection of a Qp reporter construct in two EBV(+) BL cell lines, Rael and WW2, showed that Qp activity is repressed in response to the TGF-beta treatment . This repression involves the interaction of a Smad3/Smad4 complex and the transcriptional repressor TGIF, as determined by cotransfection assay and coimmunoprecipitation analysis . Results suggest that TGF-beta may transcriptionally repress Qp through the Smad4-binding site in human BL cells .

Biochem Biophys Res Commun, 2000 Nov 2, 277(3), 611 - 6
Association of L-type calcium channels with a vacuolar H(+)-ATPase G2 subunit; Gao T et al.; The class C L-type calcium (Ca(2+)) channels have been implicated in many important physiological processes . Here, we have identified a mouse vacuolar H(+)-ATPase (V-ATPase) G2 subunit protein that bound to the C-terminal domain of the pore-forming alpha(1C) subunit using a yeast two-hybrid screen . Protein-protein interaction between the V-ATPase G subunit and the alpha(1C) subunit was confirmed using in vitro GST pull-down assays and coimmunoprecipitation from intact cells . Moreover, treatment of cells expressing L-type Ca(2+) channels with a specific inhibitor of the V-ATPase blocked proper targeting of the channels to the plasma membrane .

J Biomed Sci, 2000 Nov-Dec, 7(6), 484 - 93
Functional implication of human serine/threonine kinase, hAIK, in cell cycle progression; Yang SC et al.; Protein phosphorylation is involved in many biological activities and plays important roles in cell cycle progression . In the present study, we identified a serine/threonine kinase, hAIK, from human hepatic cells using degenerated polymerase chain reactions with a pair of primers derived from the highly conserved sequence in the catalytic domain of kinases . The full-length hAIK cDNA was then obtained, which contained 403 amino acids and was homologous to Drosophila Aurora2 and yeast Ipl1 proteins . Northern blotting analysis revealed that hAIK was highly expressed in the testis but not in other tissues . Expressions of hAIK drastically increased in cancer tissues/cell lines but not in fibroblasts or nontumorigenic cell lines . The recombinant hAIK protein phosphorylated itself and histone H1; this phosphorylation activity was totally abolished after a point mutation at the catalytic domain (hAIKm) . During the interphase cell, hAIK was found mainly in the cytoplasm; during mitosis hAIK accumulated at the centrosomes . In addition, overexpression of hAIK in cancer cell lines (HEK293T and HeLa) appeared to inhibit cell cycle progression . None of these phenomena were observed in hAIKm whose kinase activity was rendered inactive . Our results suggest that hAIK protein/activity might modulate cell cycle progression by interacting with the centrosomes and/or proteins associated with these structures .

J Biol Chem, 2001 Jan 26, 276(4), 2802 - 7 Epub 2000 Nov 01.
MAP-1, a novel proapoptotic protein containing a BH3-like motif that associates with Bax through its Bcl-2 homology domains; Tan KO et al.; A novel Bax-associating protein, named MAP-1 (Modulator of Apoptosis), has been identified in a yeast two-hybrid screen . MAP-1 contains a BH3-like (BH: Bcl-2 homology) motif and mediates caspase-dependent apoptosis in mammalian cells when overexpressed . MAP-1 homodimerizes and associates with the proapoptotic Bax and the prosurvival Bcl-2 and Bcl-X(L) of the Bcl-2 family in vitro and in vivo in mammalian cells . Mutagenesis analyses revealed that the BH3-like domain in MAP-1 is not required for its association with Bcl-X(L) but is required for association with Bax and for mediating apoptosis . Interestingly, in contrast to other Bax-associating proteins such as Bcl-X(L) and Bid, which require the BH3 and BH1 domains of Bax, respectively, for binding, the binding of MAP-1 to Bax appears to require all three BH domains (BH1, BH2, and BH3) of Bax, because point mutation of the critical amino acid in any one of these domains is sufficient to abolish its binding to MAP-1 . These data suggest that MAP-1 mediates apoptosis through a mechanism that involves binding to Bax.

J Biol Chem, 2001 Feb 9, 276(6), 3733 - 42 Epub 2000 Nov 01.
Characterization of the initiation factor eIF2B and its regulation in Drosophila melanogaster; Williams DD et al.; Eukaryotic initiation factor (eIF) 2B catalyzes a key regulatory step in the initiation of mRNA translation . eIF2B is well characterized in mammals and in yeast, although little is known about it in other eukaryotes . eIF2B is a hetropentamer which mediates the exchange of GDP for GTP on eIF2 . In mammals and yeast, its activity is regulated by phosphorylation of eIF2alpha . Here we have cloned Drosophila melanogaster cDNAs encoding polypeptides showing substantial similarity to eIF2B subunits from yeast and mammals . They also exhibit the other conserved features of these proteins . D . melanogaster eIF2Balpha confers regulation of eIF2B function in yeast, while eIF2Bepsilon shows guanine nucleotide exchange activity . In common with mammalian eIF2Bepsilon, D . melanogaster eIF2Bepsilon is phosphorylated by glycogen synthase kinase-3 and casein kinase II . Phosphorylation of partially purified D . melanogaster eIF2B by glycogen synthase kinase-3 inhibits its activity . Extracts of D . melanogaster S2 Schneider cells display eIF2B activity, which is inhibited by phosphorylation of eIF2alpha, showing the insect factor is regulated similarly to eIF2B from other species . In S2 cells, serum starvation increases eIF2alpha phosphorylation, which correlates with inhibition of eIF2B, and both effects are reversed by serum treatment . This shows that eIF2alpha phosphorylation and eIF2B activity are under dynamic regulation by serum . eIF2alpha phosphorylation is also increased by endoplasmic reticulum stress in S2 cells . These are the first data concerning the structure, function or control of eIF2B from D . melanogaster.

Proc Natl Acad Sci U S A, 2000 Nov 7, 97(23), 12902 - 7
LEUNIG, a putative transcriptional corepressor that regulates AGAMOUS expression during flower development; Conner J et al.; Regulation of homeotic gene expression is critical for proper developmental patterns in both animals and plants . LEUNIG is a key regulator of the Arabidopsis floral homeotic gene AGAMOUS . Mutations in LEUNIG cause ectopic AGAMOUS mRNA expression in the outer two whorls of a flower, leading to homeotic transformations of floral organ identity as well as loss of floral organs . We isolated the LEUNIG gene by using a map-based approach and showed that LEUNIG encodes a glutamine-rich protein with seven WD repeats and is similar in motif structure to a class of functionally related transcriptional corepressors including Tup1 from yeast and Groucho from Drosophila . The nuclear localization of LEUNIG-GFP is consistent with a role of LEUNIG as a transcriptional regulator . The detection of LEUNIG mRNA in all floral whorls at the time of their inception suggests that the restricted activity of LEUNIG in the outer two floral whorls must depend on interactions with other spatially restricted factors or on posttranslational regulation . Our finding suggests that both animals and plants use similar repressor proteins to regulate critical developmental processes.

Nucleic Acids Res, 2000 Nov 1, 28(21), 4212 - 8
A novel cytoplasmic GTPase XAB1 interacts with DNA repair protein XPA; Nitta M et al.; The xeroderma pigmentosum group A protein (XPA) plays a central role in nucleotide excision repair (NER) . To identify proteins that bind to XPA, we screened a HeLa cDNA library using the yeast two-hybrid system . Here we report a novel cytoplasmic GTP-binding protein, designated XPA binding protein 1 (XAB1) . The deduced amino acid sequence of XAB1 consisted of 374 residues with a molecular weight of 41 kDa and an isoelectric point of 4.65 . Sequence analysis revealed that XAB1 has four sequence motifs G1-G4 of the GTP-binding protein family in the N-terminal half . XAB1 also contains an acidic region in the C-terminal portion . Northern blot analysis showed that XAB1 mRNA is expressed ubiquitously, and immunofluorescence analysis revealed that XAB1 is localized mainly in the cytoplasm . Consistent with the GTP-binding motif, purified recombinant XAB1 protein has intrinsic GTPase activity . Using the yeast two-hybrid system, we elucidated that XAB1 binds to the N-terminal region of XPA . The deletion of five amino acids, residues 30-34 of XPA, required for nuclear localization of XPA abolished the interaction with XAB1 . These results suggest that XAB1 is a novel cytoplasmic GTPase involved in nuclear localization of XPA.

J Cell Sci, 2000 Nov, 113 ( Pt 22), 4121 - 35
The armadillo repeat region targets ARVCF to cadherin-based cellular junctions; Kaufmann U et al.; The cytoplasmic domain of the transmembrane protein M-cadherin is involved in anchoring cytoskeletal elements to the plasma membrane at cell-cell contact sites . Several members of the armadillo repeat protein family mediate this linkage . We show here that ARVCF, a member of the p120 (ctn) subfamily, is a ligand for the cytoplasmic domain of M-cadherin, and characterize the regions involved in this interaction in detail . Complex formation in an in vivo environment was demonstrated in (1) yeast two-hybrid screens, using a cDNA library from differentiating skeletal muscle and part of the cytoplasmic M-cadherin tail as a bait, and (2) mammalian cells, using a novel experimental system, the MOM recruitment assay . Immunoprecipitation and in vitro binding assays confirmed this interaction . Ectopically expressed EGFP-ARVCF-C11, an N-terminal truncated fragment, targets to junctional structures in epithelial MCF7 cells and cardiomyocytes, where it colocalizes with the respective cadherins, beta-catenin and p120 (ctn) . Hence, the N terminus of ARVCF is not required for junctional localization . In contrast, deletion of the four N-terminal armadillo repeats abolishes this ability in cardiomyocytes . Detailed mutational analysis revealed the armadillo repeat region of ARVCF as sufficient and necessary for interaction with the 55 membrane-proximal amino acids of the M-cadherin tail.

Int Rev Cytol, 2001, 201, 209 - 75
Mitogen-activated protein (MAP} kinase pathways in plants: versatile signaling tools; Ligterink W et al.; Mitogen-activated protein kinases (MAPKs) are important signaling tools in all eukaryotes, and function in mediating an enormous variety of external signals to appropriate cellular responses . MAPK pathways have been studied extensively in yeast and mammalian cells, and a large body of knowledge on their functioning has accumulated, which is summarized briefly . Plant MAPK pathways have attracted increasing interest, resulting in the isolation of a large number of different components of MAPK cascades . Studies on the functions of these components have revealed that MAPKs play important roles in the response to a broad variety of stresses, as well as in the signaling of most plant hormones and in developmental processes . Finally, the involvement of various plant phosphatases in the inactivation of MAPKs is discussed.

Biotechniques, 2000 Oct, 29(4), 874 - 80, 882
Automated image analysis of live/dead staining of the fungus Aureobasidium pullulans on microscope slides and leaf surfaces; Nelson CD et al.; An image analysis program and protocol for the identification and enumeration of live versus dead cells of the yeast-like fungus Aureobasidium pullulans was developed for both populations on microscope slides and leaf surfaces . Live cells took up CellTracker Blue, while nonviable cells stained with DEAD Red . Image analysis macro programs running under Optimas software were used to acquire images and to differentiate and enumerate viable from nonviable cells . The software was capable of discriminating green as a third parameter for identification and quantification of green fluorescent protein-expressing cells in a wild-type population.

J Biochem (Tokyo), 2000 Nov, 128(5), 793 - 801
Identification and characterization of novel isoforms of COP I subunits; Futatsumori M et al.; COP I-coated vesicles are involved in vesicular trafficking in the early secretory pathway . The COP I coat is composed of seven subunits, alpha-, beta-, beta'-, gamma-, delta-, epsilon-, and zeta-COPs . Evidence suggests, however, that there may be isoforms of the COP I subunits . In the present study, we identified homologs of gamma-COP (gamma2-COP; original gamma-COP is referred to as gamma1-COP in this paper) and of zeta-COP (zeta2-COP; original zeta-COP is referred to as zeta1-COP) . gamma1- and gamma2-COPs, and zeta1- and zeta2-COPs share 80 and 75%, respectively, of amino acids . mRNAs for gamma2-COP and zeta2-COP are expressed ubiquitously, suggesting their fundamental role in cellular function . Immunofluorescence analysis shows that gamma2-COP and zeta2-COP are colocalized with beta-COP in the paranuclear cis-Golgi region . Yeast two-hybrid analysis indicates that gamma1- and gamma2-COPs can directly, albeit promiscuously, interact with zeta1- and zeta2-COPs . Like gamma1-COP, gamma2-COP can form a complex with beta-COP in vivo . The gamma1-COP-containing and gamma2-COP-containing complexes can similarly interact with the cytoplasmic domain of p23 . These results indicate that gamma2-COP and zeta2-COP can form a COP I-like complex in place of gamma1-COP and zeta1-COP, respectively, and suggest that the COP I complex and the COP I-like complex are functionally redundant.

FEBS Lett, 2000 Oct 27, 484(1), 22 - 8
An acidic amino acid cluster regulates the nucleolar localization and ribosome assembly of human ribosomal protein L22; Shu-Nu C et al.; The control of human ribosomal protein L22 (rpL22) to enter into the nucleolus and its ability to be assembled into the ribosome is regulated by its sequence . The nuclear import of rpL22 depends on a classical nuclear localization signal of four lysines at positions 13-16 . RpL22 normally enters the nucleolus via a compulsory sequence of KKYLKK (I-domain, positions 88-93) . An acidic residue cluster at the C-terminal end (C-domain) plays a nuclear retention role . The retention is concealed by the N-domain (positions 1-9) which weakly interacts with the C-domain as demonstrated in the yeast two-hybrid system . Once it reaches the nucleolus, the question of whether rpL22 is assembled into the ribosome depends upon the presence of the N-domain . This suggests that the N-domain, on dissociation from its interaction with the C-domain, binds to a specific region of the 28S rRNA for ribosome assembly.

FEBS Lett, 2000 Oct 27, 484(1), 17 - 21
The MAT1 cyclin-dependent kinase-activating kinase (CAK) assembly/targeting factor interacts physically with the MCM7 DNA licensing factor; Wang Y et al.; MAT1 (menage a trois1) functions as an assembly/targeting factor of CAK (cyclin-dependent kinase-activating kinase) . In a search for MAT1-interacting proteins using yeast two-hybrid system, MCM7 (minichromosome maintenance 7), a member of a family of DNA licensing factors, was identified . The physical interaction between MAT1 and MCM7 was confirmed in vivo in yeast cells and verified with in vitro protein binding assays . Further studies showed the RING-finger motif of MAT1 is not required for the interaction with MCM7, while the C-terminal domain of MAT1 is indispensable . Immunoprecipitation of MCM7 in human osteosarcoma MG63 cells demonstrated that MCM7 associates with the CAK complex in vivo.

Genomics, 2000 Nov 1, 69(3), 305 - 13
A sequence-ready PAC contig of a 550-kb region on rat chromosome 4 including the diabetes susceptibility gene Lyp; Hornum L et al.; The Lyp locus controls diabetes development in rats . The diabetogenic allele in diabetes-prone BB rats is responsible for T cell lymphopenia characterized by the absence of regulatory T cells . We present refined genetic and radiation hybrid maps of the Lyp region on rat chromosome 4, a single 800-kb rat yeast artificial chromosome and a rat P1-derived artificial chromosome (PAC) contig corresponding to approximately 550 kb, both encompassing the entire candidate region . The contig, consisting of 48 PACs, gives 3- to 12-fold coverage . Genetic, radiation hybrid, and physical data were all in agreement and supported the same marker order . Nine genes and ESTs were identified in the contig in addition to a rat EST from the University of Iowa rat EST database-all possible candidate genes for Lyp . Alignment of our rat PAC contig with sequenced human PAC/BAC contigs confirms the position within the region of 3 of the 10 candidates and identifies an additional 8 genes/ESTs as candidates . These data will facilitate identification of Lyp .

Genomics, 2000 Nov 1, 69(3), 287 - 94
High-throughput scanning of the rat genome using interspersed repetitive sequence-PCR markers; Gosele C et al.; We report the establishment of a hybridization-based marker system for the rat genome based on the PCR amplification of interspersed repetitive sequences (IRS) . Overall, 351 IRS markers were mapped within the rat genome . The IRS marker panel consists of 210 nonpolymorphic and 141 polymorphic markers that were screened for presence/absence polymorphism patterns in 38 different rat strains and substrains that are commonly used in biomedical research . The IRS marker panel was demonstrated to be useful for rapid genome screening in experimental rat crosses and high-throughput characterization of large-insert genomic library clones . Information on corresponding YAC clones is made available for this IRS marker set distributed over the whole rat genome . The two existing rat radiation hybrid maps were integrated by placing the IRS markers in both maps . The genetic and physical mapping data presented provide substantial information for ongoing positional cloning projects in the rat .

J Cell Physiol, 2000 Dec, 185(3), 473 - 80
Investigation of the substrate spectrum of the human mismatch-specific DNA N-glycosylase MED1 (MBD4): fundamental role of the catalytic domain; Petronzelli F et al.; The human DNA repair protein MED1 (also known as MBD4) was isolated as an interactor of the mismatch repair protein MLH1 in a yeast two-hybrid screening . MED1 has a tripartite structure with an N-terminal 5-methylcytosine binding domain (MBD), a central region, and a C-terminal catalytic domain with homology to bacterial DNA damage-specific glycosylases/lyases . Indeed, MED1 acts as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil paired with guanine . The glycosylase activity of MED1 preferentially targets G:T mismatches in the context of CpG sites; this indicates that MED1 is involved in the repair of deaminated 5-methylcytosine . Interestingly, frameshift mutations of the MED1 gene have been reported in human colorectal, endometrial, and pancreatic cancers . For its putative role in maintaining genomic fidelity at CpG sites, it is important to characterize the biochemical properties and the substrate spectrum of MED1 . Here we show that MED1 works under a wide range of temperature and pH, and has a limited optimum range of ionic strength . MED1 has a weak glycosylase activity on the mutagenic adduct 3,N(4)-ethenocytosine, a metabolite of vinyl chloride and ethyl carbamate . The differences in glycosylase activity on G:U and G:T substrates are not related to differences in substrate binding and likely result from intrinsic differences in the chemical step . Finally, the isolated catalytic domain of MED1 retains the preference for G:T and G:U substrates in the context of methylated or unmethylated CpG sites . This suggests that the catalytic domain is fundamental, and the 5-methylcytosine binding domain dispensable, in determining the substrate spectrum of MED1 .

J Am Podiatr Med Assoc, 2000 Oct, 90(9), 450 - 9
Dermatophyte test medium culture versus mycology laboratory analysis for suspected onychomycosis . A study of 100 cases in a geriatric population; Scherer WP et al.; Onychomycosis is the most frequently encountered condition in podiatric practice in the United States . A variety of modalities are available to confirm the presumptive diagnosis of onychomycosis . This study was conducted to compare the results of in-office dermatophyte test medium cultures with those of mycology laboratory analysis for 100 cases of suspected onychomycosis in a geriatric population . The results demonstrated that 20% of the patients had dermatophyte involvement, 56% had saprophyte involvement, and 19% had yeast involvement . Only 50% of positive dermatophyte test medium cultures correlated with a positive microscopic fungal culture for dermatophytes . Given these results, it is questionable whether in-office dermatophyte test medium cultures should be routinely used in geriatric patients for the diagnosis of onychomycosis . The authors believe mycology laboratory testing with fluorescent potassium hydroxide preparations and microscopic fungal cultures to be superior to in-office dermatophyte test medium cultures for the diagnosis of onychomycosis in geriatric patients.

Gene, 2000 Oct 17, 257(1), 33 - 43
Evidence that dim1 associates with proteins involved in pre-mRNA splicing, and delineation of residues essential for dim1 interactions with hnRNP F and Npw38/PQBP-1; Zhang Y et al.; The small evolutionarily conserved protein Dim1p/hDim1/Dib1p/DML-1 was initially defined as a factor essential for progression through the G2/M transition, and shown to be required to maintain the steady state level of a component of the fission yeast anaphase promoting complex/cyclosome . More recently, Dib1p has been defined as a component of the U4/U6.U5 tri-snRNP, required for pre-mRNA splicing . To investigate the mechanism(s) of Dim1 function, reiterative two-hybrid screening was performed to identify interacting proteins . Proteins thus identified were solely those involved in pre-mRNA splicing or related functions, and one partner induced a striking synthetic phenotype when co-expressed with hDim1 in mammalian cells . Saturating alanine scanning mutagenesis of Dim1 allowed delineation of amino acids essential for its ability to interact with its defined partners: mapping these residues on the structural coordinates of hDim1 defined an interactive sector of the protein . Finally, depletion studies have recently shown that Dim1 function is essential for pre-mRNA splicing in yeast . We find that elimination of DML-1 expression in C . elegans by RNA interference leads to embryonal lethality during gastrulation, marked by a failure to correctly express early zygotic transcripts . These results parallel the arrest phenotypes associated with global disruption of zygotic gene expression, suggesting that Dim1 proteins maintain an essential function in gene expression in higher eukaryotes.

Gene, 2000 Oct 17, 257(1), 13 - 22
Characterization of the hCTR1 gene: genomic organization, functional expression, and identification of a highly homologous processed gene; Moller LB et al.; The human hCTR1 gene was originally identified by its ability to complement a yeast mutant deficient in high-affinity copper uptake (Zhou, B., Gitschier, J., 1997 . A human gene for copper uptake identified by complementation in yeast . Proc . Natl . Acad . Sci . USA 94, 7481-7486) . Here, we have determined the DNA sequence of the exon-intron borders of the hCTR1 structural gene and report that the coding sequence is disrupted by three introns, all of which comply with the GT/AG rule . Furthermore, human fibroblasts, transfected with hCTR1 cDNA, were shown to have a dramatically increased capacity for (64)Cu uptake, indicating that the hCtr1 protein is functional in copper uptake in human cells . In contrast, no evidence was found for involvement of the hCTR2 gene product in copper uptake . Finally, we have identified a highly homologous processed pseudogene, hCTR1psi, which was localized to chromosome 3q25/26 . The processed gene was found to be transcribed, but due to a frame shift mutation, it only had the potential to encode a truncated protein of 95 amino acid residues, and cells transfected with hCTR1psi DNA showed no increase of (64)Cu uptake.

Br J Haematol, 2000 Sep, 110(4), 819 - 25
Activation of an ataxia telangiectasia mutation-dependent intra-S-phase checkpoint by anti-tumour drugs in HL-60 and human lymphoblastoid cells; Sugimoto K et al.; In yeast cells, the intra-S-phase checkpoint slows down the rate of DNA replication in response to DNA damage . Here we showed that a similar checkpoint mechanism is present and activated by anti-tumour drugs in HL-60 and Epstein-Barr virus (EBV)-transformed human lymphoblastoid cells . Using bromodeoxyuridine (BrdU) pulse labelling combined with two-dimensional flow cytometric analysis, we clearly visualized the cell-cycle progression of the BrdU-positive population (cells originally belonging to the S phase) and detected even subtle changes in S-phase progression induced by mild drug treatment conditions free of apoptosis . The DNA topoisomerase II inhibitors, doxorubicin and etoposide (250 nmol/l and 400 nmol/l, respectively, for 8 h), retained the BrdU-positive HL-60 cells in the latter half of S and G2/M positions, and the pyrimidine analogue anti-metabolite, cytosine beta-D-arabinofuranose (Ara-C; 50 nmol/l), kept them in early-to-late S phase after 8 h of incubation . Because 10 micromol/l of caffeine added 2 h later attenuated the S-phase retardation by these drugs in HL-60 cells, slowing of the S-phase progression should be actively regulated . Furthermore, two ataxia telangiectasia (AT)-derived lymphoblastoid cell lines were impaired in the doxorubicin-induced S-phase retardation, which indicated that the process is at least partially dependent on ataxia telangiectasia mutated (ATM) gene product . The inhibitory mechanism on S-phase progression elicited by anti-tumour drugs in HL-60 and lymphoblastoid cells may therefore correspond to the intra-S-phase checkpoint of the yeast cells.

Exp Gerontol, 2000 Sep, 35(6-7), 795 - 801
Does a retrograde response in human aging and longevity exist?
De Benedictis G, Carrieri G, Garasto S, Rose G, Varcasia O, Bonafe M, Franceschi C, Jazwinski SM.
The retrograde response (RR) is a compensatory mechanism by which mutant strains of yeast are able to cope with mitochondrial DNA (mtDNA) impairments by up-regulating the expression of the stress-responder nuclear genes and significantly increasing lifespan . Starting from the observation that both mtDNA variability and Tyrosine hydroxylase (THO, stress-responder gene) variability are correlated with human longevity, we asked ourselves whether mechanisms similar to RR may exist in humans . As a first investigative step we have analyzed the distribution of the mtDNA inherited variants (haplogroups) according to THO genotypes in three sample groups of increasing ages (20-49 years; 50-80 years; centenarians) . We found that the mtDNA haplogroups and the THO genotypes are associated randomly in the first group, while in the second group, and particularly in the centenarians, a non-random association is observed between the mtDNA and nuclear DNA variability . Moreover, in centenarians the U haplogroup is over-represented (p=0.012) in subjects carrying the THO genotype unfavorable to longevity . On the whole these findings are in line with the hypothesis that longevity requires particular interactions between mtDNA and nuclear DNA and do not exclude the possibility that an RR has been maintained throughout evolution and it is present in higher organisms.

J Agric Food Chem, 2000 Oct, 48(10), 4540 - 3
Microassay for rapid screening of alpha-amylase activity; Wong DW et al.; A microassay was developed for measuring the activity of alpha-amylases in the nanogram enzyme concentration range, based on the use of dye-labeled cross-linked starch as the substrate, and the release of soluble colored fragments formed in enzyme hydrolysis . Reaction conditions were optimized to generate a linear correlation between the increase in absorbance and a reaction time of 0-10 min, as well as enzyme concentrations in the range of 0-50 ng . A standard curve for the conversion of absorbance to enzyme activity units was constructed . The protocol developed was applied to monitoring the production of ultralow concentrations of recombinant barley alpha-amylase in yeast cells.

Plant Mol Biol, 2000 Jul, 43(4), 527 - 36
Protein phosphatase 2A holoenzyme and its subunits from Medicago sativa; Toth EC et al.; We detected an about 200 kDa holoenzyme of protein phosphatase 2A (PP2A) in the crude extract of Medicago sativa microcallus cells by gel permeation chromatography . By polymerase chain reaction (PCR) we isolated two M . sativa cDNA fragments corresponding to the catalytic (C) subunit, and one each coding for the A and the B regulatory subunits of PP2A . The C subunit sequences were different from that published previously, indicating the existence of at least three different isoforms in M . sativa . Using the PCR fragments as probes, we obtained two distinct full-length clones for both the A and B subunits from an alfalfa cDNA library . Our results demonstrate that the components of the PP2A holoenzyme, namely the catalytic and regulatory subunits, are present in alfalfa in several isoforms and that their sequences are highly similar to their plant, yeast and animal counterparts . The distinct regulatory subunit genes are constitutively expressed during the cell cycle . Interestingly, two A-B subunit pairs had parallel mRNA steady-state levels in different plant tissues suggesting that not all of the possible isoform combinations are present in all tissues . The expression of the MsPP2A Bbeta subunit form was induced by abscisic acid indicating a specific function for this protein in the stress response.

Clin Cancer Res, 2000 Oct, 6(10), 3937 - 43
Functional evaluation of p53 and PTEN gene mutations in gliomas; Kato H et al.; We screened mutations of two major tumor suppressor genes, p53 and PTEN, in 66 human brain tumors using a yeast-based functional assay and cDNA-based direct sequencing, respectively . The frequency of p53 mutations was 28.8% (19 of 66) and was higher in anaplastic astrocytoma (9 of 14, 64.3%,) than in glioblastoma multiforme (GBM; 7 of 27, 25.9%,), supporting previous speculation that there are at least two genetic pathways leading to GBM, a de novo pathway without p53 mutation and a "progressive" pathway with p53 mutation . PTEN mutation was observed in 8 of 64 tumors (12.5%), mainly GBMs (7 of 26, 26.9%), both with and without p53 mutation . These results suggest that mutation of the PTEN gene is a later event than that of the p53 gene in glioma progression and is associated with both the genetic pathways . All of the detected PTEN missense mutations and an in-frame small deletion inactivated PTEN phosphoinositide phosphatase activity in vitro . Because the tumors containing PTEN mutations also showed loss of heterozygosity in the chromosome 10q23 region flanking the PTEN gene, our data clearly indicate that inactivation of both PTEN alleles occurs in a subset of high-grade gliomas, therefore confirming the previous idea that PTEN acts as a tumor suppressor gene.

J Biol Chem, 2001 Mar 2, 276(9), 6645 - 55 Epub 2000 Oct 25.
Syncoilin, a novel member of the intermediate filament superfamily that interacts with alpha-dystrobrevin in skeletal muscle; Newey SE et al.; Dystrophin coordinates the assembly of a complex of structural and signaling proteins that are required for normal muscle function . A key component of the dystrophin protein complex is alpha-dystrobrevin, a dystrophin-associated protein whose absence results in neuromuscular junction defects and muscular dystrophy . To gain further insights into the role of alpha-dystrobrevin in skeletal muscle, we used the yeast two-hybrid system to identify a novel alpha-dystrobrevin-binding partner called syncoilin . Syncoilin is a new member of the intermediate filament superfamily and is highly expressed in skeletal and cardiac muscle . In normal skeletal muscle, syncoilin is concentrated at the neuromuscular junction, where it colocalizes and coimmunoprecipitates with alpha-dystrobrevin-1 . Expression studies in mammalian cells demonstrate that, while alpha-dystrobrevin and syncoilin associate directly, overexpression of syncoilin does not result in the self-assembly of intermediate filaments . Finally, unlike many components of the dystrophin protein complex, we show that syncoilin expression is up-regulated in dystrophin-deficient muscle . These data suggest that alpha-dystrobrevin provides a link between the dystrophin protein complex and the intermediate filament network at the neuromuscular junction, which may be important for the maintenance and maturation of the synapse.

Jpn J Cancer Res, 2000 Oct, 91(10), 1007 - 14
Centrosomal kinases, HsAIRK1 and HsAIRK3, are overexpressed in primary colorectal cancers; Takahashi T et al.; Members of the recently identified family of Homo sapiens Aurora / Ipl1-related kinases (HsAIRKs), homologous to chromosome segregation kinases, fly Aurora and yeast Ipl1, are highly expressed during M phase, and have been suggested to regulate centrosome function, chromosome segregation, and cytokinesis . In the present study, immunohistochemical analyses were performed of HsAIRK1 and HsAIRK3 expression in 78 primary colorectal cancers and 36 colorectal adenomas as well as 15 normal colorectal specimens . In normal colon mucosa, some crypt cells showed weak positive staining in 10 and 12 out of 15 cases for HsAIRK1 and HsAIRK3, respectively, the remaining cases being negative . Elevated expression of HsAIRK1 was observed in 53 (67.9%) of the colorectal cancers, and of HsAIRK3 in 40 (51.3%) . Furthermore, colorectal adenomas showed high expression of HsAIRK1 and HsAIRK3 in 11 (30.6%) and 7 (19.4%) cases, respectively, thus being intermediate between colorectal cancers and normal colorectal mucosa . Interestingly, HsAIRK1 overexpression was significantly associated with pT (primary tumor invasion) and p53 accumulation in colorectal cancers . There was no significant correlation between proliferating cell nuclear antigen-labeling index (PCNA-LI) and the levels of these proteins . The results suggest that overexpression of HsAIRK1 and HsAIRK3 might be involved in tumorigenesis and / or progression of colorectal cancers.

Jpn J Cancer Res, 2000 Oct, 91(10), 987 - 93
Overexpression of DA41 in v-Ha-ras-3Y1 cells causes growth suppression; Ozaki T et al.; We have recently found that DA41 exhibits marked homology with mouse PLIC-1, PLIC-2, frog XDRP1 and yeast DSK2 . XDRP1 has been shown to be associated with cyclin A, and blocks cell division of frog embryo . In the present study, we examined the biological role(s) of DA41 in mammalian cells by overexpressing it in v-Ha-ras-transformed 3Y1 cells (ras-3Y1) . Transfectants which expressed a high level of DA41 mRNA exhibited a decrease in growth rate, a reduction in saturation density, and a suppression of colony formation in soft agar medium . To clarify the molecular mechanism(s) by which DA41 affects cell growth, the effect of DA41 expression on the levels of various cell cycle-regulatory proteins was examined . The forced expression of DA41 gene resulted in a remarkable reduction in CDK2 activity, while the amount of CDK2 did not change . These observations indicate that DA41 is involved in cell cycle regulation in ras-3Y1 cells.

Curr Biol, 2000 Oct 5, 10(19), 1172 - 81
Incenp and an aurora-like kinase form a complex essential for chromosome segregation and efficient completion of cytokinesis; Kaitna S et al.; BACKGROUND: In animal cells, cytokinesis begins shortly after the sister chromatids move to the spindle poles . The inner centromere protein (Incenp)has been implicated in both chromosome segregation and cytokinesis, but it is not known exactly how it mediates these two distinct processes . RESULTS: We identified two Caenorhabditis elegans proteins, ICP-1 and ICP-2, with significant homology in their carboxyl termini to the corresponding region of vertebrate Incenp . Embryos depleted of ICP-1 by RNA-mediated interference had defects in both chromosome segregation and cytokinesis . Depletion of the Aurora-like kinase AIR-2 resulted in a similar phenotype . The carboxy-terminal region of Incenp is also homologous to that in Sli15p, a budding yeast protein that functions with the yeast Aurora kinase Ipl1p . ICP-1 bound C . elegans AIR-2 in vitro, and the corresponding mammalian orthologs Incenp and AIRK2 could be co-immunoprecipitated from cell extracts . A significant fraction of embryos depleted of ICP-1 and AIR-2 completed one cell division over the course of several cell cycles . ICP-1 promoted the stable localization of ZEN-4 (also known as CeMKLP1), a kinesin-like protein required for central spindle assembly . CONCLUSIONS: ICP-1 and AIR-2 are part of a complex that is essential for chromosome segregation and for efficient completion of cytokinesis . We propose that this complex acts by promoting dissolution of sister chromatid cohesion and the assembly of the central spindle.

Proc Natl Acad Sci U S A, 2000 Oct 24, 97(22), 12062 - 7
Creatine kinase, an ATP-generating enzyme, is required for thrombin receptor signaling to the cytoskeleton; Mahajan VB et al.; Thrombin orchestrates cellular events after injury to the vascular system and extravasation of blood into surrounding tissues . The pathophysiological response to thrombin is mediated by protease-activated receptor-1 (PAR-1), a seven-transmembrane G protein-coupled receptor expressed in the nervous system that is identical to the thrombin receptor in platelets, fibroblasts, and endothelial cells . Once activated by thrombin, PAR-1 induces rapid and dramatic changes in cell morphology, notably the retraction of growth cones, axons, and dendrites in neurons and processes in astrocytes . The signal is conveyed by a series of localized ATP-dependent reactions directed to the actin cytoskeleton . How cells meet the dynamic and localized energy demands during signal transmission is unknown . Using the yeast two-hybrid system, we identified an interaction between PAR-1 cytoplasmic tail and the brain isoform of creatine kinase, a key ATP-generating enzyme that regulates ATP within subcellular compartments . The interaction was confirmed in vitro and in vivo . Reducing creatine kinase levels or its ATP-generating potential inhibited PAR-1-mediated cellular shape changes as well as a PAR-1 signaling pathway involving the activation of RhoA, a small G protein that relays signals to the cytoskeleton . Thrombin-stimulated intracellular calcium release was not affected . Our results suggest that creatine kinase is bound to PAR-1 where it may be poised to provide bursts of site-specific high-energy phosphate necessary for efficient receptor signal transduction during cytoskeletal reorganization.

Proc Natl Acad Sci U S A, 2000 Oct 24, 97(22), 11984 - 9
Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral; Baird GS et al.; DsRed is a recently cloned 28-kDa fluorescent protein responsible for the red coloration around the oral disk of a coral of the Discosoma genus . DsRed has attracted tremendous interest as a potential expression tracer and fusion partner that would be complementary to the homologous green fluorescent protein from Aequorea, but very little is known of the biochemistry of DsRed . We now show that DsRed has a much higher extinction coefficient and quantum yield than previously reported, plus excellent resistance to pH extremes and photobleaching . In addition, its 583-nm emission maximum can be further shifted to 602 nm by mutation of Lys-83 to Met . However, DsRed has major drawbacks, such as strong oligomerization and slow maturation . Analytical ultracentrifugation proves DsRed to be an obligate tetramer in vitro, and fluorescence resonance energy transfer measurements and yeast two-hybrid assays verify oligomerization in live cells . Also, DsRed takes days to ripen fully from green to red in vitro or in vivo, and mutations such as Lys-83 to Arg prevent the color change . Many potential cell biological applications of DsRed will require suppression of the tetramerization and acceleration of the maturation.

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