Biochem J, 2005 Jan 15, 385(Pt 2), 347 - 53 The septin Sept5/CDCrel-1 competes with alpha-SNAP for binding to the SNARE complex; Beites CL et al.; SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins are supposed to mediate the docking and/or fusion of the vesicle with the plasma membrane . However, it is not clearly understood how this process is regulated . In a search for potential SNARE regulators, we recently identified septin 5 (Sept5) as a novel SNARE interacting protein . Septins were first identified as filamentous proteins required for cytokinesis in yeast . Several septins have now been identified in mammals but little is known about their functions . We have previously shown that Sept5 is predominantly expressed in the brain, where it associates with vesicles and membranes through its interaction with the SNARE domain of syntaxin 1A . Furthermore, Sept5 appears to inhibit exocytosis, possibly by regulating vesicle targeting and/or fusion events . To gain insight into the role of Sept5, we have mapped the Sept5 domains important for syntaxin binding . We also investigated the ability of Sept5 to bind to syntaxin when in various protein complexes . Although Sept5 cannot bind an nSec1-syntaxin complex, it can bind syntaxin in a SNARE complex . This interaction is occluded by the binding of alpha-SNAP, suggesting that Sept5 may regulate the availability of SNARE proteins through its interaction with syntaxin and the 7 S complex. Annu Rev Cell Dev Biol . 2004 Jun 15; {Epub ahead of print} Retrovirus Budding; Morita E et al.; Human immunodeficiency virus (HIV) and other retroviruses acquire their envelopes and spread infection by budding through the limiting membranes of producer cells . To facilitate budding, retroviruses usurp a cellular pathway that is normally used to create vesicles that bud into late endosomal compartments called multivesicular bodies (MVB) . Research on yeast and human MVB biogenesis has led to the identification of 25 human proteins that are required for vesicle formation and for HIV-1 budding, and has produced a working model for sequential recruitment of these proteins during MVB vesicle formation . Retroviruses can redirect this machinery to the plasma membrane and leave the cell in a single step or, alternatively, can bud directly into MVB compartments and then exit cells via the exosome pathway . Remarkably, virus release from both the plasma membrane and MVB compartments can occur directionally into specialized sites of cell-to-cell contact called virological synapses . Thus retroviruses have evolved elaborate mechanisms for escaping the cell and maximizing their chances of infecting a new host . Expected online publication date for the Annual Review of Cell and Developmental Biology Volume 20 is October 6, 2004 . Please see for revised estimates. Annu Rev Entomol . 2004 Aug 5; {Epub ahead of print} Folsomia candida (Collembola): The "Standard" Soil Insect; Fountain MT et al.; Folsomia candida Willem 1902, a member of the order Collembola (colloquially called springtails), is a common and widespread arthropod that occurs in soils throughout the world . The species is parthenogenetic and is easy to maintain in the laboratory on a diet of granulated dry yeast . F . candida has been used as a "standard" test organism for more than 40 years for estimating the effects of pesticides and environmental pollutants on nontarget soil arthropods . However, it has also been employed as a model for the investigation of numerous other phenomena such as cold tolerance, quality as a prey item, and effects of microarthropod grazing on pathogenic fungi and mycorrhizae of plant roots . In this comprehensive review, aspects of the life history, ecology, and ecotoxicology of F . candida are covered . We focus on the recent literature, especially studies that have examined the effects of soil pollutants on reproduction in F . candida using the protocol published by the International Standards Organization in 1999 . Expected online publication date for the Annual Review of Entomology Volume 50 is December 3, 2004 . Please see for revised estimates. Annu Rev Genet . 2004 Jun 15; {Epub ahead of print} Closing Mitosis: The Functions of the Cdc14 Phosphatase and Its Regulation; Stegmeier F et al.; Completion of the cell cycle requires the temporal and spatial coordination of chromosome segregation with mitotic spindle disassembly and cytokinesis . In budding yeast, the protein phosphatase Cdc14 is a key regulator of these late mitotic events . Here, we review the functions of Cdc14 and how this phosphatase is regulated to accomplish the coupling of mitotic processes . We also discuss the function and regulation of Cdc14 in other eukaryotes, emphasizing conserved features . Expected online publication date for the Annual Review of Genetics Volume 38 is November 10, 2004 . Please see for revised estimates. Annu Rev Genet . 2004 May 25; {Epub ahead of print} Mobile Group II Introns; Lambowitz AM et al.; Mobile group II introns, found in bacterial and organellar genomes, are both catalytic RNAs and retrotransposable elements . They use an extraordinary mobility mechanism in which the excised intron RNA reverse splices directly into a DNA target site and is then reverse transcribed by the intron-encoded protein . After DNA insertion, the introns remove themselves by protein-assisted, autocatalytic RNA splicing, thereby minimizing host damage . Here we discuss the experimental basis for our current understanding of group II intron mobility mechanisms, beginning with genetic observations in yeast mitochondria, and culminating with a detailed understanding of molecular mechanisms shared by organellar and bacterial group II introns . We also discuss recently discovered links between group II intron mobility and DNA replication, new insights into group II intron evolution arising from bacterial genome sequencing, and the evolutionary relationship between group II introns and both eukaryotic spliceosomal introns and non-LTR-retrotransposons . Finally, we describe the development of mobile group II introns into gene-targeting vectors, "targetrons," which have programmable target specificity . Expected online publication date for the Annual Review of Genetics Volume 38 is November 10, 2004 . Please see for revised estimates. Front Biosci, 2004 Sep 01, 9, 3029 - 45 Initiation of DNA replication in xenopus egg extracts; Arias EE et al.; In the last decade, extraordinary advances in our understanding of the initiation step of eukaryotic DNA replication have been achieved . Many factors required for replication initiation have been identified, and an elegant model to explain how DNA replication is restricted to a single round per cell cycle has emerged . Of the many experimental approaches used to study DNA replication, egg extracts from Xenopus laevis are among the most powerful, since they recapitulate a complete round of cell-cycle regulated chromosomal DNA replication in vitro . In this review, we discuss current models for how DNA replication is initiated and regulated in Xenopus eggs, and we highlight similarities and differences seen between this and the other most common experimental organisms, yeast and humans. Arch Oral Biol, 2004 Nov, 49(11), 881 - 7 Tissue distribution and nucleotide sequence of bovine mRNA for salivary proline-rich protein P-B; Isemura S et al.; The tissue distribution of P-B was investigated to obtain information on the physiological significance of this proline-rich protein . To design primers and probes for a tissue distribution analysis, a polymerase chain reaction (PCR)-based cloning of bovine P-B cDNA was performed using tooth germ and the nucleotide sequence was determined . The cloned bovine P-B cDNA was composed of 356 bp and included the region corresponding to the mature P-B protein and part of the 3' non-coding sequence . This part of the sequence is identical to the corresponding region of human P-B cDNA from the submaxillary gland . DNA corresponding to the P-B mRNA was amplified by PCR using cDNAs from various bovine tissues including tooth germ, submaxillary gland, parotid gland, lachrymal gland, heart, liver, stomach, pancreas, spleen, kidney, adrenal, and ovary . A quantitative analysis indicated the heart, submaxillary gland, tooth germ and kidney to be major sites of P-B expression . The ubiquitous distribution of P-B mRNA among bovine tissues together with findings of the presence of genes hybridizable with a DNA probe for P-B among species such as human, bovine, rat, mouse, and yeast as reported previously suggested a fundamental physiological role for this protein. BMC Pharmacol . 2004 Sep 07;4(1):19. The FK506 binding protein 13 kDa (FKBP13) interacts with the C-chain of complement C1q; Neye H et al.; BACKGROUND: The pharmacological action of specific immunosuppressants is mediated by immunophilins . While cyclosporin A binds to cyclophilins, FK506/tacrolimus, rapamycin, and others bind to FK506 binding proteins (FKBPs) . Different physiological actions of immunophilins were described but their genuine function, however, remains elusive and is still under investigation . A yeast two-hybrid screen was performed using the FK506 binding protein 13 kDa (FKBP13) as a bait and a fetal liver expression library as a prey . RESULTS: The C-chain of complement C1q (C1q-C) was detected to interact with FKBP13 in the yeast two-hybrid system and in a protein complementation assay . Neither FKBP12, FKBP25, FKBP52 nor the unrelated immunophilin CypA did react with C1q-C in the yeast system stressing the specificity of the interaction . Binding of C1q-C to FKBP13 could not be prevented in the presence of FK506, demonstrating that possibly other regions than the binding pocket of the drug are responsible for the interaction of the two proteins . CONCLUSION: It is concluded that exclusively FKBP13 but no other FKBPs tested so far interact with the C-chain of complement C1q in the two different assays and further work will be initiated to investigate the physiological relevance of the interaction. Biochem Biophys Res Commun, 2004 Oct 8, 323(1), 168 - 74 Differential cooperation between dHAND and three different E-proteins; Murakami M et al.; dHAND is a transcription factor belonging to the class B basic helix-loop-helix protein family and is expressed during embryogenesis in the heart, branchial arches, limb buds, and neural crest derivatives . Despite much study, the molecular mechanisms involved in the regulation of dHAND activity are not well understood . We therefore carried out yeast two-hybrid screening using full-length dHAND as bait, which led to identification of several dHAND-binding proteins, including three E-proteins: E2A, ME2, and ALF1 . Subsequent analysis revealed that although their heterodimerization and transcriptional activities were similar, dHAND/E-protein heterodimers bind to an E-box element with differing affinities, suggesting they have distinct DNA binding specificities . Moreover, in situ hybridization showed that E-protein genes are expressed fairly ubiquitously among embryonic tissues, including the branchial arches and limb buds . By contrast, little signal was detected in the heart, suggesting that dHAND complexes with partners other than E-proteins in cardiac tissue . J Mol Biol, 2004 Sep 24, 342(4), 1197 - 208 The 1.3 A crystal structure of human mitochondrial Delta3-Delta2-enoyl-CoA isomerase shows a novel mode of binding for the fatty acyl group; Partanen ST et al.; The crystal structure of Delta3-Delta2-enoyl-CoA isomerase from human mitochondria (hmEci), complexed with the substrate analogue octanoyl-CoA, has been refined at 1.3 A resolution . This enzyme takes part in the beta-oxidation of unsaturated fatty acids by converting both cis-3 and trans-3-enoyl-CoA esters (with variable length of the acyl group) to trans-2-enoyl-CoA . hmEci belongs to the hydratase/isomerase (crotonase) superfamily . Most of the enzymes belonging to this superfamily are hexamers, but hmEci is shown to be a trimer . The mode of binding of the ligand, octanoyl-CoA, shows that the omega-end of the acyl group binds in a hydrophobic tunnel formed by residues of the loop preceding helix H4 as well as by side-chains of the kinked helix H9 . From the structure of the complex it can be seen that Glu136 is the only catalytic residue . The importance of Glu136 for catalysis is confirmed by mutagenesis studies . A cavity analysis shows the presence of two large, adjacent empty hydrophobic cavities near the active site, which are shaped by side-chains of helices H1, H2, H3 and H4 . The structure comparison of hmEci with structures of other superfamily members, in particular of rat mitochondrial hydratase (crotonase) and yeast peroxisomal enoyl-CoA isomerase, highlights the variable mode of binding of the fatty acid moiety in this superfamily. J Mol Biol, 2004 Sep 24, 342(4), 1131 - 41 Characterization of hnRNP K protein-RNA interactions; Klimek-Tomczak K et al.; The heterogeneous nuclear ribonucleoprotein K protein is an RNA-binding protein found in several subcellular compartments where it is thought to be involved in signaling multiple processes that compose gene expression . K protein contains three K homology (KH) domains that mediate RNA-binding . We used a serial analysis of gene expression (SAGE)-based strategy, yeast three-hybrid screen, RNA pull-down assays and computational analysis to characterize K protein-associated RNAs . We demonstrate that K protein interacts with many sense and antisense nuclear and mitochondrial transcripts through both direct and indirect binding . The highly specific direct binding of transcripts to K protein is mediated by a consensus sequence comprising three C-rich patches . Structural analysis suggests a three-prong interaction model whereby each of the three KH domains binds one of the C-rich patches . Genome-wide and yeast three-hybrid clone analysis revealed that these sequences are located preferentially in the 3' untranslated regions, which are known to regulate mRNA translation and processing. J Mater Sci Mater Med, 2003 Apr, 14(4), 307 - 10 Effects of dental resin metabolites on estrogenic activity in vitro; Nomura Y et al.; Three monomers (Bis-GMA, UDMA, and TEGDMA) and five polymerization initiators (CQ, BPO, DMPT, DMAEMA, and ATU) commonly used in dental composite resins were tested for estrogenic activity using a reporter gene assay (yeast two-hybrid system) in vitro, and compared with bisphenol-A (BPA) . Estrogenic activity was indicated by agonist and antagonist activity, with (+S9) and without (-S9) metabolic activation using rat liver cells . No estrogenic agonist activity was seen for each monomer and polymerization initiator in either the -S9 and +S9 tests in the concentration ranges examined in this study . On the other hand, estrogen antagonist activity was found with BPO and DMPT . BPO showed antagonist activity at a concentration of approximately 1800 nM with the -S9 test, but not with the +S9 test . With DMPT, antagonist activity was not seen with the -S9 test, but it was seen at a concentration of approximately 610 nM using the +S9 test . With BPA, the +S9 test indicated antagonist activity at a concentration of approximately 780 nM . The estrogen antagonist activities of DMPT and BPA appeared to be similar . CQ, DMAEMA, ATU, and the three monomers did not show antagonist activity as demonstrated by the -S9 or +S9 tests within the concentration range tested in this study. J Mater Sci Mater Med, 2000 Aug, 11(8), 465 - 468 Estrogenic activity of chemicals for dental and similar use in vitro; Hashimoto Y et al.; The estrogenic activities of chemicals for dental and similar use were tested by a reporter gene assay (yeast two-hybrid system) and an estrogen/estrogen receptor (ER-alpha) competition binding assay (fluorescence polarization system) . Among the 10 chemicals {bisphenol-A (BPA), bis-2-hydroxypropyl methacrylate (Bis-GMA), triethylene glycol dimethacrylate (TEGDMA), methyl methacrylate (MMA) and 2-hydroxyethyl methacrylate (HEMA), dibutyl phthalate (DBP), n-butyl benzyl phthalate (BBP), n-butyl phthalyl n-butyl glycolate (BPBG), di-2-ethylhexyl phthalate (DEHP), and di-2-ethylhexyl adipate (DOA)}, which were diluted with DMSO to concentrations ranging from 5 \times 10^{\,-\,7} to 5 \times 10^{\,-\,3}\,\hbox{M}, and 17beta-estradiol (E2) as a positive control, BPA and BBP showed estrogenic activity in these two assays, while the remaining eight chemicals did not at the concentrations tested . Additional data, together with in vivo and epidemiological examinations, are required . Such investigations should also provide information on the validity of these methods for testing the estrogenic activity of chemicals . Plant Physiol, 2004 Sep, 136(1), 2747 - 61 Epub 2004 Sep 03. Two transcription factors are negative regulators of gibberellin response in the HvSPY-signaling pathway in barley aleurone; Robertson M; SPINDLY (SPY) protein from barley (Hordeum vulgare L . cv Himalaya; HvSPY) negatively regulated GA responses in aleurone, and genetic analyses of Arabidopsis thaliana predict that SPY functions in a derepressible GA-signaling pathway . Many, if not all, GA-dependent responses require SPY protein, and to improve our understanding of how the SPY signaling pathway operates, a yeast two-hybrid screen was used to identify both upstream and downstream components that might regulate the activity of the HvSPY protein . A number of proteins from diverse classes were identified using HvSPY as bait and barley cDNA libraries as prey . Two of the HvSPY-interacting (HSI) proteins were transcription factors belonging to the myb and NAC gene families, HSImyb and HSINAC . Interaction occurred via the tetratricopeptide repeat domain of HvSPY and specificity was shown both in vivo and in vitro . Messenger RNAs for these proteins were expressed differentially in many parts of the barley plant but at very low levels . Both HSImyb and HSINAC inhibited the GA(3) up-regulation of alpha-amylase expression in aleurone, both were activators of transcription in yeast, and the green fluorescent protein-HSI fusion proteins were localized in the nucleus . These results are consistent with the model that HSI transcription factors act downstream of HvSPY as negative regulators and that they in turn could activate other negative regulators, forming the HvSPY negative regulator-signaling pathway for GA response. J Biol Chem, 2004 Nov 12, 279(46), 48205 - 13 Epub 2004 Aug 29. Different mechanisms participate in the R-dependent activity of the R2R3 MYB transcription factor C1; Hernandez JM et al.; The R2R3 MYB transcription factor C1 requires the basic helix-loop-helix factor R as an essential co-activator for the transcription of maize anthocyanin genes . In contrast, the R2R3 MYB protein P1 activates a subset of the C1-regulated genes independently of R . Substitution of six amino acids in P1 with the C1 amino acids results in P1(*), whose activity on C1-regulated and P1-regulated genes is R-dependent or R-enhanced, respectively . We have used P1(*) in combination with various promoters to uncover two mechanisms for R function . On synthetic promoters that contain only C1/P1 binding sites, R is an essential co-activator of C1 . This function of R is unlikely to simply be the result of an increase in the C1 DNA-binding affinity, since transcriptional activity of a C1 mutant that binds DNA at a higher affinity, comparable with P1, remains R-dependent . The differential transcriptional activity of C1 fusions with the yeast Gal4 DNA-binding domain in yeast and maize cells suggests that part of the function of R is to relieve C1 from a plant-specific inhibitor . A second function of R requires cis-regulatory elements in addition to the C1/P1 DNA-binding sites for R-enhanced transcription of a1 . We hypothesize that R functions in this mode by binding or recruiting additional factors to the anthocyanin regulatory element conserved in the promoters of several anthocyanin genes . Together, these findings suggest a model in which combinatorial interactions with co-activators enable R2R3 MYB factors with very similar DNA binding preferences to discriminate between target genes in vivo. J Biol Chem, 2004 Nov 5, 279(45), 46946 - 53 Epub 2004 Aug 30. CCTeta, a novel soluble guanylyl cyclase-interacting protein; Hanafy KA et al.; Nitric oxide (NO) transduces most of its biological effects through activation of the heterodimeric enzyme, soluble guanylyl cyclase (sGC) . Activation of sGC results in the production of cGMP from GTP . In this paper, we demonstrate a novel protein interaction between CCT (chaperonin containing t-complex polypeptide) subunit eta and the alpha1beta1 isoform of sGC . CCTeta was found to interact with the beta1 subunit of sGC via a yeast-two-hybrid screen . This interaction was then confirmed in vitro with a co-immunoprecipitation from mouse brain . The interaction between these two proteins was further supported by a co-localization of the proteins within rat brain . Using the yeast two-hybrid system, CCTeta was found to bind to the N-terminal portion of sGC . In vitro assays with purified CCTeta and Sf9 lysate expressing sGC resulted in a 30-50% inhibition of diethylamine diazeniumdiolate-NO-stimulated sGC activity . The same assays were then performed using BAY41-2272, an NO-independent allosteric sGC activator, and CCTeta had no effect on this activity . Furthermore, CCTeta had no effect on basal or sodium nitroprusside-stimulated alphabeta(Cys-105) sGC, a constitutively active mutant that only lacks the heme group . The N-terminal 94 amino acids of CCTeta seem to be critical for the mediation of this inhibition . Lastly, a 45% inhibition of sGC activity by CCTeta was seen in vivo in BE2 cells stably transfected with CCTeta and treated with sodium nitroprusside . These data suggest that CCTeta binds to sGC and, in cooperation with some other factor, inhibits its activity by modifying the binding of NO to the heme group or the subsequent conformational changes. DNA Seq, 2004 Apr, 15(2), 88 - 95 CDNA cloning and characterization of the Ve homologue gene StVe from Solanum torvum Swartz; Fei J et al.; Verticillium wilt is a disastrous disease causing significant yield losses of many crops . Isolation of verticillium wilt resistance gene is a fundamental work for controlling this disease through genetic engineering . In this report, we describe the cloning and characterization of a Ve like gene (StVe) from Solanum torvum Swartz . The nucleotide sequence of StVe is 3640 bp long with an open reading frame of 3414 bp encoding a protein precursor of 1138 aa . Sharing high homologies to tomato verticillium wilt disease resistance genes Ve1 and Ve2, the leucine rich (15.89%) protein StVe has a calculated molecular weight of 126.48kDa with an isoelectric point of 5.62 . It possesses a hydrophobic N-terminal signal peptide of 20 aa and 38 predicted leucine-rich repeats containing 32 potential N-glycosylation sites (28 being significant) . Fifty-seven predicted phosphorylation sites (36 for S, 8 for T and 13 for Y) distribute in StVe protein . A PEST-like sequence and a mammalian endocytosis signals YCVF are found within the C-terminal region . The C terminus of StVe concludes with the residues KKF similar to the KKX motif that confers endoplasmic reticulum localization in plants as well as mammals and yeast . The sequence analysis of the StVe gene implies that the StVe is a potential verticillium wilt disease resistance gene encoding a cell surface-like receptor protein. Acta Biochim Biophys Sin (Shanghai), 2004 Sep, 36(9), 623 - 8 Interaction between plasminogen activator inhibitor type-2 and pre-mRNA processing factor 8; Fan J et al.; The plasminogen activator inhibitor type-2 (PAI-2) dependent apoptosis protection is due to the 33 amino acids fragment located between helix C and D of PAI-2, this fragment may interact with some unknown intracellular proteins . In this study we used the fragment between helix C and D of PAI-2 as a bait to perform a yeast two-hybrid screen using a cDNA library constructed with HeLa cells during apoptosis, and retrieved a clone encoding 94 amino acid residues of C-terminus of pre-mRNA processing factor 8 (PRPF8) . Co-immunoprecipitation experiments confirmed that PAI-2 could interact with PRPF8 in vivo . PAI-2 could bind PRPF8 C-terminal in both the inside and outside of nuclear . These results suggested that the interaction between these two proteins might not be involved in the apoptosis process. Acta Biochim Biophys Sin (Shanghai), 2004 Sep, 36(9), 618 - 22 Trichostatin A extends the lifespan of Drosophila melanogaster by elevating hsp22 expression; Tao D et al.; The level of acetylation of histones in nucleosomes is related to the longevity of yeast and animals . However, the mechanisms by which acetylation and deacetylation affect longevity remain unclear . In present study, we investigated the influence of histone acetylation modification on the expression of hsp22 gene and the lifespan in Drosophila melanogaster using histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) . The results showed that TSA could extend the lifespan of Drosophila melanogaster . Furthermore, TSA significantly promoted the hsp22 gene transcription, and affected the chromatin morphology at the locus of hsp22 gene along the polytene chromosome . Present data implicate that TSA may affect the lifespan of Drosophila through changing the level of histone acetylation and influencing the expression of hsp22 gene that is related to aging. Int Arch Allergy Immunol, 2004 Oct, 135(2), 93 - 100 Epub 2004 Sep 02. Elevated levels of IgG and IgG4 to Malassezia allergens in atopic eczema patients with IgE reactivity to Malassezia; Johansson C et al.; BACKGROUND: The opportunistic yeast Malassezia is considered to be one of the factors that can contribute to atopic eczema (AE) . Elevated serum IgE levels, T-cell proliferation and positive skin prick test (SPT) and atopy patch test (APT) reactions to Malassezia are found among AE patients . METHODS: Sera from 127 AE patients, 14 patients with seborrheic dermatitis (SD) and 33 healthy controls were investigated for IgE and IgG4 to M . sympodialis extract and four recombinant Malassezia allergens; rMala s 1, rMala s 5, rMala s 6, and rMala s 9 . In addition, IgG to the recombinant allergens was analyzed . The IgG and IgG4 levels were compared to IgE levels and in vivo reactions (SPT and APT) to Malassezia . RESULTS: AE patients with serum IgE levels >0.35 kU/l to M . sympodialis extract had significantly higher IgG4 levels to M . sympodialis extract than AE patients without detectable serum IgE to M . sympodialis extract, SD patients and healthy controls . Among the AE patients with and without detectable serum IgE to M . sympodialis extract, respectively, there were no differences in IgG4 levels between patients with positive or negative in vivo reactions to M . sympodialis extract . IgG4 to the rMala s allergens was almost exclusively found among patients with IgE to the same allergen . Within the four tested rMala s allergens, most IgG4 reactions were found to rMala s 6, an allergen with homology to cyclophilin . CONCLUSIONS: Elevated serum IgG4 to M . sympodialis extract accompanies elevated serum IgE to the extract . This is further confirmed by the association between IgG/IgG4 and IgE to recombinant Malassezia allergens. Endocrinology, 2004 Dec, 145(12), 5820 - 31 Epub 2004 Sep 02. Interaction of stress-activated protein kinase-interacting protein-1 with the interferon receptor subunit IFNAR2 in uterine endometrium; Wang SZ et al.; During early pregnancy in ruminants, a type I interferon (IFN-tau) signals from the conceptus to the mother to ensure the functional survival of the corpus luteum . IFN-tau operates through binding to the type I IFN receptor (IFNR) . Here we have explored the possibility that IFNAR2, one of the two subunits of the receptor, might interact with hitherto unknown signal transduction factors in the uterus that link IFN action to pathways other than the well established Janus kinase-signal transducer and activator of transcription pathways . A yeast two-hybrid screen of an ovine (ov) endometrial cDNA library with the carboxyl-terminal 185 amino acids of ovIFNAR2 as bait identified stress-activated protein kinase-interacting protein 1 (ovSin1) as a protein that bound constitutively through its own carboxyl terminus to the receptor . ovSin1 is a little studied, 522-amino acid-long polypeptide (molecular weight, 59,200) that is highly conserved across vertebrates, but has identifiable orthologs in Drosophila and yeast . It appears to be expressed ubiquitously in mammals, although in low abundance, in a wide range of mammalian tissues in addition to endometrium . Sin1 mRNA occurs in at least two alternatively spliced forms, the smaller of which lacks a 108-bp internal exon . ovSin1, although not exhibiting features of a membrane-spanning protein, such as IFNAR2, is concentrated predominantly in luminal and glandular epithelial cells of the uterine endometrium . When ovSin1 and ovIFNAR2 are coexpressed, the two proteins can be coimmunoprecipitated and colocalized to the plasma membrane and to perinuclear structures . Sin1 provides a possible link among type I IFN action, stress-activated signaling pathways, and control of prostaglandin production. Blood . 2004 Sep 2; {Epub ahead of print} The adaptor protein 3BP2 associates with VAV guanine nucleotide exchange factors to regulate NFAT activation by the B-cell antigen receptor; Foucault I et al.; Engagement of the B-cell antigen receptor (BCR) activates kinases of the Src and Syk families and signaling complexes assembled by adaptor proteins, which dictate B-cell fate and function . The adaptor 3BP2/SH3BP2, an Abl Src homology domain 3 (SH3)-binding and Syk-kinases interacting protein, exhibits positive regulatory roles in T, natural killer (NK), and basophilic cells . However, its involvement in BCR signaling is completely unknown . Here we show that 3BP2 is tyrosine phosphorylated following BCR aggregation on B lymphoma cells, and that 3BP2 is a substrate for Syk and Fyn, but not Btk . To further explore the function of 3BP2 in B cells, we screened a yeast 2-hybrid B-lymphocyte library and found 3BP2 as a binding partner of Vav proteins . The interaction between 3BP2 and Vav proteins involved both constitutive and inducible mechanisms . 3BP2 also interacted with other components of the BCR signaling pathway, including Syk and phospholipase C gamma (PLC-gamma) . Furthermore, overexpression and RNAi blocking experiments showed that 3BP2 regulated BCR-mediated activation of nuclear factor of activated T cells (NFATs) . Finally, evidence was provided that 3BP2 functionally cooperates with Vav proteins and Rho GTPases to activate NFATs . Our results show that 3BP2 may regulate BCR-mediated gene activation through Vav proteins. J Biol . 2004;3(4):18 . Epub 2004 Aug 31. Adaptive evolution of centromere proteins in plants and animals; Talbert PB et al.; BACKGROUND: Centromeres represent the last frontiers of plant and animal genomics . Although they perform a conserved function in chromosome segregation, centromeres are typically composed of repetitive satellite sequences that are rapidly evolving . The nucleosomes of centromeres are characterized by a special H3-like histone (CenH3), which evolves rapidly and adaptively in Drosophila and Arabidopsis . Most plant, animal and fungal centromeres also bind a large protein, centromere protein C (CENP-C), that is characterized by a single 24 amino-acid motif (CENPC motif) . RESULTS: Whereas we find no evidence that mammalian CenH3 (CENP-A) has been evolving adaptively, mammalian CENP-C proteins contain adaptively evolving regions that overlap with regions of DNA-binding activity . In plants we find that CENP-C proteins have complex duplicated regions, with conserved amino and carboxyl termini that are dissimilar in sequence to their counterparts in animals and fungi . Comparisons of Cenpc genes from Arabidopsis species and from grasses revealed multiple regions that are under positive selection, including duplicated exons in some grasses . In contrast to plants and animals, yeast CENP-C (Mif2p) is under negative selection . CONCLUSIONS: CENP-Cs in all plant and animal lineages examined have regions that are rapidly and adaptively evolving . To explain these remarkable evolutionary features for a single-copy gene that is needed at every mitosis, we propose that CENP-Cs, like some CenH3s, suppress meiotic drive of centromeres during female meiosis . This process can account for the rapid evolution and the complexity of centromeric DNA in plants and animals as compared to fungi. Shi Yan Sheng Wu Xue Bao, 2002 Mar, 35(1), 58 - 61 {In silico cloning of evolutionarily conserved mouse F-LANa}; Ying H et al.; Differentially expressed genes between normal liver and hepatocellular carcinomas were investigated using differential display . In previous study, human F-LANa was identified as a differentially expressed gene, up-regulated in hepatocellular carcinoma . In this study, we developed an in silico cloning approach to rapidly and accurately characterize the mouse ortholog of the human F-LANa . Mouse F-LANa encodes a 239 aa protein exhibiting 97.9% similarity to the human ortholog gene . Homology analysis was carried out in various species and showed that F-LANa was evolutionarily conserved from yeast to human . Based on the alignment results, phylogenetic tree was established here. EMBO J, 2004 Sep 15, 23(18), 3589 - 98 Epub 2004 Sep 02. Tandem LIM domains provide synergistic binding in the LMO4:Ldb1 complex; Deane JE et al.; Nuclear LIM-only (LMO) and LIM-homeodomain (LIM-HD) proteins have important roles in cell fate determination, organ development and oncogenesis . These proteins contain tandemly arrayed LIM domains that bind the LIM interaction domain (LID) of the nuclear adaptor protein LIM domain-binding protein-1 (Ldb1) . We have determined a high-resolution X-ray crystal structure of LMO4, a putative breast oncoprotein, in complex with Ldb1-LID, providing the first example of a tandem LIM:Ldb1-LID complex and the first structure of a type-B LIM domain . The complex possesses a highly modular structure with Ldb1-LID binding in an extended manner across both LIM domains of LMO4 . The interface contains extensive hydrophobic and electrostatic interactions and multiple backbone-backbone hydrogen bonds . A mutagenic screen of Ldb1-LID, assessed by yeast two-hybrid and competition ELISA analysis, identified key features at the interface and revealed that the interaction is tolerant to mutation . These combined properties provide a mechanism for the binding of Ldb1 to numerous LMO and LIM-HD proteins . Furthermore, the modular extended interface may form a general mode of binding to tandem LIM domains. Proc Natl Acad Sci U S A, 2004 Sep 14, 101(37), 13590 - 5 Epub 2004 Sep 01. Proapoptotic N-truncated BCL-xL protein activates endogenous mitochondrial channels in living synaptic terminals; Jonas EA et al.; Neuronal death is often preceded by functional alterations at nerve terminals . Anti- and proapoptotic BCL-2 family proteins not only regulate the neuronal death pathway but also affect excitability of healthy neurons . We found that exposure of squid stellate ganglia to hypoxia, a death stimulus for neurons, causes a cysteine protease-dependent loss of full-length antiapoptotic BCL-xL, similar to previous findings in mammalian cells . Therefore, to determine the direct effect of the naturally occurring proapoptotic cleavage product of BCL-xL on mitochondria, recombinant N-truncated BCL-xL was applied to mitochondria inside the squid presynaptic terminal and to purified mitochondria isolated from yeast . N-truncated BCL-xL rapidly induced large multi-conductance channels with a maximal conductance significantly larger than those produced by full-length BCL-xL . This activity required the hydrophobic C terminus and the BH3 domain of BCL-xL . Moreover, N-truncated BCL-xL failed to produce any channel activity when applied to plasma membranes, suggesting that a component of the mitochondrial membrane is necessary for its actions . Consistent with this idea, the large channels induced by N-truncated BCL-xL are inhibited by NADH and require the presence of VDAC, a voltage-dependent anion channel present in the outer mitochondrial membrane . These observations suggest that the mitochondrial channels specific to full-length and N-truncated BCL-xL contribute to their opposite effects on synaptic transmission, and are consistent with their opposite effects on the cell death pathway. Mol Biol Evol, 2004 Dec, 21(12), 2326 - 39 Epub 2004 Sep 01. Adaptive evolution drives the diversification of zinc-finger binding domains; Schmidt D et al.; The human genome is estimated to contain 700 zinc-finger genes, which perform many key functions, including regulating transcription . The dramatic increase in the number of these genes as we move from yeast to C . elegans to Drosophila and to humans, as well as the clustered organization of these genes in humans, suggests that gene duplication has played an important role in expanding this family of genes . Using likelihood methods developed by Yang and parsimony methods introduced by Suzuki and Gojobori, we have investigated four clusters of zinc-finger genes on human chromosome 19 and found evidence that positive selection was involved in diversifying the family of zinc-finger binding motifs. Mol Biol Cell, 2004 Nov, 15(11), 4949 - 59 Epub 2004 Sep 01. Cross talk between sphingolipids and glycerophospholipids in the establishment of plasma membrane asymmetry; Kihara A et al.; Glycerophospholipids and sphingolipids are distributed asymmetrically between the two leaflets of the lipid bilayer . Recent studies revealed that certain P-type ATPases and ATP-binding cassette (ABC) transporters are involved in the inward movement (flip) and outward movement (flop) of glycerophospholipids, respectively . In this study of phytosphingosine (PHS)-resistant yeast mutants, we isolated mutants for PDR5, an ABC transporter involved in drug efflux as well as in the flop of phosphatidylethanolamine . The pdr5 mutants exhibited an increase in the efflux of sphingoid long-chain bases (LCBs) . Genetic analysis revealed that the PHS-resistant phenotypes exhibited by the pdr5 mutants were dependent on Rsb1p, a putative LCB-specific transporter/translocase . We found that the expression of Rsb1p was increased in the pdr5 mutants . We also demonstrated that expression of RSB1 is under the control of the transcriptional factor Pdr1p . Expression of Rsb1p also was enhanced in mutants for the genes involved in the flip of glycerophospholipids, including ROS3, DNF1, and DNF2 . These results suggest that altered glycerophospholipid asymmetry induces the expression of Rsb1p . Conversely, overexpression of Rsb1p resulted in increased flip and decreased flop of fluorescence-labeled glycerophospholipids . Thus, there seems to be cross talk between sphingolipids and glycerophospholipids in maintaining the functional lipid asymmetry of the plasma membrane. Mol Biol Cell, 2004 Nov, 15(11), 5092 - 100 Epub 2004 Sep 01. The interaction of neurofilaments with the microtubule motor cytoplasmic dynein; Wagner OI et al.; Neurofilaments are synthesized in the cell body of neurons and transported outward along the axon via slow axonal transport . Direct observation of neurofilaments trafficking in live cells suggests that the slow outward rate of transport is due to the net effects of anterograde and retrograde microtubule motors pulling in opposition . Previous studies have suggested that cytoplasmic dynein is required for efficient neurofilament transport . In this study, we examine the interaction of neurofilaments with cytoplasmic dynein . We used fluid tapping mode atomic force microscopy to visualize single neurofilaments, microtubules, dynein/dynactin, and physical interactions between these neuronal components . AFM images suggest that neurofilaments act as cargo for dynein, associating with the base of the motor complex . Yeast two-hybrid and affinity chromatography assays confirm this hypothesis, indicating that neurofilament subunit M binds directly to dynein IC . This interaction is blocked by monoclonal antibodies directed either to NF-M or to dynein . Together these data suggest that a specific interaction between neurofilament subunit M and cytoplasmic dynein is involved in the saltatory bidirectional motility of neurofilaments undergoing axonal transport in the neuron. J Biol Chem, 2004 Nov 5, 279(45), 46424 - 30 Epub 2004 Sep 01. The ISG15 isopeptidase UBP43 is regulated by proteolysis via the SCFSkp2 ubiquitin ligase; Tokarz S et al.; The Skp2 oncoprotein belongs to the family of F-box proteins that function as substrate recognition factors for SCF (Skp1, cullin, F-box protein) E3 ubiquitin-ligase complexes . Binding of the substrate to the SCFSkp2 complex catalyzes the conjugation of ubiquitin molecules to the bound substrate, resulting in multi-ubiquitination and rapid degradation by the 26 S proteasome . Using Skp2 as bait in a yeast two-hybrid screen, we have identified UBP43 as a novel substrate for Skp2 . UBP43 belongs to the family of ubiquitin isopeptidases and specifically cleaves ISG15, a ubiquitin-like molecule that is induced by cellular stresses, such as type 1 interferons (IFN), nephrotoxic damage, and bacterial infection . UBP43 was originally identified as an up-regulated gene in knock-in mice expressing an acute myelogenous leukemia fusion protein, AML1-ETO, as well as in melanoma cell lines treated with IFN-beta . The phenotype of UBP43 knockout mice includes shortened life span, hypersensitivity to IFN, and neuronal damage, suggesting that tight regulation of ISG15 conjugation is critical for normal cellular function . In this study, we demonstrate that UBP43 is ubiquitinated in vivo and accumulates in cells treated with proteasome inhibitors . We also show that Skp2 promotes UBP43 ubiquitination and degradation, resulting in higher levels of ISG15 conjugates . In Skp2-/- mouse cells, levels of UBP43 are consistently up-regulated, whereas levels of ISG15 conjugates are reduced . Our results demonstrate that the SCFSkp2 is involved in controlling UBP43 protein levels and may therefore play an important role in modulating type 1 IFN signaling. Am J Physiol Cell Physiol, 2005 Jan, 288(1), C148 - 55 Epub 2004 Sep 01. Serum and glucocorticoid-regulated kinase Sgk1 inhibits insulin-dependent activation of phosphomannomutase 2 in transfected COS-7 cells; Menniti M et al.; Serum- and glucocorticoid-regulated kinase (Sgk1) is considered to be an essential convergence point for peptide and steroid regulation of ENaC-mediated sodium transport . We tried to identify molecular partners of Sgk1 by yeast two-hybrid screening . Yeast two-hybrid screening showed a specific interaction between Sgk1 and phosphomannomutase (PMM)2, the latter of which is an enzyme involved in the regulation of glycoprotein biosynthesis . The interaction was confirmed in intact cells by coimmunoprecipitation and colocalization detected using confocal microscopy . We were then able to demonstrate that Sgk1 phosphorylated PMM2 in an in vitro assay . In addition, we found that the enzymatic activity of PMM2 is upregulated by insulin treatment and that Sgk1 completely inhibits PMM2 activity both in the absence and in the presence of insulin stimulation . These data provide evidence suggesting that Sgk1 may modulate insulin action on the cotranslational glycosylation of glycoproteins. Curr Biol, 2004 Sep 7, 14(17), R711 - 3 Membrane targeting: glued by a lipid to the ER; Daum G; Opi1p, a transcription regulator of phospholipid metabolism in budding yeast, is retained in the endoplasmic reticulum by association with Scs2p . New research shows that binding of phosphatidic acid to Opi1p is a prerequisite for this targeting. Curr Biol, 2004 Sep 7, 14(17), R708 - 10 MAPK signaling: Sho business; Seet BT et al.; Sho1 is a membrane protein in yeast that activates the Hog MAPK signaling pathway in response to high osmolarity . An accumulating body of work has focused on Sho1 as a model to better understand the mechanisms that dictate signaling specificity. Curr Biol, 2004 Sep 7, 14(17), R702 - 4 Microtubule dynamics: faint speckle, hidden dragon; Sawin KE; The results of recent experiments in budding and fission yeast show that there is a diversity of mechanisms for targeting proteins to the plus ends of microtubules in eukaryotic cells. Curr Biol, 2004 Sep 7, 14(17), 1598 - 603 Scc2 couples replication licensing to sister chromatid cohesion in Xenopus egg extracts; Gillespie PJ et al.; The cohesin complex is a central player in sister chromatid cohesion, a process that ensures the faithful segregation of chromosomes in mitosis and meiosis . Previous genetic studies in yeast show that Scc2/Mis4, a HEAT-repeat-containing protein, is required for the loading of cohesin onto chromatin . In this study, we have identified two isoforms of Scc2 in humans and Xenopus (termed Scc2A and Scc2B), which are encoded by a single gene but have different carboxyl termini created by alternative splicing . Both Scc2A and Scc2B bind to chromatin concomitant with cohesin during DNA replication in Xenopus egg extracts . Simultaneous immunodepletion of Scc2A and Scc2B from the extracts impairs the association of cohesin with chromatin, leading to severe defects in sister chromatid pairing in the subsequent mitosis . The loading of Scc2 onto chromatin is inhibited in extracts treated with geminin but not with p21(CIP1), suggesting that this step depends on replication licensing but not on the initiation of DNA replication . Upon mitotic entry, Scc2 is removed from chromatin through a mechanism that requires cdc2 but not aurora B or polo-like kinase . Our results suggest that vertebrate Scc2 couples replication licensing to sister chromatid cohesion by facilitating the loading of cohesin onto chromatin. Curr Biol, 2004 Sep 7, 14(17), 1569 - 75 Feo, the Drosophila homolog of PRC1, is required for central-spindle formation and cytokinesis; Verni F et al.; We performed a functional analysis of fascetto (feo), a Drosophila gene that encodes a protein homologous to the Ase1p/PRC1/MAP65 conserved family of microtubule-associated proteins (MAPs) . These MAPs are enriched at the spindle midzone in yeast and mammals and at the fragmoplast in plants, and are essential for the organization and function of these microtubule arrays . Here we show that the Feo protein is specifically enriched at the central-spindle midzone and that its depletion either by mutation or by RNAi results in aberrant central spindles . In Feo-depleted cells, late anaphases showed normal overlap of the antiparallel MTs at the cell equator, but telophases displayed thin MT bundles of uniform width instead of robust hourglass-shaped central spindles . These thin central spindles exhibited diffuse localizations of both the Pav and Asp proteins, suggesting that these spindles comprise improperly oriented MTs . Feo-depleted cells also displayed defects in the contractile apparatus that correlated with those in the central spindle; late anaphase cells formed regular contractile structures, but these structures did not constrict during telophase, leading to failures in cytokinesis . The phenotype of Feo-depleted telophases suggests that Feo interacts with the plus ends of central spindle MTs so as to maintain their precise interdigitation during anaphase-telophase MT elongation and antiparallel sliding. Structure (Camb), 2004 Sep, 12(9), 1683 - 91 Crystal structure of the carboxyltransferase domain of acetyl-coenzyme A carboxylase in complex with CP-640186; Zhang H et al.; Acetyl-coenzyme A carboxylases (ACCs) are important targets for the development of therapeutic agents against obesity, diabetes, and other diseases . CP-640186 is a potent inhibitor of mammalian ACCs and can reduce body weight and improve insulin sensitivity in test animals . It is believed to target the carboxyltransferase (CT) domain of these enzymes . Here we report the crystal structure of the yeast CT domain in complex with CP-640186 . The inhibitor is bound in the active site at the interface of a dimer of the CT domain . CP-640186 has tight interactions with the putative biotin binding site in the CT domain and demonstrates a distinct mode of inhibiting the CT activity as compared to the herbicides that inhibit plant ACCs . The affinity of inhibitors for the CT domain has been assessed using kinetic and fluorescence anisotropy binding studies . The structural information identifies three regions for drug binding in the active site of CT. Environ Sci Pollut Res Int, 2004, 11(4), 240 - 53 The identification of readily bioavailable pollutants in Lake Shkodra/Skadar using semipermeable membrane devices (SPMDs), bioassays and chemical analysis; Rastall AC et al.; GOAL, SCOPE AND BACKGROUND: Lake Shkodra/Skadar is the largest lake in the Balkans region and located on the border between Albania to the south and Montenegro to the north . Because of the wide range of endemic, rare or endangered plant and animal species it supports, Lake Shkodra/Skadar and its extensive associated wetlands are internationally recognised as a site of significance and importance (Ramsar site) . In recent years, social and economic changes in both Albania and Montenegro have lead to unprecedented levels of urban and industrial effluent entering the lake . Of particular concern is the increasing input of toxic hydrophobic organic pollutants (HOPs) into the lake and the degree to which these compounds are available for uptake by aquatic biota . Semipermeable membrane devices (SPMDs) have been shown to sample the readily bioavailable fraction (dissolved phase) of waterborne HOPs and in doing so provide relevant data for exposure assessment . The aim of the current study was to use SPMD-based sampling in conjunction with appropriate bioassays and chemical analysis to identify readily bioavailable HOPs in the lake . METHODS: SPMDs were constructed and deployed at three sites in the Albanian sector and three sites in the Montenegrin sector of Lake Skadar/Shkodra for 21 days . Following the dialytic recovery of target analytes and size exclusion chromatographic clean-up, aliquots of SPMD samples were subjected to GC-MS scan analysis for major components, GC-MS SIM analysis for 16 priority pollutant polycyclic aromatic hydrocarbons (PP-PAHs) and assayed for EROD-inducing, estrogenic and mutagenic potential using rainbow trout liver cells (RTL-W1), the yeast estrogen screen (YES) and the Ames Test, respectively . RESULTS AND DISCUSSION: A total of 39 compounds were tentatively identified in SPMD samples from the six sampling sites . Alkylated PAHs were the most abundant and ubiquitous compounds present along with various sterols and sterol derivatives . Numerous other compounds remain unidentified . 15 of the 16 targeted PP-PAHs were present in samples from one or more of the sampling sites indicating these compounds are both readily bioavailable and widely distributed in Lake Shkodra/Skadar . Total PP-PAH concentrations ranged between 3991 ng/SPMD and 10695 ng/SPMD . Bioassays carried out on SPMD samples revealed significant EROD-inducing and estrogenic potential at five of the six sampling sites indicating toxicologically relevant compounds are readily available for uptake by resident aquatic biota . EROD-inducing potential was positively correlated with targeted PP-PAH concentration (r2 = 0.74) . However, comparison of bioassay- and analytically-derived toxicity equivalents revealed targeted PP-PAHs were responsible for less than 0.06% of the total EROD-inducing potential . CONCLUSIONS AND OUTLOOK: The combination of SPMD-based sampling with appropriate bioassays and chemical analysis provided an effective tool for the identification of environmentally relevant waterborne pollutants in Lake Shkodra/Skadar . Our results show that toxicologically relevant HOPs including EROD-inducing and potentially estrogenic compounds are widely distributed in the lake and readily available for uptake by aquatic biota . Our results also suggest that alkylated PAHs rather than parent compounds may be of greater toxicological relevance in the lake . As anthropogenic influences continue to increase, SPMD-based sampling is expected to play a central role in future research concerned with the identification, monitoring and assessment of the risk posed by HOPs to Lake Shkodra/Skadar's aquatic biota. Chem Commun (Camb), 2004 Sep 7, (17), 1936 - 7 Epub 2004 Jul 29. Looking glass inhibitors: L-DMDP, a more potent and specific inhibitor of alpha-glucosidases than the enantiomeric natural product DMDP; Yu CY et al.; L-DMDP, prepared from D-gulonolactone, is a highly specific inhibitor of a number of plant and mammalian alpha-glucosidases {between 2 and 4 orders of magnitude more potent than the enantiomeric natural product DMDP} but is not an inhibitor of bacterial and yeast alpha-glucosidases . Additionally N-butyl-DMDP is a potent inhibitor of ceramide-specific glucosyltransferase but N-butyl-L-DMDP shows no inhibition. Mol Cell Biol, 2004 Sep, 24(18), 8080 - 9 Drosophila Ada2b is required for viability and normal histone H3 acetylation; Qi D et al.; Regulation of chromatin through histone acetylation is an important step in gene expression . The Gcn5 histone acetyltransferase is part of protein complexes, e.g., the SAGA complex, that interact with transcriptional activators, targeting the enzyme to specific promoters and assisting in recruitment of the basal RNA polymerase transcription machinery . The Ada2 protein directly binds to Gcn5 and stimulates its catalytic activity . Drosophila contains two Ada2 proteins, Drosophila Ada2a (dAda2a) and dAda2b . We have generated flies that lack dAda2b, which is part of a Drosophila SAGA-like complex . dAda2b is required for viability in Drosophila, and its deletion causes a reduction in histone H3 acetylation . A global hypoacetylation of chromatin was detected on polytene chromosomes in dAda2b mutants . This indicates that the dGcn5-dAda2b complex could have functions in addition to assisting in transcriptional activation through gene-specific acetylation . Although the Drosophila p53 protein was previously shown to interact with the SAGA-like complex in vitro, we find that p53 induction of reaper gene expression occurs normally in dAda2b mutants . Moreover, dAda2b mutant animals show excessive p53-dependent apoptosis in response to gamma radiation . Based on this result, we speculate that dAda2b may be necessary for efficient DNA repair or generation of a DNA damage signal . This could be an evolutionarily conserved function, since a yeast ada2 mutant is also sensitive to a genotoxic agent. J Cell Sci, 2004 Sep 15, 117(Pt 20), 4807 - 18 Epub 2004 Aug 31. Arabidopsis VARIEGATED 3 encodes a chloroplast-targeted, zinc-finger protein required for chloroplast and palisade cell development; Naested H et al.; The stable, recessive Arabidopsis variegated 3 (var3) mutant exhibits a variegated phenotype due to somatic areas lacking or containing developmentally retarded chloroplasts and greatly reduced numbers of palisade cells . The VAR3 gene, isolated by transposon tagging, encodes the 85.9 kDa VAR3 protein containing novel repeats and zinc fingers described as protein interaction domains . VAR3 interacts specifically in yeast and in vitro with NCED4, a putative polyene chain or carotenoid dioxygenase, and both VAR3 and NCED4 accumulate in the chloroplast stroma . Metabolic profiling demonstrates that pigment profiles are qualitatively similar in wild type and var3, although var3 accumulates lower levels of chlorophylls and carotenoids . These results indicate that VAR3 is a part of a protein complex required for normal chloroplast and palisade cell development. Cell Mol Life Sci, 2004 Sep, 61(17), 2264 - 73 Hcc-1 is a novel component of the nuclear matrix with growth inhibitory function; Leaw CL et al.; Hcc-1 is a novel nuclear protein containing the SAF-box DNA-binding domain . It binds to both double-stranded and single-stranded DNA with higher affinity for the single-stranded form . In addition, it also binds specifically to scaffold/matrix attachment region DNA . These nucleic acid-binding characteristics suggest a potential function for Hcc-1 as a component of the heterogeneous ribonucleoprotein complex . Using a yeast two-hybrid screen, two DEAD-box RNA helicases, BAT1 and DDX39, were identified as proteins that interact with Hcc-1 . Interactions with these RNA helicases suggested a role for Hcc-1 in nucleic acid biogenesis . Expression of Hcc-1 in the HEK293 cell line resulted in a slower growth rate compared to controls (p = 0.0173) and an accumulation of cells at the G2/M phase (p = 0.0276 compared to control HEK293 cells) . Taken together, these results suggest a role for Hcc-1 in growth regulation and nucleic acids metabolism. J Bioenerg Biomembr, 2004 Jun, 36(3), 219 - 28 Functional characterization of a Drosophila mitochondrial uncoupling protein; Fridell YW et al.; Sequence alignment of conserved signature motifs predicts the existence of the uncoupling protein 5 (UCP5)/brain mitochondrial carrier protein (BMCP1) homologue in Drosophila melanogaster . Here we demonstrate the functional characterization of the Drosophila melanogaster UCP5 protein (DmUCP5) in the heterologous yeast system, the first insect UCP reported to date . We show that physiological levels of DmUCP5 expression are responsible for an increase in state 4 respiration rates and a decrease in mitochondrial membrane potential . Furthermore, similar to UCP1, UCP2, and UCP3, the uncoupling activity of DmUCP5 is augmented by fatty acids and inhibited by the purine nucleotide GDP . Thus, DmUCP5 shares the mechanisms known to regulate the UCPs characterized to date . A lack of growth inhibition observed in DmUCP5 expressing yeast is consistent with the notion that physiological uncoupling has a minimal effect on cell growth . Finally, semiquantitative RT-PCR analysis shows a distinctive pattern of DmUCP5 expression predominantly localized in the adult head, similar to the expression pattern of its mammalian homologues . The conserved regulation of the expression of this gene from mammals to fruit flies suggests a role for UCP5 in the brain. J Biol Chem, 2004 Nov 19, 279(47), 48562 - 8 Epub 2004 Aug 27. The human FA2H gene encodes a fatty acid 2-hydroxylase; Alderson NL et al.; 2-Hydroxysphingolipids are a subset of sphingolipids containing 2-hydroxy fatty acids . The 2-hydroxylation occurs during de novo ceramide synthesis and is catalyzed by fatty acid 2-hydroxylase (also known as fatty acid alpha-hydroxylase) . In mammals, 2-hydroxysphingolipids are present abundantly in brain because the major myelin lipids galactosylceramides and sulfatides contain 2-hydroxy fatty acids . Here we report identification and characterization of a human gene that encodes a fatty acid 2-hydroxylase . Data base searches revealed a human homologue of the yeast ceramide 2-hydroxylase gene (FAH1), which we named FA2H . The FA2H gene encodes a 372-amino acid protein with 36% identity and 46% similarity to yeast Fah1p . The amino acid sequence indicates that FA2H protein contains an N-terminal cytochrome b5 domain and four potential transmembrane domains . FA2H also contains the iron-binding histidine motif conserved among membrane-bound desaturases/hydroxylases . COS7 cells expressing human FA2H contained 3-20-fold higher levels of 2-hydroxyceramides (C16, C18, C24, and C24:1) and 2-hydroxy fatty acids compared with control cells . Microsomal fractions prepared from transfected COS7 cells showed tetracosanoic acid 2-hydroxylase activities in an NADPH- and NADPH: cytochrome P-450 reductase-dependent manner . FA2H lacking the N-terminal cytochrome b5 domain had little activity, indicating that this domain is a functional component of this enzyme . Northern blot analysis showed that the FA2H gene is highly expressed in brain and colon tissues . These results demonstrate that the human FA2H gene encodes a fatty acid 2-hydroxylase . FA2H is likely involved in the formation of myelin 2-hydroxy galactosylceramides and -sulfatides. J Biol Chem, 2004 Nov 5, 279(45), 47081 - 91 Epub 2004 Aug 26. Leukemia/lymphoma-related factor, a POZ domain-containing transcriptional repressor, interacts with histone deacetylase-1 and inhibits cartilage oligomeric matrix protein gene expression and chondrogenesis; Liu CJ et al.; Mutations in the human cartilage oligomeric matrix protein (COMP) gene have been linked to the development of pseudoachondroplasia and multiple epiphyseal dysplasia . We previously cloned the promoter region of the COMP gene and delineated a minimal negative regulatory element (NRE) that is both necessary and sufficient to repress its promoter (Issack, P . S., Fang, C . H., Leslie, M . P., and Di Cesare, P . E . (2000) J . Orthop . Res . 18, 345-350; Issack, P . S., Liu, C . J., Prazak, L., and Di Cesare, P . E . (2004) J . Orthop . Res . 22, 751-758) . In this study, a yeast one-hybrid screen for proteins that associate with the NRE led to the identification of the leukemia/lymphoma-related factor (LRF), a transcriptional repressor that contains a POZ (poxvirus zinc finger) domain, as an NRE-binding protein . LRF bound directly to the NRE both in vitro and in living cells . Nine nucleotides (GAGGGTCCC) in the 30-bp NRE are essential for binding to LRF . LRF showed dose-dependent inhibition of COMP-specific reporter gene activity, and exogenous overexpression of LRF repressed COMP gene expression in both rat chondrosarcoma cells and bone morphogenetic protein-2-treated C3H10T1/2 progenitor cells . In addition, LRF also inhibited bone morphogenetic protein-2-induced chondrogenesis in high density micromass cultures of C3H10T1/2 cells, as evidenced by lack of expression of other chondrocytic markers, such as aggrecan and collagen types II, IX, X, and XI, and by Alcian blue staining . LRF associated with histone deacetylase-1 (HDAC1), and experiments utilizing the HDAC inhibitor trichostatin A revealed that LRF-mediated repression requires deacetylase activity . LRF is the first transcription factor found to bind directly to the COMP gene promoter, to recruit HDAC1, and to regulate both COMP gene expression and chondrogenic differentiation. Mol Ther, 2004 Sep, 10(3), 432 - 46 Maximizing antigen targeting to the proteasome for gene-based vaccines; Andersson HA et al.; Wild-type or immunoevasive antigens can drive weak CD8+T-cell responses against both dominant and subdominant epitopes during gene-based vaccination . For many antigens, fusion to ubiquitin (Ub) to target them to the proteasome circumvents this problem . Although this procedure works in most cases, for one subset of antigens, Ub fusion does not improve immune responses . To determine why these failures occur, we have evaluated in detail the 'rules' for proteasome targeting that have been applied in mammalian vaccine studies, but that were actually defined in yeast systems . To do this, we fused a series of engineered Ub genes to green fluorescent protein (GFP) and tested their ability to target GFP to the proteasome for enhanced antigen processing and CD8+ T-cell responses . Here we demonstrate that Ub fusion mediates enhanced CD8+ responses by proteasome targeting rather than by enhancing protein translation . We also show that several of the yeast-defined Ub constructs failed to target the proteasome in mammalian cells and likewise failed to enhance transgene-specific CD8+ T-cell responses in mice . In contrast, when mammalian-optimized constructs were applied to target the influenza virus nucleoprotein, CD8+ responses were enhanced against its refractory subdominant epitope in mice . This work demonstrates that Ub fusion has efficacy to enhance CD8+ responses, especially against subdominant antigen epitopes, provided constructs are optimized for mammalian use. Biochem Biophys Res Commun, 2004 Sep 24, 322(3), 957 - 65 ADRP is dissociated from lipid droplets by ARF1-dependent mechanism; Nakamura N et al.; Adipocyte differentiation-related protein (ADRP) is a member of PAT proteins existing in lipid droplets (LDs) . By yeast two-hybrid screening, we identified ADP-ribosylation factor 1 (ARF1) as a binding partner of ADRP . The interaction of ADRP and ARF1 was verified by GST pull-down and co-immunoprecipitation experiments . Interestingly, ADRP precipitated the GDP-bound ARF1 preferentially to the GTP-bound ARF1 . Consistent with this, either brefeldin A (BFA), a fungal metabolite to inhibit ARF-GEF, or a dominant-negative mutant of ARF1 caused dissociation of ADRP from LD . On the other hand, overexpression of wild-type ARF1 did not promote the ADRP dissociation or new LD formation . By using deletion mutants, a central domain of ADRP, which is dispensable for LD binding, was shown to bind to ARF1 . The present study showed that the GDP-bound ARF1 induces dissociation of ADRP from the LD surface, and that LD is a target of BFA action. FEMS Microbiol Lett, 2004 Sep 1, 238(1), 255 - 62 Characterisation of complex formation between members of the Mycobacterium tuberculosis complex CFP-10/ESAT-6 protein family: towards an understanding of the rules governing complex formation and thereby functional flexibility; Lightbody KL et al.; We have previously shown that the secreted M . tuberculosis complex proteins CFP-10 and ESAT-6 form a tight, 1:1 complex, which may represent their functional form . In the work reported here a combination of yeast two-hybrid and biochemical analysis has been used to characterise complex formation between two other pairs of CFP-10/ESAT-6 family proteins (Rv0287/Rv0288 and Rv3019c/Rv3020c) and to determine whether complexes can be formed between non-genome paired members of the family . The results clearly demonstrate that Rv0287/Rv0288 and Rv3019c/3020c form tight complexes, as initially observed for CFP-10/ESAT-6 . The closely related Rv0287/Rv0288 and Rv3019c/Rv3020c proteins are also able to form non-genome paired complexes (Rv0287/Rv3019c and Rv0288/Rv3020c), but are not capable of binding to the more distantly related CFP-10/ESAT-6 proteins. FEMS Microbiol Lett, 2004 Sep 1, 238(1), 79 - 84 Elucidation of polysaccharide origin in Ramalina peruviana symbiosis; Cordeiro LM et al.; A structural elucidation of polysaccharides extracted from the aposymbiotically cultured mycobiont of the lichen Ramalina peruviana was carried out in order to determine whether the polysaccharides found previously in the symbiotic thalli are produced by the mycobiont or photobiont or both . The mycobiont isolate was cultivated on a solid malt-yeast extract-medium and the freeze-dried colonies were defatted and the polysaccharides extracted successively with hot water and aq . 2% KOH, each at 100 degrees C . The alkaline extract was obtained in much higher yield (31.5%) and submitted to a freeze-thawing treatment, giving rise to a precipitate (PK2) of a mixture of (1-->3),(1-->4)-alpha-glucan (1.2:1 ratio, nigeran) and a (1-->3)-beta-glucan (laminaran) . The mother liquor was treated with Fehling solution to give a precipitate (galactomannan) . This had a (1-->6)-linked alpha-d-mannopyranosyl main chain, substituted at O-4 and in small proportion at O-2,4 by beta-Galp units . All three polysaccharides have previously been found in the symbiotic thalli of R . peruviana, showing that these are produced by the fungus, without the participation of the Trebouxia photobiont . Surprisingly, isolichenan, a cold-water soluble (1-->3),(1-->4)-alpha-linked-glucan (3:1 ratio) was not found in the isolated mycobiont, despite being the main polysaccharide found in the thalli. Curr Biol, 1993 Dec 1, 3(12), 805 - 12 Association of pRas and pRaf-1 in a complex correlates with activation of a signal transduction pathway; Finney RE et al.; Background: A key pathway for transduction of proliferative, developmental and oncogenic stimuli from receptors at the cell surface to transcription factors located in the nucleus involves the activation of pRas and pRaf-1 . Recent publications have described a physical interaction between pRas and pRaf-1, either as ectopic proteins in yeast or as recombinant proteins added to cellular extracts . Until now, however, physical complexes that include pRas and pRaf-1 have not been identified as native structures in mammalian cells . Results: We have directly identified a pRas-pRaf-1 complex in extracts of mammalian cells . Formation of the complex is augmented in neoplastically transformed cells expressing constitutively activated pRas . Moreover, the complexes form in concert with the activation of pRas during intracellular signalling through the T-cell receptor in T-leukemia cells . Conclusions: We propose that, pRas signals to pRaf-1 in vivo by means of a direct physical interaction that results in activation of the pRaf-1 protein kinase. Curr Biol, 1993 Jul 1, 3(7), 424 - 33 Simple and efficient generation of marked clones in Drosophila; Harrison DA et al.; Background: Cell lineage analysis and mosaic analysis of mutations are important techniques that are used to study the development of many organisms . Unfortunately, the methods employed for such analyses are usually inefficient, technically demanding or labor intensive . In Drosophila, the most common methodology used for the generation of mosaic animals is mitotic recombination, which is induced by X-rays . Although this technique is simple, it has the undesirable characteristics of a low efficiency and a high rate of cell death . Furthermore, although a large number of marker systems has been employed to detect mitotic recombinants, none allows easy identification of clones for all cell types . Results: A system is described here that allows a highly efficient generation of clones with the concomitant expression of an easily detectable cellular marker . This method can be applied to cell lineage and mosaic analysis in Drosophila . The site-specific yeast FLP recombinase, under the control of a heat shock-inducible promoter, efficiently catalyses mitotic recombination specifically at the site of a FLP recombination target (FRT) . In this system, recombination fuses the alpha-tubulin promoter to the lacZ gene, allowing transcription of the marker . Recombinant cells and their progeny can, therefore, be detected by standard assays for beta-galactosidase . Of particular importance is the fact that only the cells of interest stain, thus allowing their simple detection in any tissue . Conclusions: We demonstrate that, by intermolecular recombination, we can use FLIP recombinase to generate marked clones efficiently in embryonic, larval and adult tissues . This simple and efficient technique is well suited to cell-lineage analysis and can be easily extended to the generation and detection of mutant clones in mosaic animals. Curr Biol, 1993 Aug, 3(8), 498 - 506 One-step site-directed mutagenesis of the Kex2 protease oxyanion hole; Brenner C et al.; Background: Members of the subtilisin family of serine proteases usually have a conserved asparagine residue that stabilizes the oxyanion transition state of peptide-bond hydrolysis . Yeast Kex2 protease is a member of the subtilisin family that differs from the degradative subtilisin proteases in its high substrate specificity, it processes pro-alpha-factor, the precursor of the alpha-factor mating pheromone of yeast, and also removes the pro-peptide from its own precursor by an intramolecular cleavage reaction . Curiously, the mammalian protease PC2, a Kex2 homolog that is likely to be required for pro-insulin processing, has an aspartate in place of asparagine at the 'oxyanion hole' . Results: We have tested the effect of making substitutions of the conserved oxyanion-hole asparagine (Asn 314) of the Kex2 protease . To do this, we have developed a rapid method of site-directed mutagenesis, involving homologous recombination of a polymerase chain reaction product in yeast . Using this method, we have substituted alanine or aspartate for Asn 314 in a form of Kex2 engineered for secretion . Transformants expressing the two mutant enzymes could be identified by failure either to produce mature alpha-factor or to mate . The Ala 314 enzyme was unstable but the Asp 314 enzyme accumulated to a high level, so that it could be purified and its activity towards various substrates tested in vitro . We found that, with three peptides that are good substrates of wild-type Kex2, the k(cal) of the Asp 314 enzyme was reduced approximately 4500-fold and its K(M) approximately 4-fold, relative to the wild-type enzyme . For the peptide substrate corresponding to the cleavage site of pro-alpha-factor, however, k(cat) of the Asp 314 enzyme was reduced only 125-fold, while the K(m) was increased 3-fold . Despite its reduced catalytic activity, however, processing of the mutant enzyme in vivo - by the intramolecular cleavage that removes its amino-terminal pro-domain - occurs at an unchanged rate . Conclusions: The effects of the Asn 314-Asp substitution reveal contributions to the reaction specificity of the Kex2 protease of substrate residues amino-terminal to the pair of basic residues at the cleavage site . Aspartate at the oxyanion hole appears to confer k(caf) discrimination between substrates by raising the energy barrier for productive substrate binding: this may have implications for pro-insulin processing by the PC2 protease, which has an aspartate at the equivalent position . The rate of intramolecular cleavage of pro-Kex2 may be limited by a step other than catalysis, presumably protein folding. Nucleic Acids Res, 2004 Aug 27, 32(15), 4657 - 64 Print 2004. Design and synthesis of fluorescent substrates for human tyrosyl-DNA phosphodiesterase I; Rideout MC et al.; Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that acts upon protein-DNA covalent complexes . Tdp1 hydrolyzes 3'-phosphotyrosyl bonds to generate 3'-phosphate DNA and free tyrosine in vitro . Mutations in Tdp1 have been linked to patients with spinocerebellar ataxia, and over-expression of Tdp1 results in resistance to known anti-cancer compounds . Tdp1 has been shown to be involved in double-strand break repair in yeast, and Tdp1 has also been implicated in single-strand break repair in mammalian cells . Despite the biological importance of this enzyme and the possibility that Tdp1 may be a molecular target for new anti-cancer drugs, there are very few assays available for screening inhibitor libraries or for characterizing Tdp1 function, especially under pre-steady-state conditions . Here, we report the design and synthesis of a fluorescence-based assay using oligonucleotide and nucleotide substrates containing 3'-(4-methylumbelliferone)-phosphate . These substrates are efficiently cleaved by Tdp1, generating the fluorescent 4-methylumbelliferone reporter molecule . The kinetic characteristics determined for Tdp1 using this assay are in agreement with the previously published values, and this fluorescence-based assay is validated using the standard gel-based methods . This sensitive assay is ideal for kinetic analysis of Tdp1 function and for high-throughput screening of Tdp1 inhibitory molecules. J Virol, 2004 Sep, 78(18), 9697 - 704 Heat shock protein 70 protects cells from cell cycle arrest and apoptosis induced by human immunodeficiency virus type 1 viral protein R; Iordanskiy S et al.; Viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) is an accessory protein that plays an important role in viral pathogenesis . This pathogenic activity of Vpr is related in part to its capacity to induce cell cycle G2 arrest and apoptosis of target T cells . A screening for multicopy suppressors of these Vpr activities in fission yeast identified heat shock protein 70 (Hsp70) as a suppressor of Vpr-induced cell cycle arrest . Hsp70 is a member of a family of molecular chaperones involved in innate immunity and protection from environmental stress . In this report, we demonstrate that HIV-1 infection induces Hsp70 in target cells . Overexpression of Hsp70 reduced the Vpr-dependent G2 arrest and apoptosis and also reduced replication of the Vpr-positive, but not Vpr-deficient, HIV-1 . Suppression of Hsp70 expression by RNA interference (RNAi) resulted in increased apoptosis of cells infected with a Vpr-positive, but not Vpr-defective, HIV-1 . Replication of the Vpr-positive HIV-1 was also increased when Hsp70 expression was diminished . Vpr and Hsp70 coimmunoprecipitated from HIV-infected cells . Together, these results identify Hsp70 as a novel anti-HIV innate immunity factor that targets HIV-1 Vpr . J Cell Sci, 2004 Sep 1, 117(Pt 19), 4495 - 508 Intramolecular protein-protein and protein-lipid interactions control the conformation and subcellular targeting of neuronal Ykt6; Hasegawa H et al.; Although the membrane-trafficking functions of most SNAREs are conserved from yeast to humans, some mammalian SNAREs have evolved specialized functions unique to multicellular life . The mammalian homolog of the prenylated yeast SNARE Ykt6p might be one such example, because rat Ykt6 is highly expressed only in brain neurons . Furthermore, neuronal Ykt6 displayed a remarkably specialized, punctate localization that did not overlap appreciably with conventional compartments of the endomembrane system, suggesting that Ykt6 might be involved in a pathway unique to or specifically modified for neuronal function . Targeting of Ykt6 to its unique subcellular location was directed by its profilin-like longin domain . We have taken advantage of high-resolution structural data available for the yeast Ykt6p longin domain to examine mechanisms by which the mammalian longin domain controls Ykt6 conformation and subcellular targeting . We found that the overall tertiary structure of the longin domain, not sequence-specific surface features, drives direct targeting to the Ykt6 punctate structures . However, several sequence-specific surface features of the longin domain indirectly regulate Ykt6 localization through intramolecular interactions that mask otherwise-dominant targeting signals on the SNARE motif and lipid groups . Specifically, two hydrophobic binding pockets, one on each face of the longin domain, and one mixed hydrophobic/charged surface, participate in protein-protein interactions with the SNARE motif and protein-lipid interactions with the lipid group(s) at the molecule's C-terminus . One of the hydrophobic pockets suppresses protein-palmitoylation-dependent mislocalization of Ykt6 to the plasma membrane . The Ykt6 intramolecular interactions would be predicted to create a compact, closed conformation of the SNARE that prevents promiscuous targeting interactions and premature insertion into membranes . Interestingly, both protein-protein and protein-lipid interactions are required for a tightly closed conformation and normal targeting. J Cell Sci, 2004 Sep 1, 117(Pt 19), 4343 - 54 The genetics of Pak; Hofmann C et al.; p21-activated kinases (Paks) are a highly conserved family of enzymes that bind to and are activated by small GTPases of the Cdc42 and Rac families . With the notable exception of plants, nearly all eukaryotes encode one or more Pak genes, indicating an ancient origin and important function for this family of enzymes . Genetic approaches in many different experimental systems, ranging from yeast to mice, are beginning to decipher the different functions of Paks . Although some of these functions are unique to a given organism, certain common themes have emerged, such as the activation of mitogen-activated protein kinase (MAPK) cascades and the regulation of cytoskeletal structure through effects on the actin and tubulin cytoskeletons. Cancer Lett, 2004 Oct 8, 214(1), 81 - 90 The evidences of human orphan receptor COUP-TFII inhibiting telomerase activity through decreasing hTERT transcription; Wang Q et al.; Activation of hTERT, the human telomerase catalytic subunit, has been implicated as the critical event in triggering telomerase activity of cancer cells, but the details of regulation of hTERT need to be elucidated . By screening HeLa cDNA library with hTERT promoter-based yeast one-hybrid assay, one of positive clones, which potentially interacted with hTERT promoter, contained the full sequences of COUP-TFII cDNA . EMSA showed that the prepared His-tagged COUP-TFII could firmly bind to -201 to +35 fragment of hTERT promoter . The precise binding sites were confirmed by DNase I footprint analysis and proved to involve the E-box motif of hTERT promoter . Luciferase reporter assays indicated that COUP-TFII could suppress the transcription of hTERT promoter, and the inhibition to some extent could be reversed by co-transfection of c-myc . Stable introduction of COUP-TFII into HeLa cells decreased both endogenous hTERT expression and telomerase activity . The results suggested that the human COUP-TFII could specifically interact with hTERT promoter, in part via the E-box, and suppress the expression of hTERT. Biochem J, 2004 Dec 15, 384(Pt 3), 599 - 607 Domain-specific characteristics of the bifunctional key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase; Blume A et al.; UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase is a bifunctional enzyme, which initiates and regulates sialic acid biosynthesis . Sialic acids are important compounds of mammalian glycoconjugates, mediating several biological processes, such as cell-cell or cell-matrix interactions . In order to characterize the function of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, a number of deletion mutants were generated, lacking either parts of the N-terminal epimerase or the C-terminal kinase domain . N-terminal deletion of only 39 amino acids results in a complete loss of epimerase activity . Deletions in the C-terminal part result in a reduction or complete loss of kinase activity, depending on the size of the deletion . Deletions at either the N- or the C-terminus also result in a reduction of the other enzyme activity . These results indicate that a separate expression of both domains is possible, but that a strong intramolecular dependency of the two domains has arisen during evolution of the enzyme . N-terminal, as well as C-terminal, mutants tend to form trimers, in addition to the hexameric structure of the native enzyme . These results and yeast two-hybrid experiments show that structures required for dimerization are localized within the kinase domain, and a potential trimerization site is possibly located in a region between the two domains . In conclusion, our results reveal that the activities, as well as the oligomeric structure, of this bifunctional enzyme seem to be organized and regulated in a complex manner. Med Sci (Paris), 2004 Jun-Jul, 20(6-7), 669 - 73 {RNA localization in the cytoplasm}; Basyuk E et al.; RNA localization in subcytoplasmic areas is a process known for more than twenty years, and more than a hundred RNAs have now been shown to be spatially regulated . In most cases, RNA localization is involved in cell polarity, either by reading spatial clues and translating them into a spatial regulation of gene expression, or more directly by controlling cytoskeletal polarity . In this review, the various functions of RNA localization will be presented, and by analyzing two examples, Ash1 mRNA in yeast and retroviral genomic RNAs in mammals, the reader will be taken step by step into the detailed mechanisms of this fascinating process. Nature, 2004 Aug 26, 430(7003), 1044 - 8 Separase-mediated cleavage of cohesin at interphase is required for DNA repair; Nagao K et al.; Sister chromatids are held together by cohesins . At anaphase, separase is activated by degradation of its inhibitory partner, securin . Separase then cleaves cohesins, thus allowing sister chromatid separation . Fission yeast securin (Cut2) has destruction boxes and a separase (Cut1) interaction site in the amino and carboxyl terminus, respectively . Here we show that securin is essential for separase stability and also for proper repair of DNA damaged by ultraviolet, X-ray and gamma-ray irradiation . The cut2(EA2) mutant is defective in the repair of ultraviolet damage lesions, although the DNA damage checkpoint is activated normally . In double mutant analysis of ultraviolet sensitivity, checkpoint kinase chk1 (ref . 9) and excision repair rad13 (ref . 10) mutants were additive with cut2(EA2), whereas recombination repair rhp51 (ref . 11) and cohesin subunit rad21 (ref . 12) mutants were not . Cohesin was hyper-modified on ultraviolet irradiation in a Rad3 kinase-dependent way . Experiments using either mutant cohesin that cannot be cleaved by separase or a protease-dead separase provide evidence that this DNA repair function of securin-separase acts through the cleavage of cohesin . We propose that the securin-separase complex might aid DNA repair by removing local cohesin in interphase cells. EMBO J, 2004 Sep 15, 23(18), 3667 - 76 Epub 2004 Aug 26. A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication; Pacek M et al.; In vertebrates, MCM2-7 and Cdc45 are required for DNA replication initiation, but it is unknown whether they are also required for elongation, as in yeast . Moreover, although MCM2-7 is a prime candidate for the eukaryotic replicative DNA helicase, a demonstration that MCM2-7 unwinds DNA during replication is lacking . Here, we use Xenopus egg extracts to investigate the roles of MCM7 and Cdc45 in DNA replication . A fragment of the retinoblastoma protein, Rb(1-400), was used to neutralize MCM7, and antibodies were used to neutralize Cdc45 . When added immediately after origin unwinding, or after significant DNA synthesis, both inhibitors blocked further DNA replication, indicating that MCM7 and Cdc45 are required throughout replication elongation in vertebrates . We next exploited the fact that inhibition of DNA polymerase by aphidicolin causes extensive chromosome unwinding, likely due to uncoupling of the replicative DNA helicase . Strikingly, Rb(1-400) and Cdc45 antibodies both abolished unwinding by the uncoupled helicase . These results provide new support for the model that MCM2-7 is the replicative DNA helicase, and they indicate that Cdc45 functions as a helicase co-factor. Proc Natl Acad Sci U S A, 2004 Sep 7, 101(36), 13380 - 5 Epub 2004 Aug 25. A small CDC25 dual-specificity tyrosine-phosphatase isoform in Arabidopsis thaliana; Landrieu I et al.; The dual-specificity CDC25 phosphatases are critical positive regulators of cyclin-dependent kinases (CDKs) . Even though an antagonistic Arabidopsis thaliana WEE1 kinase has been cloned and tyrosine phosphorylation of its CDKs has been demonstrated, no valid candidate for a CDC25 protein has been reported in higher plants . We identify a CDC25-related protein (Arath;CDC25) of A . thaliana, constituted by a sole catalytic domain . The protein has a tyrosine-phosphatase activity and stimulates the kinase activity of Arabidopsis CDKs . Its tertiary structure was obtained by NMR spectroscopy and confirms that Arath;CDC25 belongs structurally to the classical CDC25 superfamily with a central five-stranded beta-sheet surrounded by helices . A particular feature of the protein, however, is the presence of an additional zinc-binding loop in the C-terminal part . NMR mapping studies revealed the interaction with phosphorylated peptidic models derived from the conserved CDK loop containing the phosphothreonine-14 and phosphotyrosine-15 . We conclude that despite sequence divergence, Arath;CDC25 is structurally and functionally an isoform of the CDC25 superfamily, which is conserved in yeast and in plants, including Arabidopsis and rice. J Neurosci, 2004 Aug 25, 24(34), 7491 - 502 AMPA receptor synaptic targeting regulated by stargazin interactions with the Golgi-resident PDZ protein nPIST; Cuadra AE et al.; Regulation of AMPA receptors (AMPARs) at synapses plays a critical role in alterations of synaptic strength in the brain . Stargazin, an AMPAR-interacting protein, is critical for clustering and regulation of synaptic AMPARs . Stargazin interacts with AMPARs via its extracellular domain and with PDZ {postsynaptic density-95 (PSD-95)/Discs large (Dlg)/zona occludens-1 (ZO-1)} proteins via its C-terminal PDZ-binding motif, and these interactions are necessary for stargazin and AMPAR synaptic targeting . By studying the expression of stargazin mutant constructs in cultured hippocampal neurons, we identified a novel domain corresponding to residues 243-283 within the cytoplasmic C terminus of stargazin that is also required for stargazin and AMPAR synaptic clustering . To identify proteins that interact with this stargazin synaptic clustering domain, we performed a yeast two-hybrid assay and found that this stargazin domain binds to nPIST (neuronal isoform of protein-interacting specifically with TC10), a Golgi-enriched protein implicated in trafficking of transmembrane proteins . Using in situ hybridization, immunohistochemistry, coimmunoprecipitation studies, and biochemical fractionation, we found that stargazin and nPIST colocalize and interact in the brain . Finally, by studying AMPAR clustering in transfected hippocampal neurons, we found that overexpression of nPIST enhances AMPAR synaptic clustering, whereas transfection of a dominant-negative nPIST construct attenuates AMPAR synaptic clustering . These studies identify a novel stargazin domain necessary for synaptic clustering of AMPARs and suggest that nPIST and stargazin interactions play a critical role in AMPAR trafficking to the synapse. J Mol Biol, 2004 Aug 20, 341(4), 911 - 25 Biochemical and structural insights into substrate binding and catalytic mechanism of mammalian poly(A) polymerase; Martin G et al.; Polyadenylation of messenger RNA precursors is an essential process in eukaryotes . Poly(A) polymerase (PAP), a member of the nucleotidyltransferase family that includes DNA polymerase beta, incorporates ATP at the 3' end of mRNAs in a template-independent manner . Although the structures of mammalian and yeast PAPs are known, their mechanism of ATP selection has remained elusive . In a recent bovine PAP structure complexed with an analog of ATP and Mn2+, strictly conserved residues interact selectively with the adenine base, but the nucleotide was found in a "non-productive" conformation . Here we report a second bovine crystal structure, obtained in the presence of Mg2+, where 3'-dATP adopts a "productive" conformation similar to that seen in yeast PAP or DNA polymerase beta . Mutational analysis and activity assays with ATP analogs suggest a role in catalysis for one of the two adenine-binding sites revealed by our structural data . The other site might function to prevent futile hydrolysis of ATP . In order to investigate the role of metals in catalysis we performed steady state kinetics experiments under distributive polymerization conditions . These tests suggest a sequential random mechanism in vitro in the presence of ATP and RNA, without preference for a particular order of binding of the two substrates . In vivo, however, where polyadenylation is processive and the primer does not dissociate from the enzyme, an ordered mechanism with the primer as the leading substrate is more likely . Proc Natl Acad Sci U S A, 2004 Aug 31, 101(35), 12980 - 5 Epub 2004 Aug 24. An accelerated assay for the identification of lifespan-extending interventions in Drosophila melanogaster; Bauer JH et al.; Recent advances in aging research have uncovered genes and genetic pathways that influence lifespan in such diverse organisms as yeast, nematodes, flies, and mice . The discovery of genes and drugs that affect lifespan has been delayed by the absence of a phenotype other than survivorship, which depends on the measurement of age at death of individuals in a population . The use of survivorship to identify genetic and pharmacological interventions that prolong life is time-consuming and requires a large number of homogeneous animals . Here, we report the development of an assay in Drosophila melanogaster using the expression of molecular biomarkers that accelerates the ability to evaluate potential lifespan-altering interventions . Coupling the expression of an age-dependent molecular biomarker to a lethal toxin reduces the time needed to perform lifespan studies by 80% . The assay recapitulates the effect of the three best known environmental life-span-extending interventions in the fly: ambient temperature, reproductive status, and calorie reduction . Single gene mutations known to extend lifespan in the fly such as Indy and rpd3 also extend lifespan in this assay . We used this assay as a screen to identify drugs that extend lifespan in flies . Lipoic acid and resveratrol were identified as being beneficial in our assay and shown to extend lifespan under normal laboratory conditions . We propose that this assay can be used to screen pharmacological as well as genetic interventions more rapidly for positive effects on lifespan . J Biol Chem, 2004 Oct 22, 279(43), 44639 - 44 Epub 2004 Aug 24. PratA, a periplasmic tetratricopeptide repeat protein involved in biogenesis of photosystem II in Synechocystis sp . PCC 6803; Klinkert B et al.; The light reactions of oxygenic photosynthesis are mediated by multisubunit pigment-protein complexes situated within the specialized thylakoid membrane system . The biogenesis of these complexes is regulated by transacting factors that affect the expression of the respective subunit genes and/or the assembly of their products . Here we report on the analysis of the PratA gene from the cyanobacterium Synechocystis sp . PCC 6803 that encodes a periplasmic tetratricopeptide repeat protein of formerly unknown function . Targeted inactivation of PratA resulted in drastically reduced photosystem II (PSII) content . Protein pulse labeling experiments of PSII subunits indicated that the C-terminal processing of the precursor of the reaction center protein D1 is compromised in the pratA mutant . Moreover, a direct interaction of PratA and precursor D1 was demonstrated by applying yeast two-hybrid analyses . This suggests that PratA represents a factor facilitating D1 maturation via the endoprotease CtpA . The periplasmic localization of PratA supports a model that predicts the initial steps of PSII biogenesis to occur at the plasma membrane of cyanobacterial cells. J Biol Chem, 2004 Nov 5, 279(45), 46566 - 72 Epub 2004 Aug 24. Omi/HtrA2 promotes cell death by binding and degrading the anti-apoptotic protein ped/pea-15; Trencia A et al.; ped/pea-15 is a ubiquitously expressed 15-kDa protein featuring a broad anti-apoptotic function . In a yeast two-hybrid screen, the pro-apoptotic Omi/HtrA2 mitochondrial serine protease was identified as a specific interactor of the ped/pea-15 death effector domain . Omi/HtrA2 also bound recombinant ped/pea-15 in vitro and co-precipitated with ped/pea-15 in 293 and HeLa cell extracts . In these cells, the binding of Omi/HtrA2 to ped/pea-15 was induced by UVC exposure and followed the mitochondrial release of Omi/HtrA2 into the cytoplasm . Upon UVC exposure, cellular ped/pea-15 protein expression levels decreased . This effect was prevented by the ucf-101 specific inhibitor of the Omi/HtrA2 proteolytic activity, in a dose-dependent fashion . In vitro incubation of ped/pea-15 with Omi/HtrA2 resulted in ped/pea-15 degradation . In intact cells, the inhibitory action of ped/pea-15 on UVC-induced apoptosis progressively declined at increasing Omi/HtrA2 expression . This further effect of Omi/HtrA2 was also inhibited by ucf-101 . In addition, ped/pea-15 expression blocked Omi/HtrA2 co-precipitation with the caspase inhibitor protein XIAP and caspase 3 activation . Thus, in part, apoptosis following Omi/HtrA2 mitochondrial release is mediated by reduction in ped/pea-15 cellular levels . The ability of Omi/HtrA2 to relieve XIAP inhibition on caspases is modulated by the relative levels of Omi/HtrA2 and ped/pea-15. Drug Metab Dispos, 2004 Nov, 32(11), 1213 - 7 Epub 2004 Aug 24. Mechanism-based inactivation of CYP2D6 by methylenedioxymethamphetamine; Heydari A et al.; The potency of methylenedioxymethamphetamine (MDMA) as a mechanism-based inhibitor of CYP2D6 has been defined using microsomes prepared from yeast expressing the enzyme and from three human livers . The inhibitory effect was increased by preincubation through formation of a metabolic intermediate complex . Inactivation parameters (kinact and KI), defined with respect to the O-demethylation of dextromethorphan, were 0.29 +/- 0.03 (S.E.) min(-1) and 12.9 +/- 3.6 (S.E.) microM for yeast-expressed CYP2D6, and 0.26 +/- 0.02 min(-1) and 14.4 +/- 2.5 microM, 0.15 +/- 0.01 min(-1) and 8.8 +/- 2.6 microM, and 0.12 +/- 0.05 min(-1) and 45.3 +/- 32.1 microM for the liver microsomal preparations . The rate of inactivation of CYP2D6 by MDMA decreased when quinidine, a competitive inhibitor of CYP2D6, was added to the primary incubation mixture . However, inactivation was unaffected by the addition of glutathione . The results indicate that MDMA is a potent mechanism-based inhibitor of CYP2D6, with implications for understanding its in vivo disposition and drug interaction potential . Clin Cancer Res, 2004 Aug 15, 10(16), 5630 - 9 CXCR4 and CXCL12 (SDF-1) in prostate cancer: inhibitory effects of human single chain Fv antibodies; Vaday GG et al.; PURPOSE: Metastasis is a major cause of morbidity in prostate cancer (PCa) . Several studies have shown that the chemokine receptor CXCR4 and its ligand, CXCL12 (stromal cell-derived factor-1), regulate tumor cell metastasis to specific organs . Recently, it was demonstrated that CXCL12 enhances PCa cell adhesion, migration, and invasion, implicating CXCR4 in PCa metastasis . In this study, we examined the inhibitory effects of anti-CXCR4 antibodies on CXCL12-mediated PCa cell activities . EXPERIMENTAL DESIGN: We developed fully human single chain Fv antibodies (scFv), Ab124 and Ab125, against CXCR4 using the yeast two-hybrid system . We performed immunofluorescent staining, flow cytometry, and ELISA-binding assays to measure scFv binding to PCa cells . We also examined the effects of scFv on CXCL12-mediated calcium mobilization, cell migration, and invasion . RESULTS: Our results confirmed that PCa cell lines express cell-surface CXCR4 . Real-time quantitative reverse transcription-PCR and immunohistochemical staining also verified that CXCR4 is expressed in primary cultures of prostate epithelial cells from adenocarcinomas and in human prostate tissues . Ab124 and Ab125 demonstrated specific binding to PCa cell lines by flow cytometry and in binding assays . Preincubation with scFv resulted in significant reduction of CXCL12-induced calcium mobilization in PC-3 and LNCaP cells . Ab124 and Ab125 also inhibited PCa cell migration toward CXCL12, as well as invasion through extracellular matrix gels . CONCLUSIONS: Ab124 and Ab125 inhibit CXCL12-mediated cellular activities by binding the receptor CXCR4 . Recombinant scFv are an efficient mode of targeting tumor antigens . Considering the high incidence of PCa, the development of fully human scFv may be a useful therapeutic approach in the prevention and treatment of PCa metastasis. Nucleic Acids Res, 2004 Aug 23, 32(15), 4491 - 502 Print 2004. The effect of eukaryotic release factor depletion on translation termination in human cell lines; Janzen DM et al.; Two competing events, termination and readthrough (or nonsense suppression), can occur when a stop codon reaches the A-site of a translating ribosome . Translation termination results in hydrolysis of the final peptidyl-tRNA bond and release of the completed nascent polypeptide . Alternatively, readthrough, in which the stop codon is erroneously decoded by a suppressor or near cognate transfer RNA (tRNA), results in translation past the stop codon and production of a protein with a C-terminal extension . The relative frequency of termination versus readthrough is determined by parameters such as the stop codon nucleotide context, the activities of termination factors and the abundance of suppressor tRNAs . Using a sensitive and versatile readthrough assay in conjunction with RNA interference technology, we assessed the effects of depleting eukaryotic releases factors 1 and 3 (eRF1 and eRF3) on the termination reaction in human cell lines . Consistent with the established role of eRF1 in triggering peptidyl-tRNA hydrolysis, we found that depletion of eRF1 enhances readthrough at all three stop codons in 293 cells and HeLa cells . The role of eRF3 in eukarytotic translation termination is less well understood as its overexpression has been shown to have anti-suppressor effects in yeast but not mammalian systems . We found that depletion of eRF3 has little or no effect on readthrough in 293 cells but does increase readthrough at all three stop codons in HeLa cells . These results support a direct role for eRF3 in translation termination in higher eukaryotes and also highlight the potential for differences in the abundance or activity of termination factors to modulate the balance of termination to readthrough reactions in a cell-type-specific manner. J Biol Chem, 2004 Nov 5, 279(45), 46393 - 9 Epub 2004 Aug 23. Cisplatin stabilizes a multimeric complex of the human Ctr1 copper transporter: requirement for the extracellular methionine-rich clusters; Guo Y et al.; Cisplatin is a highly effective cancer chemotherapy agent . However, acquired resistance currently limits the clinical utility of this drug . The human high affinity copper importer, hCtr1, and its yeast and murine orthologues have been shown to mediate the uptake of cisplatin . This transporter is located at the plasma membrane under low copper conditions, and excess copper concentrations stimulate its endocytosis and degradation . In this study we further examined the link between cisplatin and hCtr1 by examining whether cisplatin can also stimulate the endocytosis and degradation of hCtr1 . The steady-state location of hCtr1 and its endocytosis from the plasma membrane were not altered by cisplatin treatment . Unexpectedly, cisplatin treatment of a cell line expressing hCtr1 revealed the time- and concentration-dependent appearance of a stable hCtr1 multimeric complex, consistent with a homotrimer, which was not observed following copper treatment of these same cells . Mutagenesis studies identified two methionine-rich clusters in the extracellular amino-terminal region of hCtr1 that were required for stabilization of the hCtr1 multimer by cisplatin, suggesting that these sequences bind cisplatin and form crosslinks between hCtr1 polypeptides . Treatment with the metal chelator dimethyldithiocarbamate disassembled the hCtr1 multimer following cisplatin exposure, suggesting that platinum was an integral component of this complex . These studies provide the first evidence for a direct interaction between cisplatin and the hCtr1 protein and establish that cisplatin and copper have distinct biochemical consequences on this transporter. Int J Biochem Cell Biol, 2004 Dec, 36(12), 2491 - 502 Methods for monitoring autophagy; Mizushima N; Autophagy is an intracellular bulk degradation system that is found ubiquitously in eukaryotes . Autophagy is responsible for the degradation of most long-lived proteins and some organelles . Cytoplasmic constituents, including organelles, are sequestered into double-membraned autophagosomes, which subsequently fuse with lysosomes where their contents are degraded . This system has been implicated in various physiological processes including protein and organelle turnover, the starvation response, cellular differentiation, cell death, and pathogenesis . However, methods for monitoring autophagy have been very limited and unsatisfactory . The most standard method is conventional electron microscopy . In addition, some biochemical methods have been utilized to measure autophagic activity . Recently, the molecular basis of autophagosome formation has been extensively studied using yeast cells; these studies have provided useful marker proteins for autophagosomes . Importantly, most of these proteins are conserved in mammals . Using these probes, we can now specifically monitor autophagic activity. Biochem Biophys Res Commun, 2004 Sep 17, 322(2), 665 - 71 Dictyostelium calcium-binding protein 4a interacts with nucleomorphin, a BRCT-domain protein that regulates nuclear number; Myre MA et al.; Nucleomorphin from Dictyostelium discoideum is a nuclear calmodulin-binding protein that is a member of the BRCT-domain containing cell cycle checkpoint proteins . Two differentially expressed isoforms, NumA and NumB, share an extensive acidic domain (DEED) that when deleted produces highly multinucleated cells . We performed a yeast two-hybrid screen of a Dictyostelium cDNA library using NumA as bait . Here we show that nucleomorphin interacts with calcium-binding protein 4a (CBP4a) in a Ca(2+)-dependent manner . Further deletion analysis suggests this interaction requires residues found within the DEED domain . NumA and CBP4a mRNAs are expressed at the same stages of development . CBP4a belongs to a large family of Dictyostelium CBPs, for which no cellular or developmental functions had previously been determined . Since the interaction of CBP4a with nucleomorphin requires the DEED domain, this suggests that CBP4a may respond to Ca(2+)-signalling through modulating factors that might function in concert to regulate nuclear number. Biol Cell, 2004 Aug, 96(6), 457 - 62 Drosophila cohesins DSA1 and Drad21 persist and colocalize along the centromeric heterochromatin during mitosis; Valdeolmillos A et al.; Sister chromatid cohesion in eukaryotes is maintained mainly by a conserved multiprotein complex termed cohesin . Drad21 and DSA1 are the Drosophila homologues of the yeast Scc1 and Scc3 cohesin subunits, respectively . We recently identified a Drosophila mitotic cohesin complex composed of Drad21/DSA1/DSMC1/DSMC3 . Here we study the contribution of this complex to sister chromatid cohesion using immunofluorescence microscopy to analyze cell cycle chromosomal localization of DSA1 and Drad21 in S2 cells . We observed that DSA1 and Drad21 colocalize during all cell cycle stages in cultured cells . Both proteins remain in the centromere until metaphase, colocalizing at the centromere pairing domain that extends along the entire heterochromatin; the centromeric cohesion protein MEI-S332 is nonetheless reported in a distinct centromere domain . These results provide strong evidence that DSA1 and Drad21 are partners in a cohesin complex involved in the maintenance of sister chromatid arm and centromeric cohesion during mitosis in Drosophila. Cell Tissue Res, 2004 Oct, 318(1), 195 - 9 Epub 2004 Aug 19. The role of synphilin-1 in synaptic function and protein degradation; Kruger R; The name synphilin-1 comes from its identification as an alpha-synuclein-interacting protein (SNCAIP) in yeast two-hybrid screens . Since alpha-synuclein ( PARK1) was the first gene identified as causing inherited forms of Parkinson's disease (PD), synphilin-1 was quickly implicated in neurodegeneration in PD . Recently, the first genetic evidence for the direct contribution of synphilin-1 in the pathogenesis of PD has been defined with the identification of an R621C mutation as a susceptibility factor for PD in two German patients . Extensive in vitro studies have determined the physiological functions of synphilin-1, identified novel synphilin-1-interacting proteins, and linked synphilin-1 to ubiquitin-mediated protein degradation . The present article provides an overview of the current concepts of the role of synphilin-1 in synaptic function and protein degradation and in the molecular mechanisms leading to neurodegeneration in PD. Biosci Biotechnol Biochem, 2004 Aug, 68(8), 1808 - 10 Glutamate receptor-interacting protein 1 protein binds to the microtubule-associated protein; Seog DH; To identify the interaction proteins for the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit glutamate receptor-interacting protein 1 (GRIP1), GRIP1 interactions with microtubule-associated protein (MAP)-1B light chain (LC) were investigated . GRIP1 interacts with MAP-1A and MAP-1B in the yeast two-hybrid assay, as is indicated also by glutathione S-transferase (GST) pull-down and coimmunoprecipitation with MAP-1B LC antibody in brain fractions . These results suggest a novel mechanism for localizing AMPA receptors to synaptic sites. Proc Natl Acad Sci U S A, 2004 Aug 31, 101(35), 12928 - 33 Epub 2004 Aug 20. Asynchronous replication timing of telomeres at opposite arms of mammalian chromosomes; Zou Y et al.; Telomeres are defining structural elements of all linear chromosomes, yet information concerning the timing of their replication in higher eukaryotes is surprisingly limited . We developed an approach that allowed a study of telomere replication patterns of specific mammalian chromosomes . In the Indian muntjac (Muntiacus muntjac), replication timing between respective telomeres of homologous chromosomes was highly coordinated, but no such synchrony was evident for p- and q-arm telomeres of the same chromosome . This finding contrasts with the coordinated timing of both ends of each chromosome in yeast . Also in contrast to yeast, where replication of all telomeres is confined to late S phase, we found specific telomeres in Indian muntjac chromosomes that replicated early in S and other telomeres that replicated later . Finally, replication timing of some but not all telomeres was influenced by telomere length . Knowledge of telomere replication timing represents a first step toward understanding the relationship between telomere replication and telomerase action . The approach, which we call replicative detargeting fluorescence in situ hybridization, is widely applicable to different species and genetic loci . Mol Pharmacol, 2004 Sep, 66(3), 761 - 9 Functional role of a C-terminal Gbetagamma-binding domain of Ca(v)2.2 channels; Li B et al.; Presynaptic Ca(2+) channels are inhibited by neurotransmitters acting through G protein-coupled receptors via a membrane-delimited pathway . Inhibition is reversed by strong depolarization, resulting in prepulse facilitation . Activated G protein betagamma subunits (Gbetagamma) are required for maximal prepulse facilitation . Gbetagamma binds to multiple sites on Ca(v)2.1, Ca(v)2.2, and Ca(v)2.3 alpha1 subunits . Here we examine the functional relevance of a C-terminal binding site for Gbetagamma on Ca(v)2.2b channels, which mediate N-type Ca(2+) currents . In vitro binding studies showed that Gbetagamma subunits bind to the intracellular loop connecting domains I and II and the C-terminal domain of Ca(v)2.2b but not the intracellular loops connecting domains II and III or III and IV . Deletion analysis revealed that the binding site is located near the C terminus, within amino acid residues 2257 to 2336 . Directed yeast two-hybrid analysis confirmed this specific binding interaction in vivo in yeast cells . Ca(v)2.2b channels with this site deleted had normal function properties, and they were inhibited essentially normally by strong activation of G proteins with guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) and were facilitated nearly normally by depolarizing prepulses . Similarly deletion of this site had small, statistically insignificant effects on inhibition of Ca(2+) current and on prepulse facilitation in the presence of somatostatin to stimulate receptor-mediated activation of G proteins . In contrast, deletion of the C-terminal Gbetagamma site substantially reduced the low level of intrinsic prepulse facilitation present at the basal level of G protein activation in tsA-201 cells . Thus, this C-terminal Gbetagamma binding site contributes to the affinity or efficacy of Gbetagamma regulation at basal levels of G protein activation . The simplest interpretation of our results is that the C-terminal binding site increases the affinity of Gbetagamma for the channel but is not required for Gbetagamma action . C-terminal binding of Gbetagamma may influence the physiological responsiveness of Ca(2+) channels to low-level G protein activation. Mol Pharmacol, 2004 Sep, 66(3), 430 - 9 Mutation E522K in human DNA topoisomerase IIbeta confers resistance