Yeasts

J Insect Physiol, 2001 Dec, 47(12), 1467 - 1473
Age-specific mortality and reproduction respond to adult dietary restriction in Drosophila melanogaster; Good TP et al.; Adult dietary yeast modulates mortality rate and reproduction of the Mediterranean fruit fly, Ceratatis capitata . In the medfly, a sugar-only diet leads to low mortality rates and reduced reproduction; addition of dietary yeast increases both mortality and egg laying . In Drosophila melanogaster low availability of dietary yeast is known to increase life span and reduce the rate of reproduction . Despite these similarities, because of differences in experimental design it remains unclear whether a common physiological mechanism modulates the effect of diet on survival . Here, we investigate how mortality rate and reproduction in D . melanogaster respond to the treatment regime used to study the medfly: no-yeast versus full diet . We find that adult medfly and D . melanogaster have opposite responses to the absence of yeast: D . melanogaster have high mortality when on no-yeast diet; when switched to full diet, D . melanogaster reduce mortality rates to the level presented by females continuously maintained on yeast . This reduction in mortality is accompanied by increased fecundity . These patterns are observed in all tested wildtype stocks, but flies made sterile by mutation in the gene oo18 RNA-binding protein (orb) lack this response . D . melanogaster, unlike medflies, appear to require adult dietary yeast to maintain maximal survival, and the capacity to assimilate yeast for somatic processes is one wildtype function of the gene orb.

Curr Protein Pept Sci, 2003 Jun, 4(3), 159 - 81
Computational analyses of high-throughput protein-protein interaction data; Chen Y et al.; Protein-protein interactions play important roles in nearly all events that take place in a cell . High-throughput experimental techniques enable the study of protein-protein interactions at the proteome scale through systematic identification of physical interactions among all proteins in an organism . High-throughput protein-protein interaction data, with ever-increasing volume, are becoming the foundation for new biological discoveries . A great challenge to bioinformatics is to manage, analyze, and model these data . In this review, we describe several databases that store, query, and visualize protein-protein interaction data . Comparison between experimental techniques shows that each high-throughput technique such as yeast two-hybrid assay or protein complex identification through mass spectrometry has its limitations in detecting certain types of interactions and they are complementary to each other . In silico methods using protein/DNA sequences, domain and structure information to predict protein-protein interaction can expand the scope of experimental data and increase the confidence of certain protein-protein interaction pairs . Protein-protein interaction data correlate with other types of data, including protein function, subcellular location, and gene expression profile . Highly connected proteins are more likely to be essential based on the analyses of the global architecture of large-scale interaction network in yeast . Use of protein-protein interaction networks, preferably in conjunction with other types of data, allows assignment of cellular functions to novel proteins and derivation of new biological pathways . As demonstrated in our study on the yeast signal transduction pathway for amino acid transport, integration of high-throughput data with traditional biology resources can transform the protein-protein interaction data from noisy information into knowledge of cellular mechanisms.

Biochem Biophys Res Commun, 2003 Jun 13, 305(4), 1049 - 56
Association of hepatitis B virus polymerase with promyelocytic leukemia nuclear bodies mediated by the S100 family protein p11; Choi J et al.; Hepatitis B virus (HBV) polymerase (Pol) interacts with cellular chaperone proteins and thereby performs multiple functions necessary for viral replication . Yeast two-hybrid analysis was applied to identify additional cellular targets required for HBV Pol function . HBV Pol interacted with S100A10 (p11), a Ca(2+)-modulated protein previously shown to bind to annexin II . The interaction between HBV Pol and p11 was confirmed by co-immunoprecipitation of the two proteins synthesized either in vitro or in transfected cells and by inhibition of the DNA polymerase activity of HBV Pol by p11 . Immunofluorescence analysis of transfected human cell lines revealed that, although most HBV Pol and p11 was restricted to the cytoplasm, a small proportion of each protein colocalized as nuclear speckles; HBV Pol was not detected in the nucleus in the absence of p11 . The HBV Pol-p11 nuclear speckles coincided with nuclear bodies containing the promyelocytic leukemia protein PML . Furthermore, the association of HBV Pol-p11 with PML was increased by exposure of cells to EGTA and inhibited by valinomycin . These results suggest a role for p11 in modulation of HBV Pol function and implicate PML nuclear bodies and intracellular Ca(2+) in viral replication.

J Mol Biol, 2003 Jun 6, 329(3), 479 - 93
Isolation of proteins that interact with the signal transduction molecule Dof and identification of a functional domain conserved between Dof and vertebrate BCAP; Battersby A et al.; Dof is a large molecule essential for signal transduction by the two FGF receptors in Drosophila . It contains two ankyrin repeats and a coiled-coil region, but has no other recognisable structural motif . Dof shares these features with its closest vertebrate relatives, the B-cell signalling molecules BCAP and BANK . In addition, this family of proteins shares a region of homology upstream of the ankyrin repeats, which we call the Dof/BCAP/BANK (DBB) motif . We have identified 44 proteins that interact with Dof in a yeast two-hybrid screen . These include the Drosophila FGF-receptor Heartless and Dof itself . We show that the integrity of the DBB motif is required both for Dof and for BCAP to form dimers . Analysis of the interactions between a set of deletion constructs of Dof and the panel of interactors suggests that Dof may adopt different conformations, with a folded conformation stabilized by interactions between the DBB motif and the C-terminal part of the protein.

Biochemistry, 2003 Jun 3, 42(21), 6493 - 9
Architecture of the Qo site of the cytochrome bc1 complex probed by superoxide production; Muller FL et al.; Although several X-ray structures have been determined for the mitochondrial cytochrome (cyt) bc(1) complex, none yet shows the position of the substrate, ubiquinol, in the quinol oxidase (Q(o)) site . In this study, the interaction of molecular oxygen with the reactive intermediate Q(o) semiquinone is used to probe the Q(o) site . It has been known for some time that partial turnover of the cyt bc(1) complex in the presence of antimycin A, a Q(i) site inhibitor, results in accumulation of a semiquinone at the Q(o) site, which can reduce O(2) to superoxide (O(2)(*)(-)) . It was more recently shown that myxothiazol, which binds close to the cyt b(L) heme in the proximal Q(o) niche, also induces O(2)(*)(-) production . In this work, it is shown that, in addition to myxothiazol, a number of other proximal Q(o) inhibitors {including (E)-beta-methoxyacrylate-stilbene, mucidin, and famoxadone} also induce O(2)(*)(-) production in the isolated yeast cyt bc(1) complex, at approximately 50% of the V(max) observed in the presence of antimycin A . It is proposed that proximal Q(o) site inhibitors induce O(2)(*)(-) production because they allow formation, but not oxidation, of the semiquinone at the distal niche of the Q(o) site pocket . The apparent K(m) for ubiquinol at the Q(o) site in the presence of proximal Q(o) site inhibitors suggests that the "distal niche" of the Q(o) pocket can act as a fully independent quinol binding and oxidation site . Together with the X-ray structures, these results suggest substrate ubiquinol binds in a fashion similar to that of stigmatellin with H-bonds between H161 of the Rieske iron-sulfur protein and E272 of the cyt b protein . When modeled in this way, mucidin and ubiquinol can bind simultaneously to the Q(o) site with virtually no steric hindrance, whereas progressively bulkier inhibitors exhibit increasing overlap . The fact that partial turnover of the Q(o) site is possible even with bound proximal Q(o) site inhibitors is consistent with the participation of two separate functional Q(o) binding niches, occupied simultaneously or sequentially.

J Neurobiol, 2003 Jul, 56(1), 1 - 12
Blocking sensory inputs to identified antennal glomeruli selectively modifies odorant perception in Drosophila; Devaud JM et al.; Neural coding of sensory input is a major unsolved issue in neuroscience . Current experimental methods rely on neural activity recording or visualization following sensory stimulation . Most of them, however, do not include behavioral correlates on the actual perception by the animal . We present a novel approach to address olfaction and coding in adult Drosophila . Sensory input was selectively blocked in two subsets of sensory neurons that project to different, albeit overlapping, groups of central targets, by means of tetanus toxin expressed under the control of the yeast transcription factor Gal4 . Glomeruli DL1, DL2, VM1, and VM4 were tested following stimulation with benzaldehyde, ethyl acetate, propionic acid, butanol, or acetone at various concentrations . The behavioral response was found to be modified in an odorant-specific and a concentration-dependent manner . Sensory input to DL2 and, to a minor extent, VM1 and/or VM4, appear to be required for benzaldehyde perception, while acetone is processed through DL1 . None of these glomeruli, however, seem necessary for butanol perception . In addition, sexual differences were observed for some stimuli . These results demonstrate the behavioral relevance of odor representation as maps of glomerular activity generated in the antennal lobes following specific sensory input . The strategy used here should be useful to characterize olfactory coding, as new and selective Gal4 lines become available .

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 May, 35(5), 478 - 82
{Screening of proteins interacting with tTG in HeLa cells}; Li P et al.; Tissue transglutaminase(tTG) belongs to a class of transglutaminase family which is up-regulated in almost all cells apoptosis and is thought to be closely related to cell apoptosis . To investigate the mechanism of tTG in cell apoptosis, yeast two hybrid system was used to screen HeLa cDNA library . One of the 17 positive clones we have obtained encoded the glutamine-rich carboxyl terminus of TIAR, and this interaction between tTG and TIAR, which was finely regulated by Ca(2+), was proved in vitro by GST pull-down . These findings suggest that tTG might affect the function of TIAR by a calcium-dependent posttranslational modification and the interaction might possibly be involved in the regulation of cell apoptosis.

J Biomol NMR, 2003 Jun, 26(2), 147 - 55
Interaction of the tail with the catalytic region of a class II E2 conjugating enzyme; Merkley N et al.; Ubiquitination plays an important role in many biological processes, including DNA repair, cell cycle regulation, and protein degradation . In the latter pathway the ubiquitin-conjugating enzymes or E2 enzymes are important proteins forming a key E2-ubiquitin thiolester prior to substrate labelling . While the structure of the 150-residue catalytic domain has been well characterized, a subset of E2 enzymes (class II) carry a variable length C-terminal "tail" where structural detail is not available . The presence of this C-terminal extension plays an important role in target recognition, ubiquitin chain assembly and oligomerization . In this work NMR spectroscopy was used to determine the secondary structure of the 215-residue yeast E2 protein Ubc1 and the interactions of its C-terminus with the catalytic domain . The C-terminal tail of Ubc1 was found to contain three alpha-helices between residues D169-S176, K183-L193 and N203-L213 providing the first evidence for a well-defined secondary structure in this region . Chemical shift mapping indicated that residues in the L2 loop of the catalytic domain were most affected indicating the C-terminus of Ubc1 likely interacts with this region . This site of interaction is distinct from that observed in the E2-ubiquitin thiolester and may act to protect the catalytic C88 residue and direct the interaction of ubiquitin in the thiolester intermediate.

Biochimie, 2003 Jan-Feb, 85(1-2), 189 - 94
Specificity of interactions of galectin-3 with Chrp, a cysteine- and histidine-rich cytoplasmic protein; Bawumia S et al.; Earlier work described the cloning of a gene from murine 3T3 cells encoding a cytoplasmic protein Chrp containing a cysteine- and histidine-rich motif characteristic of Zn-finger proteins . The interaction of Chrp with murine galectin-3 first became evident in a yeast two-hybrid screen, but it was also observed in co-precipitation experiments from 3T3 cell lysates . Here, the formation of equimolar complexes by murine Chrp and hamster galectin-3 is shown . Moreover, we found that Chrp binds to the carbohydrate-recognition domain (CRD) of hamster galectin-3 and not to the N-terminal domain carrying the proline- and glycine-rich repeats characteristic of galectin-3 and absent in other galectins . However, galectin-1 does not bind to Chrp, although its CRD is homologous to the galectin-3 CRD . Finally, we report that galectin-3, in a complex with Chrp, binds to laminin in surface plasmon resonance experiments with similar kinetics and affinity as it does in the free state . The formation of higher-order complexes containing these proteins and additional binding partners may be relevant to cytoplasmic functions involving galectin-3.

Appl Microbiol Biotechnol, 2003 Jun, 61(5-6), 393 - 404 Epub 2003 Jan 28.
Catabolism of hydroxyacids and biotechnological production of lactones by Yarrowia lipolytica; Wache Y et al.; The gamma- and delta-lactones of less than 12 carbons constitute a group of compounds of great interest to the flavour industry . It is possible to produce some of these lactones through biotechnology . For instance, gamma-decalactone can be obtained by biotransformation of methyl ricinoleate . Among the organisms used for this bioproduction, Yarrowia lipolytica is a yeast of choice . It is well adapted to growth on hydrophobic substrates, thanks to its efficient and numerous lipases, cytochrome P450, acyl-CoA oxidases and its ability to produce biosurfactants . Furthermore, genetic tools have been developed for its study . This review deals with the production of lactones by Y . lipolytica with special emphasis on the biotransformation of methyl ricinoleate to gamma-decalactone . When appropriate, information from the lipid metabolism of other yeast species is presented.

Ann N Y Acad Sci, 2003 Apr, 986, 461 - 71
Characterization of PISP, a novel single-PDZ protein that binds to all plasma membrane Ca2+-ATPase b-splice variants; Goellner GM et al.; Plasma membrane Ca(2+) ATPases (PMCAs) maintain intracellular Ca(2+) homeostasis and participate in the local regulation of Ca(2+) signaling . Spatially separate demands for Ca(2+) regulation require proper membrane targeting of PMCAs, but the mechanism of PMCA targeting is unknown . Using the PMCA2b carboxyl-terminal tail as yeast two-hybrid bait, we isolated a novel PDZ domain-containing protein from a human brain cDNA library . This protein, named PISP for PMCA-interacting single-PDZ protein, consists of 140 amino acids and contains little else besides a single PDZ domain . Pulldown experiments showed that PISP interacts with all PMCA b-splice forms . PISP was found to be ubiquitously expressed and, in MDCK cells, was present in a punctate pattern throughout the cytosol and at the basolateral membrane . When added to microsomal membranes expressing PMCA4b, PISP was unable to stimulate the PMCA-dependent ATPase activity . Our data suggest that PISP is a transiently interacting partner of the PMCA b-splice forms that may play a role in their sorting to or from the plasma membrane.

Ann N Y Acad Sci, 2003 Apr, 986, 360 - 8
Ion pump-interacting proteins: promising new partners; Pagel P et al.; The sorting and regulation of the Na,K and H,K-ATPases requires that the pump proteins must associate, at least transiently, with kinases, phosphatases, scaffolding molecules, and components of the cellular trafficking machinery . The identities of these interacting proteins and the nature of their associations with the pump polypeptides have yet to be elucidated . We have begun a series of yeast two-hybrid screens employing structurally defined segments of pump polypeptides as baits in order to gain insight into the nature and function of these interacting proteins.

Front Neuroendocrinol, 2003 Apr, 24(2), 128 - 39
Molecular determinants and physiological relevance of extrasomatic RNA localization in neurons; Mohr E et al.; Specific sorting of mRNA molecules to subcellular microdomains is an evolutionarily conserved mechanism by which the polarized nature of eukayotic cells may be established and maintained . The molecular composition of the RNA localization machinery is complex . Sequence motifs within RNA molecules to be transported, called cis-acting elements, and proteins, referred to as trans-acting factors, are essential components . Transport of the resulting ribonucleoprotein complexes to distinct cytoplasmic regions occurs along the cytoskeletal network . The pathway is observed in organisms as diverse as yeast and human and it plays a critical role in development and cell differentiation . Moreover, RNA localization takes place in differentiated cell types including neurons . There is ample evidence to suggest that sorting of defined mRNA species to the neurites of nerve cells and on-site translation has an impact on various aspects of nerve cell biology.

In Silico Biol, 2003, 3(1-2), 173 - 85 Epub 2003 Mar 01.
In silico search for functionally similar proteins involved in meiosis and recombination in evolutionarily distant organisms; Bogdanov YF et al.; Evolutionarily distant organisms have not only orthologs, but also nonhomologous proteins that build functionally similar subcellular structures . For instance, this is true with protein components of the synaptonemal complex (SC), a universal ultrastructure that ensures the successful pairing and recombination of homologous chromosomes during meiosis . We aimed at developing a method to search databases for genes that code for such nonhomologous but functionally analogous proteins . Advantage was taken of the ultrastructural parameters of SC and the conformation of SC proteins responsible for these . Proteins involved in SC central space are known to be similar in secondary structure . Using published data, we found a highly significant correlation between the width of the SC central space and the length of rod-shaped central domain of mammalian and yeast intermediate proteins forming transversal filaments in the SC central space . Basing on this, we suggested a method for searching genome databases of distant organisms for genes whose virtual proteins meet the above correlation requirement . Our recent finding of the Drosophila melanogaster CG17604 gene coding for synaptonemal complex transversal filament protein received experimental support from another lab . With the same strategy, we showed that the Arabidopsis thaliana and Caenorhabditis elegans genomes contain unique genes coding for such proteins.

Oncogene, 2003 May 22, 22(21), 3231 - 42
Function of p73, not of p53, is inhibited by the physical interaction with RACK1 and its inhibitory effect is counteracted by pRB; Ozaki T et al.; The newly identified p53-related gene, p73, encodes a nuclear transcription factor . Unlike p53, p73 has various isoforms with different NH(2)- and COOH-terminal tails . p73alpha with the longest COOH-terminal extension is most abundantly expressed in many tissues and cells among those splicing isoforms of p73 and the COOH-terminal region appears to have an autoregulatory function . To isolate and characterize the cellular protein(s) that interacts with the unique COOH-terminal region of p73alpha, we employed a yeast two-hybrid screen with a human fetal brain and 293 cell cDNA libraries . We identified the receptor for activated C kinase (RACK1) as a new member of p73alpha-binding proteins . The interaction was confirmed by coimmunoprecipitation experiments, whereas RACK1 did not interact with p53 or p73beta . Ectopic overexpression of RACK1 in SAOS-2 cells reduced the p73alpha-mediated transcription from the p53/p73-responsive promoters, and inhibited the p73alpha-dependent apoptosis . On the other hand, the p53-dependent transcriptional activation as well as apoptosis was unaffected in the presence of RACK1 . Furthermore, we found that pRB physically bound to RACK1, and repressed the RACK1-dependent inhibition of p73alpha . Taken together, our observations suggest that pRB diminishes the RACK1-mediated inhibition of p73alpha activity through the interaction with RACK1.

J Biochem (Tokyo), 2003 Apr, 133(4), 493 - 500
Identification of a novel tissue-specific transcriptional activator FESTA as a protein that interacts with the transcription elongation factor S-II; Saso K et al.; Transcription elongation factor S-II was originally purified as a specific stimulator of transcription by RNA polymerase II . Recent studies suggest that S-II participates in gene-specific transcriptional activation in vivo, despite the fact that it directly binds RNA polymerase II and does not recognize specific DNA sequences . In this study, under the hypothesis that S-II requires co-factors to regulate the expression of specific-genes in vivo, we searched for factors that directly interact with S-II using a yeast two-hybrid system, and isolated a novel nuclear protein, FESTA . FESTA is expressed specifically in kidney and spleen, supporting our notion that S-II participates in gene-specific regulation . Two mRNA isoforms of FESTA encoding proteins with different sizes were identified and named FESTA-S and FESTA-L . FESTA contains a serine-rich region and a C-terminal tail that are highly similar to those of the ELL-associated factor EAF1 . Reporter gene assays indicated that both GAL4-FESTA-S and GAL4-FESTA-L fusion proteins have trans-activating ability . Furthermore, deletion of the C-terminal tail of FESTA dramatically reduced its trans-activating ability and abolished its interaction with S-II . This study is the first report of a transcriptional activator that directly interacts with S-II and contains a transcriptional activation domain that cooperates with S-II via direct interaction.

J Biochem (Tokyo), 2003 Jan, 133(1), 115 - 21
Dual subcellular distribution of cytochrome b5 in plant, cauliflower, cells; Zhao J et al.; Subfractionation studies showed that cytochrome b(5) (cyt b5), which has been considered to be a typical ER protein, was localized in both the endoplasmic reticulum membrane (ER) and the outer membrane of mitochondria in cauliflower (Brassica olracea) cells and was a component of antimycin A-insensitive NADH-cytochrome c reductase system in both membranes . When cDNA for cauliflower cyt b5 was introduced into mammalian (COS-7) and yeast cells as well as into onion cells, the expressed cytochrome was localized both in the ER and mitochondria in those cells . On the other hand, rat and yeast cyt b5s were specifically localized in the ER membranes even in the onion cells . Mutation experiments showed that cauliflower cyt b5 carries information that targets it to the ER and mitochondria within the carboxy-terminal 10 amino acids, as in the case of rat and yeast cyt b5s, and that replacement of basic amino acids in this region of cauliflower cyt b5 with neutral or acidic ones resulted in its distribution only in the ER . Together with the established findings of the importance of basic amino acids in mitochondrial targeting signals, these results suggest that charged amino acids in the carboxy-terminal portion of cyt b5 determine its location in the cell, and that the same mechanism of signal recognition and of protein transport to organelles works in mammalian, plant, and yeast cells.

J Biochem (Tokyo), 2003 Jan, 133(1), 109 - 13
CIS1 interacts with the Y532 of the prolactin receptor and suppresses prolactin-dependent STAT5 activation; Endo T et al.; Prolactin (PRL) interacts with a single-chain prolactin-specific receptor of the cytokine receptor superfamily . PRL triggers the activation of JAK2 kinase, which phosphorylates the PRL receptor itself, and of STAT5, a member of the family of signal transducers and activators of transcription (STAT) . We have shown that the STAT5-dependent immediate early gene, CIS1 (Cytokine-Inducible SH2 domain-containing protein-1), suppresses PRL-induced STAT5 activation in vitro as well as in transgenic mice . To facilitate the study of the interactions between CIS1 and the PRL receptor, we have developed the yeast tri-hybrid system, a modification of the yeast two-hybrid system . We expressed CIS1 fused to the DNA-binding domain and PRL receptor cytoplasmic domain fused to the transcription activation domain in the presence or absence of the tyrosine kinase domain of JAK2 in yeast . CIS1 bound to the PRL receptor cytoplasmic domain in a JAK2-dependent manner . Moreover, we determined that the phosphorylated Y532 of the murine PRL receptor is the binding site for CIS1 . Interestingly, Y532 has been shown to be unnecessary for STAT5 activation, although CIS1 overexpression suppressed PRL-induced STAT5 activation . These data suggest that the suppression of STAT5 activation by CIS1 is not due to a simple competition with STAT5 but rather to a modification of the receptor by CIS1 binding.

Bioinformatics, 2003 May 22, 19(8), 973 - 80
Fuzzy C-means method for clustering microarray data; Dembele D et al.; MOTIVATION: Clustering analysis of data from DNA microarray hybridization studies is essential for identifying biologically relevant groups of genes . Partitional clustering methods such as K-means or self-organizing maps assign each gene to a single cluster . However, these methods do not provide information about the influence of a given gene for the overall shape of clusters . Here we apply a fuzzy partitioning method, Fuzzy C-means (FCM), to attribute cluster membership values to genes . RESULTS: A major problem in applying the FCM method for clustering microarray data is the choice of the fuzziness parameter m . We show that the commonly used value m = 2 is not appropriate for some data sets, and that optimal values for m vary widely from one data set to another . We propose an empirical method, based on the distribution of distances between genes in a given data set, to determine an adequate value for m . By setting threshold levels for the membership values, genes which are tigthly associated to a given cluster can be selected . Using a yeast cell cycle data set as an example, we show that this selection increases the overall biological significance of the genes within the cluster . AVAILABILITY: Supplementary text and Matlab functions are available at http://www-igbmc.u-strasbg.fr/fcm/

Bioinformatics, 2003 May 22, 19(8), 923 - 9
Integrative approach for computationally inferring protein domain interactions; Ng SK et al.; MOTIVATION: The current need for high-throughput protein interaction detection has resulted in interaction data being generated en masse through such experimental methods as yeast-two-hybrids and protein chips . Such data can be erroneous and they often do not provide adequate functional information for the detected interactions . Therefore, it is useful to develop an in silico approach to further validate and annotate the detected protein interactions . RESULTS: Given that protein-protein interactions involve physical interactions between protein domains, domain-domain interaction information can be useful for validating, annotating, and even predicting protein interactions . However, large-scale, experimentally determined domain-domain interaction data do not exist . Here, we describe an integrative approach to computationally derive putative domain interactions from multiple data sources, including protein interactions, protein complexes, and Rosetta Stone sequences . We further prove the usefulness of such an integrative approach by applying the derived domain interactions to predict and validate protein-protein interactions . AVAILABILITY: A database of putative protein domain interactions derived using the method described in this paper is available at http://interdom.lit.org.sg.

Biochem J, 2003 Aug 15, 374(Pt 1), 229 - 37
Cloning of an emopamil-binding protein (EBP)-like protein that lacks sterol delta8-delta7 isomerase activity; Moebius FF et al.; EBP (emopamil-binding protein) is a high-affinity binding protein for {3H}emopamil and belongs to the family of so-called sigma receptors . Mutations that disrupt EBP's 3beta-hydroxysteroid sterol delta8-delta7 isomerase activity (EC 5.3.3.5) impair cholesterol biosynthesis and cause X-chromosomal dominant chondrodysplasia punctata . We identified a human cDNA for a novel EBPL (EBP-like protein) with a calculated mass of 23.2 kDa . Amino acid sequence alignments and phylogenetic analysis revealed that EBPL is distantly related to EBP (31% identity and 52% similarity) and found in animals but not in plants . EBPL is encoded by four exons on human chromosome 13q14.2 covering 30.7 kb, and a partially processed EBPL pseudogene was found on 16q21 . The EBPL mRNA was expressed ubiquitously and most abundant in liver, lung and kidney . Upon heterologous expression in yeast EBPL had no detectable 3beta-hydroxysteroid sterol delta8-delta7 isomerase and sigma-ligand-binding activity . Nine out of ten amino acid residues essential for catalytic activity of EBP were conserved in EBPL . Replacement of the only differing residue (EBP-Y111W) reduced catalytic activity of EBP . Transfer of the divergent residue from EBP to EBPL (EBPL-W91Y) and chimaerization of EBP and EBPL at various positions failed to restore catalytic activity of EBPL . Chemical cross-linking induced homodimerization of EBPL and EBP . Whereas mevinolin increased the mRNA for EBP and DHCR7 (delta7-sterol reductase) in HepG2 cells, it had no effect on mRNAs for EBPL and sigma1 receptor, indicating that EBP and EBPL expression are not co-ordinated . We propose that EBPL has a yet-to-be-discovered function other than cholesterol biosynthesis.

J Protein Chem, 2003 Feb, 22(2), 109 - 13
Palindromes in proteins; Giel-Pietraszuk M et al.; Palindromes in DNA consist of nucleotides sequences that read the same from the 5'-end to the 3'-end, and its double helix is related by twofold axis . They occur in genomes of all organisms and have various functions . For example, restriction enzymes often recognize palindromic sequences of DNA . Palindromes in telomeres are crucial for initiation of replication . One can ask the questions, Do palindromes occur in protein, and if so, what function they play? We have searched the protein SWISSPROT database for palindromic sequences . A great number (26%) of different protein palindromes were found . One example of such protein is systemin, an 18-amino-acid-long peptide . It contains palindrome in its beta-sheet domain that interacts with palindromic fragment of DNA . The other palindrome containing protein is cellular human tumor suppressor p53 . Oligonucleotide LTI-ITL has been observed in the crystal structure and is located close to a DNA recognizing domain . As the number of possible palindromic sequences of a given length is far much greater for proteins (20N) than for nucleic acids (4N), the study on their role seems to be an exciting challenge . Our results have clearly showed that palindromes are frequently occurring motives in proteins . Moreover, even very few examples that we have examined so far indicate the importance of further studies on protein palindromes.

J Cell Sci, 2003 Jul 1, 116(Pt 13), 2781 - 90 Epub 2003 May 20.
Temporally and spatially selective loss of Rec8 protein from meiotic chromosomes during mammalian meiosis; Lee J et al.; Sister chromatid cohesion is maintained from DNA replication to metaphase-to-anaphase transition by multisubunit protein complexes called cohesin, which include at least four proteins, SMC1alpha, SMC3, Rad21 and either SA1 or SA2, in mammalian somatic cells . We report here the first evidence of the involvement of Rec8 protein, a mammalian homolog of yeast Rec8p, in meiosis-specific chromosome behavior in mammals . In immunoblotting and immunohistochemical analysis using specific antibodies against mouse Rec8, we found that Rec8 was expressed in the testis but not in the kidney or liver; more precisely, it was expressed in spermatocytes and spermatids but not in spermatogonia or other somatic cells . We also found that Rec8 is present in both phosphorylated and dephosphorylated states in vivo . Immunoprecipitation analyses revealed that Rec8 associates with other cohesin proteins, SMC1beta (meiosis-specific protein) and SMC3 and with a component of synaptonemal complexes, SCP3, but not with SMC1alpha . In meiotic chromosome spreads, Rec8 was localized along the axial/lateral elements of the synaptonemal complexes in meiotic prophase from the leptotene to diplotene stages . At later stages, diakinesis and metaphase I, Rec8 was localized along the interstitial axes of chromosomes, including both centromere and arm regions of chromosomes . However, concomitantly with separation of homologous chromosomes in anaphase I, Rec8 was no longer detected along the arm regions, while it persisted on centromere regions up to metaphase II . In anaphase II, the centromeric signals were diminished . We propose from these results that mammalian Rec8 protein, in association with SMC3 and SMC1beta but not SMC1alpha, is involved in meiosis-specific chromosome behavior, and that homologous chromosome separation is triggered by selective loss of Rec8 from chromosome arms in meiosis I, while sister chromatid cohesion is maintained until metaphase II/anaphase II transition by centromeric Rec8 during mammalian meiosis.

Biochim Biophys Acta, 2003 May 20, 1638(1), 35 - 42
p80 coilin, a coiled body-specific protein, interacts with ataxin-1, the SCA1 gene product; Hong S et al.; Spinocerebellar ataxia type 1 (SCA1) is an autosomal-dominant neurodegenerative disorder characterized by ataxia and progressive motor deterioration . SCA1 is associated with an elongated polyglutamine tract in ataxin-1, the SCA1 gene product . Using the yeast two-hybrid system and co-immunoprecipitation experiments, we have found that p80 coilin, coiled body-specific protein, binds to ataxin-1 . In further experiments with deletion mutants, we found that the C-terminal regions of ataxin-1 and p80 coilin were essential for this interaction . In HeLa cells that have been co-transfected with ataxin-1 and p80 coilin, the p80 coilin protein co-localizes with ataxin-1 aggregates in the nucleoplasm . However, immunohistochemical analysis and immunofluorescence assays showed that mutant ataxin-1 aggregates do not redistribute p80 coilin's dot-like structures in the Purkinje cells of SCA1 transgenic mice . This feature of the interaction between ataxin-1 and p80 coilin suggests that p80 coilin might be implicated in altering the function of ataxin-1.

J Exp Biol, 2003 Jun, 206(Pt 12), 2031 - 8
Analysis of glycolytic enzyme co-localization in Drosophila flight muscle; Sullivan DT et al.; In Drosophila flight muscles, glycolytic enzymes are co-localized along sarcomeres at M-lines and Z-discs and co-localization is required for normal flight . We have extended our analysis of this phenomenon to include a set of six glycolytic enzymes that catalyze consecutive reactions along the glycolytic pathway: aldolase, glycerol-3-phosphate dehydrogenase (GPDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), triose phosphate isomerase, phosphoglycerate kinase and phosphoglycerol mutase (PGLYM) . Each of these enzymes has an identical pattern of localization . In mutants null for GPDH, localization of none of the other enzymes occurs and therefore is interdependent . In optimally fixed preparations of myofibrils, accumulation of the enzymes at M-lines is much greater than at Z-discs . However, localization at M-lines is more labile, as shown by loss of localization when fixation is delayed . We have begun to analyze the protein-protein interaction involved in glycolytic enzyme co-localization using the yeast two-hybrid system . We have identified two pair-wise interactions . One is between GPDH and GAPDH and another is between GPDH and PGLYM.

J Exp Biol, 2003 Jun, 206(Pt 12), 1977 - 84
Actin comet tails, endosomes and endosymbionts; Fehrenbacher K et al.; The Arp2/3 complex consists of seven highly conserved and tightly associated subunits, two of which are the actin-related proteins Arp2 and Arp3 . One of the best-studied functions of the Arp2/3 complex is to stimulate actin nucleation and force production at the leading edge of motile cells . What is now clear is that Arp2/3-complex-mediated force production drives many intracellular movements, including movement of bacterial pathogens in infected host cells, internalization of extracellular materials via phagocytosis and endocytosis, and movement of mitochondria during cell division in budding yeast . Here, we describe recent advances in the mechanisms underlying Arp2/3 complex-driven intracellular movement.

Eur J Biochem, 2003 Jun, 270(11), 2459 - 66
Emerin interacts in vitro with the splicing-associated factor, YT521-B; Wilkinson FL et al.; Emerin is a nuclear membrane protein that interacts with lamin A/C at the nuclear envelope . Mutations in either emerin or lamin A/C cause Emery-Dreifuss muscular dystrophy (EDMD) . The functions of emerin are poorly understood, but EDMD affects mainly skeletal and cardiac muscle . We used a high-stringency yeast two-hybrid method to screen a human heart cDNA library, with full-length emerin as bait . Four out of five candidate interactors identified were nuclear proteins: lamin A, splicing factor YT521-B, proteasome subunit PA28 gamma and transcription factor vav-1 . Specific binding between emerin and the functional C-terminal domain of YT521-B was confirmed by pull-down assays and biomolecular interaction analysis (BIAcore) . Inhibition by emerin of YT521-B-dependent splice site selection in vivo suggests that the interaction is physiologically significant . A 'bipartite' binding site for YT521-B in emerin was identified using alanine substitution or disease-associated mutations in emerin . The transcription factor GCL (germ cell-less) has previously been shown to bind to the same site . The results are consistent with an emerging view that lamins and lamina-associated proteins, like emerin, have a regulatory role, as well as a structural role in the nucleus . YT521-B joins a growing list of candidates for a role in a gene expression model of the pathogenesis of EDMD.

Prikl Biokhim Mikrobiol, 2003 May-Jun, 39(3), 335 - 40
{Optimization of conditions for storage and cultivation of the fungus Claviceps sp.--a producer of the ergot alkaloid agroclavine}; Boichenko LV et al.; Conditions of agroclavine biosynthesis by the mutant Claviceps sp . strain s 106 were studied . The content of agroclavine was maximum (1.5-2 g/l) on days 15-16 of cultivation in the complex medium T25, containing sucrose, citric acid, and yeast extract . Agroclavine was the major component of the alkaloid fraction (90-95%) . Storage of the culture at -70 degrees C in T25 supplemented by 7% glycerol provided a stable level of alkaloid formation.

J Histochem Cytochem, 2003 Jun, 51(6), 797 - 808
Single-strand DNA aptamers as probes for protein localization in cells; Stanlis KK et al.; The accurate localization of proteins in fixed cells is important for many studies in cell biology, but good fixation is often antagonistic to good immunolabeling, given the density of well-preserved cells and the size of most labeled antibody probes . We therefore explored the use of single-stranded oligonucleotides (aptamers), which can bind to proteins with very high affinity and specificity but which are only approximately 10 kD . To evaluate these probes for general protein localization, we sought an aptamer that binds to a widely used protein tag, the green fluorescent protein (GFP) . Although this quest was not successful, we were able to solve several practical problems that will confront any such labeling effort, e.g., the rates at which oligonucleotides enter fixed cells of different kinds and the extent of nonspecific oligonucleotide binding to both mammalian and yeast cell structures . Because such localization methods would be of particular value for electron microscopy of optimally fixed material, we also explored the solubility of aptamers under conditions suitable for freeze-substitution fixation . We found that aptamers are sufficiently soluble in cold organic solvents to encourage the view that this approach may be useful for the localization of specific proteins in context of cellular fine structure.

FEBS Lett, 2003 May 22, 543(1-3), 55 - 60
The A20-binding protein ABIN-2 exerts unexpected function in mediating transcriptional coactivation; Chien CY et al.; The human ABIN-2 was originally identified as an A20-associating cytosolic protein to block NF-kappaB activation induced by various stimuli . Here we report that ABIN-2 has the potential to enter the nucleus and plays a role in mediating transcriptional activation in both yeast and mammalian cells . The Gal4BD-ABIN-2 fusion protein is able to drive the expression of the GAL4-responsive reporter gene in yeast efficiently without the need of the Gal4p activation domain, suggesting that ABIN-2 functions as a transcriptional coactivator and facilitates transcription in yeast . In contrast to the activity in yeast, however, only the C-terminal fragment of ABIN-2 exerts the transactivating activity in mammalian cells but not the full-length ABIN-2 protein . This observation has led to the identification of the N-terminal 195 amino acids of ABIN-2 as a regulatory domain, which retains the full-length ABIN-2 in the cytoplasm of mammalian cells and thus cannot transactivate . We have also found that BAF60a, a component of chromatin-remodeling complex, interacts with ABIN-2 by the yeast two-hybrid analysis . Together, our results suggest that the nuclear ABIN-2 defines a novel transcriptional coactivator and acts presumably by recruiting a chromatin-remodeling complex to the site of the target gene.

Plant J, 2003 May, 34(4), 417 - 25
Cell cycle function of a rice B2-type cyclin interacting with a B-type cyclin-dependent kinase; Lee J et al.; Cyclin-dependent kinases (CDKs) are involved in the control of cell cycle progression . Plant A-type CDKs are functional homologs of yeast Cdc2/Cdc28 and are expressed throughout the cell cycle . In contrast, B-type CDK (CDKB) is a family of mitotic CDKs expressed during the S/M phase, and its precise function remains unknown . Here, we identified two B2-type cyclins, CycB2;1 and CycB2;2, as a specific partner of rice CDKB2;1 . The CDKB2;1-CycB2 complexes produced in insect cells showed a significant level of kinase activity in vitro, suggesting that CycB2 binds to and activates CDKB2 . We then expressed green fluorescent protein (GFP)-fused CDKB2;1 and CycB2;2 in tobacco BY2 cells to investigate their subcellular localization during mitosis . Surprisingly, the fluorescence signal of CDKB2;1-GFP was tightly associated with chromosome alignment as well as with spindle structure during the metaphase . During the telophase, the signal was localized to the spindle midzone and the separating sister chromosomes, and then to the phragmoplast . On the other hand, the CycB2;2-GFP fluorescence signal was detected in nuclei during the interphase and prophase, moved to the metaphase chromosomes, and then disappeared completely after the cells passed through the metaphase . Co-localization of CDKB2;1-GFP and CycB2;2-GFP on chromosomes aligned at the center of the metaphase cells suggests that the CDKB2-CycB2 complex may function in retaining chromosomes at the metaphase plate . Overexpression of CycB2;2 in rice plants resulted in acceleration of root growth without any increase in cell size, indicating that CycB2;2 promoted cell division probably through association with CDKB2 in the root meristem.

Infect Dis Clin North Am, 2003 Mar, 17(1), 21 - 40, vii
Blastomycosis; Bradsher RW et al.; Blastomycosis is an endemic mycoses in the central United States caused by a dimorphic fungus, Blastomyces dermatitidis, that exists in nature in mycelial phase and converts to yeast phase at body temperature . The organism may produce epidemics of infection following a point source of infection or sporadic endemic infection . Blastomycosis can be a subclinical illness with subsequent protection against progressive infection afforded by cellular immune mechanisms, but it may present with progressive disease with either pulmonary or extrapulmonary disease or both . Itraconazole has been shown to be the drug of choice for both infections, except in cases of life-threatening infection when amphotericin B should be used.

Mikrobiologiia, 2003 Mar-Apr, 72(2), 168 - 73
{Induction of cytochrome P-450 and ethanol oxidation in Yarrowia lipolytica}; Il'chenko AP et al.; The study of the effect of different ethanol concentrations in the medium on the growth and the activity of enzymatic systems involved in ethanol oxidation in Yarrowia lipolytica showed that the cultivation of yeast cells on 1 and 2% ethanol caused their rapid growth and a drastic increase in cell respiration and sensitivity to cyanide already in the first hours of cultivation . At the same time, during cultivation on 3, 4, and 5% ethanol, the growth and respiration of yeast cells were considerably suppressed . All of the ethanol concentrations studied induced the synthesis of cytochrome P-450, its dynamics in cells being dependent on the initial concentration of ethanol in the medium . When the initial concentration of ethanol was 1 and 2%, the content of cytochrome P-450 in cells steeply decreased after a short period of induction . But when the initial concentration of ethanol in the medium was 3 to 5%, the content of cytochrome P-450 in cells was high throughout the cultivation period . The induction of cytochrome P-450 in cells preceded the induction of the NAD-dependent enzymes alcohol dehydrogenase and catalase, which, like cytochrome P-450, are also involved in ethanol oxidation by yeasts . The activity of catalase was higher in the yeast cells grown in the presence of 3 to 5% ethanol than in the cells grown in the presence of 1 and 2% ethanol . The roles played by cytochrome P-450, alcohol dehydrogenase, and catalase in ethanol oxidation by yeast cells are discussed.

Science, 2003 May 16, 300(5622), 1152 - 5
Distinct cohesin complexes organize meiotic chromosome domains; Kitajima TS et al.; Meiotic cohesin complexes at centromeres behave differently from those along chromosome arms, but the basis for these differences has remained elusive . The fission yeast cohesin molecule Rec8 largely replaces its mitotic counterpart, Rad21/Scc1, along the entire chromosome during meiosis . Here we show that Rec8 complexes along chromosome arms contain Rec11, whereas those in the vicinity of centromeres have a different partner subunit, Psc3 . The arm associated Rec8-Rec11 complexes are critical for meiotic recombination . The Rec8-Psc3 complexes comprise two different types of assemblies . First, pericentromeric Rec8-Psc3 complexes depend on histone methylation-directed heterochromatin for their localization and are required for cohesion during meiosis II . Second, central core Rec8-Psc3 complexes form independently of heterochromatin and are presumably required for establishing monopolar attachment at meiosis I . These findings define distinct modes of assembly and functions for cohesin complexes at different regions along chromosomes.

Proc Natl Acad Sci U S A, 2003 May 27, 100(11), 6364 - 9 Epub 2003 May 15.
Genomewide demarcation of RNA polymerase II transcription units revealed by physical fractionation of chromatin; Nagy PL et al.; Epigenetic modifications of chromatin serve an important role in regulating the expression and accessibility of genomic DNA . We report here a genomewide approach for fractionating yeast chromatin into two functionally distinct parts, one containing RNA polymerase II transcribed sequences, and the other comprising noncoding sequences and genes transcribed by RNA polymerases I and III . Noncoding regions could be further fractionated into promoters and segments lacking promoters . The observed separations were apparently based on differential crosslinking efficiency of chromatin in different genomic regions . The results reveal a genomewide molecular mechanism for marking promoters and genomic regions that have a license to be transcribed by RNA polymerase II, a previously unrecognized level of genomic complexity that may exist in all eukaryotes . Our approach has broad potential use as a tool for genome annotation and for the characterization of global changes in chromatin structure that accompany different genetic, environmental, and disease states.

J Biol Chem, 2003 Aug 1, 278(31), 28823 - 30 Epub 2003 May 15.
A role for epsin N-terminal homology/AP180 N-terminal homology (ENTH/ANTH) domains in tubulin binding; Hussain NK et al.; The epsin N-terminal homology (ENTH) domain is a protein module of approximately 150 amino acids found at the N terminus of a variety of proteins identified in yeast, plants, nematode, frog, and mammals . ENTH domains comprise multiple alpha-helices folded upon each other to form a compact globular structure that has been implicated in interactions with lipids and proteins . In characterizing this evolutionarily conserved domain, we isolated and identified tubulin as an ENTH domain-binding partner . The interaction, which is direct and has a dissociation constant of approximately 1 microm, was observed with ENTH domains of proteins present in various species . Tubulin is co-immunoprecipitated from rat brain extracts with the ENTH domain-containing proteins, epsins 1 and 2, and punctate epsin staining is observed along the microtubule cytoskeleton of dissociated cortical neurons . Consistent with a role in microtubule processes, the over-expression of epsin ENTH domain in PC12 cells stimulates neurite outgrowth . These data demonstrate an evolutionarily conserved property of ENTH domains to interact with tubulin and microtubules.

Cancer Res, 2003 May 15, 63(10), 2589 - 95
Assembly of functional ALT-associated promyelocytic leukemia bodies requires Nijmegen Breakage Syndrome 1; Wu G et al.; Immortalized cells maintain telomere length through either a telomerase-dependent process or a telomerase-independent pathway termed alternative lengthening of telomeres (ALT) . Homologous recombination is implicated in the ALT pathway in both yeast and human ALT cells . In ALT cells, two types of DNA double-strand break repair and homologous recombination factors, the Rad50/Mre11/NBS1 complex and Rad51/Rad52 along with replication factors (RPA) and telomere binding proteins (TRF1 and TRF2), are associated with the ALT-associated PML body (APB) . DNA synthesis in late S-G(2) is associated with APBs, which contain telomeric DNA and, are therefore, potential sites for telomere length maintenance . Here, we show that the breast cancer susceptibility gene product, breast cancer susceptibility gene 1, and the human homologue of yeast Rap1, hRap1, are also associated with APBs specifically during late S-G(2) phase of the cell cycle . We additionally show that the localization of the double-strand break repair factors with APBs is distinct from their association with ionizing radiation-induced nuclear foci . To systematically explore the mechanism involved in the assembly of APBs, we examine the role of Nijmegen breakage syndrome 1 (NBS1) and TRF1 in this process, respectively . We demonstrated that NBS1 plays a key role in the assembly and/or recruitment of Rad50, Mre11, and breast cancer susceptibility gene 1, but not Rad51 or TRF1, to APBs . The NH(2) terminus of NBS1, specifically the BRCA1 COOH-terminal domain, is required for this activity . Although TRF1 interacts with NBS1 directly, it is dispensable for the association of either Rad50/Mre11/NBS1 or Rad51 with APBs . Perturbation of the interactions between NBS1/Mre11 and APBs correlates with reduced BrdUrd incorporation associated with APBs, consistent with decreased DNA synthesis at these sites . Taken together, these results support a model in which NBS1 has a vital role in the assembly of APBs, which function to maintain telomeres in human ALT cells.

Eur J Med Chem, 2003 Apr, 38(4), 363 - 6
Discovery of selective CYP11B2 (aldosterone synthase) inhibitors for the therapy of congestive heart failure and myocardial fibrosis; Hartmann RW et al.; An increased aldosterone concentration due to congestive heart failure leads to a further progression of the disease as well as to myocardial fibrosis . To interfere with these fatal processes selective inhibition of aldosterone synthase (CYP11B2) is required . CYP11B1, a key enzyme in glucocorticoid biosynthesis showing a high homology to the target enzyme (>93%), must not be inhibited . Screening of our P450 inhibitor library for inhibition of bovine aldosterone synthase resulted in a high number of compounds showing reasonable inhibition . In the next step substances were tested for oral absorption using two artificial membrane assays . The inhibition of human CYP11B2 was evaluated using assays in fission yeast and V79MZ cells stably expressing the active human target enzyme . For selectivity, inhibition of CYP11B1, CYP11A1, CYP17, CYP19 and CYP5 was determined . Rather potent and selective compounds obtained in this way were structurally further optimised, finally leading to inhibitors showing IC(50) values within the low nanomolar range.

Brain Res Mol Brain Res, 2003 May 12, 113(1-2), 57 - 66
Ets transcription factors ER81 and Elk1 regulate the transcription of the human presenilin 1 gene promoter; Pastorcic M et al.; We have previously defined a crucial DNA element controlling 90% of the expression of the presenilin 1 gene at (-35 to +6) . This region contains an Ets transcription factor binding motif, and a 2-base pair alteration within the core sequence (GGAA to TTAA) of the Ets consensus also reduced transcription by over 90% . We have shown that Ets1/2 transcription factors bind specifically to the -10 Ets element and activate PS1 transcription . The identification of other transcription factors recognizing specifically this promoter area should provide insights into the regulation of PS1 . We have used the -10 Ets element as a bait in yeast one hybrid screening of a human brain cDNA library . This assay selected three factors from the Ets family: Ets2, ER81 and Elk1 . We show that in vitro translated ER81 indeed binds specifically to the -10 region of the PS1 promoter and that ER81 activates by two- to threefold the basal transcription of a presenilin-1 promoter-chloramphenicol acetyltransferase reporter synthetic gene (-119, +178)PS1CAT in transient infection assays in neuroblastoma cells (SK-N-SH) . GABPalpha, a member of the Ets family closely related to Ets2 and also containing a pointed domain, only increased PS1 transcription by about twofold . Cotransfection of GABPbeta together with GABPalpha did not increase PS1 transcription . However, GABPbeta alone activated PS1 transcription by two- to threefold . In contrast, the more distantly related Ets factor Elk1 repressed PS1 transcription very effectively.

Exp Cell Res, 2003 Jun 10, 286(2), 308 - 20
Mouse NIPK interacts with ATF4 and affects its transcriptional activity; Ord D et al.; Neuronal cell death-inducible putative kinase (NIPK) is a protein with an unknown function encoded by a gene activated in neuronal cells in cell death-causing conditions (disruption of calcium homeostasis, trophic factor deprivation) . Using the yeast two-hybrid screening of an embryonic mouse cDNA library, we identified activating transcription factor 4 (ATF4) as a protein binding to mouse (m) NIPK . The critical domain for mNIPK-binding resides in a 72 amino acid stretch near the N-terminus of ATF4, covering the second leucine zipper motif and the preceding region . mNIPK expressed as fusion protein with enhanced yellow fluorescence protein (EYFP) is localized predominantly in the nucleus, and the mNIPK-ATF4 complex can be immunoprecipitated from cells cotransfected with epitope-tagged mNIPK and ATF4 constructs . The expression of both mNIPK and ATF4 is upregulated in the neuronal cell line GT1-7 in response to disruption of calcium homeostasis by thapsigargin, but ATF4 is induced more rapidly than mNIPK . The coexpression of mNIPK inhibits ATF4 CRE-dependent transcriptional activation activity in transiently transfected cells . At the same time, ATF4 degradation rate is not increased in the cells coexpressing mNIPK, and ATF4, associated to mNIPK, is able to bind to CRE . Thus, mNIPK is a novel regulator of ATF4 transcriptional activity.

IUBMB Life, 2003 Feb, 55(2), 103 - 7
Isoelectric point mobility shift assay for rapid screening of charged and uncharged ligands bound to proteins; Kempna P et al.; Three human proteins (hTAP1, hTAP2 and hTAP3) that are related to the yeast phosphatidylinositol/phosphatidylcholine transfer protein SEC14p were recently cloned in our laboratory . These proteins contain a relatively large hydrophobic pocket, the so called CRAL-TRIO domain, which is present also in other human proteins, such as CRALBP, alpha-TTP and MEG2 . The CRAL-TRIO domains in these proteins bind ligands such as retinaldehyde, tocopherols and polyphosphoinositides, respectively . To screen for potential hTAPs ligands, we developed a semi-quantitative isoelectric point mobility shift assay (IPMS-assay) that allows assessing the binding of potential hydrophobic ligands to proteins . Purified proteins occupied with a charged ligand migrate differently on isoelectric focusing gels when compared with free protein . Competition of bound charged ligands with uncharged ones reverses the mobility shift, so that the relative affinities of the two ligands to the protein can be estimated.

IUBMB Life, 2003 Feb, 55(2), 71 - 81
Plant peroxisomes, reactive oxygen metabolism and nitric oxide; del Rio LA et al.; In plant cells, as in most eukaryotic organisms, peroxisomes are probably the major sites of intracellular H2O2 production, as a result of their essentially oxidative type of metabolism . Like mitochondria and chloroplasts, peroxisomes also produce superoxide radicals (O2*-) and there are, at least, two sites of superoxide generation: one in the organelle matrix, the generating system being xanthine oxidase, and another site in the peroxisomal membranes dependent on NAD(P)H . In peroxisomal membranes, three integral polypeptides (PMPs) with molecular masses of 18, 29, and 32 kDa have been shown to generate O2*- radicals . Besides catalase, several antioxidative systems have been demonstrated in plant peroxisomes, including different superoxide dismutases, the four enzymes of the ascorbate-glutathione cycle plus ascorbate and glutathione, and three NADP-dependent dehydrogenases . A CuZn-SOD and two Mn-SODs have been purified and characterized from different types of plant peroxisomes . The presence of the enzyme nitric oxide synthase (NOS) and its reaction product, nitric oxide (NO*), has been recently demonstrated in plant peroxisomes . Different experimental evidence has suggested that peroxisomes have a ROS-mediated cellular function in leaf senescence and in stress situations induced by xenobiotics and heavy metals . Peroxisomes could also have a role in plant cells as a source of signal molecules like NO*, O2*- radicals, H2O2, and possibly S-nitrosoglutathione (GSNO) . It seems reasonable to think that a signal molecule-producing function similar to that postulated for plant peroxisomes could also be performed by human, animal and yeast peroxisomes, where research on oxy radicals, antioxidants and nitric oxide is less advanced than in plant peroxisomes.

Mycopathologia, 2002, 156(3), 163 - 9
Morphological characterization of in-vitro human hair keratinolysis, produced by identified wild strains of Chrysosporium species; Mitola G et al.; Chrysosporium species were isolated from soil and keratinized material . Primary isolation was performed following the general method of hair baiting on modified Czapek-agar media with washed, defated and sterilized human hair fragments added . Strains were maintained in test tubes of potato dextrose agar at 29 degrees C and cultivated on phytone yeast extract agar at 28 degrees C for 14 days for identification . Isolates were characterized using Van Oorschot's key . Keratinolytic activity was expressed following a subjective scale representing degree/severity of attack upon hair surface and presence of fungal structures observed in substrate . Culture results and characterization methods were effective for soil Chrysosporium strain isolation . A new hair attack mode is described . Of 71 keratinolytic fungal isolates, eight (12%) Chrysosporium species were identified . One keratinolytic Chrysosporium sp . isolate is yet to be identified.

Mol Cell Biol, 2003 Jun, 23(11), 3929 - 35
The histidine triad protein Hint is not required for murine development or Cdk7 function; Korsisaari N et al.; The histidine triad (HIT) protein Hint has been found to associate with mammalian Cdk7, as well as to interact both physically and genetically with the budding yeast Cdk7 homologue Kin28 . To study the function of Hint and to explore its possible role in modulating Cdk7 activity in vivo, we have characterized the expression pattern of murine Hint and generated Hint-deficient (Hint(-/-)) mice . Hint was widely expressed during mouse development, with pronounced expression in several neuronal ganglia, epithelia, hearts, and testes from embryonic day 15 onward . Despite this widespread expression, disruption of Hint did not impair murine development . Moreover, Hint-deficient mice had a normal life span and were apparently healthy . Histological examination of tissues with high Hint expression in wild-type animals did not show signs of abnormal pathology in Hint(-/-) mice . Functional redundancy within the HIT family was addressed by crossing Hint(-/-) mice with mice lacking the related HIT protein, Fhit, and by assaying the expression levels of the HIT protein gene family members Hint2 and Hint3 in Hint(+/+) and Hint(-/-) tissues . Finally, Cdk7 kinase activity and cell cycle kinetics were found to be comparable in wild-type and Hint(-/-) mouse embryonic fibroblasts, suggesting that Hint may not be a key regulator of Cdk7 activity.

Mol Cell Biol, 2003 Jun, 23(11), 3735 - 52
p27Kip1 inhibition of GRB2-SOS formation can regulate Ras activation; Moeller SJ et al.; p27(Kip1) (p27) is often inappropriately downregulated in aggressive human cancers . Although p27 can inhibit cyclin-dependent kinases (CDKs), low p27 does not always correlate with increased CDK activity . Furthermore, cells derived from p27(-/-) mice respond to antimitogens, maintain restriction point control, and do not deregulate CDKs . Thus, disruption of a p27 function other than CDK inhibition may contribute to the disease state . A yeast two-hybrid screen identified growth factor receptor-bound protein 2 (GRB2) as a p27 binding partner . We now demonstrate that p27 can inhibit GRB2 function by blocking its association with the guanine nucleotide exchange factor SOS . Endogenous p27 is rapidly exported from the nucleus to the cytoplasm in response to mitogen stimulation, where it binds GRB2 concomitant with a decrease in GRB2-associated SOS . As predicted, mitogen-stimulated p27(-/-) cells maintained their GRB2-SOS complexes for significantly longer . The Ras/mitogen-activated protein kinase pathway does not appear to be deregulated in cells lacking p27 despite excess GRB2-SOS, suggesting that additional control mechanisms are present . A transient-transfection approach was employed to show that p27 can inhibit Ras activation by targeting GRB2 and further revealed that the CDK and GRB2 inhibitory functions of p27 are separable and distinct . Thus, p27 downregulation may compromise control of Ras, one of the most common oncogenic events in human cancer.

J Biol Chem, 2003 Aug 1, 278(31), 29121 - 9 Epub 2003 May 14.
The role of the SAP motif in promoting Holliday junction binding and resolution by SpCCE1; Ahn JS et al.; Holliday junctions are four-way branched DNA structures that are formed during recombination and by replication fork regression . Their processing depends on helicases that catalyze junction branch migration, and endonucleases that resolve the junction into nicked linear DNAs . Here we have investigated the role of a DNA binding motif called SAP in binding and resolving Holliday junctions by the fission yeast mitochondrial resolvase SpCCE1 . Mutation or partial/complete deletion of the SAP motif dramatically impairs the ability of SpCCE1 to resolve Holliday junctions in a heterologous in vivo system . These mutant proteins retain the ability to recognize the junction structure and to distort it upon binding . However, once formed the mutant protein-junction complexes are relatively unstable and dissociate much faster than wild-type complexes . We show that binding stability is necessary for efficient junction resolution, and that this may be due in part to a requirement for maintaining the junction in an open conformation so that it can branch migrate to cleavable sites.

J Biol Chem, 2003 Jul 18, 278(29), 26879 - 87 Epub 2003 May 13.
RING finger protein AO7 supports NF-kappaB-mediated transcription by interacting with the transactivation domain of the p65 subunit; Asamitsu K et al.; In this study, a novel interactor of the p65 subunit (RelA) of NF-kappaB has been explored by performing yeast two-hybrid screen using the transactivation domain (TAD) of p65 located in the C terminus as bait . We have isolated a RING finger motif-containing protein, AO7, previously identified as an interacting protein with a ubiquitin-conjugating enzyme, Ubc5B . We confirmed the protein-protein interaction between p65 and AO7 in vitro and in vivo and found that the C-terminal region of AO7 is responsible for the interaction with p65 TAD . AO7 was predominantly localized in the nucleus and activated the NF-kappaB-dependent gene expression upon stimulation with IL-1beta or TNF or overexpression of NF-kappaB-inducing kinase . We found that both the RING finger and the C-terminal regions of AO7 were necessary for the transcriptional activation . When cotransfected with plasmids expressing Gal4-p65 fusion proteins containing various functional domains of p65, we found that p65 TAD was essential for the transcriptional activation mediated by AO7 . Furthermore, the p65-mediated transactivation was suppressed by a ubiquitination-defective AO7 mutant in which the essential Cys residue within the RING finger motif was substituted by Ser . These data suggest that AO7 interacts with the p65 TAD and modulates its transcriptional activity.

J Biol Chem, 2003 Aug 8, 278(32), 30028 - 36 Epub 2003 May 14.
Identification and characterization of a nuclear interacting partner of anaplastic lymphoma kinase (NIPA); Ouyang T et al.; Anaplastic large-cell lymphoma is a subtype of non-Hodgkin lymphomas characterized by the expression of CD30 . More than half of these lymphomas carry a chromosomal translocation t(2;5) leading to expression of the oncogenic tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) . NPM-ALK is capable of transforming fibroblasts and lymphocytes in vitro and of causing lymphomas in mice . Previously, we and others demonstrated phospholipase C-gamma and phosphatidylinositol 3-kinase as crucial downstream signaling mediators of NPM-ALK-induced oncogenicity . In this study, we used an ALK fusion protein as bait in a yeast two-hybrid screen identifying NIPA (nuclear interacting partner of ALK) as a novel downstream target of NPM-ALK . NIPA encodes a 60-kDa protein that is expressed in a broad range of human tissues and contains a classical nuclear translocation signal in its C terminus, which directs its nuclear localization . NIPA interacts with NPM-ALK and other ALK fusions in a tyrosine kinase-dependent manner and is phosphorylated in NPM-ALK-expressing cells on tyrosine and serine residues with serine 354 as a major phosphorylation site . Overexpression of NIPA in Ba/F3 cells was able to protect from apoptosis induced by IL-3 withdrawal . Mutations of the nuclear translocation signal or the Ser-354 phosphorylation site impaired the antiapoptotic function of NIPA . In NPM-ALK-transformed Ba/F3 cells, apoptosis triggered by wortmannin treatment was enhanced by overexpression of putative dominant-negative NIPA mutants . These results implicate an antiapoptotic role for NIPA in NPM-ALK-mediated signaling events.

J Biol Chem, 2003 Jul 18, 278(29), 26870 - 8 Epub 2003 May 13.
tbCPSF30 depletion by RNA interference disrupts polycistronic RNA processing in Trypanosoma brucei; Hendriks EF et al.; Gene expression in eukaryotes requires the post-transcriptional cleavage of mRNA precursors into mature mRNAs . In Trypanosoma brucei, mRNA processing is of particular importance, since most transcripts are derived from polycistronic transcription units . This organization dictates that regulated gene expression is promoter-independent and governed at the posttranscriptional level . We have identified tbCPSF30, a protein containing five CCCH zinc finger motifs, which is a homologue of the cleavage and polyadenylation specificity factor (CPSF) 30-kDa subunit, a component of the machinery required for 3'-end formation in yeast and mammals . Using gene silencing of tbCPSF30 by RNA interference, we demonstrate that this gene is essential in bloodstream and procyclic forms of T . brucei . Interestingly, tbCPSF30-specific RNA interference results in the accumulation of an aberrant tbCPSF30 mRNA species concomitant with depletion of tbCPSF30 protein . tbCPSF30 protein depletion is accompanied by the accumulation of unprocessed tubulin RNAs, implicating tbCPSF30 in polycistronic RNA processing . By genome data base mining, we also identify several other putative components of the T . brucei cleavage and polyadenylation machinery, indicating their conservation throughout eukaryotic evolution . This study is the first to identify and characterize a core component of the T . brucei CPSF and show its involvement in polycistronic RNA processing.

Arch Biochem Biophys, 2003 Jun 1, 414(1), 83 - 90
Mechanism of binding of warfarin enantiomers to recombinant domains of human albumin; Twine SM et al.; Domain fragments of human serum albumin corresponding to domains 1 and 2 (D12) and domains 2 and 3 (D23) were expressed in yeast . The kinetics of warfarin binding to these fragments were investigated using stopped-flow fluorescence spectroscopy . Binding can be characterized by a two-step process, a rapid diffusion-controlled step and a slower rate-limiting step in which a stable drug-protein complex is formed . The equilibrium constant for step 1 is greater for both D12 and D23 than for albumin, probably as a result of reduced steric hindrance offered by the domain fragments . Binding step 2, thought to be the result of a conformational change as warfarin is accommodated by the protein, is faster for D12 and D23 . Albumin and the domain fragments show an increased preference for the R enantiomer, but the preference is particularly enhanced for domain fragment D12 . These preferences can largely be explained by the domains having different rates for step 2 of the binding process.

Biochem Biophys Res Commun, 2003 May 30, 305(2), 359 - 64
The viral death protein Apoptin interacts with Hippi, the protein interactor of Huntingtin-interacting protein 1; Cheng CM et al.; Apoptin, a chicken anemia virus-encoded protein, induces apoptosis in human tumor cells but not in normal cells . The tumor-specific activity of Apoptin is correlated with its nuclear localization in tumor cells . In an attempt to elucidate the molecular mechanism of Apoptin-induced apoptosis, we identified human Hippi, the protein interactor and apoptosis co-mediator of Huntingtin interacting protein 1, as one of the Apoptin-associated proteins by yeast two-hybrid screen . We also demonstrated that Hippi could interact with Apoptin both in vitro and in human cells . Furthermore, subcellular localization studies showed that Hippi and Apoptin perfectly colocalized in the cytoplasm of normal human HEL cells, whereas in cancerous HeLa cells most Apoptin and Hippi were located separately in the nucleus and cytoplasm and, thus, showed only a modest colocalization . Mapping studies indicate that Hippi binds within the self-multimerization domain of Apoptin, and Apoptin binds to the C-terminal half of Hippi, including its death effector domain-like motif . Our results suggest that the Apoptin-Hippi interaction may play a role in the suppression of apoptosis in normal cells.

Biochem Biophys Res Commun, 2003 May 30, 305(2), 345 - 52
Interaction of the deafness-dystonia protein DDP/TIMM8a with the signal transduction adaptor molecule STAM1; Blackstone C et al.; The Mohr-Tranebjaerg-Jensen deafness-dystonia-optic atrophy protein DDP/TIMM8a is translated on cytoplasmic ribosomes but targeted ultimately to the mitochondrial intermembrane space, where it is involved in mitochondrial protein import . STAM1 is a cytoplasmic signal-transducing adaptor molecule implicated in cytokine signaling . We report here a direct interaction between DDP and STAM1, identified by yeast two-hybrid screening and confirmed by co-immunoprecipitation, fusion protein "pull downs," and nuclear redistribution assays . DDP coordinates Zn(2+), and Zn(2+) was found to stimulate the DDP-STAM1 interaction in vitro . Endogenous STAM1 localizes predominantly to early endosomes, and we found no evidence that STAM1 is imported into mitochondria in vitro . Thus, the DDP-STAM1 interaction likely occurs in the cytoplasm or at the mitochondrial outer membrane . The DDP-STAM1 interaction requires a coiled-coil region in STAM1 that overlaps with the immunoreceptor tyrosine-based activation motif (ITAM), a region previously shown to be important for interaction with Jak2/3 and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) . Thus, DDP binding may alter the interactions of STAM1 with several cytoplasmic proteins involved in cell signaling and endosomal trafficking.

Biochem Biophys Res Commun, 2003 May 30, 305(2), 311 - 4
Abolishment of the interaction between cyclin-dependent kinase 2 and Cdk-associated protein phosphatase by a truncated KAP mutant; Yeh CT et al.; The cyclin-dependent kinase (Cdk)-associated protein phosphatase (KAP) is a human dual-specificity protein phosphatase that dephosphorylates Cdk2 on a conserved threonine residue, T160, in a cyclin dependent manner . Several aberrant KAP transcripts with characteristic deletion regions have been identified in hepatocellular carcinoma tissues . In this report, we demonstrated that multiple aberrant KAP transcripts were also present in a hepatoblastoma cell line (HepG2), albeit harboring a totally different set of deletions . By performing yeast two-hybrid and co-immunoprecipitation experiments, a KAP-Cdk2 interaction domain located in the amino acid 1-34 region was identified . This interaction domain was different from the major protein interface deduced from crystal structure analysis . Using a yeast three-hybrid system, it was shown that the presence of a truncated KAP mutant encoding this interaction domain abolished the wild-type KAP-Cdk2 interaction . In conclusion, a previously unidentified KAP-Cdk2 interaction domain was discovered . Truncated KAP mutants containing this domain interfered with the wild-type KAP-Cdk2 interaction.

Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2003 Apr, 11(2), 165 - 8
{Detection of 20q(-) chromosome abnormality in myelodysplastic syndrome by interphase fluorescence in situ hybridization}; Shen YM et al.; In order to explore the value of interphase fluorescence in situ hybridization (FISH) in the detection of partial deletion of the long arm of chromosome 20 (20q(-)) in patients with myelodysplastic syndrome (MDS), spectrum Green fluorescein directly labeled yeast artificial chromosome (YAC) clone 912C3 which spans the breakpoint cluster region in band 20q12 was used as probes to perform interphase FISH on the marrow cells from 52 cases of MDS and 5 normal controls . 200 to 300 cells were scored for each case and cases which had cells with a green hybridization signal>7.16% were defined as 20q(-) positive . The results of FISH were compared with those of conventional cytogenetics (CC) assay . The results showed that among 52 cases of MDS, 7 (13.5%) cases were positive by FISH, however, of which, 4 cases were positive and the other 3 cases were negative by CC assay . It is concluded that YAC912C3 and interphase FISH providing a powerful technique in the detection of 20q(-) in MDS is an important complement to CC assay.

Mol Plant Microbe Interact, 2003 Apr, 16(4), 352 - 9
Characterization of a novel barley protein, HCP1, that interacts with the Brome mosaic virus coat protein; Okinaka Y et al.; Brome mosaic virus (BMV) requires the coat protein (CP) not only for encapsidation but also for viral cell-to-cell and long-distance movement in barley plants . This suggests that BMV infection is controlled by interactions of CP with putative host factors as well as with viral components . To identify the host factors that interact with BMV CP, we screened a barley cDNA library containing 2.4 x 10(6) independent clones, using a yeast two-hybrid system . Using full-length and truncated BMV CPs as baits, four candidate cDNA clones were isolated . One of the candidate cDNAs encodes a unique oxidoreductase enzyme, designated HCP1 . HCP1 was found predominantly in the soluble fractions after differential centrifugation of BMV-infected and mock-inoculated barley tissues . A two-hybrid binding assay using a series of truncated BMV CPs demonstrated that a C-terminal portion of CP is essential for its interaction with HCP1 . Interestingly, experiments with CP mutants bearing single amino acid substitutions at the C-terminus revealed that the capacity for mutant CP-HCP1 binding correlates well with the infectivity of the corresponding mutant viruses in barley . These results indicate that CP-HCP1 binding controls BMV infection of barley, interacting directly with CP, probably in the cell cytoplasm.

Dev Genes Evol, 2003 Jun, 213(5-6), 284 - 90 Epub 2003 May 13.
A genomewide survey of developmentally relevant genes in Ciona intestinalis . VIII . Genes for PI3K signaling and cell cycle; Kawashima T et al.; Cell growth and cell divisions are two fundamental biological processes for cells in multi-cellular organisms . The molecules involved in these biological processes are highly conserved within eukaryotes, including plants and unicellular organisms such as yeast . However, some regulatory molecules seem to be innovated during animal evolution . Therefore, to understand how the ubiquitous systems have evolved or have been conserved, we examined genes for the phosphoinositide 3-kinase (PI3K) pathway that is important for cell growth, and genes for cell cycle regulation in the genome of Ciona intestinalis . It was found that the Ciona intestinalis genome contains all the essential constituents of the PI3K pathway . In addition, the class IB PI3K catalytic and regulatory subunits, which had not previously been known in animals other than mammals, were found in the Ciona genome . Similarly, all essential cyclins and CDKs were found in the Ciona genome, while cyclin G and cyclin L were likely to be independently lost in the ascidian lineage, which may be dispensable for the cell cycle . Cyclin F, which was previously known only in vertebrates, was not found in the Ciona genome . Therefore, this gene was probably innovated during the evolution of vertebrates to be involved in vertebrate-specific cell cycle regulation . Since Ciona is regarded as one of the most primitive extant chordates, the present analysis gives us an insight into how these fundamental biological genes are evolved or are conserved during chordate evolution.

Oncogene, 2003 May 8, 22(18), 2823 - 35
SKIP3, a novel Drosophila tribbles ortholog, is overexpressed in human tumors and is regulated by hypoxia; Bowers AJ et al.; Regions of hypoxia are a hallmark of solid tumors . Tumor cells modulate the regulation of specific genes allowing adaptation and survival in the harsh hypoxic environment . We have identified SKIP3, a novel human kinase-like gene, which is overexpressed in multiple human tumors and is regulated by hypoxia . SKIP3 is an ortholog of the Drosophila tribbles, rat NIPK, dog C5FW, and human C8FW genes . Drosophila tribbles is involved in slowing cell-cycle progression during Drosophila development, but little is known regarding the function or tissue distribution of the vertebrate orthologs . We show that the normal tissue expression of SKIP3 is confined to human liver, while multiple primary human lung, colon, and breast tumors express high levels of SKIP3 transcript . Endogenous SKIP3 protein accumulates within 48 h under hypoxic growth conditions in HT-29 and PC-3 cells, with upregulation of the SKIP3 mRNA transcript by 72 h . We identified activating transcription factor 4 (ATF4) as a SKIP3-binding partner using the yeast-two-hybrid assay . Coexpression of SKIP3 and ATF4 showed that SKIP3 is associated with the proteolysis of ATF4, which can be blocked using a proteosome inhibitor . These results indicate that SKIP3 may be an important participant in tumor cell growth.

Oncogene, 2003 May 8, 22(18), 2699 - 709
EAPII interacts with ETS1 and modulates its transcriptional function; Pei H et al.; Ets proteins constitute a family of conserved sequence-specific DNA-binding proteins and function as transcription factors . ETS1 plays important roles in differentiation, lymphoid cell development, invasiveness and angiogenesis . Such diverse roles of ETS1 are likely to be dependent on its associated proteins . A yeast two-hybrid screen was conducted and here we describe a novel ETS1 interacting protein designated as ETS1-associated protein II (EAPII) . EAPII protein interacts with ETS1 and other Ets proteins (ETS2 and FLI1) both in vitro and in vivo . Indirect immunofluorescence demonstrated that EAPII is predominately localized to the nucleus of mammalian cells . EAPII negatively modulates ETS1 transcriptional activity and attenuates synergistic transactivation by ETS1 and AP-1 . Significantly, re-expression of EAPII inhibits the migration of epithelial cancer cells, but does not affect cell viability . Therefore, EAPII is a novel ETS1 modulator that regulates specific aspects of the ETS1 functions.

Biochem J, 2003 Aug 1, 373(Pt 3), 747 - 57
Zinc-fingers and homeoboxes (ZHX) 2, a novel member of the ZHX family, functions as a transcriptional repressor; Kawata H et al.; Zinc-fingers and homeoboxes (ZHX) 1 is a transcription factor that interacts with the activation domain of the A subunit of nuclear factor-Y (NF-YA) . Using a yeast two-hybrid system, a novel ubiquitous transcription factor ZHX2 as a ZHX1-interacting protein was cloned . ZHX2 consists of 837 amino acid residues and contains two zinc-finger motifs and five homeodomains (HDs) as well as ZHX1 . The mRNA is expressed among various tissues . ZHX2 not only forms a heterodimer with ZHX1, but also forms a homodimer . Moreover, ZHX2 interacts with the activation domain of NF-YA . Further analysis revealed that ZHX2 is a transcriptional repressor that is localized in the nuclei . Since ZHX2 shares a number of properties in common with ZHX1, we conclude that all these come under the ZHX family . The minimal functional domains of ZHX2 were then characterized . The dimerization domain with both ZHX1 and ZHX2 is the region containing HD1, the domain that interacts with NF-YA is the HD1 to HD2 region, the repressor domain is the HD1 to a proline-rich region . Lastly, using an immunoprecipitation assay, we showed that ZHX2 intrinsically interacts with NF-YA in HEK-293 cells and that ZHX2 represses the promoter activity of the cdc25C gene stimulated by NF-Y in Drosophila Schneider line 2 cells . Thus the ZHX family of proteins may participate in the expression of a number of NF-Y-regulated genes via a more organized transcription network.

Arch Pathol Lab Med, 2003 Jun, 127(6), 706 - 10
Rapid polymerase chain reaction-based confirmation of cat scratch disease and Bartonella henselae infection; Margolis B et al.; CONTEXT: Cat scratch disease (CSD) commonly occurs secondary to Bartonella henselae infection, and the diagnosis has traditionally been made by microscopic findings, the identification of organisms by cytochemistry, and clinical history . However, cytochemical analysis tends to be very difficult to interpret, and histology alone may be insufficient to establish a definitive diagnosis of CSD . OBJECTIVE: To demonstrate the presence of B henselae in tissue suspected of involvement by CSD, using a novel polymerase chain reaction (PCR) assay . DESIGN: Isolates of B henselae (American Tissue Culture Collection 49793) and Afipia felis (American Tissue Culture Collection 49714) were cultured on blood agar and buffered charcoal yeast extract agar, respectively . DNA was isolated from these organisms and from formalin-fixed, paraffin-embedded tissue sections with involvement by CSD (8 patients) . Negative controls included water, human placental tissue, and lymph node specimens from 6 patients with reactive lymphoid hyperplasia and from 2 patients with granulomatous lymphadenitis . A primer complementary to B henselae citrate synthase gltA gene sequence was designed to perform a seminested PCR amplification . For restriction fragment length polymorphism analysis, PCR products were digested by TaqI restriction enzyme and analyzed by gel electrophoresis . RESULTS: Seminested PCR analysis of the cultured isolates of B henselae, but not of A felis, showed specific amplification . However, nonnested PCR did not provide consistently positive results in tissue sections with CSD . Therefore, we used a seminested PCR, which revealed positivity in all of the cases with clinicopathologic diagnoses of CSD . None of the negative controls showed positivity . Restriction enzyme provided confirmation of the specific PCR amplification of the B henselae sequence . CONCLUSIONS: Since the amplification product has a low molecular size (<200 base pairs), this assay is useful for detection of B henselae in formalin-fixed, paraffin-embedded tissues . The seminested PCR protocol described here can be used for rapid and reliable confirmation of B henselae in samples that are histologically suggestive of CSD.

Biochemistry, 2003 May 20, 42(19), 5736 - 47
The Euplotes La motif protein p43 has properties of a telomerase-specific subunit; Aigner S et al.; Telomerase is a specialized reverse transcriptase synthesizing DNA repeats at telomeres . In addition to the RNA and catalytic protein components, telomerase from the ciliate Euplotes aediculatus contains the subunit p43 . This protein is homologous to the La autoantigen, functioning in maturation of RNA polymerase III transcripts . Here we provide evidence that p43 is primarily associated with the telomerase ribonucleoprotein in vivo . Recombinant p43 binds telomerase RNA with low-nanomolar affinity in vitro, recognizing stem I and adjacent nucleotides or structures in the core of the RNA . Unlike authentic La proteins, p43 does not bind strongly to RNA polymerase III precursor transcripts and does not exhibit a marked binding preference for 3'-terminal oligouridylate residues . In isolated macronuclei, p43 largely colocalizes with telomerase RNA in discrete foci . These findings suggest that p43 is not the Euplotes La protein but instead plays a dedicated role in telomerase assembly and/or function . Thus, p43 joins the telomerase reverse transcriptase and the yeast proteins Est1p and Est3p as the only telomerase-specific proteins identified so far.

Int J Cancer, 2003 Jul 10, 105(5), 644 - 53
EBV-encoded EBNA-5 associates with P14ARF in extranucleolar inclusions and prolongs the survival of P14ARF-expressing cells; Kashuba E et al.; Epstein-Barr virus (EBV) carrying lymphoblastoid cells of normal origin express the full program of all 9 virus-encoded, growth transformation associated proteins . They have an intact p53 pathway as a rule . This raises the question of whether any of the viral proteins impair the pathway functionally . Using a yeast 2-hybrid system, we have shown that EBNA-5 but not the other EBNAs interacts with the p14ARF protein, a regulator of the p53 pathway . The interaction was confirmed in vitro using a GST pull-down assay . Moreover, expression of EBNA-5 increased the survival of p14ARF-transfected cells . EBV infection of resting B cells induced the expression of p14ARF mRNA without increased level of the protein . A fraction of the p14ARF localized to the nucleoli but the bulk of the protein accumulated in nuclear but extranucleolar inclusions . Formation of the extranucleolar inclusions led to complete relocalization of EBNA-5 from nucleoplasm to these structures . The inclusions also contained p53 and HDM2, and were surrounded by PML bodies and proteasomes, which suggests that these inclusions could be targets for proteasome dependent protein degradation .

Cell Motil Cytoskeleton, 2003 Jun, 55(2), 134 - 46
Dynamic association of a tumor amplified kinase, Aurora-A, with the centrosome and mitotic spindle; Stenoien DL et al.; Aurora-A kinase, also known as STK15/BTAK kinase, is a member of a serine/threonine kinase superfamily that includes the prototypic yeast Ipl1 and Drosophila aurora kinases as well as other mammalian and non-mammalian aurora kinases involved in the regulation of centrosomes and chromosome segregation . The Aurora-A gene is amplified and overexpressed in a wide variety of human tumors . Aurora-A is centrosome-associated during interphase, and binds the poles and half-spindle during mitosis; its over-expression has been associated with centrosome amplification and multipolar spindles . GFP-Aurora-A was used to mark centrosomes and spindles, and monitor their movements in living cells . Centrosome pairs labeled with GFP-Aurora-A are motile throughout interphase undergoing oscillations and tumbling motions requiring intact microtubules and ATP . Fluorescence recovery after photobleaching (FRAP) was used to examine the relative molecular mobility of GFP-Aurora-A, and GFP-labeled alpha-tubulin, gamma-tubulin, and NuMA . GFP-Aurora-A rapidly exchanges in and out of the centrosome and mitotic spindle (t(1/2) approximately 3 sec); in contrast, both tubulins are relatively immobile indicative of a structural role . GFP-NuMA mobility was intermediate in both interphase nuclei and at the mitotic spindle (t(1/2) approximately 23-30 sec) . Deletion mapping identifies a central domain of Aurora-A as essential for its centrosomal localization that is augmented by both the amino and the carboxyl terminal ends of the protein . Interestingly, amino or carboxy terminal deletion mutants that maintained centrosomal targeting exhibited significantly slower molecular exchange . Collectively, these studies contrast the relative cellular dynamics of Aurora-A with other cytoskeletal proteins that share its micro-domains, and identify essential regions required for targeting and dynamics .

Microsc Res Tech, 2003 Jun 1, 61(2), 151 - 60
Degradation of normal and proliferated peroxisomes in rat hepatocytes: regulation of peroxisomes quantity in cells; Yokota S; Degradation and turnover of peroxisomes is reviewed . First, we describe the historical aspects of peroxisome degradation research and the two major concepts for breakdown of peroxisomes, i.e., autophagy and autolysis . Next, the comprehensive knowledge on autophagy of peroxisomes in mammalian and yeast cells is reviewed . It has been shown that proliferated peroxisomes are degraded by selective autophagy, and studies using yeast cells have been especially helpful in shedding light on the molecular mechanisms of this process . The degradation of extraperoxisomal urate oxidase crystalloid is noted . Overexpressed wild-type urate oxidase in cultured cells has been shown to be degraded through an unknown proteolytic pathway distinct from the lysosomal system including autophagy or the ubiquitin-proteasome system . Finally, peroxisome autolysis mediated by 15-lipoxygenase (15-LOX) is described . 15-LOX is integrated into the peroxisome membrane causing focal membrane disruptions . The content of the peroxisomes is then exposed to cytosol proteases and seems to be digested quickly . In conclusion, the number of peroxisomes appears to be regulated by two selective pathways, autophagy, including macro- and microautophagy, and 15-LOX-mediated autolysis .

J Biol Chem, 2003 Jul 18, 278(29), 26750 - 6 Epub 2003 May 09.
Protein kinase A-anchoring protein AKAP95 interacts with MCM2, a regulator of DNA replication; Eide T et al.; Protein kinase A (PKA)-anchoring protein AKAP95 is localized to the nucleus in interphase, where it primarily associates with the nuclear matrix . A yeast two-hybrid screen for AKAP95 interaction partners identified the minichromosome maintenance (MCM) 2 protein, a component of the pre-replication complex . AKAP95-MCM2 interaction was mapped to residues 1-195 of AKAP95 and corroborated by glutathione S-transferase precipitation and immunoprecipitation from chromatin . Disruption of AKAP95-MCM2 interaction with an AKAP95-(1-195) peptide within HeLa cell nuclei abolishes initiation of DNA replication in G1 phase and the elongation phase of replication in vitro without affecting global nuclear organization or import . Disruption of the C-terminal zinc finger of AKAP95 reduces efficiency of replication initiation . Disruption of the PKA-binding domain does not impair replication in G1- or S-phase nuclei, whereas a PKA inhibitor affects the initiation but not the elongation phase of replication . Depleting AKAP95 from nuclei partially depletes MCM2 and abolishes replication . Recombinant AKAP95 restores intranuclear MCM2 and replication in a dose-dependent manner . Our results suggest a role of AKAP95 in DNA replication by providing a scaffold for MCM2.

Int J Oncol, 2003 Jun, 22(6), 1357 - 62
NADE (p75NTR-associated cell death executor) suppresses cellular growth in vivo; Tong X et al.; NADE, a p75NTR (low-affinity neurotrophin receptor p75) -associated cell death executor, was initially cloned from a human ovarian granulosa cell cDNA library, as an unknown protein with the name, pHGR74 . It was reported to mediate nerve growth factor-induced apoptosis . We independently isolated human NADE (pHGR74) from breast cancer cell lines . Expression of NADE in various human cancer cell lines, and human and murine tissues was examined . NADE was highly expressed in human endocrine-related organs and embryotic murine tissues . Forced expression of NADE in CHO (Chinese hamster ovary) cells and MDA-MB-231 human breast cancer cells had little effect on the growth of the cells in vitro, while it dramatically suppressed cellular growth in vivo . We used the yeast two-hybrid system to search for NADE binding protein . Dynactin was identified as a candidate . The p75NTR was not found in this assay and did not co-immunoprecipitate with human NADE . Furthermore, the cells stably transfected with NADE did not respond to NGF or TNF . Thus, human and murine NADE appear to have different functions.

Proc Natl Acad Sci U S A, 2003 May 27, 100(11), 6622 - 7 Epub 2003 May 08.
Coincidence of synteny breakpoints with malignancy-related deletions on human chromosome 3; Kost-Alimova M et al.; We have found previously that during tumor growth intact human chromosome 3 transferred into tumor cells regularly looses certain 3p regions, among them the approximately 1.4-Mb common eliminated region 1 (CER1) at 3p21.3 . Fluorescence in situ hybridization analysis of 12 mouse orthologous loci revealed that CER1 splits into two segments in mouse and therefore contains a murine/human conservation breakpoint region (CBR) . Several breaks occurred in tumors within the region surrounding the CBR, and this sequence has features that characterize unstable chromosomal regions: deletions in yeast artificial chromosome clones, late replication, gene and segment duplications, and pseudogene insertions . Sequence analysis of the entire 3p12-22 revealed that other cancer-associated deletions (regions eliminated from monochromosomal hybrids carrying an intact chromosome 3 during tumor growth and homozygous deletions found in human tumors) colocalized nonrandomly with murine/human CBRs and were characterized by an increased number of local gene duplications and murine/human conservation mismatches (single genes that do not match into the conserved chromosomal segment) . The CBR within CER1 contains a simple tandem TATAGA repeat capable of forming a 40-bp-long secondary hairpin-like structure . This repeat is nonrandomly localized within the other tumor-associated deletions and in the vicinity of 3p12-22 CBRs.

Mol Endocrinol, 2003 Aug, 17(8), 1555 - 67 Epub 2003 May 08.
Computer-assisted generation of a protein-interaction database for nuclear receptors; Albert S et al.; With the increasing amount of biological data available, automated methods for information retrieval become necessary . We employed computer-assisted text mining to retrieve all protein-protein interactions for nuclear receptors from MEDLINE in a systematic way . A dictionary of protein names and of terms denoting interactions was generated, and trioccurrences of two protein names and one interaction term in one sentence were retrieved . Abstracts containing at least one such trioccurrence were manually checked by biologists to select the relevant interactions out of the automatically extracted data.In total, 4360 abstracts were retrieved containing data on protein interactions for nuclear receptors . The resulting database contains all reported protein interactions involving nuclear receptors from 1966 to September 2001 . Remarkably, the annual increase in number of reported interactors for nuclear receptors has been following an exponential growth curve in the years 1991 to 2001.Apparent in the data set is the high complexity of protein interactions for nuclear receptors . The number of interactions correlates with the number of published papers for a given receptor, suggesting that the number of reported interactors is a reflection of the intensity of research dedicated to a given receptor . Indeed, comparison of the retrieved data to a systematic yeast two-hybrid-based interaction analysis suggests that most NRs are similar with respect to the number of interacting proteins . The data set obtained serves as a source for information on NR interactions, as well as a reference data set for the improvement of advanced text-mining methods.

J Ethnopharmacol, 2003 Jun, 86(2-3), 229 - 34
Analgesic, antipyretic, anti-inflammatory effects of methanol, chloroform and ether extracts of Vernonia cinerea less leaf; Iwalewa EO et al.; The chloroform, methanolic and ether extracts of Vernonia cinerea (Asteraceae; Less) leaf (100, 200 and 400mg/kg intraperitoneally) were tested in: acetic acid-induced writhing in mice, carrageenin-induced oedema and brewer's yeast-induced pyrexia in rats to assess their analgesic, anti-inflammatory, antipyretic and behavioral activities, respectively . The changes in writhings and behavioural activities in mice, the pyrexia and paw volumes in rats were reduced significantly (P<0.05) compared to the control . There was an increase in pain threshold on the oedematous right hind limb paw of the rats . These results indicate that the extracts could possess analgesic, antipyretic and anti-inflammatory properties . All these effects and the changes in the behavioural activities could be suggested as contributory effects to the use of V . cinerea leaf in the treatment of malaria.

Int J Mol Med, 2003 Jun, 11(6), 705 - 12
Regulation of CREB-mediated transcription by association of CDK4 binding protein p34SEI-1 with CBP; Hirose T et al.; CREB binding protein (CBP) plays a central role in cell differentiation and proliferation, interacting with a large number of nuclear factors . To find novel nuclear factors associating with CBP, we have carried out yeast two-hybrid screening of human chondrocyte cDNA library using the C/H3 region of CBP as a bait and cloned CDK4 binding protein p34SEI-1, the recently found cell cycle regulator . The association of p34SEI-1 with CBP was confirmed in vitro by GST pull-down assay and in vivo by coimmunoprecipitation . Results of the immunofluorescence assay also supported the association of p34SEI-1 and CBP . In reporter assay using CRE promoter, p34SEI-1 strongly suppressed CREB-mediated transcription, and this suppression was overcome by excess amount of CBP, but not by CBPDeltaCH3 . It is suggested that the association of p34SEI-1 and CBP is not only involved in cell cycle regulation by CBP, but also have some effect on other CBP-dependent transcription.

Pediatr Res, 2003 Aug, 54(2), 224 - 9 Epub 2003 May 07.
Abnormal glycosylation of red cell membrane band 3 in the congenital disorder of glycosylation Ig; Zdebska E et al.; A description is provided of the clinical presentation in an infant of the recently described congenital disorder of glycosylation type Ig, and the changes affecting glycosylation of red cell membrane band 3, the anion exchanger . It has been shown that the condition stems from a homozygous mutation within the human ortholog of yeast ALG12 gene, which encodes a dolichol-P-mannose:Man7GlcNAc2-PP-dolichol alpha,1-6 mannosyltransferase of the endoplasmic reticulum . The clinical phenotype included prominent central and peripheral manifestations in the CNS . Although the infant studied had no anemia, band 3 abnormally separated into two fractions upon electrophoresis . The chemical composition of the glycans of both fractions was analyzed in detail . The fraction with low electrophoretic mobility was moderately hypoglycosylated (by 27%) and its mannose content was normal . The fraction with high electrophoretic mobility was deeply carbohydrate deficient (by 64%) and had 1 mol mannose in excess but only three residues of N-acetylglucosamine . Glycophorin A was hypoglycosylated with respect to O-linked glycans . Glycosphingolipids of red cells were normal . We suggest that the incomplete biosynthesis of the N-linked glycan of band 3 was largely caused by the persistence of the 3-linked mannose residue on the 6-mannose arm of the trimannosyl moiety of the glycoprotein . It is remarkable that the changes recorded in band 3 have no clinical consequences . Band 3 alteration might serve as an additional indicator (some serum N-glycoproteins of hepatic origin are also indicative) of the congenital disorder of glycosylation type Ig.

Nucleic Acids Res, 2003 May 15, 31(10), 2614 - 21
The solution structure of an essential stem-loop of human telomerase RNA; Leeper T et al.; The ribonucleoprotein enzyme telomerase maintains chromosome ends in most eukaryotes and is critical for a cell's genetic stability and its proliferative viability . All telomerases contain a catalytic protein component homologous to viral reverse transcriptases (TERT) and an RNA (TR) that provides the template sequence as well as a scaffold for ribonucleoprotein assembly . Vertebrate telomerase RNAs have three essential domains: the template, activation and stability domains . Here we report the NMR structure of an essential RNA element derived from the human telomerase RNA activation domain . The sequence forms a stem-loop structure stabilized by a GU wobble pair formed by two of the five unpaired residues capping a short double helical region . The remaining three loop residues are in a well-defined conformation and form phosphate-base stacking interactions reminiscent of other RNA loop structures . Mutations of these unpaired nucleotides abolish enzymatic activity . The structure rationalizes a number of biochemical observations, and allows us to propose how the loop may function in the telomerase catalytic cycle . The pre-formed structure of the loop exposes the bases of these three essential nucleotides and positions them to interact with other RNA sequences within TR, with the reverse transcriptase or with the newly synthesized telomeric DNA strand . The functional role of this stem-loop appears to be conserved in even distantly related organisms such as yeast and ciliates.

Nucleic Acids Res, 2003 May 15, 31(10), 2601 - 13
The high diversity of snoRNAs in plants: identification and comparative study of 120 snoRNA genes from Oryza sativa; Chen CL et al.; Using a powerful computer-assisted analysis strategy, a large-scale search of small nucleolar RNA (snoRNA) genes in the recently released draft sequence of the rice genome was carried out . This analysis identified 120 different box C/D snoRNA genes with a total of 346 gene variants, which were predicted to guide 135 2'-O-ribose methylation sites in rice rRNAs . Though not exhaustive, this analysis has revealed that rice has the highest number of known box C/D snoRNAs among eukaryotes . Interestingly, although many snoRNA genes are conserved between rice and Arabidopsis, almost half of the identified snoRNA genes are rice specific, which may highlight further the differences in rRNA methylation patterns between monocotyledons and dicotyledons . In addition to 76 singletons, 70 clusters involving 270 snoRNA genes were also found in rice . The large number of the novel snoRNA polycistrons found in the introns of rice protein-coding genes is in contrast to the one-snoRNA-per-intron organization of vertebrates and yeast, and of Arabidopsis in which only a few intronic snoRNA gene clusters were identified . Furthermore, due to a high degree of gene duplication, rice snoRNA genes are clearly redundant and exhibit great sequence variation among isoforms, allowing generation of new snoRNAs for selection . Thus, the large snoRNA gene family in plants can serve as an excellent model for a rapid and functional evolution.

Nucleic Acids Res, 2003 May 15, 31(10), 2495 - 507
RNomics in Drosophila melanogaster: identification of 66 candidates for novel non-messenger RNAs; Yuan G et al.; By generating a specialised cDNA library from four different developmental stages of Drosophila melanogaster, we have identified 66 candidates for small non-messenger RNAs (snmRNAs) and have confirmed their expression by northern blot analysis . Thirteen of them were expressed at certain stages of D.melanogaster development, only . Thirty-five species belong to the class of small nucleolar RNAs (snoRNAs), divided into 15 members from the C/D subclass and 20 members from the H/ACA subclass, which mostly guide 2'-O-methylation and pseudouridylation, respectively, of rRNA and snRNAs . These also include two outstanding C/D snoRNAs, U3 and U14, both functioning as pre-rRNA chaperones . Surprisingly, the sequence of the Drosophila U14 snoRNA reflects a major change of function of this snoRNA in Diptera relative to yeast and vertebrates . Among the 22 snmRNAs lacking known sequence and structure motifs, five were located in intergenic regions, two in introns, five in untranslated regions of mRNAs, eight were derived from open reading frames, and two were transcribed opposite to an intron . Interestingly, detection of two RNA species from this group implies that certain snmRNA species are processed from alternatively spliced pre-mRNAs . Surprisingly, a few snmRNA sequences could not be found on the published D.melanogaster genome, which might suggest that more snmRNA genes (as well as mRNAs) are hidden in unsequenced regions of the genome.

J Biol Chem, 2003 Jul 18, 278(29), 27278 - 86 Epub 2003 May 06.
Mutation of Leu-536 in human estrogen receptor-alpha alters the coupling between ligand binding, transcription activation, and receptor conformation; Zhao C et al.; The estrogen receptor (ER), of which there are two forms, ERalpha and ERbeta, is a ligand-modulated transcription factor important in both normal biology and as a target for agents to prevent and treat breast cancer . Crystallographic studies of the ERalpha ligand-binding domain suggest that Leu-536 may be involved in hydrophobic interactions at the start of a helix, "helix 12," that is crucial in the agonist-stimulated activity of ERalpha, as well as in the ability of antagonists to block the activity of ERalpha . We found that certain mutations of Leu-536 increased the ligand-independent activity of ERalpha although greatly reducing or eliminating the agonist activity of 17beta-estradiol (E2) and 4-hydroxytamoxifen (4OHT), on an estrogen response element-driven and an AP-1-driven reporter . The mutations impaired the interaction of the ER ligand-binding domain with the SRC1 receptor-interacting domain in a mammalian two-hybrid system . When tested in the yeast two-hybrid system, mutation of Leu-536 increased the basal reactivity of ERalpha to probes that recognize the agonist-bound conformation but did not significantly alter its reactivity to these probes in the presence of E2 . Most interestingly, mutation of Leu-536 reduced the interaction of the 4OHT-bound ERalpha and increased the reactivity of the raloxifene- or ICI 182,780-bound ERalpha, with probes that recognize the 4OHT-bound ERalpha conformation in a yeast two-hybrid system . These results show that Leu-536 is critical in coupling the binding of ligand to the modulation of the conformation and activity of ERalpha.

Development, 2003 Jun, 130(12), 2555 - 65
Arabidopsis MSI1 is required for epigenetic maintenance of reproductive development; Hennig L et al.; WD40 repeat proteins similar to yeast MSI1 are conserved in animals and plants, in which they participate in complexes involved in chromatin metabolism . Although MSI1-like proteins are well characterised biochemically, their function in the development of multicellular eukaryotes is not well understood . We constructed Arabidopsis plants in which the AtMSI1 protein level was altered . Strong ectopic expression of AtMSI1 produced no visible altered phenotype, but reduction of AtMSI1 dramatically affected development . The primary shoot apical meristem was unable to develop organs after the transition to flowering . Flowers that developed on floral shoots from axillary meristems experienced a progressive loss of floral morphology, including a reduction in size of the petals and stamens and the development of carpel-like sepals . Ovule development was disrupted in all flowers, resulting in complete female sterility . Molecular analysis of the mutant plants revealed that AtMSI1 is required to maintain the correct temporal and organ-specific expression of homeotic genes, including AGAMOUS and APETALA2 . In contrast, FAS1 and FAS2, which together with AtMSI1 form the chromatin assembly complex CAF-1, are not required for repression of these genes . Therefore, AtMSI1 has specific functions in addition to CAF-1-mediated chromatin assembly . Efficient formation of heterochromatin, but not methylation of centromeric DNA repeats, depends on AtMSI1 presence demonstrating a key role of AtMSI1 in maintenance of chromatin structure.

Growth Horm IGF Res, 2003 Apr-Jun, 13(2-3), 89 - 97
Type Ialpha collagen is an IGFBP-3 binding protein; Liu B et al.; Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) possesses both growth-inhibitory and -potentiating effects on cells, which are independent of IGF action and mediated through specific IGFBP-3 binding proteins/receptors located at the cell membrane, cytosol, or nuclear compartments as well as in the extracellular matrix . We here characterized type Ialpha collagen as one of these IGFBP-3 binding proteins . Human serum was fractionated over an IGFBP-3 affinity column, and bands at 70-100 kDa were eluted as IGFBP-3 ligands . The 100-kDa band was extracted, subjected to N-terminal amino acid sequencing, and identified through database searching as the N-terminal chain of type Ialpha collagen protein . In a separate screening approach, using a yeast two-hybrid system, we cloned the type Ialpha collagen cDNA from a human liver cDNA library as an IGFBP-3 protein partner . Anti-IGFBP-3 antibodies co-immunoprecipitated type Ialpha collagen and IGFBP-3 from the conditioned media of human fibroblasts and vice versa . We demonstrated through ligand dot blot analysis that type Ialpha collagen binds IGFBP-3 . IGFBP-3 mutants, with altered sequence at the nuclear localization sequence, bound type Ialpha collagen poorly . Western immunoblot showed that type Ialpha collagen binds only IGFBP-3 but not IGF-I, suggesting an IGF-I-independent mechanism of this interaction . Physiological effects of IGFBP-3-collagen interactions may include modulation of cell adhesion and migration.

J Sex Marital Ther, 2003, 29 Suppl 1, 45 - 58
Characteristics of women with vulvar pain disorders: responses to a Web-based survey; Gordon AS et al.; This article presents data contributed by 428 highly educated, internet-savvy women who frequented various vulvar pain discussion lists . The age range was in the reproductive years and older and over 90% were Caucasians . No country of origin was given . They had a number of distressing symptoms, including vulvar pain at rest and with contact, burning, itching, redness, and inflammation . Most felt that they had either vulvar vestibulitis, vulvodynia, or both, although they had other vulvar conditions as well . Many felt that yeast infections, stress, antibiotics, infections, and chemicals played a contributing role . There were a number of comorbidities, including irritable bowel syndrome, fibromyalgia, and interstitial cystitis . Sexual abuse was not a major issue . The vulvar pain destroyed or altered then sex lives, lowered their self-esteem, and affected their relationships . Often, they relied upon understanding partners, support groups, and hobbies but not the medical profession for comfort.

Biochem J, 2003 Jul 15, 373(Pt 2), 319 - 26
Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases; Kavanagh KL et al.; The co-ordinates reported have been submitted to the Protein Data Bank under accession number 1MI3 . Xylose reductase (XR; AKR2B5) is an unusual member of aldo-keto reductase superfamily, because it is one of the few able to efficiently utilize both NADPH and NADH as co-substrates in converting xylose into xylitol . In order to better understand the basis for this dual specificity, we have determined the crystal structure of XR from the yeast Candida tenuis in complex with NAD(+) to 1.80 A resolution (where 1 A=0.1 nm) with a crystallographic R -factor of 18.3% . A comparison of the NAD(+)- and the previously determined NADP(+)-bound forms of XR reveals that XR has the ability to change the conformation of two loops . To accommodate both the presence and absence of the 2'-phosphate, the enzyme is able to adopt different conformations for several different side chains on these loops, including Asn(276), which makes alternative hydrogen-bonding interactions with the adenosine ribose . Also critical is the presence of Glu(227) on a short rigid helix, which makes hydrogen bonds to both the 2'- and 3'-hydroxy groups of the adenosine ribose . In addition to changes in hydrogen-bonding of the adenosine, the ribose unmistakably adopts a 3'- endo conformation rather than the 2'- endo conformation seen in the NADP(+)-bound form . These results underscore the importance of tight adenosine binding for efficient use of either NADH or NADPH as a co-substrate in aldo-keto reductases . The dual specificity found in XR is also an important consideration in designing a high-flux xylose metabolic pathway, which may be improved with an enzyme specific for NADH.

Curr Microbiol, 2003 Jun, 46(6), 408 - 12
Neurospora crassa FKS protein binds to the (1,3)beta-glucan synthase substrate, UDP-glucose; Schimoler-O'Rourke R et al.; The essential fungal cell-wall polymer (1,3)beta-glucan is synthesized by the enzyme (1,3)beta-glucan synthase . This enzyme, which is the target of the echinocandin and pneumocandin families of fungicidal antibiotics, is a complex composed of at least two proteins, Rho1p and Fks1p . Homologs of the yeast FKS1 gene have been discovered in numerous fungi, and existing evidence points to, but has not yet proved, Fks1p being the catalytic subunit of (1,3)beta-glucan synthase . We have purified (1,3)beta-glucan synthase from Neurospora crassa approximately 400-fold enrichment and labeled the substrate-binding protein by using a UDP-glucose analog, 5-azido-{beta-(32)P}-UDP-glucose . UDP-glucose-binding proteins were photo-crosslinked to the substrate analog and identified from SDS-PAGE gels by Quadrupole time-of-flight mass spectrometry by sequencing the tryptic peptides . Two plasma membrane proteins were labeled FKS and H(+)-ATPase . These results suggest that FKS appears to be the substrate-binding subunit of (1,3)beta-glucan synthase.

J Biol Chem, 2003 Jul 18, 278(29), 27216 - 23 Epub 2003 May 05.
p116Rip is a novel filamentous actin-binding protein; Mulder J et al.; p116Rip is a ubiquitously expressed protein that was originally identified as a putative binding partner of RhoA in a yeast two-hybrid screen . Overexpression of p116Rip in neuroblastoma cells inhibits RhoA-mediated cell contraction induced by lysophosphatidic acid (LPA); so far, however, the function of p116Rip is unknown . Here we report that p116Rip localizes to filamentous actin (F-actin)-rich structures, including stress fibers and cortical microfilaments, in both serum-deprived and LPA-stimulated cells, with the N terminus (residues 1-382) dictating cytoskeletal localization . In addition, p116Rip is found in the nucleus . Direct interaction or colocalization with RhoA was not detected . We find that p116Rip binds tightly to F-actin (Kd approximately 0.5 microm) via its N-terminal region, while immunoprecipitation assays show that p116Rip is complexed to both F-actin and myosin-II . Purified p116Rip and the F-actin-binding region can bundle F-actin in vitro, as shown by electron microscopy . When overexpressed in NIH3T3 cells, p116Rip disrupts stress fibers and promotes formation of dendrite-like extensions through its N-terminal actin-binding domain; furthermore, overexpressed p116Rip inhibits growth factor-induced lamellipodia formation . Our results indicate that p116Rip is an F-actin-binding protein with in vitro bundling activity and in vivo capability of disassembling the actomyosin-based cytoskeleton.

Int J Hematol, 2003 Apr, 77(3), 259 - 62
Absence of R24C mutation of the CDK4 gene in leukemias and solid tumors; Mori N et al.; A mutation of the p16(INK4a)-binding domain of the cyclin dependent kinase 4 (CDK4) gene, R24C, has been reported in some cases of melanoma . This mutation prevented binding of the CDK4 inhibitor p16(INK48) to CDK4 . To determine the relevance of the mutation, we performed polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis in diverse types of human leukemias and solid tumors . No mobility shifts indicating sequence alterations were observed in 273 tumors and 49 cell lines from diverse kinds of tumors These results suggest that in contrast to melanoma, in many other types of human neoplasms the mutation of the CDK4 gene is very rare . To better understand these findings, we randomly mutagenized the CDK4 gene and used the yeast two-hybrid method to screen for CDK4 mutants that had lost the ability to bind to p16(INK4a) . Sequence analysis and in vitro kinase assays showed that most of the mutations that disrupted interactions with p16(INK4) also knocked out the activity of CDK4 . This result may explain the rareness of CDK4 mutations in human tumors.

J Hum Genet, 2003, 48(4), 164 - 9 Epub 2003 Mar 01.
Isolation and identification of a novel cDNA that encodes human yrdC protein; Chen J et al.; In the course of detecting the interaction protein of RBBP10 by yeast two-hybridization, we isolated a novel cDNA that encodes a putative human protein with yrdC domain . It is named human yrdC protein . Because the cDNA contains an open reading fragment (ORF) without a 5' in- frame stop codon, 5' RACE and 3' RACE were proceeded to produce the full-length cDNA . An 1825 bp cDNA was isolated from human placenta, which encodes a putative protein of 279 amino acids . The protein contains a sua5-yciO-yrdC domain . Blast analysis against the human genome database of Genbank revealed that the gene contains five exons, and assigned the gene to human chromosome 1p34.2 . A transcript about 2.5 kb is ubiquitously expressed in human tissues . The gene is highly conserved during evolution.

Int Microbiol, 2003 Mar, 6(1), 17 - 26 Epub 2003 Mar 13.
Fungus propagules in plastids: the mycosome hypothesis; Atsatt PR; A stress-induced "mycosome" phase of Aureobasidium pullulans consisting of minute reproductive propagules that may revert directly to walled yeast cells is described . Mycosomes detected by light- and electron-microscopy reproduce within senescent plant plastids, and display three developmental pathways: wall-less cells (protoplasts), yeast cells, or membrane-bounded spherules that harbor plastids . Widespread in plant and algal cells, mycosomes are produced by both ascomycete and basidiomycete fungi.

J Biol Chem, 2003 Jul 11, 278(28), 25637 - 43 Epub 2003 May 01.
NEDP1, a highly conserved cysteine protease that deNEDDylates Cullins; Mendoza HM et al.; The ubiquitin-like protein NEDD8 is essential for activity of SCF-like ubiquitin ligase complexes . Here we identify and characterize NEDP1, a human NEDD8-specific protease . NEDP1 is highly conserved throughout evolution and equivalent proteins are present in yeast, plants, insects, and mammals . Bacterially expressed NEDP1 is capable of processing NEDD8 in vitro to expose the diglycine motif required for conjugation and can deconjugate NEDD8 from modified substrates . NEDP1 appears to be specific for NEDD8 as neither ubiquitin nor SUMO bearing COOH-terminal extensions are utilized as substrates . Inhibition studies and mutagenesis indicate that NEDP1 is a cysteine protease with sequence similarities to SUMO-specific proteases and the class of viral proteases typified by the adenovirus protease . In vivo NEDP1 deconjugates NEDD8 from a wide variety of substrates including the cullin component of SCF-like complexes . Thus NEDP1 is likely to play an important role in ubiquitin-mediated proteolysis by controlling the activity of SCF complexes.

J Biol Chem, 2003 Jul 11, 278(28), 25708 - 15 Epub 2003 May 02.
Expression and functional dynamics of the XCAP-D2 condensin subunit in Xenopus laevis oocytes; Watrin E et al.; The 13 S condensin complex plays a crucial role in the condensation and segregation of the two sets of chromosomes during mitosis in vivo as well as in cell-free extracts . This complex, conserved from yeast to human, contains a heterodimer of structural maintenance of chromosome (SMC) family proteins and three additional non-SMC subunits . We have investigated the expression of the non-SMC condensin component XCAP-D2 in Xenopus laevis oocytes . When studied during meiotic maturation, XCAP-D2 starts to accumulate at the time of germinal vesicle breakdown and reaches its maximal amount in metaphase II oocytes . This accumulation is specifically blocked by injection of antisense oligonucleotides . XCAP-D2 antisense-injected oocytes progress normally through meiosis until metaphase II . At this stage, however, chromosomes exhibit architecture defaults, and resolution of sister chromatids is impaired . Surprisingly, in mitotic extracts made from XCAP-D2 knocked-down oocytes, sperm chromatin normally condenses into compacted chromosomes, whereas the amounts of both free and chromosome-bound XCAP-D2 are markedly reduced . This apparent discrepancy is discussed in light of current knowledge on chromosome dynamics.

J Biol Chem, 2003 Jul 11, 278(28), 26265 - 74 Epub 2003 May 02.
Ca2+-dependent phosphorylation of syntaxin-1A by the death-associated protein (DAP) kinase regulates its interaction with Munc18; Tian JH et al.; Syntaxin-1 is a key component of the synaptic vesicle docking/fusion machinery that binds with VAMP/synaptobrevin and SNAP-25 to form the SNARE complex . Modulation of syntaxin binding properties by protein kinases could be critical to control of neurotransmitter release . Using yeast two-hybrid selection with syntaxin-1A as bait, we have isolated a cDNA encoding the C-terminal domain of death-associated protein (DAP) kinase, a calcium/calmodulin-dependent serine/threonine protein kinase . Expression of DAP kinase in adult rat brain is restricted to particular neuronal subpopulations, including the hippocampus and cerebral cortex . Biochemical studies demonstrate that DAP kinase binds to and phosphorylates syntaxin-1 at serine 188 . This phosphorylation event occurs both in vitro and in vivo in a Ca2+-dependent manner . Syntaxin-1A phosphorylation by DAP kinase or its S188D mutant, which mimics a state of complete phosphorylation, significantly decreases syntaxin binding to Munc18-1, a syntaxin-binding protein that regulates SNARE complex formation and is required for synaptic vesicle docking . Our results suggest that syntaxin is a DAP kinase substrate and provide a novel signal transduction pathway by which syntaxin function could be regulated in response to intracellular {Ca2+} and synaptic activity.

Vet Immunol Immunopathol, 2003 May 12, 92(3-4), 113 - 24
Serum IgE and IgG responses to food antigens in normal and atopic dogs, and dogs with gastrointestinal disease; Foster AP et al.; In human food allergy, with or without concurrent atopy, there may be significant increases in serum allergen-specific IgE . Serological methods have been tried but are not currently recommended for diagnosis of suspected food allergy in dogs . The aim of this study was to investigate humoral immune responses to food antigens in dogs . Serum IgG and IgE antibodies specific for food antigens were measured by enzyme linked immunosorbent assay (ELISA) using polyclonal anti-dog IgG and IgE reagents . Antigens tested were beef, chicken, pork, lamb, chicken, turkey, white fish, whole egg, wheat, soybean, barley, rice, maize corn, potato, yeast and cow's milk . Three groups were examined: normal dogs, dogs with atopic dermatitis (AD); and dogs with one of four types of gastrointestinal (GI) disease: small intestinal bacterial overgrowth (SIBO), inflammatory bowel disease (IBD), food-responsive disease, and infectious diarrhoea . Statistically significant differences in food-specific antibodies were not detected between the GI subgroups . There were statistically significant differences in the IgE concentration between the normal dogs, and dogs with atopic or GI disease, for all of the antigens tested . There were statistically significant differences in the average IgG concentrations between the normal dogs, and dogs with atopic or GI disease, for all of the antigens tested, except egg and yeast . The relationship of antigen responses for pooled data was analysed using principle component analysis and cluster plots . Some clustering of variables was apparent for both IgE and IgG . For example, all dogs (normal and diseased) made a similar IgG antibody response to chicken and turkey . Compared with other groups, atopic dogs had more food allergen-specific IgE and this would be consistent with a Th(2) humoral response to food antigens . Dogs with GI disease had more food allergen-specific IgG compared with the other groups . This may reflect increased antigen exposure due to increased mucosal permeability which is a recognised feature of canine intestinal disease.

FEBS Lett, 2003 May 8, 542(1-3), 7 - 11
IQGAP1 as signal integrator: Ca2+, calmodulin, Cdc42 and the cytoskeleton; Briggs MW et al.; A family of proteins known as IQGAPs have been identified in yeast, amebas and mammals . IQGAPs are multidomain molecules that contain several protein-interacting motifs which mediate binding to target proteins . Mammalian IQGAP1 is a component of signaling networks that are integral to maintaining cytoskeletal architecture and cell-cell adhesion . Published data suggest that IQGAP1 is a scaffolding protein that modulates cross-talk among diverse pathways in complex regulatory circuits . These pathways include modulating the actin cytoskeleton, mediating signaling by Rho family GTPases and calmodulin, regulating E-cadherin and beta-catenin function and organizing microtubules.

Chemosphere, 2003 Jul, 52(1), 33 - 42
Estrogenic and thyroid hormone activity of a series of hydroxy-polychlorinated biphenyls; Shiraishi F et al.; A series of novel synthetic monohydroxy polychlorinated biphenyls (OH-PCBs) (5 trichloro-, 5 tetrachloro- and 5 pentachloro-compounds) have been characterized (1H and 13C NMR and high resolution MS) and their estrogenic and thyroid hormone activities assessed using a yeast two-hybrid assay, both with and without possible metabolic activation by rat liver S9 preparation . Moderate estrogenic activity was found for 2,3,4(')-trichlorobiphenyl-4-ol (compound 5) but this was eliminated when exposed to the S9 mix . 2,2('),3('),4,6-Pentachlorobiphenyl-3-ol (13) and 2('),3,3('),6-tetrachlorobiphenyl-4-ol (10) both showed weak estrogenicity in the absence of the S9 mix . The estrogenicity of compound (10) was enhanced 10-fold by exposure to S9 metabolic activation but that of compound (13) remained unchanged . 2('),4,5('),6-Tetrachlorobiphenyl-2-ol (6) showed strong thyroid hormonal activity (5% of that of T4) whereas 3('),4,6-trichlorobiphenyl-3-ol (4), compound (10) and 2,3('),4,5('),6-pentachlorobiphenyl-3-ol (14) showed moderate activity, and 2('),3,3('),5-tetrachlorobiphenyl-2-ol (8) and 3,3('),5,5('),6-pentachlorobiphenyl-2-ol (11) showed weak activity . The activity of (4) was eliminated by S9 metabolic activation whereas those of (6) and (14) were weakened and that of (10) remained unchanged.

Nat Rev Mol Cell Biol, 2003 May, 4(5), 397 - 407
Sphingosine-1-phosphate: an enigmatic signalling lipid; Spiegel S et al.; The evolutionarily conserved actions of the sphingolipid metabolite, sphingosine-1-phosphate (S1P), in yeast, plants and mammals have shown that it has important functions . In higher eukaryotes, S1P is the ligand for a family of five G-protein-coupled receptors . These S1P receptors are differentially expressed, coupled to various G proteins, and regulate angiogenesis, vascular maturation, cardiac development and immunity, and are important for directed cell movement.

Cancer Res, 2003 May 1, 63(9), 2179 - 87
The Septin 9 (MSF) gene is amplified and overexpressed in mouse mammary gland adenocarcinomas and human breast cancer cell lines; Montagna C et al.; The expression of polyomavirus middle T antigen under the control of the mouse mammary tumor virus promoter in transgenic mice results in the induction of aggressive mammary gland adenocarcinomas at an early age . We screened 26 tumors for chromosomal aneuploidies using SKY and CGH . In 70% of the tumor samples we could detect high-level copy number gains, which mapped to chromosome band 11E2, a region orthologous to human 17q25.3 . We then identified a bacterial artificial chromosome clone that labeled double-minute chromosomes found in the tumor metaphases . This bacterial artificial chromosome clone showed sequence homology to a member of the septin gene family . Real-time PCR analysis revealed a consistently increased expression of septin 9 (Sept9), not only in polyomavirus middle T antigen-induced, but in a wide variety of mouse models of breast cancer . Six of 9 human tumor cell lines also revealed elevated expression levels of Sept9 . The family of septin genes is involved in a plethora of cellular processes, including cytokinesis in yeast and vesicle transport, and possesses GTPase activity . We identified down-regulation of Thsp1- and Bax-regulated apoptotic response in those tumors with Sept9 overexpression, an effect that could be reversed by inhibiting Sept9 expression using transfection with small interference RNA . Our results now suggest that signaling via members of the septin family plays a novel and common role in breast tumorigenesis.

FEMS Immunol Med Microbiol, 2003 May 15, 36(1-2), 55 - 61
HUVEC take up opsonized zymosan particles and secrete cytokines IL-6 and IL-8 in vitro; Langeggen H et al.; Uptake of zymosan A particles by human umbilical vein endothelial cells (HUVEC) and its effect on cellular cytokine and oxygen radical production was examined . HUVEC took up more serum-opsonized than -unopsonized zymosan as demonstrated by flow cytometry with fluorescence-labeled particles . The former uptake was inhibited in the presence of anti-C3c antibodies and thus complement-mediated . It probably occurred via CR1 (CD35), although participation of other receptors cannot be ruled out . Scanning electron microscopy indicated that HUVEC with fully internalized zymosan particles were damaged . Prolonged incubation of both serum-opsonized and -unopsonized zymosan particles with HUVEC induced increased secretion of the proinflammatory cytokines IL-6 and IL-8 to the cell culture supernatants, but had no effect on production of oxygen radicals . The results confirm previous reports that EC can internalize yeast and other pathogens and points to complement as a mechanism of uptake, but illustrates that the cells may be damaged in the process . Moreover, EC may participate in the anti-infection defense effort by secreting proinflammatory and chemotactic cytokines in response to the contact with pathogens.

Gene, 2003 Apr 24, 309(1), 11 - 21
Identification and characterization of a novel gene disrupted by a pericentric inversion inv(4)(p13.1q21.1) in a family with cleft lip; Beiraghi S et al.; Cleft lip with or without cleft palate is a common birth defect affecting 1 in every 700 live births . Several genetic loci are believed to be involved in the pathogenesis of syndromic and non-syndromic clefting . We identified a pericentric inversion of chromosome 4, inv(4)(p13q21) that segregates with cleft lip in a two-generation family . By using a combination of fluorescence in situ hybridization, yeast artificial chromosome, bacterial artificial chromosome contig mapping, and database searching we mapped and sequenced the inversion breakpoint region . The pericentric inversion disrupts a gene (ACOD4) on chromosome 4q21 that codes for a novel acyl-CoA desaturase enzyme . The 3.0 kb human ACOD4 cDNA spans approximately 170 kb and is composed of five exons of ACOD4 . The inversion breakpoint is located in the second exon . The 3.0 kb mRNA is expressed at high level in fetal brain; a lower expression level was found in fetal kidney . No expression of ACOD4 was detected in fetal lung or liver or in adult tissues . The five exons code for a protein of 330 amino acids, with a predicted molecular weight of 37.5 kDa . The protein is highly similar to acyl-CoA desaturases from Drosophila melanogaster to Homo sapiens . The catalytically essential histidine clusters and the potential transmembrane domains are well conserved.

Virus Res, 2003 May, 93(1), 41 - 50
Molecular characterization of a dsRNA totivirus infecting the sclerotial parasite Coniothyrium minitans; Cheng J et al.; The complete nucleotide sequence, 4975 bp, of the double-stranded RNA (dsRNA) mycovirus infecting the sclerotial parasite Coniothyrium minitans (CmRV) was determined . Sequence analysis revealed the occurrence of two overlapping open reading frames (ORFs): the 5'-proximal large ORF (ORF1; nucleotide positions 62-2389) encodes a putative coat protein (CP) with a predicted molecular mass of 80344 Da, and the 3'-proximal ORF (ORF2, nucleotide positions 2386-4875) encodes a putative RNA dependent RNA Polymerase (RdRp) with a predicted molecular mass of 82551 Da . The tetranucleotide AUGA at nucleotide positions 2386-2389 includes the predicted start codon of ORF2, which overlaps with the stop codon for ORF1 . Based on genome organization and sequence analysis encoded proteins, the virus infecting C . minitans strain Chy-1, designated C . minitans RNA virus (CmRV), belongs to the family Totiviridae . Pairwise sequence comparisons of the deduced amino acid sequences encoded by CmRV as well as phylogentic analysis indicated that it is more closely related to the totiviruses that infect filamentous fungi than to those infecting protozoa, yeast and smut fungi . The role of CmRV in the abnormal phenotype associated with a variant of C . minitans is discussed.

Biochem Biophys Res Commun, 2003 May 16, 304(4), 740 - 6
Inhibition of endothelial cell proliferation by the recombinant kringle domain of tissue-type plasminogen activator; Kim HK et al.; Tissue-type plasminogen activator (tPA) is a multidomain serine protease that converts the zymogen plasminogen to plasmin . tPA contains two kringle domains which display considerable sequence identity with those of angiostatin, an angiogenesis inhibitor . TK1-2, a recombinant kringle domain composed of t-PA kringles 1 and 2 (Ala(90)-Thr(263)), was produced by both bacterial and yeast expression systems . In vitro, TK1-2 inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor, and epidermal growth factor . It did not inhibit proliferation of non-endothelial cells . TK1-2 also inhibited in vivo angiogenesis in the chick embryo chorioallantoic membrane model . These results suggest that the recombinant kringle domain of t-PA is a selective inhibitor of endothelial cell growth and identifies this molecule as a novel anti-angiogenic agent.

Biochem Biophys Res Commun, 2003 May 16, 304(4), 655 - 60
Ca2+/calmodulin binds and dissociates K-RasB from membrane; Sidhu RS et al.; We have investigated the interaction of calmodulin (CaM) with Ras-p21 and the significance of this association . All Ras-p21 isoforms tested (H-, K-, and N-Ras) were detected in the particulate fraction of human platelets and MCF-7 cells, a human breast cancer cell line . In MCF-7 cells, H- and N-Ras were also detected in the cytosolic fraction . K-RasB from platelet and MCF-7 cell lysates was found to bind CaM in a Ca2+ -dependent but GTPgammaS-independent manner . The yeast two-hybrid analysis demonstrated that K-RasB binds to CaM in vivo . Incubation of isolated membranes from platelet and MCF-7 cells with CaM caused dissociation of only K-RasB from membranes in a Ca2+ -dependent manner . CaM antagonist, W7, inhibited dissociation of K-RasB . Addition of platelet or MCF-7 cytosol alone to isolated platelet membranes did not cause dissociation of K-RasB and only addition of exogenous CaM caused dissociation . The results suggest a potential role for Ca2+/CaM in the regulation of K-RasB function.

Biochem Biophys Res Commun, 2003 May 16, 304(4), 599 - 604
The cargo receptor ERGIC-53 is a target of the unfolded protein response; Nyfeler B et al.; The accumulation of unfolded proteins in the ER triggers a signaling response known as unfolded protein response (UPR) . In yeast the UPR affects several hundred genes that encode ER chaperones and proteins operating at later stages of secretion . In mammalian cells the UPR appears to be more limited to chaperones of the ER and genes assumed to be important after cell recovery from ER stress that are not important for secretion . Here, we report that the mRNA of lectin ERGIC-53, a cargo receptor for the transport of glycoproteins from ER to ERGIC, and of its related protein VIP36 is induced by the known inducers of ER stress, tunicamycin and thapsigargin . In parallel, the rate of synthesis of the ERGIC-53 protein was induced by these agents . The response was due to the UPR since it was also triggered by castanospermine, a specific inducer of UPR, and inhibited by genistein . Thapsigargin-induced upregulation of ERGIC-53 could be fully accounted for by the ATF6 pathway of UPR . The results suggest that in mammalian cells the UPR also affects traffic from and beyond the ER.

Mol Cell Biol, 2003 May, 23(10), 3477 - 86
Human BAG-1 proteins bind to the cellular stress response protein GADD34 and interfere with GADD34 functions; Hung WJ et al.; The cellular stress response protein GADD34 mediates growth arrest and apoptosis in response to DNA damage, negative growth signals, and protein malfolding . GADD34 binds to protein phosphatase PP1 and can attenuate the translational elongation of key transcriptional factors through dephosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) . Recently, we reported the involvement of human SNF5/INI1 (hSNF5/INI1) protein in the functions of GADD34 and showed that hSNF5/INI1 binds GADD34 and stimulates the bound PP1 phosphatase activity . To better understand the regulatory and functional mechanisms of GADD34, we undertook a yeast two-hybrid screen with full-length GADD34 as bait in order to identify additional protein partners of GADD34 . We report here that human cochaperone protein BAG-1 interacts with GADD34 in vitro and in SW480 cells treated with the proteasome inhibitor z-LLL-B to induce apoptosis . Two other proteins, Hsp70/Hsc70 and PP1, associate reversibly with the GADD34-BAG-1 complex, and their dissociation is promoted by ATP . BAG-1 negatively modulates GADD34-bound PP1 activity, and the expression of BAG-1 isoforms can also mask GADD34-mediated inhibition of colony formation and suppression of transcription . Our findings suggest that BAG-1 may function to suppress the GADD34-mediated cellular stress response and support a role for BAG-1 in the survival of cells undergoing stress.

J Cell Sci, 2003 Jun 15, 116(Pt 12), 2453 - 60 Epub 2003 Apr 30.
Drosophila securin destruction involves a D-box and a KEN-box and promotes anaphase in parallel with Cyclin A degradation; Leismann O et al.; Sister chromatid separation during exit from mitosis requires separase . Securin inhibits separase during the cell cycle until metaphase when it is degraded by the anaphase-promoting complex/cyclosome (APC/C) . In Drosophila, sister chromatid separation proceeds even in the presence of stabilized securin with mutations in its D-box, a motif known to mediate recruitment to the APC/C . Alternative pathways might therefore regulate separase and sister chromatid separation apart from proteolysis of the Drosophila securin PIM . Consistent with this proposal and with results from yeast and vertebrates, we show here that the effects of stabilized securin with mutations in the D-box are enhanced in vivo by reduced Polo kinase function or by mitotically stabilized Cyclin A . However, we also show that PIM contains a KEN-box, which is required for mitotic degradation in addition to the D-box, and that sister chromatid separation is completely inhibited by PIM with mutations in both degradation signals.

J Biol Chem, 2003 Jul 11, 278(28), 26111 - 9 Epub 2003 Apr 29.
Characterization of insulin inhibition of transactivation by a C-terminal fragment of the forkhead transcription factor Foxo1 in rat hepatoma cells; Perrot V et al.; The transcription factor Foxo1 controls the expression of genes involved in fundamental cellular processes . In keeping with its important physiological roles, Foxo1 activity is negatively regulated in response to growth factors and cytokines that activate a phosphatidylinositol 3-kinase (PI 3-kinase) protein kinase B (PKB)/Akt pathway . PKB/Akt-mediated phosphorylation of Foxo1 has been shown to result in the inhibition of target gene transcription and to trigger the export of Foxo1 from the nucleus, which is generally believed to explain the subsequent decrease of transcription . In the present study, using a chimeric protein in which a C-terminal fragment of Foxo1 (amino acids 208-652) containing the transactivation domain is fused to the yeast Gal4 DNA binding domain, we present evidence showing that insulin can directly regulate transactivation by Foxo1 in H4IIE rat hepatoma cells . Insulin inhibition of Foxo1-(208-652)-stimulated transactivation is mediated by PI 3-kinase but in contrast to full-length Foxo1, does not require either of the two PKB/Akt phosphorylation sites (Ser253 and Ser316) present in the protein fragment . Using mutational and deletion studies, we identify two potential phosphorylation sites, Ser319 and Ser499, as well as a 15-amino acid region located between residues 350 and 364 that are critical for insulin inhibition of transactivation by Foxo1-(208-652) . We conclude that the transcriptional activity of Foxo1 is regulated at different levels by insulin: transactivation, as well as DNA binding and nuclear exclusion . These different regulatory mechanisms allow the precise control of transcription of Foxo1 target genes by insulin.

J Biol Chem, 2003 Jul 11, 278(28), 26295 - 301 Epub 2003 Apr 30.
GIPC interacts with the beta1-adrenergic receptor and regulates beta1-adrenergic receptor-mediated ERK activation; Hu LA et al.; Beta1-adrenergic receptors, expressed at high levels in the human heart, have a carboxyl-terminal ESKV motif that can directly interact with PDZ domain-containing proteins . Using the beta1-adrenergic receptor carboxyl terminus as bait, we identified the novel beta1-adrenergic receptor-binding partner GIPC in a yeast two-hybrid screen of a human heart cDNA library . Here we demonstrate that the PDZ domain-containing protein, GIPC, co-immunoprecipitates with the beta1-adrenergic receptor in COS-7 cells . Essential for this interaction is the Ser residue of the beta1-adrenergic receptor carboxyl-terminal ESKV motif . Our data also demonstrate that beta1-adrenergic receptor stimulation activates the mitogen-activated protein kinase, ERK1/2 . beta1-adrenergic receptor-mediated ERK1/2 activation was inhibited by pertussis toxin, implicating Gi, and was substantially decreased by the expression of GIPC . Expression of GIPC had no observable effect on beta1-adrenergic receptor sequestration or receptor-mediated cAMP accumulation . This GIPC effect was specific for the beta1-adrenergic receptor and was dependent on an intact PDZ binding motif . These data suggest that GIPC can regulate beta1-adrenergic receptor-stimulated, Gi-mediated, ERK activation while having no effect on receptor internalization or Gs-mediated cAMP signaling.

Ann N Y Acad Sci, 2003 Mar, 983, 151 - 60
Epigenetics and the environment; Sutherland JE et al.; DNA methylation and histone modification promote changes in chromatin structure that may affect gene expression in a heritable manner without directly altering the genome . As such, these phenomena are considered to be epigenetic in nature and are believed to contribute to the normal processes of human development but also to aberrant disease states such as cancer . Epigenetic processes probably contribute mechanistically to toxicant-induced changes in gene expression and cancer . Nickel is a potent human carcinogen that has been shown to alter DNA methylation patterns and affect histone acetylation status . Both of these changes are associated with the proximity of the affected regions to heterochromatin . The two processes probably occur in concert in mammalian cells . However, in yeast cells, DNA methylation is absent, and nickel is capable of regulating gene expression through changes in acetylation of the lysine residues in the N terminal tail of histone H4 . Arsenic is another important environmental carcinogen, and it is methylated during its metabolism . Hence, it has been proposed that arsenic metabolism may deplete intracellular methyl group stores and thereby lead to changes in DNA methylation that may be involved in carcinogenesis . However, the data concerning DNA methylation changes following arsenic exposure are equivocal, leading researchers to propose that DNA hypo- and hypermethylation are both important in the development of arsenic-induced cancers . Heightened awareness by toxicologists of the importance of epigenetics in normal human development and in carcinogenesis should lead to the identification of other toxicants that manifest their effects, at least in part, via epigenetic mechanisms.

Ann N Y Acad Sci, 2003 Mar, 983, 84 - 100
Histone deacetylases: unique players in shaping the epigenetic histone code; Thiagalingam S et al.; The epigenome is defined by DNA methylation patterns and the associated posttranslational modifications of histones . This histone code determines the expression status of individual genes dependent upon their localization on the chromatin . The silencing of gene expression is associated with deacetylated histones, which are often found to be associated with regions of DNA methylation as well as methylation at the lysine 4 residue of histone 3 . In contrast, the activation of gene expression is associated with acetylated histones and methylation at the lysine 9 residue of histone 3 . The histone deactylases play a major role in keeping the balance between the acetylated and deacetylated states of chromatin . Histone deacetylases (HDACs) are divided into three classes: class I HDACs (HDACs 1, 2, 3, and 8) are similar to the yeast RPD3 protein and localize to the nucleus; class II HDACs (HDACs 4, 5, 6, 7, 9, and 10) are homologous to the yeast HDA1 protein and are found in both the nucleus and cytoplasm; and class III HDACs form a structurally distinct class of NAD-dependent enzymes that are similar to the yeast SIR2 proteins . Since inappropriate silencing of critical genes can result in one or both hits of tumor suppressor gene (TSG) inactivation in cancer, theoretically the reactivation of affected TSGs could have an enormous therapeutic value in preventing and treating cancer . Indeed, several HDAC inhibitors are currently being developed and tested for their potency in cancer chemotherapy . Importantly, these agents are also potentially applicable to chemoprevention if their toxicity can be minimized . Despite the toxic side effects and lack of specificity of some of the inhibitors, progress is being made . With the elucidation of the structures, functions and modes of action of HDACs, finding agents that may be targeted to specific HDACs and potentially reactivate expression of only a defined set of affected genes in cancer will be more attainable.

Biochem J, 2003 Aug 1, 373(Pt 3), 775 - 83
Complexes between the nonsense-mediated mRNA decay pathway factor human upf1 (up-frameshift protein 1) and essential nonsense-mediated mRNA decay factors in HeLa cells; Schell T et al.; mRNAs harbouring premature translation-termination codons are usually degraded by the nonsense-mediated mRNA decay (NMD) pathway . Human up-frameshift protein 1 (Hupf1) is an NMD factor that is conserved between yeast and mammals . To isolate cellular complexes that are formed with Hupf1 and to explore the role of cellular proteins in NMD, we generated a HeLa cell line that stably expresses Hupf1 bearing a double-affinity tag (termed Hupf1-2tag) . Hupf1-2tag is localized in the cytoplasm similar to the endogenous Hupf1 protein, and the Hupf1-2tag cell line is fully NMD-competent . Using affinity chromatography, Hupf1-2tag-associated proteins were isolated . MS and immunoblotting identified the NMD factors Hupf2 and Hupf3a/b as interaction partners of Hupf1 . Size-exclusion chromatography indicates that the NMD factors Hupf1, Hupf2 and the large isoform of Hupf3a might exist in a stable, high-molecular-mass complex of approx . 1.3 MDa . Interestingly, the poly(A)-binding protein was also identified by MS to be associated specifically with Hupf1-2tag . In contrast with the interaction with Hupf2 and Hupf3a/b, the association of poly(A)-binding protein with Hupf1 is highly sensitive to treatment of the isolated complexes with RNase . Components of the exon-exon junction complex or the translational eukaryotic release factor (eRF) 3 were not identified in complexes associated with Hupf1-2tag . We discuss these findings in the context of current models of NMD.

Genetica, 2003 Mar, 117(2-3), 149 - 58
SU(VAR)3-9 is a conserved key function in heterochromatic gene silencing; Schotta G et al.; This review summarizes genetic, molecular and biochemical studies of the SU(VAR)3-9 protein and the evidence for its key role in heterochromatin formation and heterochromatic gene silencing . The Su(var)3-9 locus was first identified as a dominant modifier of position-effect variegation (PEV) in Drosophila melanogaster . Together with Su(var)2-5 and Su(var)3-7, Su(var)3-9 belongs to the group of haplo-suppressor loci which show a triplo-dependent enhancer effect . All three genes encode heterochromatin-associated proteins . Su(var)3-9 is epistatic to the PEV modifier effects of Su(var)2-5 and Su(var)3-7, and it also dominates the effect of the Y chromosome on PEV . These genetic data support a central role of the SU(VAR)3-9 protein in heterochromatic gene silencing, one that is correlated with its activity as a histone H3-K9 methyltransferase (HMTase) . In fact, SU(VAR)3-9 is the main chromocenter-specific HMTase of Drosophila . SU(VAR)3-9 and HP1, the product of Su(var)2-5, are main constituents of heterochromatin protein complexes and the interaction between these two proteins is interdependent . Functional analysis in fission yeast, Drosophila and mammals demonstrate that SU(VAR)3-9-dependent gene silencing processes are conserved in these organisms . This is also demonstrated by the rescue of Drosophila Su(var)3-9 mutant phenotypes with human SUV39H1 transgenes.

Biosci Biotechnol Biochem, 2003 Mar, 67(3), 556 - 62
Characterization of rice functional monosaccharide transporter, OsMST5; Ngampanya B et al.; cDNA of a monosaccharide transporter in rice, OsMST5 (Oryza sativa monosaccharide transporter 5) was cloned and its sugar transport activity was characterized by heterologous expression analysis . The amino acid sequence and topology were similar to the sequences and topology of other plant monosaccharide transporters . Yeast cells co-expressed with OsMST5 cDNA transported some monosaccharide substrates . The transport rate increased when ethanol as an electron donor was added, so the transporter was an energy-dependent active one . Most of the OsMST5 was expressed in panicles before pollination, indicating that it is associated with pollen development in rice.

Mol Biol (Mosk), 2003 Mar-Apr, 37(2), 255 - 65
{Parallel-stranded DNA with natural base sequences}; Shchelkina AK et al.; Noncanonical parallel-stranded DNA double helices (ps-DNA) comprising natural nucleotide sequences are usually second in stability to antiparallel-stranded (aps) canonical DNA structures, which ensures reliable cell functioning . However, recent data indicate a possible role of ps-DNA in DNA loops or in trinucleotide repeats connected with neurodegenerative diseases . The review surveys recent studies on the effect of nucleotide sequence on preference of one or other type of DNA duplex . (1) Ps-DNA with mixed AT/GC composition was found to have conformational and thermodynamic properties drastically different from those of Watson-Crick double helix . Its stability depends strongly on the specific sequence in a manner peculiar to the ps double helix, because of the energy disadvantage of the AT/GC contacts . The AT/GC boundary facilitated flipping of A and T out of the ps double helix . Proton acceptor groups of bases are exposed into the both grooves of the ps-DNA and are accessible to solvent and ligands, including proteins . (2) DNA regions containing natural minor bases isoguanine and isomethylcytosine were shown to form ps-DNA with transAT-, trans isoGC, and trans iso5meCG pairs exceeding in stability a related aps duplex . (3) Nucleotide sequence dG(GT)4G from yeast telomeres and microsatellites was demonstrated to form novel ps-DNA with GG and TT base pairing . Unlike d(GT)n and d(GnTm) sequences able to form quadruplexes, the dG(GT)4G sequence formed no alternative double- or multistranded structures in a wide range of experimental conditions, thus suggesting that the nucleotide context governs the observed structural polymorphism of the d(GT)n sequence . The possible biological role of ps-DNA and the prospects of its study are discussed.

J Anim Sci, 2003 Apr, 81(4), 856 - 64
Effects of supplementary selenium source on the performance and blood measurements in beef cows and their calves; Gunter SA et al.; On December 2, 1999, 120 pregnant cows were weighed, their body condition scored, and then sorted into six groups of 20 stratified by BCS, BW, breed, and age . Groups were assigned randomly to six, 5.1-ha dormant common bermudagrass (Cynodon dactylon {L.} Pers.) pastures for 2 yr to determine the effects of supplemental Se and its source on performance and blood measurements . During the winter, each group of cows had ad libitum access to bermudagrass/dallisgrass (Paspalum dilatatum Poir.) hay plus they were allowed limited access (1 to 4 d/wk) to a 2.4-ha winter-annual paddock planted in half the pasture . Treatments were assigned randomly to pastures (two pastures per treatment), and cows had ad libitum access to one of three free-choice minerals: 1) no supplemental Se, 2) 26 mg of supplemental Se from sodium selenite/kg, and 3) 26 mg of supplemental Se from seleno-yeast/kg (designed intake = 113 g/cow daily) . Data were analyzed using a mixed model; year was the random effect and treatment was the fixed effect . Selenium supplementation or its source had no effect (P > or = 0.19) on cow BW, BCS, conception rate, postpartum interval, or hay DMI . Birth date, birth weight, BW, total BW gain, mortality, and ADG of calves were not affected (P > 0.20) by Se or its source . Whole blood Se concentrations and glutathione peroxidase (GSH-Px) activity at the beginning of the trial did not differ (P > or = 0.17) between cows receiving no Se and cows supplemented with Se or between Se sources . At the beginning of the calving and breeding seasons, cows supplemented with Se had greater (P < 0.01) whole blood Se concentrations and GSH-Px activities than cows receiving no supplemental Se; cows fed selenoyeast had greater (P < or = 0.05) whole blood Se concentrations than cows fed sodium selenite, but GSH-Px did not differ (P > or = 0.60) between the two sources . At birth and on May 24 (near peak lactation), calves from cows supplemented with Se had greater (P < or = 0.06) whole blood Se concentrations than calves from cows fed no Se . At birth, calves from cows fed seleno-yeast had greater (P < or = 0.05) whole blood Se concentrations and GSH-Px activities than calves from cows fed sodium selenite . Although no differences were noted in cow and calf performance, significant increases were noted in whole blood Se concentrations and GSH-Px activities in calves at birth as a result of feeding of seleno-yeast compared to no Se or sodium selenite.

Genetika, 2003 Mar, 39(3), 357 - 61
{A method for preparation of synaptonemal complexes of meiotic chromosomes from basidial protoplasts of the common mushroom Agaricus bisporus (Lange) Imbach.}; Mazheika IS et al.; For the first time, preparations of synaptonemal complexes (SCs) were made from meiotic chromosomes of white button mushroom (Agaricus bisporus) basidia . It is the first experience of obtaining SC preparations of filamentous fungi from isolated meiosporangium protoplasts . Previously, only yeast SC preparations were obtained following this approach . The method includes four major stages: isolation of basidium protoplasts by treatment of basidia with lytic enzymes, spreading of protoplast nuclei on a filmy support by osmotic shock, staining the preparations with silver nitrate, and examination under light and electron microscopes . The structures of spread premeiotic nuclei, axial elements of chromosomes, SCs, chromatin, and nucleoli were studied at the leptotene-diplotene stage of meiotic prophase I.

Planta, 2003 May, 217(1), 158 - 67 Epub 2003 Feb 12.
Overexpression of the sucrose transporter SoSUT1 in potato results in alterations in leaf carbon partitioning and in tuber metabolism but has little impact on tuber morphology; Leggewie G et al.; The aim of this work was to examine the consequences of the heterologous expression of a spinach ( Spinacia oleracea L.) sucrose transporter ( SoSUT1) in potato ( Solanum tuberosum L.) . Many studies have indicated that reduction of the expression of this class of sucrose transporter has deleterious effects on plant growth and development; however, until now the possibility of improving plant performance by enhancing the expression of this sucrose transporter has not been reported . With this intention we constructed a chimeric construct in which SoSUT1 was cloned in-frame with the myc epitope . We confirmed that this construct, SoSUT1m, was able to mediate sucrose transport by expression in the yeast strain SUSY7 . SoSUT1m was expressed in wild-type potato in the sense orientation under the control of the cauliflower mosaic virus 35S promoter to evaluate the effect of an increased constitutive expression of a class-I sucrose transporter . We confirmed that these plants displayed expression of SoSUT1 at both the transcript and protein level and that microsomal fragments isolated from selected lines had an increased sucrose uptake capacity . Analysis of metabolism of these lines indicated that the leaves were characterised by a reduced sucrose level yet exhibited little change in photosynthetic rate . Furthermore, despite the observed increase in sugar (and reduction in amino acid) levels within the tubers, there was little change in either starch content or tuber yield in the transformants . In summary, the genetic manipulation described in this paper resulted in a shift in carbon partitioning in both leaves and tubers and an increased sucrose uptake rate in plasma-membrane vesicles isolated from these lines, but had little impact on tuber metabolism or morphology.

J Mol Med, 2003 May, 81(5), 297 - 304 Epub 2003 Apr 30.
Cloning and characterization of a novel cardiac-specific kinase that interacts specifically with cardiac troponin I; Zhao Y et al.; Cardiac-restricted genes play important roles in cardiovascular system . In an effort to identify such novel genes we identified a novel cardiac-specific kinase gene TNNI3K localized on 1p31.1 based on bioinformatics analyses . Sequence analysis suggested that TNNI3K is a distant family member of integrin-linked kinase . Northern blot and 76-tissue array analyses showed that TNNI3K is highly expressed in heart, but is undetectable in other tissues . Immunohistochemical analysis predominantly localized TNNI3K in the nucleus of cardiac myocytes . In vitro kinase assay showed that TNNI3K is a functional kinase . The yeast two-hybrid system showed that TNNI3K could directly interact with cardiac troponin I, results that were further confirmed by coimmunoprecipitation in vivo . Our data suggest that TNNI3K is a cardiac-specific kinase and play important roles in cardiac system.

Plant Cell Physiol, 2003 Apr, 44(4), 404 - 11
Up-regulation of soyasaponin biosynthesis by methyl jasmonate in cultured cells of Glycyrrhiza glabra; Hayashi H et al.; Exogenously applied methyl jasmonate (MeJA) stimulated soyasaponin biosynthesis in cultured cells of Glycyrrhiza glabra (common licorice) . mRNA level and enzyme activity of beta-amyrin synthase (bAS), an oxidosqualene cyclase (OSC) situated at the branching point for oleanane-type triterpene saponin biosynthesis, were up-regulated by MeJA, whereas those of cycloartenol synthase, an OSC involved in sterol biosynthesis, were relatively constant . Two mRNAs of squalene synthase (SQS), an enzyme common to both triterpene and sterol biosyntheses, were also up-regulated by MeJA . In addition, enzyme activity of UDP-glucuronic acid: soyasapogenol B glucuronosyltransferase, an enzyme situated at a later step of soyasaponin biosynthesis, was also up-regulated by MeJA . Accumulations of bAS and two SQS mRNAs were not transient but lasted for 7 d after exposure to MeJA, resulting in the high-level accumulation (more than 2% of dry weight cells) of soyasaponins in cultured licorice cells . In contrast, bAS and SQS mRNAs were coordinately down-regulated by yeast extract, and mRNA accumulation of polyketide reductase, an enzyme involved in 5-deoxyflavonoid biosynthesis in cultured licorice cells, was induced transiently by yeast extract and MeJA, respectively.

J Virol, 2003 May, 77(10), 5948 - 63
Reovirus sigma NS and mu NS proteins form cytoplasmic inclusion structures in the absence of viral infection; Becker MM et al.; Reovirus replication occurs in the cytoplasm of infected cells and culminates in the formation of crystalline arrays of progeny virions within viral inclusions . Two viral nonstructural proteins, sigma NS and micro NS, and structural protein sigma 3 form protein-RNA complexes early in reovirus infection . To better understand the minimal requirements of viral inclusion formation, we expressed sigma NS, mu NS, and sigma 3 alone and in combination in the absence of viral infection . In contrast to its concentration in inclusion structures during reovirus replication, sigma NS expressed in cells in the absence of infection is distributed diffusely throughout the cytoplasm and does not form structures that resemble viral inclusions . Expressed sigma NS is functional as it complements the defect in temperature-sensitive, sigma NS-mutant virus tsE320 . In both transfected and infected cells, mu NS is found in punctate cytoplasmic structures and sigma 3 is distributed diffusely in the cytoplasm and the nucleus . The subcellular localization of mu NS and sigma 3 is not altered when the proteins are expressed together or with sigma NS . However, when expressed with micro NS, sigma NS colocalizes with mu NS to punctate structures similar in morphology to inclusion structures observed early in viral replication . During reovirus infection, both sigma NS and mu NS are detectable 4 h after adsorption and colocalize to punctate structures throughout the viral life cycle . In concordance with these results, sigma NS interacts with mu NS in a yeast two-hybrid assay and by coimmunoprecipitation analysis . These data suggest that sigma NS and mu NS are the minimal viral components required to form inclusions, which then recruit other reovirus proteins and RNA to initiate viral genome replication.

J Biol Chem, 2003 Jun 27, 278(26), 23217 - 20 Epub 2003 Apr 28.
Interaction of activator of G-protein signaling 3 (AGS3) with LKB1, a serine/threonine kinase involved in cell polarity and cell cycle progression: phosphorylation of the G-protein regulatory (GPR) motif as a regulatory mechanism for the interaction of GPR motifs with Gi alpha; Blumer JB et al.; Activator of G-protein signaling 3 (AGS3) has a modular domain structure consisting of seven tetratricopeptide repeats (TPRs) and four G-protein regulatory (GPR) motifs . Each GPR motif binds to the alpha subunit of Gi/Go (Gialpha > Goalpha) stabilizing the GDP-bound conformation of Galpha and apparently competing with Gbetagamma for GalphaGDP binding . As an initial approach to identify regulatory mechanisms for AGS3-G-protein interactions, a yeast two-hybrid screen was initiated using the TPR and linker region of AGS3 as bait . This screen identified the serine/threonine kinase LKB1, which is involved in the regulation of cell cycle progression and polarity . Protein interaction assays in mammalian systems using transfected cells or brain lysate indicated the regulated formation of a protein complex consisting of LKB1, AGS3, and G-proteins . The interaction between AGS3 and LKB1 was also observed with orthologous proteins in Drosophila where both proteins are involved in cell polarity . LKB1 immunoprecipitates from COS7 cells transfected with LKB1 phosphorylated the GPR domains of AGS3 and the related protein LGN but not the AGS3-TPR domain . GPR domain phosphorylation was completely blocked by a consensus GPR motif peptide, and placement of a phosphate moiety within a consensus GPR motif reduced the ability of the peptide to interact with G-proteins . These data suggest that phosphorylation of GPR domains may be a general mechanism regulating the interaction of GPR-containing proteins with G-proteins . Such a mechanism may be of particular note in regard to localized signal processing in the plasma membrane involving G-protein subunits and/or intracellular functions regulated by heterotrimeric G-proteins that occur independently of a typical G-protein-coupled receptor.

J Biol Chem, 2003 Jul 11, 278(28), 25526 - 33 Epub 2003 Apr 28.
Rh-RhAG/ankyrin-R, a new interaction site between the membrane bilayer and the red cell skeleton, is impaired by Rh(null)-associated mutation; Nicolas V et al.; Several studies suggest that the Rh complex represents a major interaction site between the membrane lipid bilayer and the red cell skeleton, but little is known about the molecular basis of this interaction . We report here that ankyrin-R is capable of interacting directly with the C-terminal cytoplasmic domain of Rh and RhAG polypeptides . We first show that the primary defect of ankyrin-R in normoblastosis (nb/nb) spherocytosis mutant mice is associated with a sharp reduction of RhAG and Rh polypeptides . Secondly, our flow cytometric analysis of the Triton X-100 extractability of recombinant fusion proteins expressed in erythroleukemic cell lines suggests that the C-terminal cytoplasmic domains of Rh and RhAG are sufficient to mediate interaction with the erythroid membrane skeleton . Using the yeast two-hybrid system, we demonstrate a direct interaction between the cytoplasmic tails of Rh and RhAG and the second repeat domain (D2) of ankyrin-R . This finding is supported by the demonstration that the substitution of Asp-399 in the cytoplasmic tail of RhAG, a mutation associated with the deficiency of the Rh complex in one Rhnull patient, totally impaired interaction with domain D2 of ankyrin-R . These results identify the Rh/RhAG-ankyrin complex as a new interaction site between the red cell membrane and the spectrin-based skeleton, the disruption of which might result in the stomato-spherocytosis typical of Rhnull red cells.

Biophys J, 2003 May, 84(5), 3155 - 67
Cooperative regulation of myosin-actin interactions by a continuous flexible chain I: actin-tropomyosin systems; Smith DA et al.; We present a model for cooperative myosin binding to the regulated actin filament, where tropomyosins are treated as a weakly-confined continuous flexible chain covering myosin binding sites . Thermal fluctuations in chain orientation are initially required for myosin binding, leaving kinked regions under which subsequent myosins may bind without further distortion of the chain . Statistical mechanics predicts the fraction of sites with bound myosin-S1 as a function of their affinities . Published S1 binding curves to regulated filaments with different tropomyosin isoforms are fitted by varying the binding constant, chain persistence length nu (in actin monomers), and chain kink energy A from a single bound S1 . With skeletal tropomyosin, we find an S1 actin-binding constant of 2.2 x 10(7) M(-1), A = 1.6 k(B)T and nu = 2.7 . Similar persistence lengths are found with yeast tropomyosin . Larger values are found for tropomyosin-troponin in the presence of calcium (nu = 3.7) and tropomyosins from smooth muscle and fibroblasts (nu = 4.5) . The relationship of these results to structural information and the rigid-unit model of McKillop and Geeves is discussed.

Biophys J, 2003 May, 84(5), 2981 - 9
Comparison of the TIM and TOM channel activities of the mitochondrial protein import complexes; Muro C et al.; Water-filled channels are central to the process of translocating proteins since they provide aqueous pathways through the hydrophobic environment of membranes . The Tom and Tim complexes translocate precursors across the mitochondrial outer and inner membranes, respectively, and contain channels referred to as TOM and TIM (previously called PSC and MCC) . In this study, little differences were revealed from a direct comparison of the single channel properties of the TOM and TIM channels of yeast mitochondria . As they perform similar functions in translocating proteins across membranes, it is not surprising that both channels are high conductance, voltage-dependent channels that are slightly cation selective . Reconstituted TIM and TOM channel activities are not modified by deletion of the outer membrane channel VDAC, but are similarly affected by signal sequence peptides.

Acta Dermatovenerol Croat, 2003, 11(1), 10 - 6
Identification of Malassezia species isolated from scalp skin of patients with psoriasis and healthy subjects; Prohic A; The role of Malassezia species in psoriasis is still undetermined, but several reports have associated these lipophilic yeasts with the development of skin lesions in psoriasis . The aim of our study was to analyze the prevalence of Malassezia species in the scalp lesions of patients with psoriasis and assess the distribution of the species according to patient sex, age, and duration of the disease . Forty psoriatic patients with scalp involvement and the same number of clinically healthy individuals were included in the study . The samples were obtained by scraping the skin surface of the scalp of all subjects and then incubated on modified Dixon agar . The yeasts isolated were identified by their morphological and physiological properties according to Guillot et al method . M.globosa in its yeast phase was a predominant species (55%), followed by M.slooffiae (18%) and M.restricta (10%), the latter being the most common species isolated from healthy scalp skin . We found significant difference in the distribution of Malassezia species between psoriatic and healthy scalp skin and in the distribution of Malassezia species according to the severity of the scalp involvement.

Zhonghua Gan Zang Bing Za Zhi, 2003 Apr, 11(4), 203 - 5
{Long-term efficacy of infant hepatitis B immunization program}; Gong J et al.; OBJECTIVE: To evaluate the long-term efficacy of infant hepatitis B (HB) immunization program on preventing hepatitis B virus (HBV) infection, and to assess its impact on the incidence of HB in children . METHODS: Since 1986, the universal HB vaccination for newborn babies with standard, pediatric dose had been launched without serologic prescreening of pregnant women for HBsAg, in a high endemic county of Long-An . A hepatitis surveillance system was set up to evaluate the possible impact on the incidence of hepatitis B . To serologically evaluate the effectiveness of the program, a stratified random sampling of 1000 children in 1987 birth cohorts, who received plasma-derived HB vaccine, was recruited for long-term follow up at the age of 1 to 13 years . A cross-sectional seroepidemiological survey was conducted in the county in 1985, before the program, and in 2001, for 1551 children born in 1996-2000 who were administered yeast recombinant HB vaccine . RESULTS: During the 1 to 13 years after the program, the rates of HBsAg-positive were 0.7% to 2.9% with an average of 1.7% and the protective rates were 83.5% to 96.6% . HBV infection rates were 1.1% tp 5.1% with an average of 2.4% and the protective rates were 93.5% to 98.4% . For the population aged 1 to 4 years who were immunized with recombinant HB vaccine, HBsAg positive rates were 1.8% to 2.4% with an average of 2.0% and the protective rates were 78.4 to 85.2% . 14 years after the program, the cumulative incidence of acute hepatitis B in the children aged 1 to 14 years fell to 1.5 cases per 100,000 children, down 91.8% as compared with that in 1985 to 1987 . However, the cumulative incidence of 14.4 cases per 100,000 population in unvaccinated children was not significantly different from that in the history controls . Acute hepatitis B children had not been reported, showing that the vaccination program was 100% protective in children . CONCLUSION: The universal infant HB vaccination program in a hyperendemic area has proved to be effective in controlling HBV infection and decreasing the incidence of acute hepatitis B in children . Booster dose is unnecessary in 13 years after the immunization . The protective efficacy of yeast recombinant HB vaccine is similar to that of plasma-derived HB vaccine.

Rev Iberoam Micol, 2002 Mar, 19(1), 40 - 43
{Diminution of antifungal activity of fluconazole associated with ibuprofen and piroxicam in experimental histoplasmosis of hamsters (Mesocricetus auratus)}; Finquelievich JL et al.; The aim of this study was to determine if tha association of non-steroid antiinflammatory drugs (piroxicam and ibuprofen) with fluconazole, affects the antifungal activity of the azole compound, in an experimental model histoplasmosis in hamsters (Mesocricetus auratus) . Sixty hamsters were intracardially inoculated with 4x10(6) yeasts of Histoplasma capsulatum var . capsulatum . Treatments began one week after the challenge and continued for three weeks . The hamsters were divided in six groups of ten animals each and received the following treatment: 1- fluconazole 8 mg/kg/day; 2- ibuprofen 20 mg/kg/day; 3- piroxicam 20 mg/kg/day; 4- fluconazole+ibuprofen; 5- fluconazole+piroxicam and 6- only received the solvent of these drugs . One week after ending the treatment, all the animals were sacrified and the evaluation of the treatments was based on the results of blood cultures, on the determination of colony forming units per gram of spleen, and the histopathologic studies of the same organ . The animals treated with fluconazole plus ibuprofen or piroxicam showed more colony colony forming units per gram (3.9x10(7) and 3.3x10(7)) when compared with the animals treated with fluconazole alone (0.9x10(7)) . The histopathologic results of the hamsters that received fluconazole showed well-organized granulomas with few yeast-like elements inside the macrophages . In contrast, those which received fluconazole associated with antiinflammatory drugs presented lax granulomas containing numerous yeast-like elements . These findings let us to conclude that non-steroids antiinflammatory drugs diminish the antifungal efficacy of fluconazole in this animal model.

Science, 2003 Apr 25, 300(5619), 647 - 50
Requirement of Cks2 for the first metaphase/anaphase transition of mammalian meiosis; Spruck CH et al.; We generated mice lacking Cks2, one of two mammalian homologs of the yeast Cdk1-binding proteins, Suc1 and Cks1, and found them to be viable but sterile in both sexes . Sterility is due to failure of both male and female germ cells to progress past the first meiotic metaphase . The chromosomal events up through the end of prophase I are normal in both CKS2-/- males and females, suggesting that the phenotype is due directly to failure to enter anaphase and not a consequence of a checkpoint-mediated metaphase I arrest.

Wei Sheng Yan Jiu, 1999 May 30, 28(3), 158 - 61
{Priority of selenium incorporation into selenoproteins during selenium depletion in rats}; Piao J et al.; Male weanling Wistar rats were fed with either a basal selenium deficient diet (a Torula yeast based semisynthetic diet, containing Se 0.01 mg/kg) or a selenium sufficient diet supplemented with Se as Na2SeO3 (containing Se 0.5 mg/kg) . Rats were killed after different weeks(0,1,2,4,8,12,15,17,19,20 and 24 respectively) . Their organs were taken to observe the kinetic change of selenium concentration, the activities of intracellular glutathione peroxidase (cGPX), extracellular glutathione peroxidase (eGPX), and phospholipid hydroperoxide glutathione peroxidase (PHGPX) in different organs . The results showed that selenium levels and the activities of selenoenzyme in testis and pituitary were more resistant to selenium deficiency than other organs . During selenium deficiency, the utilization of selenium by PHGPX and deiodinase was prior to eGPX and cGPX, which suggested that the function of PHGPX and deiodinase were more important than that of eGPX and cGPX.

Wei Sheng Yan Jiu, 1999 May 30, 28(3), 155 - 8
{Selenoproteins in rats with chronic selenium intoxication}; Zhang Z et al.; Weaning male Wistar rats were fed with a Torula-yeast based semisynthetic diet supplemented with Na2SeO3 to provide selenium (Se) 0.2 or 0.5 mg/kg (adequate Se or high Sediet) respectively for 20 weeks . By the end of experiment, rats were sacrificed and various tissue of rats were collected to determine the activities of Se-containing enzymes and the mRNAs level of selenoprotein P and selenoprotein W and Se concentration . Livers were examined for pathological changes . It was found that the gain of body weight of the high Se group was significantly lower than that of adequate Se group . Much more Se was accumulated in the tissue of high Se group . The activities of eGPX in plasma, cGPX in kidney, heart and testis, ID I in liver, kidney and thyroid and PHGPX in heart and testis in the high Se group were significantly lower than those in the adequate Se group . However, no specific pathological changes have been found in the liver of both groups . The results suggested that these enzymatic changes could be used as early biochemical parameters for chronic selenium intoxication.

Arch Environ Contam Toxicol, 2003 May, 44(4), 502 - 9
Immune function of cryopreserved avian peripheral white blood cells: potential biomarkers of contaminant effects in wild birds; Finkelstein M et al.; Contaminants can cause detrimental effects in wild birds . However, these effects are difficult to measure in all but the most severe cases . Immune function is a sensitive and meaningful biological marker of contaminant-induced effects in captive birds but has more limitations in wild birds due in part to the lack of a proven blood preservation method . We developed methods to assess ex vivo immune function in wild birds using cryopreserved peripheral white blood cells (WBCs) . We assessed the effects of cryopreservation on WBC viability and functionality in two immunoassays (concavalin A-induced T lymphocyte proliferation and macrophage phagocytosis) in domestic chickens (Gallus spp.: white Wyandottes and Dominiques) and validated this approach on cryopreserved WBC samples from wild American coots (Fulicia americana) . Cryopreservation of chicken WBCs caused a slight but significant decrease in cell viability (99% +/- 0.2 SE for fresh cells versus 84% +/- 2 SE for cryopreserved cells, p = 0.001, Mann-Whitney U, n = 8) . No difference was detected in viability between cells that were cryopreserved for less than 10 days (88% +/- 3.7 SE) and more than 50 days (89% +/- 1.3 SE) (n = 6) . Overall, there was no statistical difference in the performance of cryopreserved cells compared to fresh cells . Across multiple experiments, cryopreserved T lymphocytes exhibited 200-900% stimulated proliferation above nonstimulated cells, and 40-80% of cryopreserved macrophages ingested yeast . 9,10,Dimethyl-1,2-benz-anthracene (DMBA) reduced proliferation and phagocytosis in cryopreserved cells over an ex vivo exposure range of 0-170 microM DMBA . Tests of immune function on American coot WBCs cryopreserved for up to 10 months (viability of 72% +/- 2.5 SE, n = 24) were similar to the cryopreserved chicken WBCs . This study will facilitate greater use of ex vivo immune function assays as tools to study effects of contaminant exposure in wildlife by demonstrating the viability and functionality of cryopreserved avian cells.

Pharmacology, 2003 Jun, 68(2), 96 - 104
Anti-inflammatory effect and low ulcerogenic activity of etodolac, a cyclooxygenase-2 selective non-steroidal anti-inflammatory drug, on adjuvant-induced arthritis in rats; Tachibana M et al.; Adjuvant arthritic rats are known to be more susceptible to gastric damage induced by non-steroidal anti-inflammatory drugs (NSAIDs) than are normal rats . We compared the relative gastric safety profile of etodolac with those of meloxicam, diclofenac sodium and indometacin in adjuvant arthritic rats and normal rats or mice . As a measure of the safety profiles of NSAIDs, we used the safety index, the ratio of the dose that elicits gastric mucosal lesions to the effective dose as an anti-inflammatory or analgesic compound . The anti-inflammatory or analgesic effects of NSAIDs were assessed by paw swelling in adjuvant arthritic rats, and either carrageenin-induced paw edema or brewer's yeast-induced hyperalgesia, as well as acetic acid-induced writhing, in normal rats or mice . In addition, we also investigated the effects of these NSAIDs on human COX-1 and COX-2 activity . Etodolac and other NSAIDs inhibited paw swelling and caused gastric mucosal lesions in adjuvant arthritic rats in a dose-dependent manner . Etodolac showed the highest UD(50) value and safety index among these NSAIDs in arthritic rats . In normal rats, etodolac also showed the highest UD(50) value and safety index, except when its effects were assessed by acetic acid-induced writhing . Etodolac and meloxicam showed selectivity for human COX-2 over COX-1 . In contrast, diclofenac sodium and indometacin were selective for COX-1 . These results suggest that etodolac, a COX-2 selective NSAID, has anti-inflammatory effects with a better safety profile for the stomach than do non-selective NSAIDs, including diclofenac sodium and indometacin, in adjuvant arthritic as well as normal rats .

J Cell Sci, 2003 Jun 1, 116(Pt 11), 2361 - 73 Epub 2003 Apr 23.
Differential requirements of novel A1PiZ degradation deficient (ADD) genes in ER-associated protein degradation; Palmer EA et al.; In the eukaryotic cell, a protein quality control process termed endoplasmic reticulum-associated degradation (ERAD) rids the ER of aberrant proteins and unassembled components of protein complexes that fail to reach a transport-competent state . To identify novel genes required for ERAD, we devised a rapid immunoassay to screen yeast lacking uncharacterized open reading frames that were known targets of the unfolded protein response (UPR), a cellular response that is induced when aberrant proteins accumulate in the ER . Six genes required for the efficient degradation of the Z variant of the alpha1-proteinase inhibitor (A1PiZ), a known substrate for ERAD, were identified, and analysis of other ERAD substrates in the six A1PiZ-degradation-deficient (add) mutants suggested diverse requirements for the Add proteins in ERAD . Finally, we report on bioinformatic analyses of the new Add proteins, which will lead to testable models to elucidate their activities.

Nucleic Acids Res, 2003 May 1, 31(9), 2408 - 16
Assembly and isolation of intermediate steps of transcription complexes formed on the human 5S rRNA gene; Weser S et al.; By employing purified transcription factors and RNA polymerase III (pol III), we generated active pol III transcription complexes on the human 5S rRNA gene . These large complexes were separated by size exclusion chromatography from non- incorporated proteins . In addition, we succeeded in isolating specific intermediate stages of complex formation . Such isolated partial complexes require complementation with the missing activities for full transcription activity . One central finding is that a 5S DNA-TFIIIA-TFIIIC2-TFIIIBbeta complex could be isolated which had been assembled in the absence of the general pol III transcription factor IIIC1 . Thus TFIIIC1 is not an assembly factor for other transcription factors . Although pol III has the potential to bind unspecifically to DNA, such polymerase molecules cannot be rendered initiation competent by direct recruitment to a 5S DNA-TFIIIA-TFIIIC2- TFIIIBbeta complex, but this process strictly requires additional TFIIIC1 activity . This clearly demonstrates that in contrast to yeast cells, hTFIIIB(beta), although required, does not suffice for the functional recruitment of polymerase III . These data document that TFIIIC1 is the second transcription factor required for the recruitment of pol III in mammalian cells.

J Biol Chem, 2003 Jul 25, 278(30), 28038 - 44 Epub 2003 Apr 23.
Sequence-specific binding to telomeric DNA by CEH-37, a homeodomain protein in the nematode Caenorhabditis elegans; Kim SH et al.; Caenorhabditis elegans can serve as a model system to study telomere functions due to its similarity to higher organisms in telomere structures . We report here the identification of the nematode homeodomain protein CEH-37 as a telomere-binding protein using a yeast one-hybrid screen . The predicted three-dimensional model of the homeodomain of CEH-37, which has a typical helix-loop-helix structure, was similar to that of the Myb domain of known telomere-binding proteins, which is also a helix-loop-helix protein, despite little amino acid sequence similarity . We demonstrated the specific binding of CEH-37 to the nematode telomere sequences in vitro by competition assays . We determined that CEH-37 binding required at least 1.5 repeats of TTAGGC and that the core sequence for binding was GGCTTA . We found that CEH-37 had an ability to bend telomere sequence-containing DNA, which is the case for other known telomere-binding proteins such as TRF1 and RAP1, indicating that CEH-37 may be involved in establishing or maintaining a secondary structure of the telomeres in vivo . We also demonstrated that CEH-37 was primarily co-localized to the chromosome ends in vivo, indicating that CEH-37 may play roles in telomere functions . Consistent with this, a ceh-37 mutation resulting in a truncated protein caused a weak high incidence of male phenotype, which may have been caused by chromosome instability . The identification of CEH-37 as a telomere-binding protein may represent an evolutionary conservation of telomere-binding proteins in terms of tertiary protein structure rather than primary amino acid sequence.

Gene, 2003 Apr 10, 308, 53 - 65
A refined molecular karyotype for the reference strain of the Trypanosoma cruzi genome project (clone CL Brener) by assignment of chromosome markers; Porcile PE et al.; We present a useful refinement of the molecular karyotype of clone CL Brener, the reference clone of the Trypanosoma cruzi Genome Project . The assignment of 210 genetic markers (142 expressed sequence tags (ESTs), seven cDNAs, 32 protein-coding genes, eight sequence tagged sites (STSs), 21 repetitive sequences) to the chromosomal bands separated by pulsed field gel electrophoresis (PFGE) identified 61 chromosome-specific markers, two size-polymorphic chromosomes and seven linkage groups . Fourteen new repetitive elements were isolated in this work and mapped to the chromosomal bands . We found that at least ten repetitive elements can be mapped to each chromosomal band, which may render the whole genome sequence assembly a difficult task . To construct the integrated map of chromosomal band XX, we used yeast artificial chromosome (YAC) overlapping clones and a variety of probes (i.e . known gene sequences, ESTs, STSs generated from the YAC ends) . The total length covered by the YAC contig was approximately 1.3 Mb, covering 37% of the entire chromosome . We found some degree of polymorphism among YACs derived from band XX . These results are in agreement with data from phylogenetic analysis of T . cruzi which suggest that clone CL Brener is a hybrid genotype {Mol . Biochem . Parasitol . 92 (1998) 253; Proc . Natl . Acad . Sci . USA 98 (2001) 7396} . The physical map of the chromosomal bands, together with the isolation of specific chromosomal markers, will contribute in the global effort to sequence the nuclear genome of this parasite.

Biochem Biophys Res Commun, 2003 May 2, 304(2), 326 - 32
Ngly1, a mouse gene encoding a deglycosylating enzyme implicated in proteasomal degradation: expression, genomic organization, and chromosomal mapping; Suzuki T et al.; In species as diverse as yeast and mammals, peptide:N-glycanase (PNG1 in yeast; Ngly1 in mouse) is believed to play a key role in the degradation of misfolded glycoproteins by the proteasome . In this study, we report the genomic organization and mRNA distribution of the mouse Ngly1 . Mouse Ngly1 spans 61kb and is composed of 12 exons, the organization of which is conserved throughout vertebrates . Comparison of the mouse and human genomic sequence identifies a conserved gene structure with significant sequence similarity extending into introns . A 2.6kb Ngly1 message was detected in all mouse tissues examined, with the highest abundance in the testis . In addition, a lower molecular weight transcript of 2.4kb was detected in the testis . From analysis of dbESTs the alternative transcript of Ngly1 is predicted to be present in the human placenta . Given the key role Ngly1 plays in glycoprotein degradation, we predict that Ngly1 may be a contributing factor in "disease" susceptibility . To begin to address this question, we used radiation hybrid mapping to localize mouse Ngly1 to chromosome 14 and the human orthologue to chromosome 3 with a strong link with known genes.

Trends Genet, 2003 May, 19(5), 286 - 93
Class II histone deacetylases: versatile regulators; Verdin E et al.; Histone acetylation and deacetylation play essential roles in modifying chromatin structure and regulating gene expression in eukaryotes . Histone deacetylases (HDACs) catalyze the deacetylation of lysine residues in the histone N-terminal tails and are found in large multiprotein complexes with transcriptional co-repressors . Human HDACs are grouped into three classes based on their similarity to known yeast factors: class I HDACs are similar to the yeast transcriptional repressor yRPD3, class II HDACs to yHDA1 and class III HDACs to ySIR2 . In this review, we focus on the biology of class II HDACs . These newly discovered enzymes have been implicated as global regulators of gene expression during cell differentiation and development . We discuss their emerging biological functions and the molecular mechanisms by which they are regulated.

Exp Mol Pathol, 2003 Apr, 74(2), 123 - 8
The role of p55CDC in cell cycle control and mammalian cell proliferation, differentiation, and apoptosis; Lin M et al.; The p55CDC (cell division cycle) protein is a key regulator of the cell cycle . p55CDC is related to both the CDC20 and the CDH1 proteins in yeast . p55CDC has been shown to activate the ubiquitin ligase anaphase promoting complex (APC), which is involved in degradation of proteins that control mitosis . To define the role of p55CDC during the mammalian cell cycle, we overexpressed this protein in the murine myeloid cell line 32Dcl3 . 32Dcl3 cells are an ideal model system because these cells can be induced to proliferate, differentiate, or activate cellular programs leading to apoptosis . Our work suggests that p55CDC participates in cell growth, maturation, and death . Thus, p55CDC may play a more diverse role in modulating cellular functions in addition to controlling the cell cycle.

Int J Syst Evol Microbiol, 2003 Mar, 53(Pt 2), 607 - 16
Molecular systematics of the dimorphic ascomycete genus Taphrina; Rodrigues MG et al.; The ascomycete genus Taphrina Fries comprises nearly 100 species recognized by their mycelial states when parasitic on different vascular plants . Whereas the filamentous state is strictly phytoparasitic, the yeast state is saprobic and can be cultured on artificial media . Taphrina species are differentiated mainly on the basis of host range and geographical distribution, type and site of infection and morphology of the sexual stage in infected tissue . However, there has been little progress in the systematics of the genus in recent years, mainly because of the scarcity of molecular studies and available cultures . The main aim of the present study was the reappraisal of species boundaries in Taphrina based on the genetic characterization of cultures (yeast states) that represent about one-third of the currently recognized species . The molecular methods used were (i) PCR fingerprinting using single primers for microsatellite regions and (ii) determination of nucleotide sequences of two approx . 600 bp nuclear rDNA regions, the 5' end of the 26S rRNA gene (D1/D2 domains) and the internal transcribed spacer region (which includes the 5.8S rRNA gene) . Sequencing results confirmed the monophyly of the genus (with the probable exclusion of Taphrina vestergrenii) and the combined analysis of the two methods corroborated, in most cases, separation of species defined on the basis of conventional criteria . However, genetic heterogeneity was found within some species and conspecificity was suggested for strains that have been deemed to represent distinct species . Sequences from the ITS region displayed a higher degree of divergence than those of the D1/D2 region between closely related species, but were relatively conserved within species (> 99% identity) and were thus more useful for the effective differentiation of Taphrina species . The results further allowed other topics to be addressed such as the correlation between the molecular phylogenetic clustering of certain species and the respective host plant family and the significance of molecular methods in the accurate diagnosis of the different diseases caused by Taphrina species.

J Biol Chem, 2003 Jul 4, 278(27), 24697 - 704 Epub 2003 Apr 22.
Differential regulation of nramp and irt metal transporter genes in wild type and iron uptake mutants of tomato; Bereczky Z et al.; Metal transporters regulated by iron can transport a variety of divalent metals, suggesting that iron regulation is important for specificity of iron transport . In plants, the iron-regulated broad-range metal transporter IRT1 is required for uptake of iron into the root epidermis . Functions of other iron-regulated plant metal transporters are not yet established . To deduce novel plant iron transport functions we studied the regulation of four tomato metal transporter genes belonging to the nramp and irt families with respect to environmental and genetic factors influencing iron uptake . We isolated Lenramp1 and Lenramp3 from tomato and demonstrate that these genes encode functional NRAMP metal transporters in yeast, where they were iron-regulated and localized mainly to intracellular vesicles . Lenramp1 and Leirt1 revealed both root-specific expression and up-regulation by iron deficiency, respectively, in contrast to Leirt2 and Lenramp3 . Lenramp1 and Leirt1, but not Lenramp3 and Leirt2, were down-regulated in the roots of fer mutant plants deficient in a bHLH gene regulating iron uptake . In chloronerva mutant plants lacking the functional enzyme for synthesis of the plant-specific metal chelator nicotianamine Leirt1 and Lenramp1 were up-regulated despite sufficient iron supply independent of a functional fer gene . Lenramp1 was expressed in the vascular root parenchyma in a similar cellular pattern as the fer gene . However, the fer gene was not sufficient for inducing Lenramp1 and Leirt1 when ectopically expressed . Based on our results, we suggest a novel function for NRAMP1 in mobilizing iron in the vascular parenchyma upon iron deficiency in plants . We discuss fer/nicotianamine synthase-dependent and -independent regulatory pathways for metal transporter gene regulation.

J Environ Qual, 2003 Mar-Apr, 32(2), 417 - 22
Polycyclic aromatic hydrocarbons (PAHs) and estrogenic compounds in experimental flue gas streams; Muthumbi W et al.; The importance of combustion processes as a source of substances with estrogenic activity in the environment was investigated . Wood (nontreated and treated with wood preservatives), barbecue charcoal, meat, and kitchen waste were combusted in a laboratory-scale incinerator . Flue gas emissions (particulates and gaseous pollutants) were trapped in polyurethane foam cartridges . The cartridges were subjected to Soxhlet extraction and part of the extracts redissolved in dimethylsulfoxide (DMSO) for analyses of estrogenic activity by means of the yeast-based human estrogen receptor (hER) bioassay . A synthetic estrogen, 17-alpha-ethinylestradiol (EE2), was used as the reference estrogenic compound . Part of the extracts was analyzed for the 16 USEPA priority polycyclic aromatic hydrocarbons (PAHs) . Estrogenic compounds in the flue gas (wood) were as high as 234 +/- 25 ng m(-3) EE2 equivalent compared with 27 to 81 ng m(-3) EE2 equivalent in flue gas from combustion of barbecue charcoal . Concentrations of polycyclic aromatic hydrocarbons in both flue gas streams were in the range of 21,000 +/- 2000 and 240 +/- 110 ng m(-3), respectively . In general, the concentrations of EE2 equivalent in the flue gas samples were at least a factor of 1000 lower than total PAH concentration . The EE2 levels were not related to the concentration of PAHs in any flue gas sample.

J Cell Biol, 2003 Apr 28, 161(2), 403 - 16 Epub 2003 Apr 21.
Defining desmosomal plakophilin-3 interactions; Bonne S et al.; Plakophilin 3 (PKP3) is a recently described armadillo protein of the desmosomal plaque, which is synthesized in simple and stratified epithelia . We investigated the localization pattern of endogenous and exogenous PKP3 and fragments thereof . The desmosomal binding properties of PKP3 were determined using yeast two-hybrid, coimmunoprecipitation and colocalization experiments . To this end, novel mouse anti-PKP3 mAbs were generated . We found that PKP3 binds all three desmogleins, desmocollin (Dsc) 3a and -3b, and possibly also Dsc1a and -2a . As such, this is the first protein interaction ever observed with a Dsc-b isoform . Moreover, we determined that PKP3 interacts with plakoglobin, desmoplakin (DP) and the epithelial keratin 18 . Evidence was found for the presence of at least two DP-PKP3 interaction sites . This finding might explain how lateral DP-PKP interactions are established in the upper layers of stratified epithelia, increasing the size of the desmosome and the number of anchoring points available for keratins . Together, these results show that PKP3, whose epithelial and epidermal desmosomal expression pattern and protein interaction repertoire are broader than those of PKP1 and -2, is a unique multiprotein binding element in the basic architecture of a vast majority of epithelial desmosomes.

J Mol Biol, 2003 May 2, 328(3), 567 - 79
Motif refinement of the peroxisomal targeting signal 1 and evaluation of taxon-specific differences; Neuberger G et al.; Eukaryote peroxisomes, plant glyoxysomes and trypanosomal glycosomes belong to the microbody family of organelles that compartmentalise a variety of biochemical processes . The interaction between the PTS1 signal and its cognate receptor Pex5 initiates the major import mechanism for proteins into the matrix of these organelles . Relying on the analysis of amino acid sequence variability of known PTS1-targeted proteins and PTS1-containing peptides that interact with Pex5 in the yeast two-hybrid assay, on binding site studies of the Pex5-ligand complex crystal structure, 3D models and sequences of Pex5 proteins from various taxa, we derived the requirements for a C-terminal amino acid sequence to interact productively with Pex5 . We found evidence that, at least the 12 C-terminal residues of a given substrate protein are implicated in PTS1 signal recognition . This motif can be structurally and functionally divided into three regions: (i) the C-terminal tripeptide, (ii) a region interacting with the surface of Pex5 (about four residues further upstream), and (iii) a polar, solvent-accessible and unstructured region with linker function (the remaining five residues) . Specificity differences are confined to taxonomic subgroups (metazoa and fungi) and are connected with amino acid type preferences in region 1 and deviating hydrophobicity patterns in region 2.

Int J Food Microbiol, 2003 Jun 15, 83(2), 219 - 25
Development of a method for direct visual determination of aflatoxin production by colonies of the Aspergillus flavus group; Jaimez Ordaz J et al.; This report describes a simple, rapid and reliable method for screening the aflatoxin production by moulds of the Aspergillus flavus group . Strains were cultivated on yeast extract agar to which methylated beta-cyclodextrin derivative plus sodium desoxycholate was added . Production of aflatoxins was readily detectable by direct visualisation of a beige ring surrounding colonies after an incubation time of 3 days at 28 degrees C . When this ring was examined under UV light, it exhibited blue fluorescence . The presence of aflatoxins was confirmed by extracting the medium with chloroform and examining the extracts by HPLC with fluorescence detection.

Biochem Biophys Res Commun, 2003 Apr 25, 304(1), 125 - 9
Beacon interacts with cdc2/cdc28-like kinases; Kantham L et al.; Previously we found elevated beacon gene expression in the hypothalamus of obese Psammomys obesus . Beacon administration into the lateral ventricle of P . obesus stimulated food intake and body weight gain . In the current study we used yeast two-hybrid technology to screen for proteins in the human brain that interact with beacon . CLK4, an isoform of cdc2/cdc28-like kinase family of proteins, was identified as a strong interacting partner for beacon . Using active recombinant proteins and a surface plasmon resonance based detection technique, we demonstrated that the three members of this subfamily of kinases (CLK1, 2, and 4) all interact with beacon . Based on the known sequence and functional properties of beacon and CLKs, we speculate that beacon could either modulate the function of key regulatory molecules such as PTP1B or control the expression patterns of specific genes involved in the central regulation of energy metabolism.

J Biol Chem, 2003 Jun 27, 278(26), 24139 - 52 Epub 2003 Apr 18.
FHL3 is an actin-binding protein that regulates alpha-actinin-mediated actin bundling: FHL3 localizes to actin stress fibers and enhances cell spreading and stress fiber disassembly; Coghill ID et al.; Four and a half LIM domain (FHL) proteins are members of the LIM protein superfamily . Several FHL proteins function as co-activators of CREM/CREB transcription factors and the androgen receptor . FHL3 is highly expressed in skeletal muscle, but its function is unknown . FHL3 localized to the nucleus in C2C12 myoblasts and, following integrin engagement, exited the nucleus and localized to actin stress fibers and focal adhesions . In mature skeletal muscle FHL3 was found at the Z-line . Actin was identified as a potential FHL3 binding partner in yeast two-hybrid screening of a skeletal muscle library . FHL3 complexed with actin both in vitro and in vivo as shown by glutathione S-transferase pull-down assays and co-immunoprecipitation of recombinant and endogenous proteins . FHL3 promoted cell spreading and when overexpressed in spread C2C12 cells disrupted actin stress fibers . Increased FHL3 expression was detected in highly motile cells migrating into an artificial wound, compared with non-motile cells . The molecular mechanism by which FHL3 induced actin stress fiber disassembly was demonstrated by low speed actin co-sedimentation assays and electron microscopy . FHL3 inhibited alpha-actinin-mediated actin bundling . These studies reveal FHL3 as a significant regulator of actin cytoskeletal dynamics in skeletal myoblasts.

J Biol Chem, 2003 Jun 27, 278(26), 23561 - 9 Epub 2003 Apr 17.
The F-actin cross-linking and focal adhesion protein filamin A is a ligand and in vivo substrate for protein kinase C alpha; Tigges U et al.; Filamin A is an established structural component of cell-matrix adhesion sites . In addition, it serves as a scaffold for the subcellular targeting of different signaling molecules . Protein kinase C (PKC) has been found associated with filamin; however, details about this interaction and its significance for cell-matrix adhesion-dependent signaling have remained elusive . We performed a yeast two-hybrid analysis using protein kinase Calpha as a bait and identified filamin as a direct binding partner . The interaction was confirmed in transfected HeLa cells, and serial truncation fragments of filamin A were employed to identify two binding sites on filamin . In vitro ligand binding assays revealed a Ca2+ and phospholipid-dependent association of the regulatory domain of protein kinase C with these sites . Phosphorylation of filamin was found to be isoform-restricted, leading to phosphate incorporation in the C termini of filamin A and C, but not B . PKC-dependent phosphorylation of filamin was also detected in cells . Our data suggest an intimate interaction between filamin and PKC in cell signaling.

Proc Natl Acad Sci U S A, 2003 Apr 29, 100(9), 5431 - 6 Epub 2003 Apr 17.
Characterization of cells and gene-targeted mice deficient for the p53-binding kinase homeodomain-interacting protein kinase 1 (HIPK1); Kondo S et al.; The tumor suppressor p53 is regulated in part by binding to cellular proteins . We used p53 as bait in the yeast two-hybrid system and isolated homeodomain-interacting protein kinase 1 (HIPK1) as a p53-binding protein . Deletion analysis showed that amino acids 100-370 of p53 and amino acids 885-1093 of HIPK1 were sufficient for HIPK1-p53 interaction . HIPK1 was capable of autophosphorylation and specific serine phosphorylation of p53 . The HIPK1 gene was highly expressed in human breast cancer cell lines and oncogenically transformed mouse embryonic fibroblasts . HIPK1 was localized to human chromosome band 1p13, a site frequently altered in cancers . Gene-targeted HIPK1-/- mice were grossly normal but oncogenically transformed HIPK1 -/- mouse embryonic fibroblasts exhibited reduced transcription of Mdm2 and were more susceptible than transformed HIPK1+/+ cells to apoptosis induced by DNA damage . Carcinogen-treated HIPK1 -/- mice developed fewer and smaller skin tumors than HIPK1+/+ mice . HIPK1 may thus play a role in tumorigenesis, perhaps by means of the regulation of p53 and/or Mdm2.

J Biol Chem, 2003 Jul 11, 278(28), 25289 - 94 Epub 2003 Apr 17.
Identification of catalytic residues of Ca2+-independent 1,2-alpha-D-mannosidase from Aspergillus saitoi by site-directed mutagenesis; Tatara Y et al.; The roles of six conserved active carboxylic acids in the catalytic mechanism of Aspergillus saitoi 1,2-alpha-d-mannosidase were studied by site-directed mutagenesis and kinetic analyses . We estimate that Glu-124 is a catalytic residue based on the drastic decrease of kcat values of the E124Q and E124D mutant enzyme . Glu-124 may work as an acid catalyst, since the pH dependence of its mutants affected the basic limb . D269N and E411Q were catalytically inactive, while D269E and E411D showed considerable activity . This indicated that the negative charges at these points are essential for the enzymatic activity and that none of these residues can be a base catalyst in the normal sense . Km values of E273D, E414D, and E474D mutants were greatly increased to 17-31-fold wild type enzyme, and the kcat values were decreased, suggesting that each of them is a binding site of the substrate . Ca2+, essential for the mammalian and yeast enzymes, is not required for the enzymatic activity of A . saitoi 1,2-alpha-d-mannosidase . EDTA inhibits the Ca2+-free 1,2-alpha-d-mannosidase as a competitive inhibitor, not as a chelator . We deduce that the Glu-124 residue of A . saitoi 1,2-alpha-d-mannosidase is directly involved in the catalytic mechanism as an acid catalyst, whereas no usual catalytic base is directly involved . Ca2+ is not essential for the activity . The catalytic mechanism of 1,2-alpha-d-mannosidase may deviate from that typical glycosyl hydrolase.

Cancer Res, 2003 Apr 15, 63(8), 1954 - 61
Psoriasin interacts with Jab1 and influences breast cancer progression; Emberley ED et al.; Psoriasin (S100A7) is expressed at low levels in normal breast epithelial cells but is highly expressed in preinvasive ductal carcinoma in situ . Persistent psoriasin expression occurs in some invasive carcinomas and is associated with poor prognostic factors . Whereas there is evidence that secreted psoriasin can act as a chemotactic factor for CD-4-positive lymphocytes in psoriatic skin lesions, an intracellular biological function is unknown . We have found that psoriasin physically interacts with Jab1 (c-jun activation-domain binding protein 1) in the yeast two-hybrid assay and confirmed this by coimmunoprecipitation assay in breast cancer cells . Psoriasin-transfected breast cancer cells showed increased nuclear Jab1 and demonstrated several features consistent with an alteration in Jab1 activity including an increase in activator protein-1 (AP-1) activity, increased expression of AP-1 and HIF-1-dependent genes, and reduced expression of the cell-cycle inhibitor p27(Kip1) . Psoriasin overexpression was also associated with alteration of cellular functions that are associated with increased malignancy, including increased growth, decreased adhesion, and increased invasiveness in vitro, as well as increased tumorigenicity in vivo in nude mice . We conclude that intracellular psoriasin influences breast cancer progression and that this may occur through stimulation of Jab1 activity.

FEMS Yeast Res, 2001 Apr, 1(1), 23 - 31
Glucose-induced and nitrogen-starvation-induced peroxisome degradation are distinct processes in Hansenula polymorpha that involve both common and unique genes; Bellu AR et al.; In the methylotrophic yeast Hansenula polymorpha non-selective autophagy, induced by nitrogen starvation, results in the turnover of cytoplasmic components, including peroxisomes . We show that the uptake of these components occurs by invagination of the vacuolar membrane without their prior sequestration and thus differs from the mechanism described for bakers yeast . A selective mode of autophagy in H . polymorpha, namely glucose-induced peroxisome degradation, involves sequestration of individual peroxisomes tagged for degradation by membrane layers that subsequently fuse with the vacuole where the organelle is digested . H . polymorpha pdd mutants are blocked in selective peroxisome degradation . We observed that pdd1-201 is also impaired in non-selective autophagy, whereas this process still normally functions in pdd2-4 . These findings suggest that mechanistically distinct processes as selective and non-selective autophagy involve common but also unique genes.

FEMS Yeast Res, 2001 Dec, 1(3), 195 - 204
Disruption of gene YlODC reveals absolute requirement of polyamines for mycelial development in Yarrowia lipolytica; Jimenez-Bremont JF et al.; Polyamines are required for cellular growth and differentiation . In mammals and fungi they are synthesized via a pathway involving ornithine decarboxylase (ODC), which transforms ornithine into putrescine . We have cloned and disrupted the gene coding for ODC in Yarrowia lipolytica to analyze the role of polyamines in dimorphism of this fungus . Substrate- and cofactor-binding motifs, as well as two putative PEST boxes were identified in the amino acid sequence . A single transcript 1.7 kb in size was identified by Northern hybridization, and confirmed by rapid amplification of cDNA ends (RACE) . Null mutants lacked ODC activity and behaved as polyamine auxotrophs . When low levels of polyamines were supplied to the null mutant, only yeast-like, but not mycelial growth was sustained . This phenomenon was confirmed by introduction of the YlODC gene under the control of an inducible promoter into the null mutant.

FEMS Yeast Res, 2002 Jan, 1(4), 257 - 63
Isolation of mutants of Hansenula polymorpha defective in the assembly of octameric alcohol oxidase; van Dijk R et al.; Alcohol oxidase (AO) is a peroxisomal enzyme that catalyses the first step in methanol metabolism in yeast . Monomeric, inactive AO protein is synthesised in the cytosol and subsequently imported into peroxisomes, where the enzymatically active, homo-octameric form is found . The mechanisms involved in AO octamer assembly are largely unclear . Here we describe the isolation of Hansenula polymorpha mutants specifically affected in AO assembly . These mutants are unable to grow on methanol and display reduced AO activities . Based on their phenotypes, three major classes of mutants were isolated . Three additional mutants were isolated that each displayed a unique phenotype . Complementation analysis revealed that the isolated AO assembly mutants belonged to 10 complementation groups.

FEMS Yeast Res, 2002 May, 2(2), 203 - 13
Identification and analysis of genes involved in the control of dimorphism in Mucor circinelloides (syn . racemosus); Wolff AM et al.; Mucor circinelloides (syn . racemosus) is a non-pathogenic dimorphic fungus belonging to the class of zygomycetes . We are developing a novel system for heterologous protein production exploiting the dimorphic growth characteristics of M . circinelloides . In order to identify potential genetic regulators of morphology we have initiated a characterisation of key genes involved in signal transduction in Mucor . We have cloned and characterised pkaR and pkaC encoding the regulatory subunit (PKAR) and the catalytic subunit (PKAC), respectively, of the cAMP-dependent protein kinase A (PKA) of M . circinelloides . In anaerobically grown yeast cells, the levels of expression of both pkaR and pkaC were significantly higher than the levels of expression in aerobically grown mycelium . However, during the dimorphic shift, i.e . during the transition from anaerobic yeast growth to aerobic filamentous growth, the expression of pkaR was found to increase approximately two-fold . These results indicate that regulation of PKA activity is conferred at different levels according to growth and environmental conditions . Overexpression of pkaR resulted in a multi-branched colony phenotype on solid medium indicating that PKAR plays a role in filamentation and branching . Fragments of genes encoding factors of the mitogen-activated protein (MAP) kinase (MAPK) pathway have also been cloned: mpk1 (mitogen-activated protein kinase 1) encoding a MAPK homologue and ste12 encoding a transcription factor.

FEMS Yeast Res, 2002 Aug, 2(3), 349 - 61
Strain and process development for the production of human cytokines in Hansenula polymorpha; Degelmann A et al.; The early status of strain development for the production of interleukin (IL)-6, IL-8, IL-10, and interferon (IFN) gamma is described . The general approach to generating such strains was to amplify gene sequences encoding the mature forms of the various cytokines by PCR from commercially available cDNA sources . The design of the amplificates allowed an in-frame fusion to an MFalpha1 leader segment contained in two basic expression vectors, pFPMT121-MFalpha1 and pTPSMT-MFalpha1 . The two vectors differ in that one harbors the methanol-inducible FMD promoter and the other the constitutive TPS1 promoter as control elements for heterologous gene expression . The most advanced process development example is that of IFNalpha-2a . Here, the MOX promoter derived from another key gene of methanol metabolism is used for expression control . The successful development of a production process for Hansenula polymorpha-derived IFNalpha-2a is summarized . This was achieved by combining genetic engineering of suitable production strains with improved processing capabilities for the secreted cytokine, and by purification procedures from cultures grown in yeast extract-peptone-glycerol-based media.

Clin Genet, 2003 Apr, 63(4), 297 - 302
Molecular characterization of a unique de novo 15q deletion associated with Prader-Willi syndrome and central visual impairment; Windpassinger C et al.; We report a 2-year-old boy with Prader-Willi Syndrome (PWS) caused by a deletion of the PWS critical region as a result of an unbalanced translocation t(3;15) . Additional features, including central visual impairment, relative macrocephaly, retrognathia, preauricular tags, and bilateral club-feet, were noticed . The extension of the deletion was determined by fluorescence in situ hybridization (FISH) analysis using 11 region-specific YAC clones . Nine YACs were found to be deleted, allowing us to determine that the deletion is larger than in patients with typical PWS deletions . The karyotype of this patient can thus be designated: 45,XY,-15,der(3)t(3;15)(qter;q14).ish der(3)t(3;15)(qter;q14) (wcp3+,wcp15+,D15S10-,PML+,D15Z1-,D3S4560+,801_f_9x1, 815_e_6x2) de novo . Molecular analyses using seven polymorphic markers helped to narrow down the breakpoint between marker ACTC.PC3 and the distal end of the YAC 815_e_6 . These results provide evidence that haploinsufficiency for genes in 15q13-q14, not affected in common PWS deletions, is associated with the additional features found in the patient, including a central visual impairment.

Arch Microbiol, 2003 Jun, 179(6), 394 - 401 Epub 2003 Apr 08.
Palaeococcus helgesonii sp . nov., a facultatively anaerobic, hyperthermophilic archaeon from a geothermal well on Vulcano Island, Italy; Amend JP et al.; A novel, hyperthermophilic archaeon was isolated from a shallow geothermal well that taps marine waters on the Island of Vulcano in the southern Tyrrhenian Sea, Italy . The cells were irregular cocci, 0.6-1.5 microm in diameter, with multiple polar flagella . Growth was observed at temperatures from 45 to 85 degrees C (optimum at approximately 80 degrees C), pH 5-8 (optimum at 6.5), and 0.5-6.0% NaCl (optimum at approximately 2.8%) . The minimum doubling time was 50 min . The isolate was obligately chemoheterotrophic, utilizing complex organic compounds including yeast or beef extract, peptone, tryptone, or casein for best growth . The presence of elemental sulfur enhanced growth . The isolate grew anaerobically as well as microaerobically . The G+C content of the genomic DNA was 42.5 mol% . The 16S rRNA sequence indicated that the new isolate was a member of the Thermococcales within the euryarchaeota, representing the second species in the genus Palaeococcus . Its physiology and phylogeny differed in several key characteristics from those of Palaeococcus ferrophilus, justifying the establishment of a new species; the name Palaeococcus helgesonii sp . nov . is proposed, type strain PI1 (DSM 15127).

J Biol Chem, 2003 Jun 13, 278(24), 21576 - 83 Epub 2003 Apr 07.
Agonist-induced phosphorylation of the serotonin 5-HT2C receptor regulates its interaction with multiple PDZ protein 1; Parker LL et al.; Multiple PDZ domain protein 1 (MUPP1), a putative scaffolding protein containing 13 PSD-95, Dlg, ZO-1 (PDZ) domains, was identified by a yeast two-hybrid screen as a serotonin2C receptor (5-HT2C R)-interacting protein (Ullmer, C., Schmuck, K., Figge, A., and Lubbert, H . (1998) FEBS Lett . 424, 63-68) . MUPP1 PDZ domain 10 (PDZ 10) associates with Ser458-Ser-Val at the carboxyl-terminal tail of the 5-HT2C R . Both Ser458 and Ser459 are phosphorylated upon serotonin stimulation of the receptor (Backstrom, J . R., Price, R . D., Reasoner, D . T., and Sanders-Bush, E . (2000) J . Biol . Chem . 275, 23620-23626) . To investigate whether phosphorylation of these serines in the receptor regulates MUPP1 interaction, we used several approaches . First, we substituted the serines in the receptor carboxyl tail with aspartates to mimic phosphorylation (S458D, S459D, or S458D/S459D) . Pull-down assays demonstrated that Asp mutations at Ser458 significantly decreased receptor tail interaction with PDZ 10 . Next, serotonin treatment of 5-HT2C R/3T3 cells resulted in a dose-dependent reduction of receptor interaction with PDZ 10 . Effects of serotonin on receptor-PDZ 10 binding could be blocked by pretreatment with a receptor antagonist . Alkaline phosphatase treatment reverses the effect of serotonin, indicating that agonist-induced phosphorylation at Ser458 resulted in a loss of MUPP1 association and also revealed a significant amount of basal phosphorylation of the receptor . We conclude that 5-HT2C R interaction with MUPP1 is dynamically regulated by phosphorylation at Ser458.

J Lipid Res, 2003 Jun, 44(6), 1174 - 81 Epub 2003 Apr 16.
Increased expression of the SNARE accessory protein Munc18c in lipid-mediated insulin resistance; Schlaepfer IR et al.; Fatty acids inhibit insulin-mediated glucose metabolism in skeletal muscle, an effect largely attributed to defects in insulin-mediated glucose transport . Insulin-resistant mice transgenic for the overexpression of lipoprotein lipase (LPL) in skeletal muscle were used to examine the molecular mechanism(s) in more detail . Using DNA gene chip array technology, and confirmation by RT-PCR and Western analysis, increases in the yeast Sec1p homolog Munc18c mRNA and protein were found in the gastrocnemius muscle of transgenic mice, but not other tissues . Munc18c has been previously demonstrated to impair insulin-mediated glucose transport in mammalian cells in vitro . Of interest, stably transfected C2C12 cells overexpressing LPL not only demonstrated increases in Munc18c mRNA and protein but also in transcription rates of the Munc18c gene . To confirm the relevance of fatty acid metabolism and insulin resistance to the expression of Munc18c in vivo, a 2-fold increase in Munc18c protein was demonstrated in mice fed a high-fat diet for 4 weeks . Together, these data are the first to implicate in vivo increases in Munc18c as a potential contributing mechanism to fatty acid-induced insulin resistance.

J Biol Chem, 2003 Jun 27, 278(26), 23553 - 60 Epub 2003 Apr 16.
Genetic interaction between a chaperone of small nucleolar ribonucleoprotein particles and cytosolic serine hydroxymethyltransferase; Yang Y et al.; Srp40p is a nonessential yeast nucleolar protein proposed to function as a chaperone for over 100 small nucleolar ribonucleoprotein particles that are required for rRNA maturation . To verify and expand on its function, genetic screens were performed for the identification of genes that were lethal when mutated in a SRP40 null background (srp40Delta) . Unexpectedly, mutation of both cytosolic serine hydroxymethyltransferase (SHM2) and one-carbon tetrahydrofolate synthase (ADE3) was required to achieve synthetic lethality with srp40Delta . Shm2p and Ade3p are cytoplasmic enzymes producing 5,10-methylene tetrahydrofolate in convergent pathways as the primary source for cellular one-carbon groups . Nonetheless, point mutants of Shm2p that were catalytically inactive (i.e . failed to rescue the methionine auxotrophy of a shm2Delta ade3 strain) complemented the synthetic lethal phenotype, thus revealing a novel metabolism-independent function of Shm2p . The same Shm2p mutants exacerbated a giant cell phenotype observed in the shm2Delta ade3 strain suggesting a catalysis-independent role for Shm2p in cell size control, possibly through regulation of ribosome biogenesis via SRP40 . Additionally, we show that the Sm-like protein Lsm5p, which as part of Lsm complexes participates in cytosolic and nuclear RNA processing and degradation pathways, is a multicopy suppressor of the synthetic lethality and of the specific depletion of box H/ACA small nucleolar RNAs from the srp40Delta shm2 ade3 strain . Finally, rat Nopp140 restored growth and stability of box H/ACA snoRNAs after genetic depletion of SRP40 in the synthetic lethal strain indicating that it is indeed the functional homolog of yeast Srp40p.

Curr Biol, 2003 Apr 15, 13(8), R323 - 5
Protein homeostasis: a degrading role for Int6/eIF3e; von Arnim AG et al.; Similarities between the three related "PCI" complexes--eIF3, the COP9 signalosome and the proteasome lid--have hinted at novel pathways controlling protein homeostasis . Recent experiments with fission yeast have begun to weigh in with genetic evidence.

Curr Biol, 2003 Apr 15, 13(8), 627 - 37
ATX-1, an Arabidopsis homolog of trithorax, activates flower homeotic genes; Alvarez-Venegas R et al.; BACKGROUND: The genes of the trithorax (trxG) and Polycomb groups (PcG) are best known for their regulatory functions in Drosophila, where they control homeotic gene expression . Plants and animals are thought to have evolved multicellularity independently . Although homeotic genes control organ identity in both animals and plants, they are unrelated . Despite this fact, several plant homeotic genes are negatively regulated by plant genes similar to the repressors from the animal PcG . However, plant-activating regulators of the trxG have not been characterized . RESULTS: We provide genetic, molecular, functional, and biochemical evidence that an Arabidopsis gene, ATX1, which is similar to the Drosophila trx, regulates floral organ development . The effects are specific: structurally and functionally related flower homeotic genes are under different control . We show that ATX1 is an epigenetic regulator with histone H3K4 methyltransferase activity . This is the first example of this kind of enzyme activity reported in plants, and, in contrast to the Drosophila and the yeast trithorax homologs, ATX1 can methylate in the absence of additional proteins . In its ability to methylate H3K4 as a recombinant protein, ATX1 is similar to the human homolog . CONCLUSIONS: ATX1 functions as an activator of homeotic genes, like Trithorax in animal systems . The histone methylating activity of the ATX1-SET domain argues that the molecular basis of these effects is the ability of ATX1 to modify chromatin structure . Our results suggest a conservation of trxG function between the animal and plant kingdoms despite the different structural nature of their targets.

Hum Genet, 2003 Jul, 113(2), 154 - 61 Epub 2003 Apr 16.
Translocation breakpoint in two unrelated Tourette syndrome cases, within a region previously linked to the disorder; Crawford FC et al.; Tourette syndrome (TS) is a complex neuropsychiatric disorder characterized by both motor and vocal tics . The etiology of TS is poorly understood; however, evidence of genetic transmission arises from family and twin studies . A complex mode of inheritance has been suggested, likely involving contributions of several genes with different effect size . We describe here two unrelated families wherein balanced t(6;8) chromosomal translocations occur in individuals diagnosed with TS . In one of these families, the transmission of the translocation is associated with learning and behavioral difficulties; in the other family, one parent is unaffected and the other cannot be traced, thus transmission cannot be demonstrated and it is possible that the translocation may have occurred de novo . The breakpoint on chromosome 8 occurs within the q13 band in both families, suggesting that a gene or genes in this region might contribute to the TS phenotype . Existing linkage and cytogenetic data, suggesting involvement of chromosome 8 in TS families and individuals, further support this hypothesis . We have identified two YAC clones mapping distal and proximal to the chromosome 8 translocation site, as determined by fluorescent in situ hybridization (FISH) . PCR amplification of genetic markers in this region, using isolated chromosomes from one of the patients, followed by BAC screening with the closest flanking genetic markers, has identified a 200-kb BAC, which, by FISH, we have demonstrated encompasses the chromosome 8 breakpoint in both families . The fact that the chromosomal breaks in the TS cases from both families occur within such a small region of chromosome 8 further supports the hypothesis that disruption of a gene or genes in this part of chromosome 8 contributes to the clinical phenotype.

Mol Cell Biol, 2003 May, 23(9), 3363 - 72
The Grb10/Nedd4 complex regulates ligand-induced ubiquitination and stability of the insulin-like growth factor I receptor; Vecchione A et al.; The adapter protein Grb10 belongs to a superfamily of related proteins, including Grb7, -10, and -14 and Caenorhabditis elegans Mig10 . Grb10 is an interacting partner of the insulin-like growth factor I receptor (IGF-IR) and the insulin receptor (IR) . Previous work showed an inhibitory effect of mouse Grb10 (mGrb10alpha) on IGF-I-mediated mitogenesis (A . Morrione et al., J . Biol . Chem . 272:26382-26387, 1997) . With mGrb10alpha as bait in a yeast two-hybrid screen, mouse Nedd4 (mNedd4-1), a ubiquitin protein ligase, was previously isolated as an interacting protein of Grb10 (A . Morrione et al., J . Biol . Chem . 274:24094-24099, 1999) . However, Grb10 is not ubiquitinated by Nedd4 in cells . Here we show that in mouse embryo fibroblasts overexpressing Grb10 and the IGF-IR (p6/Grb10), there is a strong ligand-dependent increase in ubiquitination of the IGF-IR compared with that in parental cells (p6) . This increased ubiquitination is associated with a shorter half-life and increased internalization of the IGF-IR . The IGF-IR is stabilized following treatment with both MG132 and chloroquine, indicating that both the proteasome and lysosomal pathways mediate degradation of the receptor . Ubiquitination of the IGF-IR likely occurs at the plasma membrane, prior to the formation of endocytic vesicles, as it is insensitive to dansylcadaverine, an inhibitor of early endosome formation in IGF-IR endocytosis . Grb10 coimmunoprecipitates with the IGF-IR and endogenous Nedd4 in p6/Grb10 cells, suggesting the presence of a Grb10/Nedd4/IGF-IR complex . Ubiquitination of the IGF-IR in p6/Grb10 cells is severely impaired by overexpression of a catalytically inactive Nedd4 mutant (Nedd4-CS), which also stabilizes the receptor . Likewise, overexpression of a Grb10 mutant lacking the Src homology 2 (SH2) domain impaired ubiquitination of the IGF-IR in parental p6 and p6/Grb10 cells, indicating that Grb10 binding to Nedd4 is critical for ubiquitination of the receptor . These results suggest a role for the Grb10/Nedd4 complex in regulating ubiquitination and stability of the IGF-IR, and they suggest that Grb10 serves as an adapter to form a bridge between Nedd4 and the IGF-IR . This is the first demonstration of regulation of stability of a tyrosine kinase receptor by the Nedd4 (HECT) family of E3 ligases.

Mol Cell Biol, 2003 May, 23(9), 3305 - 19
Two Drosophila Ada2 homologues function in different multiprotein complexes; Kusch T et al.; The reversible acetylation of the N-terminal tails of histones is crucial for transcription, DNA repair, and replication . The enzymatic reaction is catalyzed by large multiprotein complexes, of which the best characterized are the Gcn5-containing N-acetyltransferase (GNAT) complexes . GNAT complexes from yeast to humans share several conserved subunits, such as Ada2, Ada3, Spt3, and Tra1/TRRAP . We have characterized these factors in Drosophila and found that the flies have two distinct Ada2 variants (dAda2a and dAda2b) . Using a combination of biochemical and cell biological approaches we demonstrate that only one of the two Drosophila Ada2 homologues, dAda2b, is a component of Spt-Ada-Gcn5-acetyltransferase (SAGA) complexes . The other Ada2 variant, dAda2a, can associate with dGcn5 but is not incorporated into dSAGA-type complexes . This is the first example of a complex-specific association of the Ada-type transcriptional adapter proteins with GNATs . In addition, dAda2a is part of Gcn5-independent complexes, which are concentrated at transcriptionally active regions on polytene chromosomes . This implicates novel functions for dAda2a in transcription . Humans and mice also possess two Ada2 variants with high homology to dAda2a and dAda2b, respectively . This suggests that the mammalian and fly homologues of the transcriptional adapter Ada2 form two functionally distinct subgroups with unique characteristics.

Mol Cell Biol, 2003 May, 23(9), 3173 - 85
Role for human SIRT2 NAD-dependent deacetylase activity in control of mitotic exit in the cell cycle; Dryden SC et al.; Studies of yeast have shown that the SIR2 gene family is involved in chromatin structure, transcriptional silencing, DNA repair, and control of cellular life span . Our functional studies of human SIRT2, a homolog of the product of the yeast SIR2 gene, indicate that it plays a role in mitosis . The SIRT2 protein is a NAD-dependent deacetylase (NDAC), the abundance of which increases dramatically during mitosis and is multiply phosphorylated at the G(2)/M transition of the cell cycle . Cells stably overexpressing the wild-type SIRT2 but not missense mutants lacking NDAC activity show a marked prolongation of the mitotic phase of the cell cycle . Overexpression of the protein phosphatase CDC14B, but not its close homolog CDC14A, results in dephosphorylation of SIRT2 with a subsequent decrease in the abundance of SIRT2 protein . A CDC14B mutant defective in catalyzing dephosphorylation fails to change the phosphorylation status or abundance of SIRT2 protein . Addition of 26S proteasome inhibitors to human cells increases the abundance of SIRT2 protein, indicating that SIRT2 is targeted for degradation by the 26S proteasome . Our data suggest that human SIRT2 is part of a phosphorylation cascade in which SIRT2 is phosphorylated late in G(2), during M, and into the period of cytokinesis . CDC14B may provoke exit from mitosis coincident with the loss of SIRT2 via ubiquitination and subsequent degradation by the 26S proteasome.

Mol Cell Biol, 2003 May, 23(9), 3031 - 42
Replication proteins influence the maintenance of telomere length and telomerase protein stability; Dahlen M et al.; We investigated the effects of fission yeast replication genes on telomere length maintenance and identified 20 mutant alleles that confer lengthening or shortening of telomeres . The telomere elongation was telomerase dependent in the replication mutants analyzed . Furthermore, the telomerase catalytic subunit, Trt1, and the principal initiation and lagging-strand synthesis DNA polymerase, Polalpha, were reciprocally coimmunoprecipitated, indicating these proteins physically coexist as a complex in vivo . In a polalpha mutant that exhibited abnormal telomere lengthening and slightly reduced telomere position effect, the cellular level of the Trt1 protein was significantly lower and the coimmunoprecipitation of Trt1 and Polalpha was severely compromised compared to those in the wild-type polalpha cells . Interestingly, ectopic expression of wild-type polalpha in this polalpha mutant restored the cellular Trt1 protein to the wild-type level and shortened the telomeres to near-wild-type length . These results suggest that there is a close physical relationship between the replication and telomerase complexes . Thus, mutation of a component of the replication complex can affect the telomeric complex in maintaining both telomere length equilibrium and telomerase protein stability.

J Biol Chem, 2003 Jul 11, 278(28), 26015 - 20 Epub 2003 Apr 15.
Phosphorylation of phosphatase inhibitor-2 at centrosomes during mitosis; Leach C et al.; Inhibitor-2 (I-2) is a regulator of protein phosphatase type-1 (PP1), known to be phosphorylated in vitro by multiple kinases . In particular Thr72 is a Thr-Pro phosphorylation site conserved from yeast to human, but there is no evidence that this phosphorylation responds to any physiological signals . Here, we used electrophoretic mobility shift and immunoblotting with a site-specific phospho-Thr72 antibody to establish Thr72 phosphorylation in HeLa cells and show a 25-fold increase in phosphorylation during mitosis . Mass spectrometry demonstrated I-2 in actively growing HeLa cells was also phosphorylated at three other sites, Ser120, Ser121, and an additional Ser located between residues 70 and 90 . In vitro kinase assays using recombinant I-2 as a substrate showed that the Thr72 kinase(s) was activated during mitosis, and sensitivity to kinase inhibitors indicated that the principal I-2 Thr72 kinase was not GSK3 but instead a member of the cyclin-dependent protein kinase family . Immunocytochemistry confirmed Thr72 phosphorylation of I-2 during mitosis, with peak intensity at prophase, and revealed subcellular concentration of the phospho-Thr72 I-2 at centrosomes . Together, the data show dynamic changes in I-2 phosphorylation during mitosis and localization of phosphorylated I-2 at centrosomes, suggesting involvement in mammalian cell division.

Endocrinology, 2003 May, 144(5), 1675 - 85
Cell context-dependent differences in the induction of E2F-1 gene expression by 17 beta-estradiol in MCF-7 and ZR-75 cells; Ngwenya S et al.; 17 beta-Estradiol (E2) induces E2F-1 gene expression in ZR-75 and MCF-7 human breast cancer cells . Analysis of the E2F-1 gene promoter in MCF-7 cells previously showed that hormone-induced transactivation required interactions between estrogen receptor alpha (ER alpha)/Sp1 bound to upstream GC-rich sites and NFYA bound to downstream CCAAT sites within the -169 to -54 region of the promoter . This same region of the E2F-1 promoter was also E2 responsive in ER alpha-positive ZR-75 cells; however, further analysis of the promoter showed that cooperative ER alpha/Sp1/NFY interactions were not necessary for hormone-induced transactivation in ZR-75 cells . The upstream GC-rich motifs (-169 to -111) are activated independently by ER alpha/Sp1 in ZR-75 but not MCF-7 cells, and a construct (pE2F-1j(m1)) containing the -122 to -54 downstream CCAAT site that bound NFYA was also E2 responsive . E2 also induced reporter gene activity in ZR-75 cells transfected with an expression plasmid for a chimeric protein containing the DNA-binding domain of the yeast GAL4 protein fused to NFYA (pM-NFYA) and a construct containing five tandem GAL4 response elements . Subsequent studies showed that hormonal activation of pE2F-1j(m1) and pM-NFYA are dependent on nongenomic pathways in which E2 activates cAMP/protein kinase A . Hormone-dependent regulation of E2F-1 gene expression in ZR-75 and MCF-7 involves the same cis elements and interacting transcription factors but different mechanisms, demonstrating the importance of cell context on transactivation pathways, even among ER-positive breast cancer cell lines.

Water Res, 2003 Apr, 37(8), 1972 - 5
Removal of estrogenic activities of 17beta-estradiol and ethinylestradiol by ligninolytic enzymes from white rot fungi; Suzuki K et al.; We investigated whether manganese peroxidase (MnP) and the laccase-mediator system with 1-hydroxybenzotriazole (HBT) as mediator can remove the estrogenic activities of the steroidal hormones 17beta-estradiol (E(2)) and ethinylestradiol (EE(2)) . Using the yeast two-hybrid assay system, we confirmed that the estrogenic activities of E(2) and EE(2) are much higher than those of bisphenol A and nonylphenol . Greater than 80% of the estrogenic activities of E(2) and EE(2) were removed following 1-h treatment with MnP or the laccase-HBT system; extending the treatment time to 8h removed the remaining estrogenic activity of both steroidal hormones . HPLC analysis demonstrated that E(2) and EE(2) had disappeared almost completely in the reaction mixture after a 1-h treatment . These results strongly suggest that these ligninolytic enzymes are effective in removing the estrogenic activities of E(2) and EE(2).

Anal Biochem, 2003 May 1, 316(1), 23 - 33
Mass spectrometric quantification of acetylation at specific lysines within the amino-terminal tail of histone H4; Smith CM et al.; Electrospray ionization mass spectrometry, a leading method for the quantification of biomolecules, is useful for the analysis of posttranslational modifications of proteins . Here we describe a mass spectrometric approach for determining levels of acetylation at individual lysine residues within the amino-terminal tail of histone H4 . Because of the high density of acetylatable lysine residues within this short span of amino acids, collision-induced dissociation tandem mass spectrometry was required . In addition, it was necessary to develop an algorithm to determine the fraction of acetylation at specific lysine residues from fragment ions containing more than one lysine residue . This is the first report of direct measurement of endogeneous levels of acetylation at individual lysine residues within the amino-terminal tail of yeast histone H4 and is the first use of tandem mass spectrometry for quantification of peptides containing multiple sites of modification.

J Oral Pathol Med, 2003 May, 32(5), 271 - 4
Phagocytic functions of salivary neutrophils in oral mucous membrane diseases; Lukac J et al.; BACKGROUND AND PURPOSE: Phagocytic functions of salivary polymorphonuclear neutrophils (sPMNs) have not been comprehensively studied in patients with oral mucous membrane diseases, although available data suggest the role of immunity in their pathogenesis . SUBJECTS AND METHODS: Phagocytic functions of sPMN were determined in 15 patients with acute recurrent aphthous ulceration (RAU), 11 patients with oral lichen planus (OLP) and 20 healthy volunteers . In healthy subjects, the same parameters were also determined in peripheral blood polymorphonuclear neutrophils (bPMNs) . Phagocytic activity (proportion of ingesting cells, PA), ingestion ability (number of ingested targets per 100 phagocytes, IA) and intracellular microbicidity (proportion of killed targets, IM) of PMNs separated from peripheral blood and the whole unstimulated saliva were determined by acridine orange method with living yeast cells as targets . RESULTS: Salivary PMNs in healthy individuals showed significant reduction in PA (33% vs . 76%; P < 0.009) and IA (0.47% vs . 2.93%; P < 0.009) and significant increase in IM (12.0% vs . 5.5%; P = 0.011) in comparison with bPMNs . In RAU patients, reduced PA (27% vs . 37%; P = 0.035) and IA (0.25% vs . 0.47%; P = 0.05) were detected, while in OLP patients enhanced IM was detected (12% vs . 19%; P = 0.033) in comparison with healthy controls . CONCLUSION: Salivary PMNs present functional features distinct from those in peripheral blood . Some phagocytic functions of sPMNs are reduced in RAU and enhanced in OLP, indicating their role in pathogenesis or reflecting clinical changes in these conditions.

Biochemistry, 2003 Apr 22, 42(15), 4560 - 8
Inter-domain redox communication in flavoenzymes of the quiescin/sulfhydryl oxidase family: role of a thioredoxin domain in disulfide bond formation; Raje S et al.; Flavoproteins of the quiescin/sulfhydryl oxidase (QSOX) family catalyze oxidation of peptide and protein thiols to disulfides with the reduction of oxygen to hydrogen peroxide . QSOX family members contain several domains, including an N-terminal thioredoxin domain (Trx) and an FAD-binding-domain (ERV) toward the C-terminus . Partial proteolysis of avian QSOX leads to two fragments, designated 30 and 60 kDa from their apparent mobilities on SDS-PAGE . The 30 kDa fragment is a monomer under nondenaturing conditions and contains a Trx domain with a CxxC sequence typical of protein disulfide isomerase (WCGHC) . This QSOX fragment is not detectably glycosylated, contains no detectable FAD, and shows undetectable sulfhydryl oxidase activity . In contrast, the 60 kDa fragment is a dimeric glycoprotein that binds FAD tightly and oxidizes dithiothreitol about 1000-fold slower than intact QSOX . Reduced RNase is not a significant substrate of the 60 kDa fragment . The redox behavior of the 60 kDa flavoprotein fragment is profoundly different from that of intact QSOX . Thus, dithionite or photochemical reduction of the 60 kDa fragment leads to two-electron reduction of the FAD without subsequent reduction of the other two CxxC motifs or the appearance of a thiolate to flavin charge-transfer complex . Further characterization of the fragments and insights gained from the crystal structure of yeast ERV2p (Gross, E., Sevier, C . S., Vala, A., Kaiser, C . A., and Fass, D . (2002) Nat . Struct . Biol . 9, 61-67) suggest that the flow of reducing equivalents in intact avian QSOX is dithiol substrate --> C80/83 --> C519/522 --> C459/462 --> FAD --> oxygen . The ancient fusion of thioredoxin domains to a catalytically more limited ERV domain has produced an efficient catalyst for the direct introduction of disulfide bonds into a wide range of proteins and peptides in multicellular organisms.

Indian J Exp Biol, 2002 Oct, 40(10), 1195 - 7
Depurination of ribosomal RNA and inhibition of viral RNA translation by an antiviral protein of Celosia cristata; Baranwal VK et al.; An antiviral protein (25 kD) isolated from leaves of Celosia cristata (CCP 25) was tested for depurination study on ribosomal RNA from yeast . Ribosomal RNA yielded 360 nucleotide base fragment after treatment with CCP 25 indicating that CCP 25 was a ribosome inactivating protein . CCP 25 also inhibited translation of brome mosaic virus (BMV) and pokeweed mosaic virus (PMV) RNAs in rabbit reticulocyte translation system . The radioactive assay showed that incorporation of {35S}-methionine was less in translation proteins of BMV nucleic acid when CCP 25 was added to translation system . This indicated that antiviral protein from Celosia cristata not only depurinated ribosomal RNA but also inhibited translation of viral RNA in vitro.

DNA Res, 2003 Feb 28, 10(1), 19 - 25
Use of gene networks from full genome microarray libraries to identify functionally relevant drug-affected genes and gene regulation cascades; Savoie CJ et al.; We developed an extensive yeast gene expression library consisting of full-genome cDNA array data for over 500 yeast strains, each with a single-gene disruption . Using this data, combined with dose and time course expression experiments with the oral antifungal agent griseofulvin, whose exact molecular targets were previously unknown, we used Boolean and Bayesian network discovery techniques to determine the gene expression regulatory cascades affected directly by this drug . Using this method we identified CIK1 as an important affected target gene related to the functional phenotype induced by griseofulvin . Cellular functional analysis of griseofulvin showed similar tubulin-specific morphological effects on mitotic spindle formation to those of the drug, in agreement with the known function of CIK1p . Further, using the nonparametric, nonlinear Bayesian gene networks we were able to identify alternative ligand-dependant transcription factors and G protein homologues upstream of CIK1 that regulate CIK1 expression and might therefore serve as alternative molecular targets to induce the same molecular response as griseofulvin.

DNA Res, 2003 Feb 28, 10(1), 1 - 8
Discovery of novel transcription control relationships with gene regulatory networks generated from multiple-disruption full genome expression libraries; Aburatani S et al.; Gene regulatory networks elucidated from strategic, genome-wide experimental data can aid in the discovery of novel gene function information and expression regulation events from observation of transcriptional regulation among genes of known and unknown biological function . To create a reliable and comprehensive data set for the elucidation of transcription regulation networks, we conducted systematic genome-wide disruption expression experiments of yeast on 118 genes with known involvement in transcription regulation . We report several novel regulatory relationships between known transcription factors and other genes with previously unknown biological function discovered with this expression library . Here we report the downstream regulatory subnetworks for UME6 and MET28 . The elucidated network topology among these genes demonstrates MET28's role as a nodal point between genes involved in cell division and those involved in DNA repair mechanisms.

Plant Physiol, 2003 Apr, 131(4), 1756 - 64
Accumulation of ferrous iron in Chlamydomonas reinhardtii . Influence of CO2 and anaerobic induction of the reversible hydrogenase; Semin BK et al.; The green alga, Chlamydomonas reinhardtii, can photoproduce molecular H(2) via ferredoxin and the reversible {Fe}hydrogenase enzyme under anaerobic conditions . Recently, a novel approach for sustained H(2) gas photoproduction was discovered in cell cultures subjected to S-deprived conditions (A . Melis, L . Zhang, M . Forestier, M.L . Ghirardi, M . Seibert {2000} Plant Physiol 122: 127-135) . The close relationship between S and Fe in the H(2)-production process is of interest because Fe-S clusters are constituents of both ferredoxin and hydrogenase . In this study, we used Mossbauer spectroscopy to examine both the uptake of Fe by the alga at different CO(2) concentrations during growth and the influence of anaerobiosis on the accumulation of Fe . Algal cells grown in media with (57)Fe(III) at elevated (3%, v/v) CO(2) concentration exhibit elevated levels of Fe and have two comparable pools of the ion: (a) Fe(III) with Mossbauer parameters of quadrupole splitting = 0.65 mm s(-1) and isomeric shift = 0.46 mm s(-1) and (b) Fe(II) with quadrupole splitting = 3.1 mm s(-1) and isomeric shift = 1.36 mm s(-1) . Disruption of the cells and use of the specific Fe chelator, bathophenanthroline, have demonstrated that the Fe(II) pool is located inside the cell . The amount of Fe(III) in the cells increases with the age of the algal culture, whereas the amount of Fe(II) remains constant on a chlorophyll basis . Growing the algae under atmospheric CO(2) (limiting) conditions, compared with 3% (v/v) CO(2), resulted in a decrease in the intracellular Fe(II) content by a factor of 3 . Incubating C . reinhardtii cells, grown at atmospheric CO(2) for 3 h in the dark under anaerobic conditions, not only induced hydrogenase activity but also increased the Fe(II) content in the cells up to the saturation level observed in cells grown aerobically at high CO(2) . This result is novel and suggests a correlation between the amount of Fe(II) cations stored in the cells, the CO(2) concentration, and anaerobiosis . A comparison of Fe-uptake results with a cyanobacterium, yeast, and algae suggests that the intracellular Fe(II) pool in C . reinhardtii may reside in the cell vacuole.

Plant Physiol, 2003 Apr, 131(4), 1511 - 7
Phloem-localizing sulfate transporter, Sultr1;3, mediates re-distribution of sulfur from source to sink organs in Arabidopsis; Yoshimoto N et al.; For the effective recycling of nutrients, vascular plants transport pooled inorganic ions and metabolites through the sieve tube . A novel sulfate transporter gene, Sultr1;3, was identified as an essential member contributing to this process for redistribution of sulfur source in Arabidopsis . Sultr1;3 belonged to the family of high-affinity sulfate transporters, and was able to complement the yeast sulfate transporter mutant . The fusion protein of Sultr1;3 and green fluorescent protein was expressed by the Sultr1;3 promoter in transgenic plants, which revealed phloem-specific expression of Sultr1;3 in Arabidopsis . Sultr1;3-green fluorescent protein was found in the sieve element-companion cell complexes of the phloem in cotyledons and roots . Limitation of external sulfate caused accumulation of Sultr1;3 mRNA both in leaves and roots . Movement of (35)S-labeled sulfate from cotyledons to the sink organs was restricted in the T-DNA insertion mutant of Sultr1;3 . These results provide evidence that Sultr1;3 transporter plays an important role in loading of sulfate to the sieve tube, initiating the source-to-sink translocation of sulfur nutrient in Arabidopsis.

Bioinformatics, 2003 Apr 12, 19(6), 756 - 63
Construction of reliable protein-protein interaction networks with a new interaction generality measure; Saito R et al.; MOTIVATION: Recent screening techniques have made large amounts of protein-protein interaction data available, from which biologically important information such as the function of uncharacterized proteins, the existence of novel protein complexes, and novel signal-transduction pathways can be discovered . However, experimental data on protein interactions contain many false positives, making these discoveries difficult . Therefore computational methods of assessing the reliability of each candidate protein-protein interaction are urgently needed . RESULTS: We developed a new 'interaction generality' measure (IG2) to assess the reliability of protein-protein interactions using only the topological properties of their interaction-network structure . Using yeast protein-protein interaction data, we showed that reliable protein-protein interactions had significantly lower IG2 values than less-reliable interactions, suggesting that IG2 values can be used to evaluate and filter interaction data to enable the construction of reliable protein-protein interaction networks.

Cancer Lett, 2003 Apr 10, 193(1), 41 - 7
Down-regulation in human cancers of DRHC, a novel helicase-like gene from 17q25.1 that inhibits cell growth; Nagai H et al.; Frequent observations of allelic loss in chromosomal band 17q25.1 in a variety of human cancers have suggested that one or more tumor suppressor genes are normally present in this region . Moreover, a locus responsible for hereditary focal non-epidermolytic palmoplantar keratoderma (tylosis oesophageal cancer; TOC), a condition associated with esophageal cancer, has been mapped to the same band . During efforts to sequence, by shot-gun methods, a 1 Mb target region that we had defined as the DNA segment harboring the putative tumor suppressor gene(s) involved in these events, we identified a novel cDNA, DRHC (down-regulated in human cancers), that showed reduced expression in 28 of 95 (29%) cell lines derived from a variety of human cancers . The full-length cDNA, 6275 bp long, was expressed predominantly in thymus and brain . The predicted 1942-amino-acid product exhibited significant sequence homology to yeast enzymes belonging to the DEAD-helicase superfamily, and appeared to be a Uvr/Rep helicase with a DEXDc consensus domain . Transfection of a DRHC expression vector inhibited growth of cancer cells in liquid medium or soft agar . The results suggest that loss of expression of DRHC may play a role in human carcinogenesis.

Gene Expr, 2003, 11(1), 13 - 21
Modulation of splicing events in histone deacetylase 3 by various extracellular and signal transduction pathways; Gray SG et al.; Within the context of the chromatin environment histone deacetylases are important transcriptional regulators . Three classes of human histone deacetylases have currently been identified on the basis of their similarity to yeast proteins . The class I enzymes contain four members: HDACs 1-3 and HDAC8 . Of these, HDAC3 is known to generate transcript variants with altered amino-terminal regions . Here we describe the identification of a novel splice variant of HDAC3, in which exon 3 is alternatively spliced from the messenger RNA transcript . We show that this human HDAC3 splice transcript is upregulated by treatments with histone deacetylase inhibitors . We also demonstrate evidence of splicing events in murine HDAC3 as a response to various signals, including switching between splice transcript isoforms following treatments with kinase inhibitors or by osmotic shock . In contrast, such switching events were not observed in human cells . These results indicate that differential pathways in mouse and human may control the regulation of HDAC3, and that splice variants may play important roles in responding to exogenous stimuli that act via signal transduction pathways.

Mycoses, 2002, 45 Suppl 3, 22 - 6
{Candida pneumonia in patients without definitive immunodeficiency}; Blaschke S et al.; The occurrence of community-acquired pneumonia due to yeast-like fungi of the genus Candida in patients without manifest immunodeficiency has previously been discounted . However, such pneumonias may indeed occur in patients with chronic parenchymal lung damage, e.g . from nicotine . Candida pneumonia can be triggered in these patients for example by trivial viral infections . Three corresponding cases are discussed.

J Biol Chem, 2003 Jun 27, 278(26), 23915 - 21 Epub 2003 Apr 10.
CD47 and the 19 kDa interacting protein-3 (BNIP3) in T cell apoptosis; Lamy L et al.; CD47 is a surface receptor that induces either coactivation or apoptosis in lymphocytes, depending on the ligand(s) bound . Interestingly, the apoptotic pathway is independent of caspase activation and cytochrome c release and is accompanied by early mitochondrial dysfunction with suppression of mitochondrial membrane potential (Deltapsim) . Using CD47 as bait in a yeast two-hybrid system, we identified the Bcl-2 homology 3 (BH3)-only protein 19 kDa interacting protein-3 (BNIP3), a pro-apoptotic member of the Bcl-2 family, as a novel partner . Interaction between CD47 and the BH3-only protein was confirmed by immunoprecipitation analysis, and CD47-induced apoptosis was inhibited by attenuating BNIP3 expression with antisense oligonucleotides . Finally, we showed that the C-terminal domain of thrombospondin-1 (TSP-1), but not signal-regulatory protein (SIRPalpha1), is the ligand for CD47 involved in inducing cell death . Immunofluorescence analysis of CD47 and BNIP3 revealed a partial colocalization of both molecules under basal conditions . After T cell stimulation via CD47, BNIP3 translocates to the mitochondria to induce apoptosis . These results show that the BH3-dependent apoptotic pathways, previously shown to be activated by intracellular pro-apoptotic events, can also be turned on by surface receptors . This new pathway results in a fast induction of cell death resembling necrosis, which is likely to play an important role in lymphocyte regulation at inflammatory sites and/or in the vicinity of thrombosis.

Diabetes Metab, 2002 Dec, 28(6 Pt 2), 3S25 - 8; discussion 3S108-12
Expression of a thioredoxin peroxidase in insulin-producing cells; Boschero AC et al.; The presence of thioredoxin peroxidase (TPx), also known as thiol specific antioxidant (TSA), was investigated in neonatal and adult rat islets, and in the beta-cell line HIT-T15 . Western blotting of extracts from neonatal and adult pancreatic islets and from the tumoral cell line HIT-T15 revealed the presence of a 25 kDa protein that comigrated with purified yeast TPx . Endocrine pancreatic TPx accounted for approximately 0.01% of the total protein content . Treatment with H2O2 for 3 h increased the expression of TPx in HIT-T15 cells . The distribution of TPx throughout the islet cells was confirmed by immunocytochemistry . Since pancreatic beta-cells possess a weak antioxidant enzyme defense system, especially with regard to hydrogen peroxidase-decomposing enzymes, the presence of a TPx analog in islets suggests that this enzyme may play a role in protecting pancreatic cells against reactive oxygen species.

J Clin Microbiol, 2003 Apr, 41(4), 1753 - 5
Molecular detection of Histoplasma capsulatum var . capsulatum in human clinical samples; Bracca A et al.; We developed a seminested PCR for the diagnosis of histoplasmosis that amplifies a portion of the Histoplasma capsulatum H antigen gene . This assay is highly sensitive and specific, being able to detect genomic material corresponding to less than 10 yeast cells without cross-reaction against other bacterial or fungal pathogens.

FEBS Lett, 2003 Apr 10, 540(1-3), 77 - 80
Interaction of Sedlin with chloride intracellular channel proteins; Fan L et al.; Sedlin is an evolutionarily conserved protein encoded by the causative gene SEDL for spondyloepiphyseal dysplasia tarda . Nevertheless, how Sedlin mutations cause the disease remains unknown . Here, the intracellular chloride channel protein CLIC1 was shown to associate with Sedlin by yeast two-hybrid screening . Green fluorescence protein-CLIC1 readily co-immunoprecipitated with FLAG-Sedlin . In addition, both proteins colocalized extensively in cytoplasmic vesicular/reticular structures in COS-7 cells, suggesting their interaction at intracellular membranous organelles . Sedlin also associated with CLIC2 in yeast two-hybrid assays . The link between Sedlin and the intracellular chloride channels is the first step to understand their functional interplays .

Oncogene, 2003 Apr 10, 22(14), 2110 - 20
The mammalian mismatch repair protein MSH2 is required for correct MRE11 and RAD51 relocalization and for efficient cell cycle arrest induced by ionizing radiation in G2 phase; Franchitto A et al.; In yeast, MSH2 plays an important role in mismatch repair (MMR) and recombination, whereas the function of the mammalian MSH2 protein in recombinational repair is not completely established . We examined the cellular responses of MSH2-deficient mouse cells to X-rays to clarify the role of MSH2 in recombinational repair . Cell survival, checkpoint functions and relocalization of the recombination-related proteins MRE11 and RAD51 were analysed in embryonic fibroblasts derived from MSH2(+/+) and MSH2(-/-) mice, and in MSH2-proficient and deficient mouse colorectal carcinoma cells . Loss of MSH2 function was found to be associated with reduction in cell survival following radiation, absence of either MRE11 or RAD51 relocalization and a higher level of X-ray-induced chromosomal damage specifically in G2-phase cells . Finally, MSH2(-/-) cells showed an inefficient early G2/M checkpoint, being arrested only transiently after irradiation before progressing into mitosis . Consistent with the premature release from the G2-phase arrest, activation of CHK1 was transient and CHK2 was not phosphorylated in synchronized MSH2-null cells . Our data suggest that an active MSH2 is required for a correct response to ionizing radiation-induced DNA damage in the G2 phase of the cell cycle, possibly connecting DSB repair to checkpoint signalling.

Mol Biol Cell, 2003 Apr, 14(4), 1638 - 51
Human MPS1 kinase is required for mitotic arrest induced by the loss of CENP-E from kinetochores; Liu ST et al.; We have determined that the previously identified dual-specificity protein kinase TTK is the human orthologue of the yeast MPS1 kinase . Yeast MPS1 (monopolar spindle) is required for spindle pole duplication and the spindle checkpoint . Consistent with the recently identified vertebrate MPS1 homologues, we found that hMPS1 is localized to centrosomes and kinetochores . In addition, hMPS1 is part of a growing list of kinetochore proteins that are localized to nuclear pores . hMPS1 is required by cells to arrest in mitosis in response to spindle defects and kinetochore defects resulting from the loss of the kinesin-like protein, CENP-E . The pattern of kinetochore localization of hMPS1 in CENP-E defective cells suggests that their interaction with the kinetochore is sensitive to microtubule occupancy rather than kinetochore tension . hMPS1 is required for MAD1, MAD2 but not hBUB1, hBUBR1 and hROD to bind to kinetochores . We localized the kinetochore targeting domain in hMPS1 and found that it can abrogate the mitotic checkpoint in a dominant negative manner . Last, hMPS1 was found to associate with the anaphase promoting complex, thus raising the possibility that its checkpoint functions extend beyond the kinetochore.

J Biol Chem, 2003 Jun 20, 278(25), 23163 - 70 Epub 2003 Apr 09.
X-ray absorption spectroscopy of the copper chaperone HAH1 reveals a linear two-coordinate Cu(I) center capable of adduct formation with exogenous thiols and phosphines; Ralle M et al.; The human copper chaperone HAH1 transports copper to the Menkes and Wilson proteins, which are copper-translocating P-type ATPases located in the trans-Golgi apparatus and believed to provide copper for important enzymes such as ceruloplasmin, tyrosinase, and peptidylglycine monooxygenase . Although a substantial amount of structural data exist for HAH1 and its yeast and bacterial homologues, details of the copper coordination remain unclear and suggest the presence of two protein-derived cysteine ligands and a third exogenous thiol ligand . Here we report the preparation and reconstitution of HAH1 with Cu(I) using a protocol that minimizes the use of thiol reagents believed to be the source of the third ligand . We show by x-ray absorption spectroscopy that this reconstitution protocol generates an occupied Cu(I) binding site with linear biscysteinate coordination geometry, as evidenced by (i) an intense edge absorption centered at 8982.5 eV, with energy and intensity identical to the rigorously linear two-coordinate model complex bis-2,3,5,6-tetramethylbenzene thiolate Cu(I) and (ii) an EXAFS spectrum that could be fit to two Cu-S interactions at 2.16 A, a distance typical of digonal Cu(I) coordination . Binding of exogenous ligands (GSH, dithiothreitol, and tris-(2-carboxyethyl)-phosphine) to the Cu(I) was investigated . When GSH or dithiothreitol was added to the chaperone during the reconstitution procedure, the resulting Cu(I)- HAH1 remained two-coordinate, whereas the addition of the phosphine during reconstitution elicited a three-coordinate species . When the exogenous ligands were titrated into the Cu(I)-HAH1, all formed three-coordinate adducts but with differing affinities . Thus, GSH and dithiothreitol showed weaker binding, with estimated KD values in the range 10-25 mm, whereas tris-(2-carboxyethyl)-phosphine showed stronger affinity, with a KD value of <5 mm . The implications of these findings for mechanisms of copper transport are discussed.

Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 30 - 5
Reaction mechanism and regulation of cystathionine beta-synthase; Banerjee R et al.; In mammals, cystathionine beta-synthase catalyzes the first step in the transsulfuration pathway which provides an avenue for the conversion of the essential amino acid, methionine, to cysteine . Cystathionine beta-synthase catalyzes a PLP-dependent condensation of serine and homocysteine to cystathionine and is unique in also having a heme cofactor . In this review, recent advances in our understanding of the kinetic mechanism of the yeast and human enzymes as well as pathogenic mutants of the human enzyme and insights into the role of heme in redox sensing are discussed from the perspective of the crystal structure of the catalytic core of the human enzyme.

J Biol Chem, 2003 Jul 4, 278(27), 24688 - 96 Epub 2003 Apr 08.
Isoform heterogeneity of the human gephyrin gene (GPHN), binding domains to the glycine receptor, and mutation analysis in hyperekplexia; Rees MI et al.; Gephyrin (GPHN) is an organizational protein that clusters and localizes the inhibitory glycine (GlyR) and GABAA receptors to the microtubular matrix of the neuronal postsynaptic membrane . Mice deficient in gephyrin develop a hereditary molybdenum cofactor deficiency and a neurological phenotype that mimics startle disease (hyperekplexia) . This neuromotor disorder is associated with mutations in the GlyR alpha1 and beta subunit genes (GLRA1 and GLRB) . Further genetic heterogeneity is suspected, and we hypothesized that patients lacking mutations in GLRA1 and GLRB might have mutations in the gephyrin gene (GPHN) . In addition, we adopted a yeast two-hybrid screen, using the GlyR beta subunit intracellular loop as bait, in an attempt to identify further GlyR-interacting proteins implicated in hyperekplexia . Gephyrin cDNAs were isolated, and subsequent RT-PCR analysis from human tissues demonstrated the presence of five alternatively spliced GPHN exons concentrated in the central linker region of the gene . This region generated 11 distinct GPHN transcript isoforms, with 10 being specific to neuronal tissue . Mutation analysis of GPHN exons in hyperekplexia patients revealed a missense mutation (A28T) in one patient causing an amino acid substitution (N10Y) . Functional testing demonstrated that GPHNN10Y does not disrupt GlyR-gephyrin interactions or collybistininduced cell-surface clustering . We provide evidence that GlyR-gephyrin binding is dependent on the presence of an intact C-terminal MoeA homology domain . Therefore, the N10Y mutation and alternative splicing of GPHN transcripts do not affect interactions with GlyRs but may affect other interactions with the cytoskeleton or gephyrin accessory proteins.

J Mol Biol, 2003 Apr 18, 328(1), 147 - 56
The Caenorhabditis elegans homologue of Down syndrome critical region 1, RCN-1, inhibits multiple functions of the phosphatase calcineurin; Lee JI et al.; A conserved family of calcineurin-regulating proteins whose members have been implicated in several disease models such as Down syndrome, Alzheimer's disease, and cardiac hypertrophy has been identified in several organisms including yeast, mice, and humans . We have characterized Caenorhabditis elegans rcn-1, which belongs to this family of calcineurin regulators, and shows approximately 40% identity with the human homologue DSCR-1 . rcn-1 is expressed in hypodermal cells, nerve cords and various neurons, vulva epithelial and muscle cells, marginal cells of the pharynx, and structures of the male tail . rcn-1 expression is upregulated by calcineurin activity . RCN-1 binds to calcineurin A from C.elegans lysate in a calcium-dependent manner, and inhibits bovine calcineurin phosphatase activity dose-dependently . In addition, overexpression of RCN-1 results in calcineurin-deficient phenotypes such as small body size, cuticle defects, fertility defects, slow growth, and serotonin-resistant egg-laying defects . Moreover, phenotypes observed in gain-of-function calcineurin mutant animals were restored to normal by RCN-1 overexpression . These results demonstrate an effective and specific inhibition of calcineurin in vitro as well as in vivo by RCN-1 .

Reproduction, 2003 Apr, 125(4), 447 - 56
Mining meiosis and gametogenesis with DNA microarrays; Schlecht U et al.; Gametogenesis is a key developmental process that involves complex transcriptional regulation of numerous genes including many that are conserved between unicellular eukaryotes and mammals . Recent expression-profiling experiments using microarrays have provided insight into the co-ordinated transcription of several hundred genes during mitotic growth and meiotic development in budding and fission yeast . Furthermore, microarray-based studies have identified numerous loci that are regulated during the cell cycle or expressed in a germ-cell specific manner in eukaryotic model systems like Caenorhabditis elegans, Mus musculus as well as Homo sapiens . The unprecedented amount of information produced by post-genome biology has spawned novel approaches to organizing biological knowledge using currently available information technology . This review outlines experiments that contribute to an emerging comprehensive picture of the molecular machinery governing sexual reproduction in eukaryotes.

J Cell Sci, 2003 May 15, 116(Pt 10), 2067 - 72 Epub 2003 Apr 01.
A hydrophilic lamin-binding domain from the Drosophila YA protein can target proteins to the nuclear envelope; Mani SS et al.; The nuclear lamina provides an architectural framework for the nuclear envelope and an attachment site for interphase chromatin . In Drosophila eggs and early embryos its major constituent, lamin Dm0, interacts with a lamina protein called YA . When the lamin-interaction region of YA is deleted, YA still enters nuclei but fails to localize to nuclear envelopes, suggesting that lamin interaction targets YA to the nuclear envelope . Here, we show that C-terminal lamin-interacting region of YA is sufficient to target the heterologous soluble protein GFP-NLS to the nuclear periphery in Drosophila tissue culture cells . Yeast two-hybrid analysis and transient transfection assays further defined this domain: residues 556-696 of YA are sufficient for both lamin Dm0 interaction and the targeting of GFP-NLS to the nuclear periphery . This region of YA is hydrophilic and lacks any transmembrane domain or known membrane-targeting motifs . We propose that the localization of YA to the nuclear lamina involves interaction with polymerized lamin Dm0 mediated by the lamin-targeting domain of YA . This hydrophilic YA domain might provide a useful molecular tool for targeting heterologous non-membrane-associated proteins to the nuclear envelope.

J Biol Chem, 2003 Jun 6, 278(23), 20821 - 7 Epub 2003 Apr 04.
Kinetic analysis of the interaction of the copper chaperone Atox1 with the metal binding sites of the Menkes protein; Strausak D et al.; Excess copper is effluxed from mammalian cells by the Menkes or Wilson P-type ATPases (MNK and WND, respectively) . MNK and WND have six metal binding sites (MBSs) containing a CXXC motif within their N-terminal cytoplasmic region . Evidence suggests that copper is delivered to the ATPases by Atox1, one of three cytoplasmic copper chaperones . Attempts to monitor a direct Atox1-MNK interaction and to determine kinetic parameters have not been successful . Here we investigated interactions of Atox1 with wild-type and mutated pairs of the MBSs of MNK using two different methods: yeast two-hybrid analysis and real-time surface plasmon resonance (SPR) . A copper-dependent interaction of Atox1 with the MBSs of MNK was observed by both approaches . Cys to Ser mutations of conserved CXXC motifs affected the binding of Atox1 underlining the essentiality of Cys residues for the copper-induced interaction . Although the yeast two-hybrid assay failed to show an interaction of Atox1 with MBS5/6, SPR analysis clearly demonstrated a copper-dependent binding with all six MBSs highlighting the power and sensitivity of SPR as compared with other, more indirect methods like the yeast two-hybrid system . Binding constants for copper-dependent chaperone-MBS interactions were determined to be 10-5-10-6 m for all the MBSs representing relatively low affinity binding events . The interaction of Atox1 with pairs of the MBSs was non-cooperative . Therefore, a functional difference of the MBSs in the MNK N terminus cannot be attributed to cooperativity effects or varying affinities of the copper chaperone Atox1 with the MBSs.

Curr Med Chem, 2003 May, 10(9), 741 - 8
Nucleo-cytoplasmic transport of proteins as a target for therapeutic drugs; Yashiroda Y et al.; Recruitment of cytoplasmic signaling proteins into the nucleus is an essential step in the activation of gene expression in response to an extracellular signal . Nucleo-cytoplasmic transport of macromolecules is mediated by the transport receptors of an importin beta family . Post-translational modifications and masking/unmasking of specific signal sequences responsible for nuclear import and export are important for the coordinated control of the nucleo-cytoplasmic transport . Malfunctioning of the nucleo-cytoplasmic transport is profoundly involved in a number of diseases including cancer . Leptomycin B (LMB) is a Streptomyces metabolite that causes specific inhibition of the cell cycle of fission yeast and mammalian cells . The target molecule of LMB has been shown by genetic and biochemical analyses to be CRM1, a highly conserved protein in eukaryotes . CRM1 was shown to be a member of the importin beta family and a receptor for the nuclear export signal (NES) of proteins in both yeast and mammalian cells . LMB binds directly to CRM1, which results in dissociation of the NES from the nuclear export machinery containing CRM1 . Thus, LMB serves as a potent tool for understanding the molecular mechanisms of nucleo-cytoplasmic transport of proteins and a potential therapeutic drug for diseases caused by mislocalization of regulatory proteins.

Curr Med Chem Anti-Canc Agents, 2002 Jul, 2(4), 553 - 66
Cross-talk between cellular stress, cell cycle and anticancer agents: mechanistic aspects; Tiligada E et al.; Environmental conditions such as temperature, radiation, hypoxia, nutrients and drugs stimulate the adaptive sensory and signaling machinery of the cell . This stress response may influence cell cycle regulation, cellular differentiation, oncogenic transforma-tion, cell survival and apoptosis . The impact of the cytoprotective reprogramming in cancer pharmacology is presented by the recent extensive literature regarding the interplay between stress tolerance and anticancer drug effectiveness and resistance, relying on the dominating intrinsic pathways, which are simultaneously activated and regulate the death process either positively or negatively . This review presents the data that argue for the emergence of either common or specific mechanisms depending on the type, duration and severity of stress in all eukaryotic organisms from yeast to mammals . The understanding of the complexity and the balance between noxious and protective signal transduction pathways would contribute to a more explicit evaluation of the current therapeutic regiments and to the development of new leads targeting malignancy.

Cell Mol Life Sci, 2003 Feb, 60(2), 401 - 12
Novel complexes of cyclin-dependent kinases and a cyclin-like protein from Arabidopsis thaliana with a function unrelated to cell division; Barroco RM et al.; Although the majority of cyclin-dependent kinases (CDKs) play a key role in cell cycle progression, recent evidence has shown that CDKs are also implicated in transcription regulation . Here, we describe two Arabidopsis CDKs designated Arath;CDKC;1 and Arath; CDKC;2 . These CDKs share a PITAIRE signature in the cyclin-binding domain and the structural characteristics of mammalian CDK9 . Yeast two-hybrid screens and immunoprecipitation assays identified CDKC-interacting proteins with homology to the animal cyclin T/cyclin K group . We suggest that these Arabidopsis CDKCs may be part of a kinase complex similar to the animal positive transcription elongation factor b, whose activity is essential for transcription control . Expression studies showed that Arath; CDKC transcripts are mainly confined to epidermal tissues and are most abundant in flower tissues . No expression was detected in actively dividing Arabidopsis tissues, suggesting a role for the CDKC proteins in differentiated cells.

Mol Endocrinol, 2003 Jul, 17(7), 1216 - 29 Epub 2003 Apr 03.
The Formin family protein, formin homolog overexpressed in spleen, interacts with the insulin-responsive aminopeptidase and profilin IIa; Tojo H et al.; Insulin stimulates translocation of glucose transporter isoform type 4 (GLUT4) and the insulin-responsive aminopeptidase (IRAP) from an intracellular storage pool to the plasma membrane in muscle and fat cells . A role for the cytoskeleton in insulin action has been postulated, and the insulin signaling pathway has been well investigated; however, the molecular mechanism by which GLUT4/IRAP-containing vesicles move from an interior location to the cell surface in response to insulin is incompletely understood . Here, we have screened for IRAP-binding proteins using a yeast two-hybrid system and have found that the C-terminal domain of FHOS (formin homolog overexpressed in spleen) interacts with the N-terminal cytoplasmic domain of IRAP . FHOS is a member of the Formin/Diaphanous family of proteins that is expressed most abundantly in skeletal muscle . In addition, there are two novel types of FHOS transcripts generated by alternative mRNA splicing . FHOS78 has a 78-bp insertion and it is expressed mainly in skeletal muscle where it may be the most abundant isoform in humans . The ubiquitously expressed FHOS24 has a 24-bp insertion encoding an in-frame stop codon that results in a truncated polypeptide . It is known that some formin family proteins interact with the actin-binding profilin proteins . Both FHOS and FHOS78 bound to profilin IIa via their formin homology 1 domains, but neither bound profilin I or IIb . Overexpression of FHOS and FHOS78 resulted in enhanced insulin-stimulated glucose uptake in L6 cells to similar levels . However, overexpression of FHOS24, lacking the IRAP-binding domain, did not affect insulin-stimulated glucose uptake . These findings suggest that FHOS mediates an interaction between GLUT4/IRAP-containing vesicles and the cytoskeleton and may participate in exocytosis and/or retention of this membrane compartment.

Genomics, 2003 Apr, 81(4), 349 - 55
Hypermutable minisatellites, a human affair?
Bois PR.
Minisatellites are a class of highly polymorphic GC-rich tandem repeats . They include some of the most variable loci in the human genome, with mutation rates ranging from 0.5% to >20% per generation . Structurally, they consist of 10- to 100-bp intermingled variant repeats, making them ideal tools for dissecting mechanisms of instability at tandem repeats . Distinct mutation processes generate rare intra-allelic somatic events and frequent complex conversion-like germline mutations in these repeats . Furthermore, turnover of repeats at human minisatellites is controlled by intense recombinational activity in DNA flanking the repeat array . Surprisingly, whereas other mammalian genomes possess minisatellite-like sequences, hypermutable loci have not been identified that suggest human-specific turnover processes at minisatellite arrays . Attempts to transfer minisatellite germline instability to the mouse have failed . However, yeast models are now revealing valuable information regarding the mechanisms regulating instability at these tandem repeats . Finally, minisatellites and tandem repeats provide exquisitely sensitive molecular tools to detect genomic insults such as ionizing radiation exposure . Surprisingly, by a mechanism that remains elusive, there are transgenerational increases in minisatellite instability.

J Invertebr Pathol, 2003 Mar, 82(3), 139 - 47
Characterization of Beauveria bassiana and Metarhizium anisopliae isolates for management of tarnished plant bug, Lygus lineolaris (Hemiptera: Miridae); Liu H et al.; Selected morphological and physiological characteristics of four Beauveria bassiana (Balsamo) Vuillemin isolates and one Metarhizium anisopliae (Metschnikoff) Sorokin isolate, which are highly pathogenic to Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), were determined . There were significant differences in conidial size, viability, spore production, speed of germination, relative hyphal growth, and temperature sensitivity . Spore viability after incubation for 24h at 20 degrees C ranged from 91.4 to 98.6% for the five isolates tested . Spore production on quarter-strength Sabouraud dextrose agar plus 0.25% (w/v) yeast extract after 10 days incubation at 20 degrees C ranged from 1.6x10(6) to 15.5x10(6)conidia/cm(2) . One B . bassiana isolate (ARSEF 1394) produced significantly more conidia than the others . Spore germination was temperature-dependant for both B . bassiana and M . anisopliae . The time required for 50% germination (TG(50)) ranged from 25.0 to 30.9, 14.0 to 16.6, and 14.8 to 18.0h at 15, 22, and 28 degrees C, respectively . Only the M . anisopliae isolate (ARSEF 3540) had significant spore germination at 35 degrees C with a TG(50) of 11.8h . A destructive sampling method was used to measure the relative hyphal growth rate among isolates . Exposure to high temperature (40-50 degrees C) for 10min had a negative effect on conidial viability . The importance of these characteristics in selecting fungal isolates for management of L . lineolaris is discussed.

Mol Cell Neurosci, 2003 Feb, 22(2), 271 - 83
Association of the type 1 inositol (1,4,5)-trisphosphate receptor with 4.1N protein in neurons; Maximov A et al.; The type 1 inositol (1,4,5)-trisphosphate receptor (InsP(3)R1) is an intracellular calcium (Ca(2+)) release channel that plays an important role in neuronal function . In yeast two-hybrid screen of rat brain cDNA library with the InsP(3)R1 carboxy-terminal bait we isolated multiple clones of neuronal cytoskeletal protein 4.1N . We mapped the 4.1N-interaction site to a short fragment (50 amino acids) within the carboxy-terminal tail of the InsP(3)R1 and the InsP(3)R1-interaction site to the carboxy-terminal domain (CTD) of 4.1N . We established that InsP(3)R1 carboxy-terminal binds selectively to the CTDDelta alternatively spliced form of the 4.1N protein . In biochemical experiments we demonstrated that 4.1N and InsP(3)R1 specifically associate in vitro . We showed that both 4.1N and InsP(3)R1 were enriched in synaptic locations and immunoprecipitated the 4.1N-InsP(3)R1 complex from rat brain synaptosomes . In biochemical experiments we demonstrated a possibility of InsP(3)R1-4.1N-CASK-syndecan-2 quaternary complex formation . From our findings we hypothesize that InsP(3)R1-4.1N association may play a role in InsP(3)R1 localization or Ca(2+) signaling in neurons.

Int J Biochem Cell Biol, 2003 Jun, 35(6), 992 - 1002
Endocytosis of the AT1A angiotensin receptor is independent of ubiquitylation of its cytoplasmic serine/threonine-rich region; Mihalik B et al.; Agonist-induced internalisation of the rat type 1A (AT(1A)) angiotensin II receptor is associated with phosphorylation of a serine/threonine-rich region in its cytoplasmic tail . In yeast, hyperphosphorylation of the alpha-factor pheromone receptor regulates endocytosis of the receptor by facilitating the monoubiquitylation of its cytoplasmic tail on lysine residues . The role of receptor ubiquitylation in AT(1A) receptor internalisation was evaluated by deletion or replacement of lysine residues in its agonist-sensitive serine/threonine-rich region . Expression of such receptor mutants in CHO cells showed that these modifications had no detectable effect on the angiotensin II-induced endocytosis of the AT(1A) receptor . Furthermore, fusion of ubiquitin in-frame to an internalisation-deficient AT(1A) receptor mutant with a truncated carboxyl-terminal tail did not restore the endocytosis of the resulting chimeric receptor . No impairment of receptor internalisation was observed after substitution of all lysine residues in the serine/threonine-rich region at saturating angiotensin II concentrations, where endocytosis occurs by a beta-arrestin and dynamin independent mechanism . Taken together, these data demonstrate that ubiquitylation of the cytoplasmic serine/threonine-rich region of the AT(1A) receptor on lysine residues is not required for its agonist-induced internalisation, and suggest that endocytosis of mammalian G protein-coupled receptors (GPCRs) occurs by a different mechanism than that of yeast GPCRs.

Curr Biol, 2003 Apr 1, 13(7), 590 - 7
S . pombe aurora kinase/survivin is required for chromosome condensation and the spindle checkpoint attachment response; Petersen J et al.; The spindle checkpoint inhibits anaphase until all chromosomes have established bipolar attachment . Two kinetochore states trigger this checkpoint . The absence of microtubules activates the attachment response, while the inability of attached microtubules to generate tension triggers the tension/orientation response . The single aurora kinase of budding yeast, Ipl1, is required for the tension/orientation, but not attachment, response . In contrast, we find that the single aurora kinase of fission yeast, Ark1, is required for the attachment response . Having established that the initiator codon assigned to ark1(+) was incorrect and that Ark1-associated kinase activity depended upon survivin function and phosphorylation, we found that the loss of Ark1 from kinetochores by either depletion or use of a survivin mutant overides the checkpoint response to microtubule depolymerization . Ark1/survivin function was not required for the association of Bub1 or Mad3 with the kinetochores . However, it was required for two aspects of Mad2 function that accompany checkpoint activation: full-scale association with kinetochores and formation of a complex with Mad3 . Neither the phosphorylation of histone H3 that accompanies chromosome condensation nor condensin recruitment to mitotic chromatin were seen when Ark1 function was compromised . Cytokinesis was not affected by Ark1 depletion or expression of the "kinase dead" ark1.K118R mutant.

Kidney Int, 2003 May, 63(5), 1632 - 44
The Rho family of small GTPases is involved in epithelial cystogenesis and tubulogenesis; Rogers KK et al.; BACKGROUND: Epithelial cyst and tubule formation represent critical processes for the development of many mammalian organs and involve transient, highly choreographed changes in cell polarity . The Rho family of small GTPases, whose prototypes are RhoA, Rac1, and Cdc42, regulate many biologic processes, including cell polarization and morphogenesis . The exocyst is a conserved eight-subunit protein complex involved in the biogenesis of polarity; in yeast, it is a downstream effector for several Rho family proteins, and, in mammals, plays a central role in cystogenesis and tubulogenesis . METHODS: Inducible cell lines expressing mutant forms of RhoA, Rac1, and Cdc42 and an in vitro model of cystogenesis and tubulogenesis were used to examine the effects of Rho family proteins on cyst and tubule formation . A series of pulse-chase assays, using basolateral, apical, and secretory proteins, were performed to examine the synthesis and membrane trafficking profile of the various Rho family mutant proteins . RESULTS: We show that expression of mutant RhoA, Rac1, and Cdc42 proteins all result in abnormal cyst and tubule formation . Furthermore, with respect to cystogenesis and tubulogenesis, the phenotypic effects of expressing each mutant Rho family protein are different . Specifically, cyst and, therefore, tubule formation is completely inhibited in the presence of constitutively active RhoA and tubulogenesis is inhibited in the presence of dominant negative Rac1 . Reversal of cyst polarity is seen in the presence of dominant negative RhoA, dominant negative Rac1, and both dominant negative and constitutively active Cdc42 . The series of synthesis and delivery assays, using basolateral, apical, and secretory proteins, revealed that Rho family mutant proteins display an exocyst-like trafficking profile . CONCLUSION: The differential effects suggest that RhoA, Rac1, and Cdc42 all act to control cyst and tubule formation and may act in concert to control these higher-order processes . The exocyst-like membrane trafficking profile displayed by the Rho family mutant proteins raises the possibility that Rho family proteins interact, either directly or indirectly, with the exocyst to control cyst and tubule formation.

Biol Chem, 2003 Feb, 384(2), 243 - 6
Different isoforms of the non-integrin laminin receptor are present in mouse brain and bind PrP; Simoneau S et al.; The prion protein (PrP) plays a central role in prion diseases, and identifying its cellular receptor appears to be of crucial interest . We previously showed in the yeast two-hybrid system that PrP interacts with the 37 kDa precursor (LRP) of the high affinity 67 kDa laminin receptor (LR), which acts as the cellular receptor of PrP in cellular models . However, among the various isoforms of the receptor that have been identified so far, those which are present in the central nervous system and which bind PrP are still unknown . In this study, we have purified mouse brain fractions enriched in the laminin receptor and have performed overlay assays in order to identify those isoforms that interact with the prion protein . We demonstrate (i) the presence, in mouse brain, of several isoforms of the LRP/LR corresponding to different maturation states of the receptor (44, 60, 67 and 220 kDa) and (ii) the binding of all of these isoforms to PrP . Our data strongly support a physiological role of the laminin receptor/PrP interaction in the brain and highlight its relevance for transmissible spongiform encephalopathies.

Neurochem Res, 2003 Apr, 28(3-4), 551 - 5
Selection and characterization of the choline transport mutation suppressor from Torpedo electric lobe, CTL1; O'Regan S et al.; The presumptive choline transporter, CTL1, was initially identified through functional complementation of a triple yeast mutant (ctr ise URA3delta) with deficiencies in both choline transport and choline neosynthesis under selective conditions that cause perturbations in membrane synthesis and growth . After transformation of these yeasts with a heterologous yeast expression library made from Torpedo electric lobe cDNAs, several colonies showed increased growth but only one clone increased the accumulation of external choline . The corresponding full-length cDNA was isolated and encodes a protein with 10 transmembrane domains . Northern analysis of Torpedo mRNA indicates that CTL1 is expressed at high levels in the spinal cord and brain . In Xenopus oocytes, Torpedo CTL1 expression was associated with the appearance of sodium independent high-affinity choline uptake . We propose that CTL1 plays a role in providing choline for membrane synthesis in the nervous system.

Biol Pharm Bull, 2003 Apr, 26(4), 448 - 52
Activation of the human Ah receptor by aza-polycyclic aromatic hydrocarbons and their halogenated derivatives; Saeki K et al.; Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor through which dioxins and carcinogenic polycyclic aromatic hydrocarbons cause altered gene expression and toxicity . Ten aza-polycyclic aromatic hydrocarbons (aza-PAHs), consisting of nitrogen substituted naphthalenes, phenanthrenes, chrysenes, and benzo{a}pyrenes (BaPs), were subjected to analysis of their structure-activity relationships as an AhR ligand by using a yeast AhR signaling assay, in which AhR ligand activity was evaluated as lacZ units . Most of the aza-PAHs showed similar or more potent AhR ligand activities than the corresponding parent PAHs . About a 100-fold increased in ligand activity was observed in 10-azaBaP compared with BaP . Halogen-substitution effects on AhR ligand activity in aza-polycyclic aromatics were also investigated with quinoline, benzo{f}quinoline (BfQ), benzo{h}quinoline (BhQ) and 1,7-phenanthroline (1,7-Phe) . Position-specific induction of AhR ligand activity was observed in aza-tricyclic aromatic compounds, BfQ, BhQ, and 1,7-Phe, and the ratio of the ligand activities (lacZ units/microM) of monochlorinated and monobrominated aza-tricyclic aromatic compounds to those of the corresponding parent non-halogenated compounds ranged from 2.2- to 254-fold . Greatest enhancement of ligand activity was observed in 2-brominated BfQ (2-Br-BfQ), and its ligand activity was higher than that of BaP . These results suggest that even monohalogenation markedly enhances AhR ligand activity in aza-PAHs.

Proc Natl Acad Sci U S A, 2003 Apr 15, 100(8), 4574 - 9 Epub 2003 Apr 02.
Processing, localization, and requirement of human separase for normal anaphase progression; Chestukhin A et al.; In all eukaryotes, anaphase is triggered by the activation of a protease called separase . Once activated, separase cleaves a subunit of cohesin, a complex that links replicated chromatids before anaphase . Separase and cohesin are conserved from yeasts to humans . Although the machinery for dissolving sister cohesion is conserved, the regulation of this process appears to be more complex in higher eukaryotes than in yeast . Here we report the cloning of full-length human separase cDNA and the characterization of the encoded protein . Human separase was observed at the poles of the mitotic spindle until anaphase, at which time its association with the mitotic spindle was abruptly lost . The dynamic pattern of localization of human separase during cell cycle progression differs from that of fungal separases . Human separase also appears to undergo an autocatalytic processing on anaphase entry . The processed forms of human separase were isolated and the identity of the cleavage sites was determined by N-terminal sequencing and site-directed mutagenesis . The processed catalytic domain was found to be stably associated with the processed N-terminal fragment . Finally, by depletion of endogenous separase with antisense oligonucleotides, we report direct evidence that separase is required for high-fidelity chromosome separation in human cells.

J Biol Chem, 2003 Jun 13, 278(24), 21685 - 92 Epub 2003 Apr 02.
Abl interactor 1 promotes tyrosine 296 phosphorylation of mammalian enabled (Mena) by c-Abl kinase; Tani K et al.; Mammalian Enabled (Mena) is a mammalian homologue of Drosophila Enabled (Ena), which genetically interacts with Drosophila Abl tyrosine kinase . The signaling pathway involving c-Abl and Mena (Ena) is not fully understood . To find molecules that participate in the c-Abl/Mena pathway, we searched for Mena-binding proteins using a yeast two-hybrid system . We identified Abl interactor 1 (Abi-1), which is known to interact with c-Abl, as a binding protein for Mena . Binding analysis revealed that the Ena/Vasp homology 1 domain of Mena and the polyproline structure of Abi-1 are necessary for the interaction . The interaction between Mena and Abi-1 was also observed in a mammalian expression system . Importantly, Abi-1 dramatically promoted c-Abl-mediated tyrosine phosphorylation of Mena but not other substrates such as c-Cbl . Mutational analysis demonstrated that the phosphorylation site of Mena is Tyr-296 . Our results suggest that Abi-1 regulates c-Abl-mediated phosphorylation of Mena by interacting with both proteins.

J Biol Chem, 2003 Jun 13, 278(24), 22136 - 43 Epub 2003 Apr 02.
Novel localization of the DNA-PK complex in lipid rafts: a putative role in the signal transduction pathway of the ionizing radiation response; Lucero H et al.; Increased sensitivity to ionizing radiation (IR) has been shown to be due to defects in DNA double-strand break repair machinery . The major pathway in mammalian cells dedicated to the repair of DNA double-strand breaks is by the nonhomologous end-joining machinery . Six components function in this pathway, of which three (Ku70, Ku86, and DNA-PKcs) constitute a protein complex known as DNA-dependent protein kinase (DNA-PK) . However, it is now recognized that the cellular radiation response is complex, and radiosensitivity may be also regulated at different levels in the radiation signal transduction pathway . In addition to DNA damage, exposure to IR triggers intracellular signaling cascades that overlap with pathways initiated by ligand engagement to a receptor . In this study, we provide evidence for the novel localization of the DNA-PK complex in lipid rafts . We also show this property is not a generalized characteristic of all DNA repair proteins . Furthermore, we have detected Ku86 in yeast lipid rafts . Our results suggest that the components of this complex might be recruited separately to the plasma membrane by tethering with raft-resident proteins . In addition, we found an irradiation-induced differential protein phosphorylation pattern dependent upon DNA-PKcs in lipid rafts . Thus, we speculate that another role for the DNA-PKcs subunit and perhaps for the holoenzyme is in the signal transduction of IR response.

Mol Biochem Parasitol, 2003 Apr 3, 127(2), 113 - 20
Identification of Entamoeba histolytica thiol-specific antioxidant as a GalNAc lectin-associated protein; Hughes MA et al.; Entamoeba histolytica is a human intestinal parasite that causes amebic dysentery . A cell surface amebic adhesin, the galactose and N-acetyl-D-galactosamine inhibitable (GalNAc) lectin mediates amebic adherence to and contact-dependent killing of host cells . Previous work has suggested that the GalNAc lectin transduces signals via protein interactions with its short cytoplasmic domain . We used a yeast two-hybrid system to screen an E . histolytica cDNA library for proteins that interact with the GalNAc lectin cytoplasmic domain . One isolate was the E . histolytica thiol-specific antioxidant (TSA) . TSA is an enzyme that detoxifies hydrogen peroxide . TSA did not interact in yeast two-hybrid experiments with a mutant version of the lectin cytoplasmic domain, confirming the specificity of the lectin-TSA interaction . Furthermore, mutational analyses of the TSA isolate demonstrated that an in-frame five amino acid sequence introduced between amino acids 61-62 yielded a TSA mutant that did not interact with the lectin cytoplasmic domain upon expression in the yeast two-hybrid system . The association of TSA and GalNAc lectin was further supported by co-immunoaffinity purification . Confocal microscopy demonstrated co-localization of TSA and GalNAc lectin at sites of ameba:host cell contact . Recruitment of TSA by the GalNAc lectin suggests a novel mechanism of parasite defense against reactive oxygen intermediates generated by host peripheral mononuclear cells.

EMBO Rep, 2003 Apr, 4(4), 381 - 6
ORFB is a subunit of F1F(O)-ATP synthase: insight into the basis of cytoplasmic male sterility in sunflower; Sabar M et al.; ORFB is the product of a gene that is conserved in plant mitochondrial genomes, and which, on the basis of sequence motif and structural similarity, is predicted to be the homologue of yeast and mammalian ATP8, part of the F(O) component of the F1F(O)-ATP synthase . We have shown that, in sunflower, orfB transcripts are edited, increasing the similarity of the predicted protein to ATP8 proteins from non-plant species . Blue-native polyacrylamide gel electrophoresis and peptide sequencing confirm that ORFB localizes to the ATP synthase complex . The predicted amino-terminal 19 amino acids of ORFB are identical to those in the chimeric mitochondrial ORF522 protein, which is associated with cytoplasmic male sterility (CMS) in sunflower . Assays comparing respiratory complexes from a male-sterile line expressing ORF522 with those from a male-fertile line show a specific decrease in ATP hydrolysis by the ATP synthase . These observations allow us to propose a mechanism underlying CMS that is associated with the expression of chimeric open reading frames containing part of the orfB gene.

J Biol Chem, 2003 Jun 13, 278(24), 21930 - 7 Epub 2003 Apr 01.
The BAL-binding protein BBAP and related Deltex family members exhibit ubiquitin-protein isopeptide ligase activity; Takeyama K et al.; Members of the DTX (Deltex) family act as Notch signaling modifiers and may also regulate transcription through interactions with specific transcription factors . DTX proteins have a basic N terminus; a central proline-rich region; and a C-terminal RING finger domain, a motif often found in ubiquitin-protein isopeptide ligases (E3) . Recently, we identified and characterized a unique diffuse large B-cell lymphoma risk-related gene named BAL (B aggressive lymphoma) . Using a yeast two-hybrid screen for BAL-binding partners, we have now identified a novel protein termed BBAP (B-lymphoma- and BAL-associated protein) . Although BBAP has a unique N terminus, the C-terminal region is highly homologous to that of DTX family members . Herein, we report that BBAP and the human family of DTX proteins (DTX1, DTX2, and DTX3) function as E3 ligases based on their capacity for self-ubiquitination . DTX family members homodimerize and heterodimerize in vivo, suggesting that physical interactions between various DTX family members modify E3 activity and/or substrate availability . Consistent with this idea, BBAP and DTX1 associate via their unique N termini, resulting in enhanced self-ubiquitination.

Int J Parasitol, 2003 Mar, 33(3), 301 - 12
Identification and molecular characterisation of a gene encoding a member of the insulin receptor family in Echinococcus multilocularis; Konrad C et al.; Receptor kinases play a key role in the communication of cells with their environment and could be important mediators of the effects of host cytokines on endoparasitic organisms . In this paper we describe, for the first time, the characterisation of a receptor tyrosine kinase of the insulin receptor family from a parasitic helminth . Using a degenerative PCR approach, we identified and completely characterised the 5.5kb coding DNA for an Echinococcus multilocularis factor (EmIR) which displays significant homologies to insulin receptors of different phylogenetic origin . EmIR exhibited a domain structure which is typical for the protein family and contained all catalytically important residues at corresponding positions . One striking difference between EmIR and known insulin receptors was the presence of a 172 amino acid insert in the tyrosine kinase region of, as yet, unknown function . In yeast two-hybrid analyses, the ligand binding domains of the human insulin receptor and of EmIR showed comparable affinity to human insulin . The EmIR encoding chromosomal locus (emir) was characterised and comprised 16.5kb . Southern blot hybridisations demonstrated that emir is present as a single copy locus in E . multilocularis . Furthermore, structural comparisons indicated that emir and the insulin receptor genes from mammals and insects derive from a common ancestor . Based on reverse transcriptase-polymerase chain reaction analyses, emir was found to be expressed in the two larval stages metacestode and protoscolex . EmIR is, therefore, likely to play an important role in echinococcal development and possibly also in the interaction with the mammalian host.

Biochem Biophys Res Commun, 2003 Apr 11, 303(3), 908 - 13
Interaction between protein phosphatase 2A and members of the importin beta superfamily; Lubert EJ et al.; While performing a yeast two-hybrid library screen to uncover novel PP2A-interacting proteins, we discovered a specific interaction between a member of the importin beta/karyopherin beta superfamily, importin 9, and the A subunit of PP2A (PR65) . This interaction between importin 9 and the A subunit was confirmed by in vitro pulldown, immunoprecipitation, and microcystin-Sepharose chromatography . We also found that another family member, importin beta, interacted specifically with the A subunit of PP2A . Finally, we showed that treatment of cells with a concentration of okadaic acid known to inhibit PP2A impeded the nuclear localization of an NLS-containing protein . These results provide evidence that these importins can exist in a native complex with endogenous PP2A and that this serine/threonine phosphatase plays a role in regulating the nuclear import of NLS-containing proteins in vivo.

Eur J Neurosci, 2003 Mar, 17(6), 1150 - 8
A tripartite motif protein TRIM11 binds and destabilizes Humanin, a neuroprotective peptide against Alzheimer's disease-relevant insults; Niikura T et al.; Humanin (HN) is a newly identified neuroprotective peptide that specifically suppresses Alzheimer's disease (AD)-related neurotoxicity . HN peptide has been detected in the human AD brain as well as in mouse testis and colon by immunoblot and immunohistochemical analyses . By means of yeast two-hybrid screening, we identified TRIM11 as a novel HN-interacting protein . TRIM11, which is a member of protein family containing a tripartite motif (TRIM), is composed of a RING finger domain, which is a putative E3 ubiquitin ligase, a B-box domain, a coiled-coil domain and a B30.2 domain . Deletion of the B30.2 domain in TRIM11 abolished the interaction with HN, whereas the B30.2 domain alone did not interact with HN . For their interaction, at least the coiled-coil domain was indispensable together with the B30.2 domain . The intracellular level of glutathione S-transferase-fused or EGFP-fused HN peptides or plain HN was drastically reduced by the coexpression of TRIM11 . Disruption of the RING finger domain by deleting the first consensus cysteine or proteasome inhibitor treatment significantly diminished the effect of TRIM11 on the intracellular level of HN . These results suggest that TRIM11 plays a role in the regulation of intracellular HN level through ubiquitin-mediated protein degradation pathways.

J Cell Sci, 2003 May 15, 116(Pt 10), 1949 - 57 Epub 2003 Mar 26.
Identification of SAP97 as an intracellular binding partner of TACE; Peiretti F et al.; Tumor necrosis factor alpha converting enzyme (TACE) is the metalloprotease-disintegrin responsible for the ectodomain shedding of several proteins, including tumor necrosis factor alpha . Using the yeast two-hybrid system, we identified the scaffolding protein synapse associated protein 97 (SAP97) as a binding partner of the cytoplasmic domain of TACE . By deletions and site-directed mutagenesis, we demonstrated that this interaction involved the PDZ3 domain of SAP97 and the extreme C-terminal amino-acid sequence of TACE . This interaction as well as the identification of the specific domains involved was confirmed in vitro by affinity purification and in mammalian cells by co-immunoprecipitation and alteration of localization analyzed by immunofluorescence microscopy . In addition, confocal microscopy showed that endogenous TACE and SAP97 colocalized in some intracellular areas of COS-7 cells and CACO-2 cells . Furthermore, overexpression of SAP97, unlike that of a mutant form of SAP97 deleted for its PDZ3 domain, altered the ability of TACE to release its substrates . Altogether, these results demonstrate an interaction between TACE and SAP97, which may have a functional implication for the regulation of TACE shedding activity.

J Cell Biol, 2003 Mar 31, 160(7), 1139 - 50
The cytosolic entry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex; Ratts R et al.; In vitro delivery of the diphtheria toxin catalytic (C) domain from the lumen of purified early endosomes to the external milieu requires the addition of both ATP and a cytosolic translocation factor (CTF) complex . Using the translocation of C-domain ADP-ribosyltransferase activity across the endosomal membrane as an assay, the CTF complex activity was 650-800-fold purified from human T cell and yeast extracts, respectively . The chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase were identified by mass spectrometry sequencing in CTF complexes purified from both human T cell and yeast . Further analysis of the role played by these two proteins with specific inhibitors, both in the in vitro translocation assay and in intact cell toxicity assays, has demonstrated their essential role in the productive delivery of the C-domain from the lumen of early endosomes to the external milieu . These results confirm and extend earlier observations of diphtheria toxin C-domain unfolding and refolding that must occur before and after vesicle membrane translocation . In addition, results presented here demonstrate that thioredoxin reductase activity plays an essential role in the cytosolic release of the C-domain . Because analogous CTF complexes have been partially purified from mammalian and yeast cell extracts, results presented here suggest a common and fundamental mechanism for C-domain translocation across early endosomal membranes.

J Cell Biol, 2003 Mar 31, 160(7), 1029 - 40
An essential role for hGle1 nucleocytoplasmic shuttling in mRNA export; Kendirgi F et al.; Gle1 is required for mRNA export in yeast and human cells . Here, we report that two human Gle1 (hGle1) isoforms are expressed in HeLa cells (hGle1A and B) . The two encoded proteins are identical except for their COOH-terminal regions . hGle1A ends with a unique four-amino acid segment, whereas hGle1B has a COOH-terminal 43-amino acid span . Only hGle1B, the more abundant isoform, localizes to the nuclear envelope (NE) and pore complex . To test whether hGle1 is a dynamic shuttling transport factor, we microinjected HeLa cells with recombinant hGle1 and conducted photobleaching studies of live HeLa cells expressing EGFP-hGle1 . Both strategies show that hGle1 shuttles between the nucleus and cytoplasm . An internal 39-amino acid domain is necessary and sufficient for mediating nucleocytoplasmic transport . Using a cell-permeable peptide strategy, we document a role for hGle1 shuttling in mRNA export . An hGle1 shuttling domain (SD) peptide impairs the export of both total poly(A)+ RNA and the specific dihydrofolate reductase mRNA . Coincidentally, SD peptide-treated cells show decreased endogenous hGle1 localization at the NE and reduced nucleocytoplasmic shuttling of microinjected, recombinant hGle1 . These findings pinpoint the first functional motif in hGle1 and link hGle1 to the dynamic mRNA export mechanism.

Trends Cell Biol, 2003 Apr, 13(4), 187 - 94
Nogo and its paRTNers; Oertle T et al.; Reticulons (RTNs) are a relatively new eukaryotic gene family with unknown functions but broad expression and peculiar topological features . RTNs are widely distributed in plants, yeast and animals and are characterized by a approximately 200-amino-acid C-terminal domain, including two long hydrophobic sequences . Nogo/RTN4 can inhibit neurite growth from the cell surface via specific receptors, whereas more general, 'ancestral', RTN functions might relate to those of the endoplasmic reticulum - for example, intracellular trafficking, cell division and apoptosis . Here, we review the taxonomic distribution and tissue expression of RTNs, summarize recent discoveries about RTN localization and membrane topology, and discuss the possible functions of RTNs.

Zhonghua Xue Ye Xue Za Zhi, 2002 Dec, 23(12), 621 - 3
{Determination of ETO interaction domain within nuclear receptor co-repressor}; Wang M et al.; OBJECTIVE: To determine the ETO-interaction domain of nuclear receptor co-repressor (N-CoR) for abolishing the biological function of AML1-ETO . METHODS: Ten different regions of N-CoR (N-CoRYs) were generated by means of polymerase chain reaction (PCR), and cloned into yeast expression plasmid pGADGL to construct pGADGL/N-CoRYs . The yeast two-hybrid technique and X-gal staining were used to determine the binding between the 10 different regions of N-CoR and ETO . RESULTS: It was shown that the co-existence of 988-1,126 and 1,551-1,803 amino acid residues of N-CoRY was the ETO-interaction domains required for the binding with ETO . CONCLUSION: Two domains of N-CoR that interact with two zinc fingers of ETO, and keep stable binding between the two proteins were identified.

Dev Dyn, 2003 Apr, 226(4), 604 - 17
Identification and characterization of Grainyhead-like epithelial transactivator (GET-1), a novel mammalian Grainyhead-like factor; Kudryavtseva EI et al.; LMO-4 is an LIM-only factor that is highly expressed in many epithelial cells, including those of the epidermis and hair follicles . Because LMOs may function by interacting with DNA-binding proteins, we have used the yeast two-hybrid system to screen mouse skin libraries for LMO-4-interacting DNA-binding proteins . In this screen, we isolated a novel LMO-4-interacting factor highly related to the Drosophila gene Grainyhead . Grainyhead is epidermally expressed and carries out important functions in cuticular formation in the fly embryo . With the identification of this novel mammalian Grainyhead-like gene, referred to as Grainyhead-like epithelial transactivator 1 (GET-1), the known members of the mammalian Grainyhead-like gene family are extended to six, falling into two classes based on sequence homology . Of interest, the expression pattern of GET-1 is similar to that of Drosophila Grainyhead with highest expression in the somatic ectoderm/epidermis, but GET-1 is additionally expressed in epithelial cells of gastrointestinal, genitourinary, and respiratory tracks . The GET-1 protein localizes to the nucleus and binds to at least one Grainyhead DNA-binding site . The GET-1 DNA-binding domain maps to a region containing homology to the Drosophila Grainyhead DNA-binding domain . GET-1 homodimerizes in solution by means of a short C-terminally located domain that is homologous to other Grainyhead-like genes . A short domain located between amino acids 100 and 190, which bears no homology to known transactivation domains, is sufficient to confer transactivation to the heterologous GAL4 DNA-binding domain . In addition, GET-1 appears to contain repression domains consistent with the observation that Grainyhead and other mammalian Grainyhead-like genes can act both as activators and repressors . These data suggest that GET-1 is a transcriptional regulator that may perform important functions in epithelial tissues of mammals .

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Dec, 16(4), 351 - 3
{Screening and cloning gene of hepatocyte protein interacting with hepatitis C virus core protein}; Li K et al.; OBJECTIVE: To clone the unknown gene of hepatocyte protein interacting with hepatitis C virus core protein . METHODS: Using the yeast dual hybrid system 3, bait plasmids of hepatitis C virus core were constructed . After identifying hepatitis C virus core protein that could stably expressed in AH109 yeast strains, we performed yeast two hybrid by mating AH109 with Y187 that transformed with liver cDNA library plasmids pACT2 and then plated on quadrople dropout (QDO) medium and assayed for alpha-gal activity . The genes of yeast colonies that could grow on QDO and had alpha-gal activity were sequenced . RESULTS: Among the 30 positive colonies, we blasted the gene of the sixth colony; we coined human hepatitis C virus binding protein 6(Hu Hcbp6) with Genbank, realized that the Hu Hcbp6 shares as much as 98% homology with two cDNA without knowing functions . We have proved that Hu Hcbp6 could interact with hepatitis C virus core protein . CONCLUSIONS: Hepatitis C virus core binding protein (Hu Hcbp 6 Genbank number: AY032594) was successfully cloned and identified . The study partly paved the way for investigating physiological function of the Hu Hcbp6.

Mol Cell Biol, 2003 Apr, 23(8), 2834 - 43
MCAF mediates MBD1-dependent transcriptional repression; Fujita N et al.; DNA methylation is involved in a variety of genome functions, including gene control and chromatin dynamics . MBD1 is a transcriptional regulator through the cooperation of a methyl-CpG binding domain, cysteine-rich CXXC domains, and a transcriptional repression domain . A yeast two-hybrid screen was performed to investigate the role of MBD1 in methylation-based transcriptional repression . We report a mediator, MBD1-containing chromatin-associated factor (MCAF), that interacts with the transcriptional repression domain of MBD1 . MCAF harbors two conserved domains that allow it to interact with MBD1 and enhancer-like transactivator Sp1 . MCAF possesses a coactivator-like activity, and it seems to facilitate Sp1-mediated transcription . In contrast, the MBD1-MCAF complex blocks transcription through affecting Sp1 on methylated promoter regions . These data provide a mechanistic basis for direct inhibition of gene expression via methylation-dependent and histone deacetylation-resistant processes.

Mol Cell Biol, 2003 Apr, 23(8), 2709 - 19
Endogenous assays of DNA methyltransferases: Evidence for differential activities of DNMT1, DNMT2, and DNMT3 in mammalian cells in vivo; Liu K et al.; While CpG methylation can be readily analyzed at the DNA sequence level in wild-type and mutant cells, the actual DNA (cytosine-5) methyltransferases (DNMTs) responsible for in vivo methylation on genomic DNA are less tractable . We used an antibody-based method to identify specific endogenous DNMTs (DNMT1, DNMT1b, DNMT2, DNMT3a, and DNMT3b) that stably and selectively bind to genomic DNA containing 5-aza-2'-deoxycytidine (aza-dC) in vivo . Selective binding to aza-dC-containing DNA suggests that the engaged DNMT is catalytically active in the cell . DNMT1b is a splice variant of the predominant maintenance activity DNMT1, while DNMT2 is a well-conserved protein with homologs in plants, yeast, Drosophila, humans, and mice . Despite the presence of motifs essential for transmethylation activity, catalytic activity of DNMT2 has never been reported . The data here suggest that DNMT2 is active in vivo when the endogenous genome is the target, both in human and mouse cell lines . We quantified relative global genomic activity of DNMT1, -2, -3a, and -3b in a mouse teratocarcinoma cell line . DNMT1 and -3b displayed the greatest in vivo binding avidity for aza-dC-containing genomic DNA in these cells . This study demonstrates that individual DNMTs can be tracked and that their binding to genomic DNA can be quantified in mammalian cells in vivo . The different DNMTs display a wide spectrum of genomic DNA-directed activity . The use of an antibody-based tracking method will allow specific DNMTs and their DNA targets to be recovered and analyzed in a physiological setting in chromatin.

Proc Natl Acad Sci U S A, 2003 Apr 15, 100(8), 4586 - 91 Epub 2003 Mar 27.
Mammalian Erv46 localizes to the endoplasmic reticulum-Golgi intermediate compartment and to cis-Golgi cisternae; Orci L et al.; Yeast endoplasmic reticulum (ER) vesicle protein Erv46p is a novel membrane protein involved in transport through the early secretory pathway . Investigation of mammalian Erv46 (mErv46) reveals that it is broadly expressed in tissues and protein-secreting cells . By immunofluorescence microscopy, mErv46 displays a crescent-shaped perinuclear staining pattern that is characteristic of the Golgi complex . Quantitative immunoelectron microscopy indicates that mErv46 is restricted to the cis face of the Golgi apparatus and to vesicular tubular structures between the transitional ER and cis-Golgi . Minor amounts of mErv46 reside in ER membranes and later Golgi cisternae . On Brefeldin A treatment, mErv46 redistributes to punctate structures that costain for ERGIC53 . Depletion of mErv46 protein by RNA interference caused no apparent structural changes in the intermediate compartment or Golgi complex . These findings place mErv46 in a group of itinerant proteins that cycle between the ER and Golgi compartments such as ERGIC53 and the p24 proteins.

J Biol Chem, 2003 May 30, 278(22), 20332 - 7 Epub 2003 Mar 27.
The Hermansky-Pudlak syndrome 1 (HPS1) and HPS4 proteins are components of two complexes, BLOC-3 and BLOC-4, involved in the biogenesis of lysosome-related organelles; Chiang PW et al.; Hermansky-Pudlak syndrome (HPS) is a genetic disease of lysosome, melanosome, and granule biogenesis . Mutations of six different loci have been associated with HPS in humans, the most frequent of which are mutations of the HPS1 and HPS4 genes . Here, we show that the HPS1 and HPS4 proteins are components of two novel protein complexes involved in biogenesis of melanosome and lysosome-related organelles: biogenesis of lysosome-related organelles complex-(BLOC) 3 and BLOC-4 . The phenotypes of Hps1-mutant (pale-ear; ep) and Hps4-mutant (light-ear; le) mice and humans are very similar, and cells from ep and le mice exhibit similar abnormalities of melanosome morphology . HPS1 protein is absent from ep-mutant cells, and HPS4 from le-mutant cells, but le-mutant cells also lack HPS1 protein . HPS4 protein seems to be necessary for stabilization of HPS1, and the HPS1 and HPS4 proteins co-immunoprecipitate, indicating that they are in a complex . HPS1 and HPS4 do not interact directly in a yeast two-hybrid system, although HPS4 interacts with itself . In a partially purified vesicular/organellar fraction, HPS1 and HPS4 are both components of a complex with a molecular mass of approximately 500 kDa, termed BLOC-3 . Within BLOC-3, HPS1 and HPS4 are components of a discrete approximately 200-kDa module termed BLOC-4 . In the cytosol, HPS1 (but not HPS4) is part of yet another complex, termed BLOC-5 . We propose that the BLOC-3 and BLOC-4 HPS1.HPS4 complexes play a central role in trafficking cargo proteins to newly formed cytoplasmic organelles.

Genetics, 2003 Mar, 163(3), 1047 - 60
Genetic selection for modulators of a retinoic-acid-responsive reporter in human cells; Richards B et al.; We used a genetic screening methodology, a human cell line bearing a retinoic-acid-responsive enhanced GFP reporter, and a flow sorter to recover dominant modulators of reporter expression . Four inducers and three suppressors that were fused to the C terminus of a protein scaffold for stability were isolated and their mechanisms of action studied . Mutagenesis experiments indicated that six of these dominant agents exerted their effects at the protein level . The single cDNA coding fragment that was isolated comprised the central 64-amino-acid section of human cyclophilin B, which contained its peptidyl-prolyl isomerase domain; this cyclophilin fragment repressed expression of the retinoic-acid-responsive reporter . The remaining clones encoded peptides shorter than 30 amino acids unrelated to known gene open reading frames . Genetic epistasis studies between the strongest inducer, R3, and a dominant-negative mutant of RARalpha suggest that the two factors function in the same pathway . Transcript microarray analyses suggest that R3 induced a subset of the retinoid-responsive genes in melanoma cells . Finally, yeast two-hybrid assays and co-immunoprecipitation studies of human cell extracts identified PAT1 as a protein that interacts with R3.

Genetics, 2003 Mar, 163(3), 973 - 82
The Drosophila chk2 gene loki is essential for embryonic DNA double-strand-break checkpoints induced in S phase or G2; Masrouha N et al.; Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the occurrence of the next cell cycle transition . The Chk2 family of kinases is known to play a central role in mediating the cellular responses to DNA damage or DNA replication blocks in various organisms . Here we show through a phylogenetic study that the Drosophila melanogaster serine/threonine kinase Loki is the homolog of the yeast Mek1p, Rad53p, Dun1p, and Cds1 proteins as well as the human Chk2 . Functional analyses allowed us to conclude that, in flies, chk2 is involved in monitoring double-strand breaks (DSBs) caused by irradiation during S and G2 phases . In this process it plays an essential role in inducing a cell cycle arrest in embryonic cells . Our results also show that, in contrast to C . elegans chk2, Drosophila chk2 is not essential for normal meiosis and recombination, and it also appears to be dispensable for the MMS-induced DNA damage checkpoint and the HU-induced DNA replication checkpoint during larval development . In addition, Drosophila chk2 does not act at the same cell cycle phases as its yeast homologs, but seems rather to be involved in a pathway similar to the mammalian one, which involves signaling through the ATM/Chk2 pathway in response to genotoxic insults . As mutations in human chk2 were linked to several cancers, these similarities point to the usefulness of the Drosophila model system.

Diagn Microbiol Infect Dis, 2003 Mar, 45(3), 217 - 20
Development and evaluation of the nuclisens basic kit NASBA for the detection of RNA from Candida species frequently resistant to antifungal drugs; Loeffler J et al.; We describe a Nucleic Acid Sequence Based Amplification (NASBA) protocol to detect 6 different Candida species (Candida krusei, Candida glabrata, Candida inconspicua, Candida dubliniensis, Candida norvegensis, Candida lusitaniae) and compare it to a PCR assay . NASBA showed a sensitivity of 1 Colony Forming Unit and detected RNA from all 6 Candida species within 1 working day . All 5 patients with documented candidiasis showed identical results by both methods . This assay offers a sensitive, specific and fast possibility to detect yeast RNA.

J Mol Biol, 2003 Apr 11, 327(5), 919 - 23
How reliable are experimental protein-protein interaction data?
Sprinzak E, Sattath S, Margalit H.
Data of protein-protein interactions provide valuable insight into the molecular networks underlying a living cell . However, their accuracy is often questioned, calling for a rigorous assessment of their reliability . The computation offered here provides an intelligible mean to assess directly the rate of true positives in a data set of experimentally determined interacting protein pairs . We show that the reliability of high-throughput yeast two-hybrid assays is about 50%, and that the size of the yeast interactome is estimated to be 10,000-16,600 interactions.

Plant J, 2003 Apr, 34(1), 13 - 26
Transport of cytokinins mediated by purine transporters of the PUP family expressed in phloem, hydathodes, and pollen of Arabidopsis; Burkle L et al.; Nucleobases and derivatives like cytokinins and caffeine are translocated in the plant vascular system . Transport studies in cultured Arabidopsis cells indicate that adenine and cytokinin are transported by a common H+-coupled high-affinity purine transport system . Transport properties are similar to that of Arabidopsis purine transporters AtPUP1 and 2 . When expressed in yeast, AtPUP1 and 2 mediate energy-dependent high-affinity adenine uptake, whereas AtPUP3 activity was not detectable . Similar to the results from cell cultures, purine permeases (PUP) mediated uptake of adenine can be inhibited by cytokinins, indicating that cytokinins are transport substrates . Direct measurements demonstrate that AtPUP1 is capable of mediating uptake of radiolabeled trans-zeatin . Cytokinin uptake is strongly inhibited by adenine and isopentenyladenine but is poorly inhibited by 6-chloropurine . A number of physiological cytokinins including trans- and cis-zeatin are also efficient competitors for AtPUP2-mediated adenine uptake, suggesting that AtPUP2 is also able to mediate cytokinin transport . Furthermore, AtPUP1 mediates transport of caffeine and ribosylated purine derivatives in yeast . Promoter-reporter gene studies point towards AtPUP1 expression in the epithem of hydathodes and the stigma surface of siliques, suggesting a role in retrieval of cytokinins from xylem sap to prevent loss during guttation . The AtPUP2 promoter drives GUS reporter gene activity in the phloem of Arabidopsis leaves, indicating a role in long-distance transport of adenine and cytokinins . Promoter activity of AtPUP3 was only found in pollen . In summary, three closely related PUPs are differentially expressed in Arabidopsis and at least two PUPs have properties similar to the adenine and cytokinin transport system identified in Arabidopsis cell cultures.

Biochem J, 2003 Jun 15, 372(Pt 3), 871 - 9
Cloning and expression of the liver and muscle isoforms of ovine carnitine palmitoyltransferase 1: residues within the N-terminus of the muscle isoform influence the kinetic properties of the enzyme; Price NT et al.; The nucleotide sequence data reported will appear in DDBJ, EMBL, GenBank(R) and GSDB Nucleotide Sequence Databases; the sequences of ovine CPT1A and CPT1B cDNAs have the accession numbers Y18387 and AJ272435 respectively and the partial adipose tissue and liver CPT1A clones have the accession numbers Y18830 and Y18829 respectively . Fatty acid and ketone body metabolism differ considerably between monogastric and ruminant species . The regulation of the key enzymes involved may differ accordingly . Carnitine palmitoyltransferase 1 (CPT 1) is the key locus for the control of long-chain fatty acid beta-oxidation and liver ketogenesis . Previously we showed that CPT 1 kinetics in sheep and rat liver mitochondria differ . We cloned cDNAs for both isoforms {liver- (L-) and muscle- (M-)} of ovine CPT 1 in order to elucidate the structural features of these proteins and their genes ( CPT1A and CPT1B ) . Their deduced amino acid sequences show a high degree of conservation compared with orthologues from other mammalian species, with the notable exception of the N-terminus of ovine M-CPT 1 . These differences were also present in bovine M-CPT 1, whose N-terminal sequence we determined . In addition, the 5'-end of the sheep CPT1B cDNA suggested a different promoter architecture when compared with previously characterized CPT1B genes . Northern blotting revealed differences in tissue distribution for both CPT1A and CPT1B transcripts compared with other species . In particular, ovine CPT1B mRNA was less tissue restricted, and the predominant transcript in the pancreas was CPT1B . Expression in yeast allowed kinetic characterization of the two native enzymes, and of a chimaera in which the distinctive N-terminal segment of ovine M-CPT 1 was replaced with that from rat M-CPT 1 . The ovine N-terminal segment influences the kinetics of the enzyme for both its substrates, such that the K (m) for palmitoyl-CoA is decreased and that for carnitine is increased for the chimaera, relative to the parental ovine M-CPT 1.

J Nat Prod, 2003 Mar, 66(3), 419 - 22
New cytotoxic lupane triterpenoids from the twigs of Coussarea paniculata; Prakash Chaturvedula VS et al.; Bioassay-guided fractionation of a CH(2)Cl(2)-MeOH extract of the twigs of Coussarea paniculata using a yeast-based assay for potential DNA-damaging agents resulted in the isolation of three new lupane triterpenoids, 1-3, in addition to eight known triterpenoids, lupeol (4), lupeyl acetate (5), betulin (6), betulinic acid (7), 3-epi-betulinic acid (8), 3-epi-betulinaldehyde (9), oleanolic acid (10), and ursolic acid (11) . The structures of the new compounds were established as lup-20(29)-en-3beta,25-diol (1), lup-20(29)-en-11alpha-ol-25,3beta-lactone (2), and 3-deoxybetulonic acid (3), on the basis of extensive 1D and 2D NMR spectroscopic data interpretation and chemical conversion.

Oncogene, 2003 Mar 27, 22(12), 1749 - 57
Binding of the corepressor TLE1 to Qin enhances Qin-mediated transformation of chicken embryo fibroblasts; Sonderegger CK et al.; The oncoprotein Qin is a member of the winged helix family of transcriptional regulators . The region C-terminal to its winged helix DNA-binding domain is required for transformation of chicken embryo fibroblasts . We isolated the corepressor TLE1 as a binding partner for Qin in a yeast two-hybrid screen and localized the TLE1-binding region to a 60 amino-acid stretch directly C-terminal of the winged helix domain of Qin . We show in vivo interaction of full-length Qin and TLE1 in a mammalian two-hybrid system . Coexpression of TLE1-binding Qin and TLE1 induces phosphorylation of TLE1 . The DNA-binding activity of Qin is not required for this function . Binding of Qin to TLE1 correlates with Qin-induced transformation . Addition of the TLE1-binding motif WRPW to the C-terminus of a transformation-defective Qin deletion mutant restores binding to TLE1 and significantly enhances transformation . Expression of TLE1 in CEF by the retroviral vector RCAS enhances cell growth and induces formation of agar colonies.

Nature, 2003 Apr 3, 422(6931), 534 - 9 Epub 2003 Mar 26.
Crystal structure of a transcription factor IIIB core interface ternary complex; Juo ZS et al.; Transcription factor IIIB (TFIIIB), consisting of the TATA-binding protein (TBP), TFIIB-related factor (Brf1) and Bdp1, is a central component in basal and regulated transcription by RNA polymerase III . TFIIIB recruits its polymerase to the promoter and subsequently has an essential role in the formation of the open initiation complex . The amino-terminal half of Brf1 shares a high degree of sequence similarity with the polymerase II general transcription factor TFIIB, but it is the carboxy-terminal half of Brf1 that contributes most of its binding affinity with TBP . The principal anchoring region is located between residues 435 and 545 of yeast Brf1, comprising its homology domain II . The same region also provides the primary interface for assembling Bdp1 into the TFIIIB complex . We report here a 2.95 A resolution crystal structure of the ternary complex containing Brf1 homology domain II, the conserved region of TBP and 19 base pairs of U6 promoter DNA . The structure reveals the core interface for assembly of TFIIIB and demonstrates how the loosely packed Brf1 domain achieves remarkable binding specificity with the convex and lateral surfaces of TBP.

EMBO J, 2003 Apr 1, 22(7), 1478 - 87
Functional expression of the epithelial Ca(2+) channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex; van de Graaf SF et al.; TRPV5 and TRPV6 constitute the Ca(2+) influx pathway in a variety of epithelial cells . Here, we identified S100A10 as the first auxiliary protein of these epithelial Ca(2+) channels using yeast two-hybrid and GST pull-down assays . This S100 protein forms a heterotetrameric complex with annexin 2 and associates specifically with the conserved sequence VATTV located in the C-terminal tail of TRPV5 and TRPV6 . Of these five amino acids, the first threonine plays a crucial role since the corresponding mutants (TRPV5 T599A and TRPV6 T600A) exhibited a diminished capacity to bind S100A10, were redistributed to a subplasma membrane area and did not display channel activity . Using GST pull-down and co-immunoprecipitation assays we demonstrated that annexin 2 is part of the TRPV5-S100A10 complex . Furthermore, the S100A10-annexin 2 pair colocalizes with the Ca(2+) channels in TRPV5-expressing renal tubules and TRPV6-expressing duodenal cells . Importantly, downregulation of annexin 2 using annexin 2-specific small interfering RNA inhibited TRPV5 and TRPV6-mediated currents in transfected HEK293 cells . In conclusion, the S100A10-annexin 2 complex plays a crucial role in routing of TRPV5 and TRPV6 to plasma membrane.

Biochem Biophys Res Commun, 2003 Apr 4, 303(2), 713 - 20
Stimulation of a mitochondrial endo-exonuclease from Podospora anserina by PCNA; Laquel-Robert P et al.; In Podospora anserina we have described the purification of an endo-exonuclease (Biochim . Biophys . Acta 1574 (1) (2002) 72) . Given the description of several nucleases addressed to the mitochondria and known to interact with the PCNA, we sought a possible effect of PCNA on the mt nuclease . A significant stimulation of the nuclease activity with PCNA was observed with double-stranded and flap structure DNA . Immuno-Western blotting experiments realized with monoclonal antibodies raised against the PCNA specifically revealed the presence of a single band of 30 kDa in the mitochondria from the filamentous fungus and yeast . A potential PCNA binding motif was found in the sequences of several mt nucleases and mt proteins involved in the maintenance of the mt DNA with respect to the consensus described by Warbrick . The hypothetical role of the PCNA as a potential regulator of the repair/recombination processes in the maintenance of the mt genome is discussed.

Biochem Biophys Res Commun, 2003 Apr 4, 303(2), 419 - 26
Estrogenic activity of an environmental pollutant, 2-nitrofluorene, after metabolic activation by rat liver microsomes; Fujimoto T et al.; In this study, the metabolic activation of 2-nitrofluorene (NF) to estrogenic compounds was examined . NF was negative in estrogen reporter assays using estrogen-responsive yeast and human breast cancer cell line MCF-7 . However, the compound exhibited estrogenic activity after incubation with liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH . Minor estrogenic activity was observed when liver microsomes of untreated or phenobarbital-treated rats were used instead of those from 3-methylcholanthrene-treated rats . When the compound was incubated with the liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH, 7-hydroxy-2-nitrofluorene (7-OH-NF) was formed as a major metabolite . However, little of the metabolite was formed by liver microsomes of untreated or phenobarbital-treated rats . Rat recombinant cytochrome P450 1A1 exhibited a significant oxidase activity toward NF, affording 7-OH-NF . Liver microsomes of phenobarbital-treated rats also enhanced oxidase activity toward NF . In this case, 9-hydroxy-2-nitrofluorene was formed . 7-OH-NF exhibited a significant estrogenic activity, while the activity of 9-hydroxy-2-nitrofluorene was much lower . These results suggest that the estrogenic activity of NF was due to formation of the 7-hydroxylated metabolite by liver microsomes.

J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Apr 5, 787(1), 19 - 27
Protein micro- and macroarrays: digitizing the proteome; Lopez MF et al.; The early applications of microarrays and detection technologies have been centered on DNA-based applications . The application of array technologies to proteomics is now occurring at a rapid rate . Numerous researchers have begun to develop technologies for the creation of microarrays of protein-based screening tools . The stability of antibody molecules when bound to surfaces has made antibody arrays a starting point for proteomic microarray technology . To minimize disadvantages due to size and availability, some researchers have instead opted for antibody fragments, antibody mimics or phage display technology to create libraries for protein chips . Even further removed from antibodies are libraries of aptamers, which are single-stranded oligonucleotides that express high affinity for protein molecules . A variation on the theme of protein chips arrayed with antibody mimics or other protein capture ligand is that of affinity MS where the protein chips are directly placed in a mass spectrometer for detection . Other approaches include the creation of intact protein microarrays directly on glass slides or chips . Although many of the proteins may likely be denatured, successful screening has been demonstrated . The investigation of protein-protein interactions has formed the basis of a technique called yeast two-hybrid . In this method, yeast "bait" proteins can be probed with other yeast "prey" proteins fused to DNA binding domains . Although the current interpretation of protein arrays emphasizes microarray grids of proteins or ligands on glass slides or chips, 2-D gels are technically macroarrays of authentic proteins . In an innovative departure from the traditional concept of protein chips, some researchers are implementing microfluidic printing of arrayed chemistries on individual protein spots blotted onto membranes . Other researchers are using in-jet printing technology to create protein microarrays on chips . The rapid growth of proteomics and the active climate for new technology is driving a new generation of companies and academic efforts that are developing novel protein microarray techniques for the future .

J Neurosci, 2003 Mar 15, 23(6), 2284 - 93
Macrophage-derived factors stimulate optic nerve regeneration; Yin Y et al.; After optic nerve injury in mature mammals, retinal ganglion cells (RGCs) are normally unable to regenerate their axons and undergo delayed apoptosis . However, if the lens is damaged at the time of nerve injury, many RGCs survive axotomy and regenerate their axons into the distal optic nerve . Lens injury induces macrophage activation, and we show here that factors secreted by macrophages stimulate RGCs to regenerate their axons . When macrophages were activated by intravitreal injections of Zymosan, a yeast cell wall preparation, the number of RGC axons regenerating into the distal optic nerve was even greater than after lens injury . These effects were further enhanced if Zymosan was injected 3 d after nerve crush . In a grafting paradigm, intravitreal Zymosan increased the number of RGCs that regenerated their axons through a 1.5 cm peripheral nerve graft twofold relative to uninjected controls and threefold if injections were delayed 3 d . In cell culture, media conditioned by activated macrophages stimulated adult rat RGCs to regenerate their axons; this effect was potentiated by a low molecular weight factor that is constitutively present in the vitreous humor . After gel-filtration chromatography, macrophage-derived proteins > or =30 kDa were found to be toxic to RGCs, whereas proteins <30 kDa reversed this toxicity and promoted axon regeneration . The protein(s) that stimulated axon growth is distinct from identified polypeptide trophic factors that were tested . Thus, macrophages produce proteins with both positive and negative effects on RGCs, and the effects of macrophages can be optimized by the timing of their activation.

J Biol Chem, 2003 Jun 6, 278(23), 21003 - 11 Epub 2003 Mar 25.
Synergistic activation of seed storage protein gene expression in Arabidopsis by ABI3 and two bZIPs related to OPAQUE2; Lara P et al.; The expression of many seed storage protein genes in cereals relies on transcription factors of the bZIP class, belonging to the maize OPAQUE2 family . Here, we describe a survey of such factors in the genome of Arabidopsis thaliana, and the characterization of two of them, AtbZIP10 and AtbZIP25 . Expression analysis by in situ hybridization shows that the occurrence of their mRNAs in the seed starts from early stages of development, peaks at maturation, and declines later in seed development, matching temporally and spatially those of the seed storage protein genes encoding 2S albumins and cruciferins . Gel mobility shift assays showed that AtbZIP10 and AtbZIP25 bind the ACGT boxes present in At2S and CRU3 promoters . Moreover, using the yeast two-hybrid system we show that AtbZIP10 and AtbZIP25 can interact in vivo with ABI3, an important regulator of gene expression in the seed of Arabidopsis . Transient expression analyses of a reporter gene under the control of the At2S1 promoter in transgenic plants overexpressing ectopically AtbZIP10, AtbZIP25, and ABI3 reveal that none of these factors could activate significantly the reporter gene when expressed individually . However, co-expression of AtbZIP10/25 with ABI3 resulted in a remarkable increase in the activation capacity over the At2S1 promoter, suggesting that they are part of a regulatory complex involved in seed-specific expression . This study shows a common mechanism of ABI3 in regulating different seed-specific genes through combinatorial interactions with particular bZIP proteins and a conserved role of O2-like bZIPs in monocot and dicot species.

J Biol Chem, 2003 Jun 6, 278(23), 21155 - 61 Epub 2003 Mar 25.
Enterophilin-1, a new partner of sorting nexin 1, decreases cell surface epidermal growth factor receptor; Pons V et al.; We previously described enterophilin-1 (Ent-1), a new intestinal protein bearing an extended leucine zipper and a B30.2 domain . Ent-1 expression is associated with growth arrest and enterocyte differentiation . To investigate the importance of Ent-1 in the differentiation, we performed a yeast two-hybrid screening . We identified sorting nexin 1 (SNX1) as a novel partner of Ent-1 and confirmed the specificity of interaction by co-immunoprecipitation experiments in mammalian cells . SNX1 is associated with endosomal membranes and triggers the endosome-to-lysosome pathway of epidermal growth factor receptor (EGFR) . We observe by immunofluorescence microscopy that Ent-1 and SNX1 are co-localized on vesicular and tubulovesicular structures, which are different from early endosome antigen 1-containing endosomes . By gel filtration chromatography, we show that Ent-1 and SNX1 co-eluted in macromolecular complexes containing part of EGFR . Furthermore, overexpressed Ent-1 decreases cell surface EGFR . Ent-1 and SNX1 co-overexpression strongly extends EGFR diminution, indicating a synergetic effect of both proteins on cell surface EGFR removal . Interestingly, the increase of endogenous Ent-1 expression correlates with the decrease of EGFR during spontaneous differentiation of Caco-2 cells . We thus propose a role of Ent-1 in the trafficking of EGFR to down-regulate intestinal mitogenic signals, highlighting the mechanisms of cell growth arrest associated with enterocytic differentiation.

J Biol Chem, 2003 Jun 13, 278(24), 21709 - 14 Epub 2003 Mar 25.
Interaction of lp-dlg/KIAA0583, a membrane-associated guanylate kinase family protein, with vinexin and beta-catenin at sites of cell-cell contact; Wakabayashi M et al.; Vinexin is a recently identified cytoskeletal protein and plays a key role in the regulation of cytoskeletal organization and signal transduction . Vinexin localizes at sites of cell-extracellular matrix adhesion in NIH3T3 fibroblasts and at sites of cell-cell contact in epithelial LLC-PK1 cells . Expression of vinexin promotes the formation of actin stress fiber, but the role of vinexin at sites of cell-cell contact is unclear . Here we identified lp-dlg/KIAA0583 as a novel binding partner for vinexin by using yeast two-hybrid screening . lp-dlg/KIAA0583 has a NH2-terminal coiled-coil-like domain, in addition to four PDZ domains, an Src homology (SH) 3 domain, and a guanylate kinase domain, which are conserved structures in membrane-associated guanylate kinase family proteins . The third SH3 domain of vinexin bound to the region between the second and third PDZ domain of lp-dlg, which contains a proline-rich sequence . lp-dlg colocalized with vinexin at sites of cell-cell contact in LLC-PK1 cells . Furthermore, lp-dlg colocalized with beta-catenin, a major adherens junction protein, in LLC-PK1 cells . Co-immunoprecipitation experiments revealed that both endogenous and epitope-tagged deletion mutants of lp-dlg/KIAA0583 associated with beta-catenin . We also showed that these three proteins could form a ternary complex . Together these findings suggest that lp-dlg/KIAA0583 is a novel scaffolding protein that can link the vinexin-vinculin complex and beta-catenin at sites of cell-cell contact.

J Biol Chem, 2003 Jun 6, 278(23), 20507 - 13 Epub 2003 Mar 25.
Human PLU-1 Has transcriptional repression properties and interacts with the developmental transcription factors BF-1 and PAX9; Tan K et al.; PLU-1 is a large (1544 amino acids) nuclear protein that is highly expressed in breast cancers and is proposed to function as a regulator of gene expression . A yeast two-hybrid screen using PLU-1 as bait has identified two unrelated PLU-1 interacting proteins, namely brain factor-1 (BF-1) and paired box 9 (PAX9), both of which are developmental transcription factors . BF-1 and PAX9 interact with PLU-1 via a novel conserved sequence motif (Ala-X-Ala-Ala-X-Val-Pro-X4-Val-Pro-X8-Pro, termed the VP motif), because deletion or site-directed mutagenesis of this motif in either protein abolishes PLU-1 interaction in vivo . In a reporter assay system, PLU-1 has potent transcriptional repression activity . BF-1 and PAX9 also represses transcription in the same assay, but co-expression of PLU-1 with BF-1 or PAX9 significantly enhances this repression . Mutation of the PLU-1 binding motifs in BF-1 and PAX9 abolishes the observed PLU-1 co-repression activity . These data support a role for PLU-1 acting as a transcriptional co-repressor of two unrelated developmental transcription factors . Because both BF-1 and PAX proteins interact with members of the groucho co-repressor family, it is plausible that PLU-1 has a role in groucho-mediated transcriptional repression.

J Biol Chem, 2003 Jun 6, 278(23), 20865 - 73 Epub 2003 Mar 25.
Isolation and characterization of a novel GRAS gene that regulates meiosis-associated gene expression; Morohashi K et al.; GRAS protein is a family of plant-specific proteins that plays a role in various developmental processes . Here we report a novel GRAS protein from lily, designated LlSCL (Lilium longiflorum Scarecrow-like), dominantly expressed at the premeiotic phase within anthers . The LlSCL protein has two highly basic regions, and transient expression analyses of dissected GFP-LlSCL fusion proteins showed that both basic regions are important for the nuclear localization . A series of transcriptional activation experiments of truncated LlSCL proteins fused to the yeast GAL4 DNA-binding domain clearly demonstrated that the amino terminus of the LlSCL protein has a strong activity of transcriptional activation in the yeast as well as in the plant cell . Further investigation on the effect of the LlSCL protein on the transcriptional activity of the meiosis-associated promoter revealed that in pollen mother cells of the lily, the activity of the meiosis-associated promoter is specifically enhanced by LlSCL protein co-expression . These results suggest that LlSCL is involved in transcriptional regulation during microsporogenesis within the lily anther.

Mol Microbiol, 2003 Apr, 48(1), 237 - 51
Light-induced carotenogenesis in Myxococcus xanthus: functional characterization of the ECF sigma factor CarQ and antisigma factor CarR; Browning DF et al.; Illumination of dark-grown Myxococcus xanthus with blue light leads to the induction of carotenoid synthesis . Central to this response is the activation of the light-inducible promoter, PcarQRS, and the transcription of three downstream genes, carQ, carR and carS . Sequence analysis predicted that CarQ is a member of the ECF (extracytoplasmic function) subfamily of RNA polymerase sigma factors, and that CarR is an inner membrane protein . Genetic analysis strongly implied that CarR is an antisigma factor that sequesters CarQ in a transcriptionally inactive complex . Using in vitro transcription run-off assays, we present biochemical evidence that CarQ functions as a bacterial sigma factor and is responsible for transcription initiation at PcarQRS . Similar experiments using the crtI promoter failed to implicate CarQ in direct transcription of the crtI gene . Experiments using the yeast two-hybrid system demonstrated a protein-protein interaction between CarQ and CarR, providing evidence of a CarQ-CarR complex . The yeast two-hybrid system data also indicated that CarR is capable of oligomerization . Fractionation of M . xanthus membranes with the detergent sarkosyl showed that CarR was associated with the inner membrane . Furthermore, CarR was found to be unstable in illuminated stationary phase cells, providing a possible mechanism by which the CarR-CarQ complex is disrupted.

Biochem Soc Symp, 2002, (69), 59 - 72
Genomic analysis of C-type lectins; Drickamer K et al.; Many biological effects of complex carbohydrates are mediated by lectins that contain discrete carbohydrate-recognition domains . At least seven structurally distinct families of carbohydrate-recognition domains are found in lectins that are involved in intracellular trafficking, cell adhesion, cell-cell signalling, glycoprotein turnover and innate immunity . Genome-wide analysis of potential carbohydrate-binding domains is now possible . Two classes of intracellular lectins involved in glycoprotein trafficking are present in yeast, model invertebrates and vertebrates, and two other classes are present in vertebrates only . At the cell surface, calcium-dependent (C-type) lectins and galectins are found in model invertebrates and vertebrates, but not in yeast; immunoglobulin superfamily (I-type) lectins are only found in vertebrates . The evolutionary appearance of different classes of sugar-binding protein modules parallels a development towards more complex oligosaccharides that provide increased opportunities for specific recognition phenomena . An overall picture of the lectins present in humans can now be proposed . Based on our knowledge of the structures of several of the C-type carbohydrate-recognition domains, it is possible to suggest ligand-binding activity that may be associated with novel C-type lectin-like domains identified in a systematic screen of the human genome . Further analysis of the sequences of proteins containing these domains can be used as a basis for proposing potential biological functions.

Bioessays, 2003 Apr, 25(4), 305 - 8
A small issue addressed; Gumienny TL et al.; Cell size is an important determinant of body size . While the genetic mechanisms of cell size regulation have been well studied in yeast, this process has only recently been addressed in multicellular organisms . One recent report by Wang et al . (2002) shows that in the nematode C . elegans, the TGFbeta-like pathway acts in the hypodermis to regulate cell size and consequently body size.1 This finding is an exciting step in discovering the molecular mechanisms that control cell and body size .

J Gen Virol, 2003 Apr, 84(Pt 4), 959 - 64
The BZLF1 promoter of Epstein-Barr virus is controlled by E box-/HI-motif-binding factors during virus latency; Thomas C et al.; The BZLF1 open reading frame of Epstein-Barr virus (EBV) encodes an important transactivator of replication . During latency, transcription of this gene is switched off . HI motifs have been shown to cause negative regulation of the promoter . Using yeast one-hybrid assays, we isolated the E box-binding protein, E2-2, interacting with these motifs . Electrophoretic mobility shift assays demonstrated that E2-2 binds to HI alpha, HI beta and HI gamma, which contain E box consensus binding sites . Deletion of the HI-associated E boxes and overexpression of E2-2 in transfection assays revealed that these elements act as repressors in lymphoid cells . In contrast, in epithelial cells they contribute to the increased responsiveness of the promoter to transactivation by the BZLF1 protein . The data presented are in accord with an alternative and exclusive binding of different cell type- and differentiation-specific factors, such as E2-2, to the HI-associated E boxes in lymphoid and epithelial cells . This implies a role in cell type-specific virus replication.

Proc Natl Acad Sci U S A, 2003 Apr 15, 100(8), 4843 - 8 Epub 2003 Mar 24.
Translational control of inducible nitric oxide synthase expression by arginine can explain the arginine paradox; Lee J et al.; L-Arginine is the only endogenous nitrogen-containing substrate of NO synthase (NOS), and it thus governs the production of NO during nervous system development as well as in disease states such as stroke, multiple sclerosis, Parkinson's disease, and HIV dementia . The "arginine paradox" refers to the dependence of cellular NO production on exogenous L-arginine concentration despite the theoretical saturation of NOS enzymes with intracellular L-arginine . Herein, we report that decreased availability of L-arginine blocked induction of NO production in cytokine-stimulated astrocytes, owing to inhibition of inducible NOS (iNOS) protein expression . However, activity of the promoter of the iNOS gene, induction of iNOS mRNA, and stability of iNOS protein were not inhibited under these conditions . Our results indicate that inhibition of iNOS activity by arginine depletion in stimulated astrocyte cultures occurs via inhibition of translation of iNOS mRNA . After stimulation by cytokines, uptake of L-arginine negatively regulates the phosphorylation status of the eukaryotic initiation factor (eIF2 alpha), which, in turn, regulates translation of iNOS mRNA . eIF2 alpha phosphorylation correlates with phosphorylation of the mammalian homolog of yeast GCN2 eIF2 alpha kinase . As the kinase activity of GCN2 is activated by phosphorylation, these findings suggest that GCN2 activity represents a proximal step in the iNOS translational regulation by availability of l-arginine . These results provide an explanation for the arginine paradox for iNOS and define a distinct mechanism by which a substrate can regulate the activity of its associated enzyme.

Genes Cells, 2003 Apr, 8(4), 371 - 8
Structure-function analysis of human Spt4: evidence that hSpt4 and hSpt5 exert their roles in transcriptional elongation as parts of the DSIF complex; Kim DK et al.; BACKGROUND: The human Spt4/Spt5 complex, termed DRB-sensitivity inducing factor (DSIF) is a dual regulator of transcription that stimulates, or, when cooperating with negative elongation factor (NELF), represses RNA polymerase II (RNAPII) elongation . Spt4 and Spt5 are also thought to be involved in mRNA capping, homologous DNA recombination, and transcription-coupled DNA repair . As a first step to understanding how these proteins regulate diverse cellular processes, we investigated the structure and function of hSpt4 in vitro . RESULTS: Immunodepletion of hSpt5 from HeLa nuclear extracts resulted in the efficient co-depletion of hSpt4 . Using DSIF-depleted nuclear extracts and a series of Spt4 mutants, we examined the amino acid sequence of hSpt4 which was important for hSpt5 binding and for transcriptional repression and activation by DSIF . Unexpectedly, the zinc finger of hSpt4, which is critical for the yeast counterpart to function in vivo, was dispensable for hSpt5 binding and for transcriptional regulation in vitro . CONCLUSION: These and other results suggest: (i) that the central region of hSpt4 is necessary and sufficient for its function in vitro and (ii) that there is no free hSpt4 or hSpt5 in cells . We propose that hSpt4 and hSpt5 exert their roles in transcriptional regulation, and possibly in other nuclear processes, as parts of the DSIF complex.

Genes Cells, 2003 Apr, 8(4), 325 - 39
A SWI2/SNF2-type ATPase/helicase protein, mDomino, interacts with myeloid zinc finger protein 2A (MZF-2A) to regulate its transcriptional activity; Ogawa H et al.; BACKGROUND: The myeloid zinc finger protein 2A (MZF-2A) is a Kruppel-type C2H2 zinc finger transcription factor expressed in myeloid cells and involved in the growth, differentiation and tumorigenesis of myeloid progenitors . Previously we identified a 180 amino acid domain in MZF-2A which is responsible for the transcriptional activation of MZF-2A . To understand the mechanism of the MZF-2A-dependent transcriptional activation, we screened for molecules that interact with the transactivation domain (TAD) of MZF-2A . RESULTS: By using the yeast Ras recruitment two-hybrid screening, we identified a novel SWI2/SNF2-related protein, termed mammalian Domino (mDomino), as an MZF-2A-binding partner . The mDomino protein, which shows a marked similarity to the Drosophila Domino protein, contains a SWI2/SNF2-type ATPase/helicase domain, a SANT domain, and a glutamine-rich (Q-rich) domain . The C-terminal Q-rich domain of mDomino physically associates with the TAD of MZF-2A in mammalian cells as well as in yeast . Expression of the mDomino Q-rich domain, together with MZF-2A in myeloid LGM-1 cells, enhanced the MZF-2A-mediated activation of a reporter gene . CONCLUSIONS: These results strongly suggest that an ATP-dependent chromatin-remodelling complex containing mDomino interacts with MZF-2A to regulate gene expression in myeloid cells.

Br J Dermatol, 2003 Mar, 148(3), 479 - 88
Atopy patch test reactions to Malassezia allergens differentiate subgroups of atopic dermatitis patients; Johansson C et al.; BACKGROUND: The yeast Malassezia is considered to be one of the factors that can contribute to atopic dermatitis (AD) . OBJECTIVES: To investigate the reactivity to Malassezia allergens, measured as specific serum IgE, positive skin prick test and positive atopy patch test (APT), in adult patients with AD . METHODS: In total, 132 adult patients with AD, 14 with seborrhoeic dermatitis (SD) and 33 healthy controls were investigated for their reactions to M . sympodialis extract and three recombinant Malassezia allergens (rMal s 1, rMal s 5 and rMal s 6) . RESULTS: Sixty-seven per cent of the AD patients, but only one of the SD patients and none of the healthy controls, showed a positive reaction to at least one of the Malassezia allergens (extract and/or recombinant allergens) in at least one of the tests . The levels of M . sympodialis-specific IgE in serum correlated with the total serum IgE levels . Elevated serum levels of M . sympodialis-specific IgE were found in 55% and positive APT reactions in 41% of the AD patients with head and neck dermatitis . A relatively high proportion of patients without head and neck dermatitis and patients with low total serum IgE levels had a positive APT for M . sympodialis, despite lower proportions of individuals with M . sympodialis-specific IgE among these groups of patients . CONCLUSIONS: These results support that Malassezia can play a role in eliciting and maintaining eczema in patients with AD . The addition of an APT to the test battery used in this study reveals a previously overlooked impact of Malassezia hypersensitivity in certain subgroups of AD patients.

Biochem Soc Trans, 2003 Apr, 31(2), 470 - 3
Proteasomal interactors control activities as diverse as the cell cycle and glutaminergic neurotransmission; Rezvani K et al.; The six regulatory non-redundant ATPases in the base of the 19 S regulator of the 26 S proteasome belong to the AAA superfamily of ATPases . Yeast two-hybrid genetic screens, biochemical analyses and cell biological studies have identified and characterized new interactors of the human S6 (rpt3) and S8 (rpt6) ATPases of the 19 S regulator of the 26 S proteasome . The S6 ATPase interacts with gankyrin . This protein is found in purified human 26 S proteasomes and in a smaller complex(es) containing CDK4 and free S6 ATPase . Gankyrin overexpression causes the phosphorylation of the retinoblastoma protein (pRb) and the release of E2F transcription factor to trigger the expression of DNA synthesis genes . Gankyrin is oncogenic in nude mice and is overexpressed in hepatocellular carcinoma cells (HCCs) . The S8 ATPase interacts with members of the large Homer-3 protein family . There are three Homer genes; the Homer 1 and 2 gene products control trafficking and calcium-store-related functions of metabotropic glutamate receptors (e.g . mGluR1alpha) . Homer-3A11 by binding to the S8 ATPase brings mGluR1alpha to the 26 S proteasome for degradation . The degradation of mGluR1alpha is blocked by proteasomal inhibitors and by overexpression of the N-terminus of Homer which binds to the receptor . The S8 ATPase and mGluR1alpha are co-localized in Purkinje dendrites in rat cerebellum . The data are discussed in terms of the regulation of the cell cycle and glutaminergic receptor functions by the 26 S proteasome.

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