FEBS Lett, 2004 Jul 16, 570(1-3), 143 - 8 Direct interaction between BKCa potassium channel and microtubule-associated protein 1A; Park SM et al.; The BKCa channel, a potassium channel that is allosterically activated by voltage and calcium, is expressed in both excitable and non-excitable cells . The channel plays an important role in regulating membrane excitability . The channel activity can be modulated by post-translational modifications such as phosphorylation . Recently, hippocampal BKCa channels were shown to be directly modulated by assembly/disassembly of the submembranous actin cytoskeleton . Here, we report that the BKCa channel physically interacts with the light chain of microtubule associated protein 1A (MAP1A) . The light chain was isolated in a yeast two-hybrid screen of a human brain cDNA library . The specificity of the interaction was demonstrated in biochemical experiments utilizing GST fusion protein pulldown assays and reciprocal co-immunoprecipitations from rat brain . Furthermore, utilizing immunofluorescence, the BKCa channel and MAP1A co-localize in the Purkinje cell layer of the cerebellum . These studies identify a novel interaction between the C-terminal tail of the BKCa channel and the light chain of MAP1A, which enables channel association with and modulation by the cytoskeleton. Brief Funct Genomic Proteomic, 2002 Feb, 1(1), 40 - 52 The jury is out on "guilt by association" trials; Semple JI et al.; The availability of comprehensive protein-protein interaction maps will significantly enhance medical research and aid the functional characterisation of novel genes . To date, the largest scale studies of protein-protein interactions have used the yeast two hybrid method . In this review we take a closer look at the different approaches used in these studies and discuss some key considerations that should be taken into account when designing high throughput interaction mapping projects. Mem Inst Oswaldo Cruz, 2004 Mar, 99(2), 147 - 52 Epub 2004 Jun 24. Genetic variability analysis among clinical Candida spp . isolates using random amplified polymorphic DNA; Pinto PM et al.; The patterns of genetic variation of samples of Candida spp . isolated from patients infected with human immunodeficiency virus in Vitoria, state of Espirito Santo, Brazil, were examined . Thirty-seven strains were isolated from different anatomical sites obtained from different infection episodes of 11 patients infected with the human immunodeficiency virus (HIV) . These samples were subjected to randomly amplified polymorphic DNA (RAPD) analysis using 9 different primers . Reproducible and complex DNA banding patterns were obtained . The experiments indicated evidence of dynamic process of yeast colonization in HIV-infected patients, and also that certain primers are efficient in the identification of species of the Candida genus . Thus, we conclude that RAPD analysis may be useful in providing genotypic characters for Candida species typing in epidemiological investigations, and also for the rapid identification of pathogenic fungi. Proc Natl Acad Sci U S A, 2004 Jul 20, 101(29), 10709 - 14 Epub 2004 Jul 12. The Treacher Collins syndrome (TCOF1) gene product is involved in ribosomal DNA gene transcription by interacting with upstream binding factor; Valdez BC et al.; Treacher Collins syndrome (TCS) is an autosomal dominant disorder characterized by an abnormality of craniofacial development that arises during early embryogenesis . TCS is caused by mutations in the gene TCOF1, which encodes the nucleolar phosphoprotein treacle . Even though the genetic alterations causing TCS have been uncovered, the mechanism underlying its pathogenesis and the function of treacle remain unknown . Here, we show that treacle is involved in ribosomal DNA gene transcription by interacting with upstream binding factor (UBF) . Immunofluorescence labeling shows treacle and UBF colocalize to specific nucleolar organizer regions and cosegregate within nucleolar caps of actinomycin d-treated HeLa cells . Biochemical analysis shows the association of treacle and UBF with chromatin . Immunoprecipitation and the yeast two-hybrid system both suggest physical interaction of the two nucleolar phosphoproteins . Down-regulation of treacle expression using specific short interfering RNA results in inhibition of ribosomal DNA transcription and cell growth . A similar correlation is observed in Tcof(+/-) mouse embryos that exhibit craniofacial defects and growth retardation . Thus, treacle haploinsufficiency in TCS patients might result in abnormal development caused by inadequate ribosomal RNA production in the prefusion neural folds during the early stages of embryogenesis . The elucidation of a physiological function of treacle provides important information of relevance to the molecular dissection of the biochemical pathology of TCS. Proc Natl Acad Sci U S A, 2004 Jul 20, 101(29), 10596 - 601 Epub 2004 Jul 12. The Arabidopsis AtRAD51 gene is dispensable for vegetative development but required for meiosis; Li W et al.; The maintenance of genome integrity and the generation of biological diversity are important biological processes, and both involve homologous recombination . In yeast and animals, homologous recombination requires the function of the RAD51 recombinase . In vertebrates, RAD51 seems to have acquired additional functions in the maintenance of genome integrity, and rad51 mutations cause lethality, but it is not clear how widely these functions are conserved among eukaryotes . We report here a loss-of-function mutant in the Arabidopsis homolog of RAD51, AtRAD51 . The atrad51-1 mutant exhibits normal vegetative and flower development and has no detectable abnormality in mitosis . Therefore, AtRAD51 is not necessary under normal conditions for genome integrity . In contrast, atrad51-1 is completely sterile and defective in male and female meioses . During mutant prophase I, chromosomes fail to synapse and become extensively fragmented . Chromosome fragmentation is suppressed by atspo11-1, indicating that AtRAD51 functions downstream of AtSPO11-1 . Therefore, AtRAD51 likely plays a crucial role in the repair of DNA double-stranded breaks generated by AtSPO11-1 . These results suggest that RAD51 function is essential for chromosome pairing and synapsis at early stages in meiosis in Arabidopsis . Furthermore, major aspects of meiotic recombination seem to be conserved between yeast and plants, especially the fact that chromosome pairing and synapsis depend on the function of SPO11 and RAD51. Biochem Biophys Res Commun, 2004 Aug 6, 320(4), 1063 - 8 Lbc proto-oncogene product binds to and could be negatively regulated by metastasis suppressor nm23-H2; Iwashita S et al.; Lbc was identified as transforming gene from human leukemic cells and encodes Rho type guanine nucleotide exchange factor with 47kDa molecular weight . We isolated overlapping cDNAs of Lbc from human lung tissue . Full-length Lbc cDNA encodes 309kDa huge protein with Ht31 PKA anchoring motif, Dof domain, C1 domain, and coiled-coil structure . In order to analyze the regulatory mechanism of its activity, we searched for binding proteins . By yeast two-hybrid screening, we identified metastasis suppressor nm23-H2 as binding protein, which interacts with amino-terminal region of Lbc containing Dof domain . nm23 gene family encodes nucleoside diphosphate kinase, however, the binding of nm23-H2 to Lbc was independent of kinase activity . nm23-H1, which binds to Rac-specific GEF Tiam1, could not bind to Lbc suggesting nm23-H2 would be specific regulator for Lbc . Expression of nm23-H2 in cells leads to decrease the amount of GTP-bound Rho and suppress stress fiber formation stimulated by expression of Lbc . Our data suggest that metastasis suppressor nm23-H2 could regulate Lbc negatively by binding to amino-terminal region of Lbc proto-oncogene product. Mol Cell Endocrinol, 2004 Jul 30, 222(1-2), 83 - 91 Id2 is a primary partner for the E2-2 basic helix-loop-helix transcription factor in the human placenta; Liu YP et al.; We screened a term placental cDNA library by the yeast two-hybrid approach with Id2, a negative regulator of basic helix-loop-helix (bHLH) factors . Of the clones obtained, approximately one-third were the E2-2 bHLH transcription factor . Id2 and E2-2 were shown to interact in direct two-hybrid assays in yeast cells, as well as immunoprecipitation assays in mammalian cells . Immunohistochemical analysis demonstrated co-localization of both Id2 and E2-2 in placental trophoblasts . Co-transfection of JEG-3 cells with E2-2 and Id2, and a luciferase reporter construct under the control of the human chorionic gonadotropin alpha-subunit promoter revealed that E2-2 had a negative effect on CGalpha-subunit transcription, which could be relieved by overexpression of Id2 . The library was in turn rescreened with E2-2, and Id2 and Id1 were essentially the only clones obtained . We conclude that Id2 is a primary binding partner for the bHLH transcription factor E2-2 in the human placenta. Crit Rev Eukaryot Gene Expr, 2004, 14(3), 147 - 69 Structure and function of histone methyltransferases; Trievel RC; Histones are the major protein constituent of chromatin in the eukaryotic nucleus . These proteins undergo a host of different post-translational modifications, including phosphorylation, acetylation, and methylation, which have profound effects on the remodeling of chromatin . Histone modifications can function either individually or combinatorially to govern such processes as transcription, replication, DNA repair, and apoptosis . Recent studies have focused on histone arginine and lysine methylation and the roles of these modifications in transcriptional regulation and the establishment of heterochromatin . Concomitantly, several families of histone methyltransferases (HMTs) have been identified that catalyze the methylation of specific arginines or lysines in histones H3 and H4 . Not surprisingly, many of these methyltransferase genes had been previously identified as important genetic regulators in organisms such as yeast and Drosophila, which underscores the importance of histone methylation in transcriptional control and chromatin remodeling . Structures of several representatives of these HMT families have recently been determined, yielding insight into their catalytic mechanism and histone substrate specificity . The focus of this review is to briefly summarize the roles of histone methylation in chromatin remodeling and to discuss the structures, substrate specificities, and mechanisms of the different classes of HMTs. Biochemistry, 2004 Jul 20, 43(28), 9225 - 33 Sequences of B-chain/domain 1-10/1-9 of insulin and insulin-like growth factor 1 determine their different folding behavior; Chen Y et al.; Although insulin and insulin-like growth factor-1 (IGF-1) belong to one family, insulin folds into one thermodynamically stable structure, while IGF-1-folds into two thermodynamically stable structures (native and swap forms) . We have demonstrated previously that the bifurcating folding behavior of IGF-1 is mainly controlled by its B-domain . To further elucidate which parts of the sequences determine their different folding behavior, by exchanging the N-terminal sequences of mini-IGF-1 and recombinant porcine insulin precursor (PIP), we prepared four peptide models: {1-9}PIP, {1-10}mini-IGF-1, {1-4}PIP, and {1-5}mini-IGF-1 by means of protein engineering, and their disulfide rearrangement, V8 digestion, circular dichroic spectra, disulfide stability, and in vitro refolding were investigated . Among them only {1-9}PIP, like mini-IGF-1/IGF-1, was expressed in yeast as two isomers: isomer 1 (corresponding to swap IGF-1) and isomer 2 (corresponding to native IGF-1), which are supported by the experimental results of disulfide rearrangements, peptide mapping of V8 endoprotenase digests, circular dichroic analysis, in vitro refolding, and disulfide stability analysis . The other peptide models, {1-10}mini-IGF-1, {1-4}PIP, and {1-5}mini-IGF-1, fold into one stable structure as PIP does, which indicates that sequence 1-4 of mini-IGF-1 is important for the folding behavior of mini-IGF-1/IGF-1 but not sufficient to lead to a bifurcating folding . The results demonstrated that the folding information, by which mini-IGF-1/IGF-1-folds into two thermodynamically structures, is encoded/written in its sequence 1-9, while sequences 1-10 of B chain in insulin/PIP play an important role in the guide of its unique disulfide pairing during the folding process. Nat Cell Biol, 2004 Aug, 6(8), 777 - 83 Epub 2004 Jul 11. The CDC-14 phosphatase controls developmental cell-cycle arrest in C . elegans; Saito RM et al.; Temporal control of cell division is critical for proper animal development . To identify mechanisms involved in developmental arrest of cell division, we screened for cell-cycle mutants that disrupt the reproducible pattern of somatic divisions in the nematode C . elegans . Here, we show that the cdc-14 phosphatase is required for the quiescent state of specific precursor cells . Whereas budding yeast Cdc14p is essential for mitotic exit, inactivation of C . elegans cdc-14 resulted in extra divisions in multiple lineages, with no apparent defects in mitosis or cell-fate determination . CDC-14 fused to the green fluorescent protein (GFP-CDC-14) localized dynamically and accumulated in the cytoplasm during G1 phase . Genetic interaction and transgene expression studies suggest that cdc-14 functions upstream of the cki-1 Cip/Kip inhibitor to promote accumulation of CKI-1 in the nucleus . Our data support a model in which CDC-14 promotes a hypophosphorylated and stable form of CKI-1 required for developmentally programmed cell-cycle arrest. Nat Cell Biol, 2004 Aug, 6(8), 763 - 9 Epub 2004 Jul 11. The mammalian retromer regulates transcytosis of the polymeric immunoglobulin receptor; Verges M et al.; Epithelial cells have separate apical and basolateral plasma membrane domains with distinct compositions . After delivery to one surface, proteins can be endocytosed and then recycled, degraded or transcytosed to the opposite surface . Proper sorting into the transcytotic pathway is essential for maintaining polarity, as most proteins are endocytosed many times during their lifespan . The polymeric immunoglobulin receptor (pIgR) transcytoses polymeric IgA (pIgA) from the basolateral to the apical surface of epithelial cells and hepatocytes . However, the molecular machinery that controls polarized sorting of pIgR-pIgA and other receptors is only partially understood . The retromer is a multimeric protein complex, originally described in yeast, which mediates intracellular sorting of Vps10p, a receptor that transports vacuolar enzymes . The yeast retromer contains two sub-complexes . One includes the Vps5p and Vps17p subunits, which provide mechanical force for vesicle budding . The other is the Vps35p-Vps29p-Vps26p subcomplex, which provides cargo specificity . The mammalian retromer binds to the mannose 6-phosphate receptor, which sorts lysosomal enzymes from the trans-Golgi network to the lysosomal pathway . Here, we show a function for the mammalian Vps35-Vps29-Vps26 retromer subcomplex in promoting pIgR-pIgA transcytosis. Oncogene, 2004 Aug 26, 23(39), 6654 - 65 SP100 expression modulates ETS1 transcriptional activity and inhibits cell invasion; Yordy JS et al.; The ETS1 transcription factor is a member of the Ets family of conserved sequence-specific DNA-binding proteins . ETS1 has been shown to play important roles in various cellular processes such as proliferation, differentiation, lymphoid development, motility, invasion and angiogenesis . These diverse roles of ETS1 are likely to be dependent on specific protein interactions . To identify proteins that interact with ETS1, a yeast two-hybrid screen was conducted . Here, we describe the functional interaction between SP100 and ETS1 . SP100 protein interacts with ETS1 both in vitro and in vivo . SP100 is localized to nuclear bodies and ETS1 expression alters the nuclear body morphology in living cells . SP100 negatively modulates ETS1 transcriptional activation of the MMP1 and uPA promoters in a dose-dependent manner, decreases the expression of these endogenous genes, and reduces ETS1 DNA binding . Expression of SP100 inhibits the invasion of breast cancer cells and is induced by Interferon-alpha, which has been shown to inhibit the invasion of cancer cells . These data demonstrate that SP100 modulates ETS1-dependent biological processes. RNA, 2004 Aug, 10(8), 1266 - 76 Epub 2004 Jul 09. Multimerization of poly(rC) binding protein 2 is required for translation initiation mediated by a viral IRES; Bedard KM et al.; The cellular protein, poly(rC) binding protein 2 (PCBP2), is known to function in picornavirus cap-independent translation . We have further examined the RNA binding properties and protein-protein interactions of PCBP2 necessary for translation . We have studied its putative multimerization properties utilizing the yeast two-hybrid assay and in vitro biochemical methods, including glutathione S-transferase (GST) pull-down assays and gel filtration . Through genetic analysis, the multimerization domain has been localized to the second K-homologous (KH) RNA binding domain of the protein between amino acids 125 and 158 . To examine the function of multimerization in poliovirus translation, we utilized the truncated protein, DeltaKH1-PCBP2, which is capable of multimer formation, but does not bind poliovirus stem-loop IV RNA (an interaction required for translation) . Utilizing RNA binding and in vitro translation assays, this protein was shown to act as a dominant negative, suggesting that PCBP2 multimerization functions in poliovirus translation and RNA binding . Additionally, PCBP2 containing a deletion in the multimerization domain (DeltaKH2-PCBP2) was not able to bind poliovirus stem-loop IV RNA and could not rescue translation in extracts that were depleted of endogenous PCBP2 . Results from these experiments suggest that the multimerization of PCBP2 is required for efficient RNA binding and cap-independent translation of poliovirus RNA . By examining the functional interactions of the cellular protein PCBP2, we have discovered a novel determinant in the mechanism of picornavirus cap-independent translation. Plant Physiol, 2004 Jul, 135(3), 1231 - 42 Epub 2004 Jul 09. Novel biosynthetic pathway of castasterone from cholesterol in tomato; Kim TW et al.; Endogenous brassinosteroids (BRs) in tomato (Lycopersicon esculentum) seedlings are known to be composed of C27- and C28-BRs . The biosynthetic pathways of C27-BRs were examined using a cell-free enzyme solution prepared from tomato seedlings that yielded the biosynthetic sequences cholesterol --> cholestanol and 6-deoxo-28-norteasterone <--> 6-deoxo-28-nor-3-dehydroteasterone <--> 6-deoxo-28-nortyphasterol --> 6-deoxo-28-norcastasterone --> 28-norcastasterone (28-norCS) . Arabidopsis CYP85A1 that was heterologously expressed in yeast mediated the conversion of 6-deoxo-28-norCS to 28-norCS . The same reaction was catalyzed by an enzyme solution from wild-type tomato but not by an extract derived from a tomato dwarf mutant with a defect in CYP85 . Furthermore, exogenously applied 28-norCS restored the abnormal growth of the dwarf mutant . These findings indicate that the C-6 oxidation of 6-deoxo-28-norCS to 28-norCS in tomato seedlings is catalyzed by CYP85, just as in the conversion of 6-deoxoCS to CS . Additionally, the cell-free solution also catalyzed the C-24 methylation of 28-norCS to CS in the presence of NADPH and S-adenosylmethionine (SAM), a reaction that was clearly retarded in the absence of NADPH and SAM . Thus it seems that C27-BRs, in addition to C28-BRs, are important in the production of more active C28-BRs and CS, where a SAM-dependent sterol methyltransferase appears to biosynthetically connect C27-BRs to C28-BRs . Moreover, the tomato cell-free solution converted CS to 26-norCS and {2H6}CS to {2H3}28-norCS, suggesting that C-28 demethylation is an artifact due to an isotope effect . Although previous feeding experiments employing {2H6}CS suggested that 28-norCS was synthesized from CS in certain plant species, this is not supported in planta . Altogether, this study demonstrated for the first time, to our knowledge, that 28-norCS is not synthesized from CS but from cholesterol . In addition, CS and {2H6}CS were not converted into BL and {2H6}BL, respectively, confirming an earlier finding that the active BR in tomato seedlings is not BL but CS . In conclusion, the biosynthesis of 28-norBRs appears to play a physiologically important role in maintaining homeostatic levels of CS in tomato seedlings. Plant Physiol, 2004 Jul, 135(3), 1457 - 70 Epub 2004 Jul 09. Role of Hsp17.4-CII as coregulator and cytoplasmic retention factor of tomato heat stress transcription factor HsfA2; Port M et al.; HsfA2 is a heat stress (hs)-induced Hsf in peruvian tomato (Lycopersicon peruvianum) and the cultivated form Lycopersicon esculentum . Due to the high activator potential and the continued accumulation during repeated cycles of heat stress and recovery, HsfA2 becomes a dominant Hsf in thermotolerant cells . The formation of heterooligomeric complexes with HsfA1 leads to nuclear retention and enhanced transcriptional activity of HsfA2 . This effect seems to represent one part of potential molecular mechanisms involved in its activity control . As shown in this paper, the activity of HsfA2 is also controlled by a network of nucleocytoplasmic small Hsps influencing its solubility, intracellular localization and activator function . By yeast two-hybrid interaction and transient coexpression studies in tobacco (Nicotiana plumbaginifolia) mesophyll protoplasts, we found that tomato (Lycopersicon esculentum) Hsp17.4-CII acts as corepressor of HsfA2 . Given appropriate conditions, both proteins together formed large cytosolic aggregates which could be solubilized in presence of class CI sHsps . However, independent of the formation of aggregates or of the nucleocytoplasmic distribution of HsfA2, its transcriptional activity was specifically repressed by interaction of Hsp17.4-CII with the C-terminal activator domain . Although not identical in all aspects, the situation with the highly expressed, heat stress-inducible Arabidopsis HsfA2 was found to be principally similar . In corresponding reporter assays its activity was repressed in presence of AtHsp17.7-CII but not of AtHsp17.6-CII or LpHsp17.4-CII. J Biol Chem, 2004 Sep 17, 279(38), 39846 - 55 Epub 2004 Jul 06. Mitochondrial bound hexokinase activity as a preventive antioxidant defense: steady-state ADP formation as a regulatory mechanism of membrane potential and reactive oxygen species generation in mitochondria; da-Silva WS et al.; Brain hexokinase is associated with the outer membrane of mitochondria, and its activity has been implicated in the regulation of ATP synthesis and apoptosis . Reactive oxygen species (ROS) are by-products of the electron transport chain in mitochondria . Here we show that the ADP produced by hexokinase activity in rat brain mitochondria (mt-hexokinase) controls both membrane potential (Deltapsi(m)) and ROS generation . Exposing control mitochondria to glucose increased the rate of oxygen consumption and reduced the rate of hydrogen peroxide generation . Mitochondrial associated hexokinase activity also regulated Deltapsi(m), because glucose stabilized low Deltapsi(m) values in state 3 . Interestingly, the addition of glucose 6-phosphate significantly reduced the time of state 3 persistence, leading to an increase in the Deltapsi(m) and in H(2)O(2) generation . The glucose analogue 2-deoxyglucose completely impaired H(2)O(2) formation in state 3-state 4 transition . In sharp contrast, the mt-hexokinase-depleted mitochondria were, in all the above mentioned experiments, insensitive to glucose addition, indicating that the mt-hexokinase activity is pivotal in the homeostasis of the physiological functions of mitochondria . When mt-hexokinase-depleted mitochondria were incubated with exogenous yeast hexokinase, which is not able to bind to mitochondria, the rate of H(2)O(2) generation reached levels similar to those exhibited by control mitochondria only when an excess of 10-fold more enzyme activity was supplemented . Hyperglycemia induced in embryonic rat brain cortical neurons increased ROS production due to a rise in the intracellular glucose 6-phosphate levels, which were decreased by the inclusion of 2-deoxyglucose, N-acetyl cysteine, or carbonyl cyanide p-trifluoromethoxyphenylhydrazone . Taken together, the results presented here indicate for the first time that mt-hexokinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism. J Biol Chem, 2004 Sep 10, 279(37), 38626 - 35 Epub 2004 Jul 07. Homo-oligomerization of ALS2 through its unique carboxyl-terminal regions is essential for the ALS2-associated Rab5 guanine nucleotide exchange activity and its regulatory function on endosome trafficking; Kunita R et al.; Mutations in the ALS2 gene have been known to account for a juvenile recessive form of amyotrophic lateral sclerosis (ALS2), a rare juvenile recessive form of primary lateral sclerosis, and a form of hereditary spastic paraplegia (HSP), indicating that the ALS2 protein is essential for the maintenance of motor neurons . Recently, we have demonstrated that the ALS2 protein specifically binds to the small GTPase Rab5 and acts as a GEF (guanine nucleotide exchange factor) for Rab5 . We have also shown that its Rab5GEF-requisite domain resides within the C-terminal 640-amino acid region spanning membrane occupation and recognition nexus motifs and the vacuolar protein sorting 9 domain . Transiently expressed ALS2 localized onto early endosomal compartments and stimulated endosome fusions in neuronal and non-neuronal cells in an Rab5GEF activity-dependent manner . These results indicate that the C-terminal region of ALS2 plays a crucial role in endosomal dynamics by its Rab5GEF activity . Here we delineate a molecular feature of the ALS2-associated function through the C-terminal region-mediated homo-oligomerization . A yeast two-hybrid screen for interacting proteins with the ALS2 C-terminal portion identified ALS2 itself . ALS2 forms a homophilic oligomer through its distinct C-terminal regions . This homo-oligomerization is crucial for the Rab5GEF activity in vitro and the ALS2-mediated endosome enlargement in the cells . Taken together, these results indicate that oligomerization of the ALS2 protein is one of the fundamental features for its physiological function involving endosome dynamics in vivo. Bioinformatics, 2004 Dec 12, 20(18), 3346 - 52 Epub 2004 Jul 09. Conserved network motifs allow protein-protein interaction prediction; Albert I et al.; MOTIVATION: High-throughput protein interaction detection methods are strongly affected by false positive and false negative results . Focused experiments are needed to complement the large-scale methods by validating previously detected interactions but it is often difficult to decide which proteins to probe as interaction partners . Developing reliable computational methods assisting this decision process is a pressing need in bioinformatics . RESULTS: We show that we can use the conserved properties of the protein network to identify and validate interaction candidates . We apply a number of machine learning algorithms to the protein connectivity information and achieve a surprisingly good overall performance in predicting interacting proteins . Using a 'leave-one-out' approach we find average success rates between 20 and 40% for predicting the correct interaction partner of a protein . We demonstrate that the success of these methods is based on the presence of conserved interaction motifs within the network . AVAILABILITY: A reference implementation and a table with candidate interacting partners for each yeast protein are available at http://www.protsuggest.org. Trends Cell Biol, 2004 Jul, 14(7), 359 - 68 Building the centromere: from foundation proteins to 3D organization; Amor DJ et al.; At each mitosis, accurate segregation of every chromosome is ensured by the assembly of a kinetochore at each centromeric locus . Six foundation kinetochore proteins that assemble hierarchically and co-dependently have been identified in vertebrates . CENP-A, Mis12, CENP-C, CENP-H and CENP-I localize to a core domain of centromeric chromatin . The sixth protein, CENP-B, although not essential in higher eukaryotes, has homologues in fission yeast that bind pericentric DNA and are essential for heterochromatin formation . Foundation kinetochore proteins have various roles and mutual interactions, and their associations with centromeric DNA and heterochromatin create structural domains that support the different functions of the centromere . Advances in molecular and microscopic techniques, coupled with rare centromere variants, have enabled us to gain fresh insights into the linear and 3D organization of centromeric chromatin. Trends Cell Biol, 2004 Jul, 14(7), 352 - 8 Seeing is believing: imaging actin dynamics at single sites of endocytosis; Merrifield CJ; Endocytosis is characterized by movement and precisely controlled changes in membrane geometry during vesicle formation . Recent developments in live-cell imaging have enabled such movements to be monitored in vivo and correlated with the recruitment and dismissal of fluorescently labeled proteins . This experimental strategy has revealed the sequential recruitment of proteins that are involved in actin polymerization, and actin to single sites of endocytosis in both yeast and mammalian cells . Actin polymerization is correlated with the inward movements of endocytic organelles, which suggests that actin polymerization has a conserved role in this process . In this article, I will discuss three models for the role of actin polymerization in endocytosis. Trends Cell Biol, 2004 Jul, 14(7), 331 - 4 Linking tumor suppression, DNA damage and the anaphase-promoting complex; Jackson PK; A recent study shows that the RASSF1A tumor suppressor functions as a regulator of the ordered proteolytic steps that organize mitosis . By controlling the stability of microtubules and the activity of the anaphase-promoting complex (APC), RASSF1A might provide a crucial link between mechanisms of tumor suppression and mitotic cell division . Furthermore, another recent study shows that protein kinase A, which is a key growth regulator, inhibits the APC during mitosis in yeast. Trends Biotechnol, 2004 Jul, 22(7), 319 - 21 Silence of the centromeres--not; Cooke HJ; Centromeres are a conundrum; although many proteins associated with centomeres are conserved from yeast to humans, the underlying DNA sequence is not . A proposed solution to this problem is that an epigenetic, largely heterochromatic, state be imposed by these proteins . Recent analysis of a human neocentromere and the complete sequence of a rice centromere suggest that this epigenetic state can enable transcription of at least some genes within a centromere. Int J Tissue React, 2003, 25(4), 159 - 65 Assessment of residual immunoreactivity in red or white wines clarified with pea or lupin extracts; Cattaneo A et al.; Vegetable proteins could be a suitable alternative to animal proteins in the clarification of wine, but their residues could represent a risk for subjects with food allergy or intolerance . The aim of this study was to investigate the presence of specific immunoreactivity in red and white wines treated, as must or wine, with vegetable proteins in the clarification process . The proteins considered were prepared from lupins and peas, which are not included among the allergens listed in annex Illbis of Directive 2003/89/EC . The presence of residual immunoreactivity to specific rabbit anti-lupin and anti-pea polyclonal antibodies in treated wines was assessed by electrophoresis (SDS-PAGE) and immunoblotting . Residual protein was not detectable in red wines clarified with lupin, pea or a mixture of pea and lupin proteins or in white wines clarified with pea proteins . A small number of musts treated with lupin or pea proteins and white wines treated with lupin proteins yielded equivocal results, probably because of the presence of interfering material (e.g., sugar-rich proteins from grape and yeast) . The use of bentonite as a secondary clarifying agent is therefore recommended since its combination with vegetable proteins is particularly effective in removing overall protein immunoreactivity. J Clin Microbiol, 2004 Jul, 42(7), 3363 - 5 Fungemia caused by Zygoascus hellenicus in an allogeneic stem cell transplant recipient; Brandt ME et al.; Zygoascus hellenicus (Candida hellenica) was isolated from a blood culture from a patient who had received an allogeneic stem cell transplant . The isolate displayed an antifungal susceptibility pattern of decreased susceptibility to fluconazole and itraconazole, high susceptibility to voriconazole, and low susceptibility to caspofungin . The organism was misidentified by a commercial yeast identification system . This is the first reported case of human infection with this rare ascomycetous yeast. Exp Cell Res, 2004 Aug 1, 298(1), 239 - 48 The suppressor of cytokine signaling (SOCS)-7 interacts with the actin cytoskeleton through vinexin; Martens N et al.; To understand the function of the suppressor of cytokine signaling (SOCS)-7, we have looked for proteins interacting with SOCS-7 in a stringent yeast two-hybrid screen of a human leukocyte cDNA-library . We identified the cytoskeletal molecule vinexin as a partner interacting with SOCS-7 . Tests with deletion mutants of SOCS-7 demonstrated that a central region of the molecule containing several proline-rich regions, N-terminal to the SH2 domain, was responsible for the binding to vinexin . It is thus likely that one of the SH3 domains of vinexin interacts with a poly-proline region of SOCS-7 . The interaction with vinexin was confirmed biochemically as vinexin-alpha was co-precipitated with SOCS-7 . Confocal laser-scanning microscopy in HEK293T, MCF-7, and 3T3-L1 cells showed that part of the transfected SOCS-7-green fluorescent protein (GFP) molecules merged with vinexin and with actin . Taken together, our data indicate that SOCS-7 interacts with vinexin and the actin cytoskeleton. Exp Cell Res, 2004 Aug 1, 298(1), 197 - 206 Interferon induces the interaction of prothymosin-alpha with STAT3 and results in the nuclear translocation of the complex; Yang CH et al.; Interferons (IFNs) play critical roles in host defense by modulating the expression of various genes via tyrosine phosphorylation of STAT transcription factors . Many cytokines including IFNs induce tyrosine phosphorylation of the STAT3 transcription factor, which regulates acute phase gene expression . Using the yeast two-hybrid interaction trap, in which a tyrosine kinase is introduced into the yeast to allow tyrosine phosphorylation of bait proteins, prothymosin-alpha (ProTalpha) was identified to interact with the amino terminal half of tyrosine-phosphorylated STAT3 . ProTalpha is a small, acidic, extremely abundant, and essential protein that may play a role in chromatin remodeling, and has been implicated in regulating the growth and survival of mammalian cells . Besides the interaction of tyrosine-phosphorylated STAT3 with ProTalpha in yeast cells, IFN induced the interaction of ProTalpha with STAT3 in mammalian cells, and this interaction was dependent on the tyrosine phosphorylation of STAT3 . Moreover, IFNalpha induces the translocation of STAT3 and ProTalpha from the cytoplasm to the nucleus where these proteins colocalize . Since ProTalpha has an extremely strong nuclear localization and STAT proteins apparently lack any nuclear localization signals, the association of STAT3 with ProTalpha may provide a mechanism to result in STAT localization in the nucleus. Mol Plant Microbe Interact, 2004 Jul, 17(7), 711 - 9 The arabidopsis TIR-NB-LRR gene RAC1 confers resistance to Albugo candida (white rust) and is dependent on EDS1 but not PAD4; Borhan MH et al.; Resistance to Albugo candida isolate Acem1 is conferred by a dominant gene, RAC1, in accession Ksk-1 of Arabidopsis thaliana . This gene was isolated by positional cloning and is a member of the Drosophila toll and mammalian interleukin-1 receptor (TIR) nucleotide-binding site leucine-rich repeat (NB-LRR) class of plant resistance genes . Strong identity of the TIR and NB domains was observed between the predicted proteins encoded by the Ksk-1 allele and the allele from an Acem1-susceptible accession Columbia (Col) (99 and 98%, respectively) . However, major differences between the two predicted proteins occur within the LRR domain and mainly are confined to the beta-strand/beta-turn structure of the LRR . Both proteins contain 14 imperfect repeats . RAC1-mediated resistance was analyzed further using mutations in defense regulation, including: pad4-1, eds1-1, and NahG, in the presence of the RAC1 allele from Ksk-1 . White rust resistance was completely abolished by eds1-1 but was not affected by either pad4-1 or NahG. Planta Med, 2004 Jun, 70(6), 483 - 8 Clinical and mycological evaluation of therapeutic effectiveness of Solanum chrysotrichum standardized extract on patients with Pityriasis capitis (dandruff) . A double blind and randomized clinical trial controlled with ketoconazole; Herrera-Arellano A et al.; Dandruff (also called Pityriasis capitis) is a seborrhoeic dermatitis of the scalp . It has been correlated with the pathological colonization of the scalp with yeast from the genus Malassezia; this illness has a worldwide distribution and represents 25% of all scalp dermatosis cases . It has been demonstrated that the extract obtained from leaves of the plant Solanum chrysotrichum possesses biological activity against dermatophytes and yeast . Different steroidal saponins with antimycotic activity have been isolated from the active extract . Clinical trials with standardized extracts prepared with this vegetal species report high rates of clinical and mycological effectiveness in the treatment of Tinea pedis,without producing secondary effects . The aim of this randomized, double blind and controlled clinical study, was to compare the therapeutic effectiveness and tolerability of a shampoo containing a standardized extract of S . chrysotrichum (applied every third day, for 4 weeks), against 2% ketoconazole in the topical treatment of Pityriasis capitis . From a total of 120 patients with the clinical diagnosis of Pityriasis capitis, 14 subjects were eliminated because the presence of Malassezia was not proved, an-other two patients withdrew from the study due to non-medical causes and one more withdrew because Tinea capitis was diagnosed . Therefore, the final analysis included 51 subjects in the experimental group and 52 in the control; in 45.6% of the cases M . furfur was identified as the pathogenic agent, in 44.66% M . globosa was isolated, and 9.71 % of the patients had a mixed infestation . At the end of the treatment period, the prepared phytopharmaceutical with the standardized extract from S . chrysotrichum achieved a clinical effectiveness (total absence of signs and symptoms produced by Pityriasis capitis) of 92.16%;the mycological effectiveness (absence of Malassezia spp . in the direct examination and culture) was 68.63 %; whilst the tolerability (absence of side effects that prompt subjects to abandon the treatment) was 100% . The therapeutic success (clinical and mycological effectiveness plus tolerability) was 64.71% . The comparison of these results with that obtained from the group treated with 2% ketoconazole, showed no significant differences (Z2, p >0.23) . These results show the therapeutic effectiveness and tolerability of the standardized extract from S . chrysotrichum on the local treatment of Pityriasis capitis associated with the yeast of the genus Malassezia. EMBO J, 2004 Aug 4, 23(15), 3144 - 53 Epub 2004 Jul 08. Preferential cleavage of chromatin-bound cohesin after targeted phosphorylation by Polo-like kinase; Hornig NC et al.; The final irreversible step in the duplication and dissemination of eukaryotic genomes takes place when sister chromatid pairs split and separate in anaphase . This is triggered by the protease separase that cleaves the Scc1 subunit of 'cohesin', the protein complex responsible for holding sister chromatids together in metaphase . Only part of cellular cohesin is bound to chromosomes in metaphase, and it is unclear whether and how separase specifically targets this fraction for cleavage . We established an assay to compare cleavage of chromatin-bound versus soluble budding yeast cohesin . Scc1 in chromosomal cohesin is significantly preferred by separase over Scc1 in soluble cohesin . The difference is most likely due to preferential phosphorylation of chromatin-bound Scc1 by Polo-like kinase . Site-directed mutagenesis of 10 Polo phosphorylation sites in Scc1 slowed cleavage of chromatin-bound cohesin, and hyperphosphorylation of soluble Scc1 by Polo overexpression accelerated its cleavage to levels of chromosomal cohesin . Polo is bound to chromosomes independently of cohesin's presence, providing a possible explanation for chromosome-specific cohesin modification and targeting of separase cleavage. EMBO J, 2004 Jul 21, 23(14), 2765 - 76 Epub 2004 Jul 08. A soluble SNARE drives rapid docking, bypassing ATP and Sec17/18p for vacuole fusion; Thorngren N et al.; Membrane fusion requires priming, the disassembly of cis-SNARE complexes by the ATP-driven chaperones Sec18/17p . Yeast vacuole priming releases Vam7p, a soluble SNARE . Vam7p reassociation during docking allows trans-SNARE pairing and fusion . We now report that recombinant Vam7p (rVam7p) enters into complex with other SNAREs in vitro and bypasses the need for Sec17p, Sec18p, and ATP . Thus, the sole essential function of vacuole priming in vitro is the release of Vam7p from cis-SNARE complexes . In 'bypass fusion', without ATP but with added rVam7p, there are sufficient unpaired vacuolar SNAREs Vam3p, Vti1p, and Nyv1p to interact with Vam7p and support fusion . However, active SNARE proteins are not sufficient for bypass fusion . rVam7p does not bypass requirements for Rho GTPases,Vps33p, Vps39p, Vps41p, calmodulin, specific lipids, or Vph1p, a subunit of the V-ATPase . With excess rVam7p, reduced levels of PI(3)P or functional Ypt7p suffice for bypass fusion . High concentrations of rVam7p allow the R-SNARE Ykt6p to substitute for Nyv1p for fusion; this functional redundancy among vacuole SNAREs may explain why nyv1Delta strains lack the vacuole fragmentation seen with mutants in other fusion catalysts. Proc Natl Acad Sci U S A, 2004 Jul 13, 101(28), 10266 - 71 Epub 2004 Jul 06. Switching desaturase enzyme specificity by alternate subcellular targeting; Heilmann I et al.; The functionality, substrate specificity, and regiospecificity of enzymes typically evolve by the accumulation of mutations in the catalytic portion of the enzyme until new properties arise . However, emerging evidence suggests enzyme functionality can also be influenced by metabolic context . When the plastidial Arabidopsis 16:0Delta7 desaturase FAD5 (ADS3) was retargeted to the cytoplasm, regiospecificity shifted 70-fold, Delta7 to Delta9 . Conversely, retargeting of two related cytoplasmic 16:0Delta9 Arabidopsis desaturases (ADS1 and ADS2) to the plastid, shifted regiospecificity approximately 25-fold, Delta9 to Delta7 . All three desaturases exhibited Delta9 regiospecificity when expressed in yeast, with desaturated products found predominantly on phosphatidylcholine . Coexpression of each enzyme with cucumber monogalactosyldiacylglycerol (MGDG) synthase in yeast conferred Delta7 desaturation, with 16:1Delta7 accumulating specifically on the plastidial lipid MGDG . Positional analysis is consistent with ADS desaturation of 16:0 on MGDG . The lipid headgroup acts as a molecular switch for desaturase regiospecificity . FAD5 Delta7 regiospecificity is thus attributable to plastidial retargeting of the enzyme by addition of a transit peptide to a cytoplasmic Delta9 desaturase rather than the numerous sequence differences within the catalytic portion of ADS enzymes . The MGDG-dependent desaturase activity enabled plants to synthesize 16:1Delta7 and its abundant metabolite, 16:3Delta(7,10,13) . Bioinformatics analysis of the Arabidopsis genome identified 239 protein families that contain members predicted to reside in different subcellular compartments, suggesting alternative targeting is widespread . Alternative targeting of bifunctional or multifunctional enzymes can exploit eukaryotic subcellular organization to create metabolic diversity by permitting isozymes to interact with different substrates and thus create different products in alternate compartments. Mol Biol Cell, 2004 Sep, 15(9), 4337 - 46 Epub 2004 Jul 07. The growth-regulatory protein HCRP1/hVps37A is a subunit of mammalian ESCRT-I and mediates receptor down-regulation; Bache KG et al.; The biogenesis of multivesicular bodies and endosomal sorting of membrane cargo are driven forward by the endosomal sorting complexes required for transport, ESCRT-I, -II, and -III . ESCRT-I is characterized in yeast as a complex consisting of Vps23, Vps28, and Vps37 . Whereas mammalian homologues of Vps23 and Vps28 (named Tsg101 and hVps28, respectively) have been identified and characterized, a mammalian counterpart of Vps37 has not yet been identified . Here, we show that a regulator of proliferation, hepatocellular carcinoma related protein 1 (HCRP1), interacts with Tsg101, hVps28, and their upstream regulator Hrs . The ability of HCRP1 (which we assign the alternative name hVps37A) to interact with Tsg101 is conferred by its mod(r) domain and is shared with hVps37B and hVps37C, two other mod(r) domain-containing proteins . HCRP1 cofractionates with Tsg101 and hVps28 by size exclusion chromatography and colocalizes with hVps28 on LAMP1-positive endosomes . Whereas depletion of Tsg101 by siRNA reduces cellular levels of both hVps28 and HCRP1, depletion of HCRP1 has no effect on Tsg101 or hVps28 . Nevertheless, HCRP1 depletion strongly retards epidermal growth factor (EGF) receptor degradation . Together, these results indicate that HCRP1 is a subunit of mammalian ESCRT-I and that its function is essential for lysosomal sorting of EGF receptors. J Immunol, 2004 Jul 15, 173(2), 1118 - 28 A short consensus repeat-containing complement regulatory protein of lamprey that participates in cleavage of lamprey complement 3; Kimura Y et al.; The prototype of the short consensus repeat (SCR)-containing C regulatory protein is of interest in view of its evolutionary significance with regard to the origin of the C regulatory system . Lamprey is an agnathan fish that belongs to the lowest class of vertebrates . Because it does not possess lymphocytes, it lacks Ig and consequently the classical C pathway . We identified an SCR-containing C regulatory protein from the lamprey . The primary structure predicted from the cDNA sequence showed that this is a secretary protein consisting of eight SCRs . This framework is similar to the alpha-chain of C4b-binding protein (C4bp) . SCR2 and -3 of human C4bp are essential for C4b inactivation, and this region is fairly well conserved in the lamprey protein . However, the other SCRs of this protein are similar to those of other human C regulatory proteins . The lamprey protein binds to the previously reported lamprey C3b/C3bi deposited on yeast and cleaves lamprey C3b-like C3 together with a putative serum protease . The scheme resembles the C regulatory system of mammals, where factor I and its cofactor inactivate C3b . Unlike human cofactors, the lamprey protein requires divalent cations for C3b-like C3 cleavage . Its artificial membrane-anchored form protects host cells from lamprey C attack via the lectin pathway . Thus, the target of this protein appears to be C3b and/or its family . We named this protein Lacrep, the lamprey C regulatory protein . Lacrep is a member of SCR-containing C regulators, the first of its kind identified in the lowest vertebrates. J Immunol, 2004 Jul 15, 173(2), 797 - 806 Mechanism by which orally administered beta-1,3-glucans enhance the tumoricidal activity of antitumor monoclonal antibodies in murine tumor models; Hong F et al.; Antitumor mAb bind to tumors and activate complement, coating tumors with iC3b . Intravenously administered yeast beta-1,3;1,6-glucan functions as an adjuvant for antitumor mAb by priming the inactivated C3b (iC3b) receptors (CR3; CD11b/CD18) of circulating granulocytes, enabling CR3 to trigger cytotoxicity of iC3b-coated tumors . Recent data indicated that barley beta-1,3;1,4-glucan given orally similarly potentiated the activity of antitumor mAb, leading to enhanced tumor regression and survival . This investigation showed that orally administered yeast beta-1,3;1,6-glucan functioned similarly to barley beta-1,3;1,4-glucan with antitumor mAb . With both oral beta-1,3-glucans, a requirement for iC3b on tumors and CR3 on granulocytes was confirmed by demonstrating therapeutic failures in mice deficient in C3 or CR3 . Barley and yeast beta-1,3-glucan were labeled with fluorescein to track their oral uptake and processing in vivo . Orally administered beta-1,3-glucans were taken up by macrophages that transported them to spleen, lymph nodes, and bone marrow . Within the bone marrow, the macrophages degraded the large beta-1,3-glucans into smaller soluble beta-1,3-glucan fragments that were taken up by the CR3 of marginated granulocytes . These granulocytes with CR3-bound beta-1,3-glucan-fluorescein were shown to kill iC3b-opsonized tumor cells following their recruitment to a site of complement activation resembling a tumor coated with mAb. Biochem Biophys Res Commun, 2004 Jul 30, 320(3), 1034 - 42 RICH-1 has a BIN/Amphiphysin/Rvsp domain responsible for binding to membrane lipids and tubulation of liposomes; Richnau N et al.; RhoGAP interacting with CIP4 homologs-1 (RICH-1) was previously found in a yeast two-hybrid screen for proteins interacting with the SH3 domain of the Cdc42-interacting protein 4 (CIP4) . RICH-1 was shown to be a RhoGAP for Cdc42 and Rac . In this study, we show that the BIN/Amphiphysin/Rvsp (BAR) domain in RICH-1 confers binding to membrane lipids, and has the potential to deform spherical liposomes into tubes . In accordance with previous findings for the BAR domains in endophilin and amphiphysin, RICH-1-induced tubes appeared striated . We propose that these striated structures are formed by oligomerization of RICH-1 through a putative coiled-coil region within the BAR domain . In support of this notion, we show that RICH-1 forms oligomers in the presence of the chemical cross-linker BS3 . These results point to an involvement of RICH-1 in membrane deformation events. Biochem Biophys Res Commun, 2004 Jul 30, 320(3), 664 - 71 Isolation and functional characterization of a dynamin-like gene from Plasmodium falciparum; Li H et al.; A novel dynamin-like GTPase gene, Pfdyn1, was cloned from an asexual stage cDNA library of Plasmodium falciparum Dd2 strain . Pfdyn1 contains a highly conserved N-terminal tripartite GTPase domain, a coiled-coil region, and a C-terminal 129 aa unknown function domain . Like yeast Vps1p, it lacks pleckstrin homology domain and proline-rich region . Western blot analysis showed that Pfdyn1 is a Triton X-100 insoluble protein expressed only in the mature sub-stage . Morphological studies indicated that Pfdyn1 is partly co-localized with PfGRP, a known ER-resident protein, and localizes diffusely with several membrane structures and a 60-100 nm vesicle both inside and on surface of the parasites and also in the cytoplasm of infected erythrocytes . The dsRNA originated by C-terminus fragment of Pfdyn1 inhibits markedly the growth of P . falciparum parasite at the erythrocyte stage . Those data showed that Pfdyn1 is a conservative, membrane related protein and plays an essential role for the survival of Plasmodium parasite. Cell Signal, 2004 Oct, 16(10), 1105 - 12 The Rheb family of GTP-binding proteins; Aspuria PJ et al.; Rheb proteins represent a novel and unique family of the Ras superfamily GTP-binding proteins that is conserved from yeast to human . Biochemical studies establish that they bind and hydrolyze GTP . Molecular modeling studies reveal a few structural differences between Rheb and Ras, which may suggest that residues involved in biochemical activities differ between the two G-proteins . The function of Rheb has been studied in a number of organisms that point to the involvement of Rheb in cell growth and cell cycle progression . In addition, studies in fungi suggest that Rheb is involved in arginine uptake . Further studies in Drosophila and mammalian cells have shown that the effects of Rheb on growth and cell cycle progression are mediated by the effect on the insulin/TOR/S6K signaling pathway . These studies have also shown that a complex consisting of the tuberous sclerosis gene products, Tsc1/Tsc2, serves as a GTPase activating protein (GAP) for Rheb, implying Rheb's role in this genetic disorder . Finally, Rheb proteins have been shown to be farnesylated and small molecule inhibitors of protein farnesyltransferase can block the ability of Rheb to activate the TOR/S6K signaling. Brief Funct Genomic Proteomic, 2003 Apr, 2(1), 57 - 71 Transposable elements as tools for genomics and genetics in Drosophila; Ryder E et al.; The P-element has been the workhorse of Drosophila genetics since it was developed as a tool for transgenesis in 1982; the subsequent development of a variety of systems based on the transposon have provided a range of powerful and flexible tools for genetics and genomics applications . P-element insertions are frequently used as starting-points for generating chromosomal deletions to remove flanking genes, either by screening for imprecise excision events or by selecting for male recombination events . Elements that utilise the yeast FLP/FLP recombination target (FRT) site-specific recombination system have been widely used to generate molecularly marked mitotic clones for mosaic analysis, extending the reach of this powerful genetic tool to virtually all areas of developmental biology . P-elements are still widely used as traditional mutagenesis reagents and form the backbone of projects aimed at generating insertions in every predicted gene in the fly genome . In addition, vectors based on the FLP/FRT system are being used for genome-wide applications, including the development of molecularly-mapped deletion and duplication kits . In addition to these 'traditional' genetic approaches, a variety of engineered elements have been developed for a wide range of transgenic applications, including enhancer trapping, gene-tagging, targeted misexpression, RNA interference (RNAi) delivery and homologous recombination/gene replacement . To complement the use of P-elements, alternative transposon vectors have been developed . The most widely used of these are the lepidopteran element piggyBac and a Drosophila hydei transposon, Minos . In total, a range of transposon vectors offers the Drosophila biologist considerable flexibility and sophistication in manipulating the genome of the fly and has allowed rapid advances in all areas of developmental biology and genome science. Biochem J, 2004 Oct 15, 383(Pt 2), 303 - 9 Copper homoeostasis in Drosophila melanogaster S2 cells; Southon A et al.; Copper homoeostasis was investigated in the Drosophila melanogaster S2 cell line to develop an insect model for the study of copper regulation . Real-time PCR studies have demonstrated expression in S2 cells of putative orthologues of human Cu regulatory genes involved in the uptake, transport, sequestration and efflux of Cu . Drosophila orthologues of the mammalian Cu chaperones, ATOX1 (a human orthologue of yeast ATX1), CCS (copper chaperone for superoxide dismutase), COX17 (a human orthologue of yeast COX17), and SCO1 and SCO2, did not significantly respond transcriptionally to increased Cu levels, whereas MtnA, MtnB and MtnD (Drosophila orthologues of human metallothioneins) were up-regulated by Cu in a time- and dose-dependent manner . To examine the effect on Cu homoeostasis, expression of several key copper homoeostasis genes was suppressed using double-stranded RNA interference . Suppression of the MTF-1 (metal-regulatory transcription factor 1), reduced both basal and Cu-induced gene expressions of MtnA, MtnB and MtnD, significantly reducing the tolerance of these cells to increased Cu . Suppression of either Ctr1A (a Drosophila orthologue of yeast CTR1) or Ctr1B significantly reduced Cu uptake from media, demonstrating that both these proteins function to transport Cu into S2 cells . Significantly, Cu induced Ctr1B gene expression, and this could be prevented by suppressing MTF-1, suggesting that Ctr1B might be involved in Cu detoxification . Suppression of DmATP7, the putative homologue of human Cu transporter genes ATP7A and ATP7B, significantly increased Cu accumulation, demonstrating that DmATP7 is essential for efflux of excess Cu . This work is consistent with previous studies in mammalian cells, validating S2 cells as a model system for studying Cu transport and identifying novel Cu regulatory mechanisms. Genetics, 2004 Jun, 167(2), 645 - 61 The PGL family proteins associate with germ granules and function redundantly in Caenorhabditis elegans germline development; Kawasaki I et al.; PGL-1 is a constitutive protein component of C . elegans germ granules, also known as P granules . Maternally supplied PGL-1 is essential for germline development but only at elevated temperature, raising the possibility that redundant factors provide sufficient function at lower temperatures . We have identified two PGL-1-related proteins, PGL-2 and PGL-3, by sequence analysis of the C . elegans genome and by a yeast two-hybrid screen for proteins that interact with PGL-1 . PGL-3 is associated with P granules at all stages of development, while PGL-2 is associated with P granules only during postembryonic development . All three PGL proteins interact with each other in vitro . Furthermore, PGL-1 and PGL-3 are co-immunoprecipitated from embryo extracts, indicating that they are indeed in the same protein complex in vivo . Nevertheless, each PGL protein localizes to P granules independently of the other two . pgl-2 or pgl-3 single-mutant worms do not show obvious defects in germline development . However, pgl-1; pgl-3 (but not pgl-2; pgl-1) double-mutant hermaphrodites and males show significantly enhanced sterility at all temperatures, compared to pgl-1 alone . Mutant hermaphrodites show defects in germline proliferation and in production of healthy gametes and viable embryos . Our findings demonstrate that both PGL-2 and PGL-3 are components of P granules, both interact with PGL-1, and at least PGL-3 functions redundantly with PGL-1 to ensure fertility in both sexes of C . elegans. J Occup Environ Hyg, 2004 Jul, 1(7), 442 - 7 An investigation into techniques for cleaning mold-contaminated home contents; Wilson SC et al.; This study examined the efficacy of the following treatments to reduce selected fungal spore and mycotoxin levels on materials commonly found in home contents: (1) gamma irradiation at a 10-13 kiloGray exposure, (2) a detergent/bleach wash, and (3) a steam cleaning technique . A minimum of six replicates were performed per treatment . Paper, cloth, wood, and carpet were inoculated with either fungal spores (Stachybotrys chartarum, Aspergillus niger, Penicillium chrysogenum, or Chaetomium globosum) at 240,000 spores/2.54 cm2 of material or with the mycotoxins roridin A, T-2, and verrucarin A at 10 microg per 2.54 cm2 of material . Treatments were evaluated with an agar plating technique for fungal spores and a yeast toxicity culture assay for mycotoxins . Results showed that gamma irradiation inactivated fungal spores, but the treatment was not successful in inactivating mycotoxins . The washing technique completely inactivated or removed spores on all materials except for C . globosum, which was reduced on all items except paper (p < 0.05) . Washing inactivated all mycotoxins on paper and cloth but not on carpet or untreated wood (p < 0.001) . The steam cleaning treatment did not completely eliminate any fungal spores; however, it reduced P . chrysogenum numbers on all materials, C . globosum was reduced on wood and carpet, and S . chartarum was reduced on wood (p < 0.05) . Steam cleaning was unsuccessful in inactivating any of the tested mycotoxins . These results show that the bleach/detergent washing technique was more effective overall in reducing spore and mycotoxin levels than gamma irradiation or steam cleaning . However, the other examined techniques were successful in varying degrees . Arch Pharm (Weinheim), 2004 Jul, 337(7), 411 - 6 Synthesis of amidinohydrazones and evaluation of their inhibitory effect towards aldosterone synthase (CYP11B2) and the formation of selected steroids; Voets M et al.; The synthesis and biological evaluation of a series of amidinohydrazones (3a-h, 6a-c, 8 and 9) as potential nonsteroidal inhibitors of aldosterone synthase (CYP11B2) are described . The compounds were tested in vitro using CYP11B2-expressing fission yeast; they showed only marginal inhibitory effect . Compound 6c was evaluated for its effect on the formation of aldosterone, cortisol, androstenedione, and DHEA in the adrenocortical tumor cell line NCI-H295R . It exhibited no significant effect on the production of these products. Exp Gerontol, 2004 Jul, 39(7), 985 - 98 RNAi: ancient mechanism with a promising future; Geley S et al.; RNA interference (RNAi) is a gene silencing mechanism that has been conserved in evolution from yeast to man . Double stranded RNA, which is either expressed by cellular genes for small non-coding RNAs, by parasitic nucleic acids, such as viruses or transposons, or is expressed as an experimental tool, becomes processed into small RNAs, which induce gene silencing by a variety of different means . RNAi-induced gene silencing controls gene expression at all levels, including transcription, mRNA stability and translation . We are only beginning to understand the physiological roles of the RNAi pathway and the function of the many small non-coding RNA species, which are found in eukaryotic genomes . Here we review the known functions of genes in RNAi in various species, the experimental use and design of small RNAs as a genetic tool to dissect the function of mammalian genes and their potential as therapeutic agents to modulate gene expression in patients. Chromosoma, 2004 Aug, 113(1), 1 - 15 Epub 2004 Jul 03. Kinetochore localization and microtubule interaction of the human spindle checkpoint kinase Mps1; Stucke VM et al.; Members of the Mps1 protein kinase family have been implicated in the regulation of the kinetochore-mediated spindle assembly checkpoint in species ranging from yeast to man . However, conflicting data have been reported on the subcellular localization of vertebrate Mps1 kinases and their possible roles in centrosome duplication . Moreover, little is presently known about the regulation of Mps1 kinases during the cell cycle . Here, we have used immunofluorescence microscopy, immunoblotting and siRNA-mediated depletion of hMps1 to re-investigate the subcellular localization of this kinase . Our data confirm the kinetochore association of hMps1 but suggest that the centrosome staining produced by some anti-hMps1 antibodies could be due to cross-reactivity with other proteins . We also show that the kinetochore association of hMps1 is mediated by the amino-terminal, non-catalytic domain and specifically requires the presence of the Hec1/Ndc80-Nuf2 complex at the kinetochore . Finally, we have combined in vitro binding studies and kinase assays to explore the influence of microtubules on hMps1 activity . Our data indicate that the catalytic domain of hMps1 displays affinity for microtubules and that microtubule binding could contribute to the regulation of kinase activity. J Biol Chem, 2004 Sep 3, 279(36), 37559 - 65 Epub 2004 Jul 02. Importin alpha1 (Rch1) mediates nuclear translocation of thioredoxin-binding protein-2/vitamin D(3)-up-regulated protein 1; Nishinaka Y et al.; Thioredoxin-binding protein-2 (TBP-2)/vitamin D(3) up-regulated protein 1 is an endogenous molecule interacting with thioredoxin (TRX), negatively regulating TRX function, and being implicated in the suppression of tumor development and metastasis . We found that TBP-2 ectopically expressed in the breast cancer cell line MCF-7 was localized predominantly in the nucleus exhibiting growth suppressive activity . The nuclear accumulation of endogenous TBP-2 protein was also demonstrated when the cells were treated with an anti-cancer drug, suberoylanilide hydroxamic acid . To investigate the mechanism underlying the nuclear localization, we performed a yeast two-hybrid screening and identified importin alpha(1) (Rch1) as a protein interacting with TBP-2 . The physical interaction between TBP-2 and Rch1 was confirmed with a glutathione S-transferase pull-down assay . The interaction of TBP-2 was specific to Rch1 among other importin alpha subfamilies (Qip1 and NPI-1), and amino acids 1-227 of TBP-2 were sufficient for both the interaction with Rch1 and the nuclear localization, although there is no typical nuclear localization signal in this sequence . The expression of short interfering RNA of Rch1 suppressed suberoylanilide hydroxamic acid-induced nuclear accumulation of TBP-2 . Collectively, our results strongly suggest that an interaction with importin system is required for TBP-2 nuclear translocation and growth control tightly associated with TRX-dependent redox regulation of transcription factors. Mol Cell Neurosci, 2004 Jul, 26(3), 406 - 17 The GABA transporter GAT1 and the MAGUK protein Pals1: interaction, uptake modulation, and coexpression in the brain; McHugh EM et al.; GABAergic signaling in the CNS is terminated in part through uptake of GABA by GABA transporters . We used the yeast two-hybrid system to identify proteins that associate with the carboxy-terminus of the neuronal GABA transporter GAT1 . We found an interaction between GAT1 and the MAGUK protein Pals1 . When coexpressed in COS-7 cells, Pals1 co-immunoprecipitates with GAT1 . We demonstrate cellular coexpression of GAT1 and Pals1 in the mouse hippocampus and striatum . Functionally, coexpression of GAT1 and Pals1 in COS-7 cells increases {3H}-GABA uptake by GAT1 . The mechanism underlying increased uptake is increased levels of GAT1 protein . We hypothesize that Pals1 contributes to the stability of the GAT1, thus promoting the expression level of the transporter protein . In the CNS, Pals1 may stabilize GAT1 at appropriate levels in specific GABAergic neurons . Chemosphere, 2004 Aug, 56(7), 717 - 23 Reduction in labile copper in the 7-day Ceriodaphnia dubia toxicity test due to the interaction with zooplankton food; Hauri JF et al.; Due to the increased popularity of zooplankton toxicity tests, it is important to investigate potential confounding factors . Though zooplankton food has been studied extensively to meet the nutritional needs of the zooplankton, less research has been done on whether food addition reduces the toxicity of metals in the tested sample . This investigation combines toxicity tests and metal speciation analysis to determine whether the EPA recommended food of YCT (yeast, cerophyll, and trout chow) and Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) reduces copper toxicity by decreasing the concentration of labile copper . Toxicity tests were performed with Ceriodaphnia dubia on culture water spiked with 0, 787, and 1574 nM copper with five different food levels . A Chelex-100 cation exchange resin and a graphite furnace-atomic absorption spectrophotometer were used in conjunction with the toxicity tests to measure the amount of labile copper in the culture water . At the EPA recommended food dosage, the C . dubia food has a chelating capacity of approximately 500 nM Cu . For both concentrations of spiked culture water, the toxicity to C . dubia was reduced with increasing food level, which seemed to be both from a decrease in labile copper concentration and an increase in the nutritional condition of the zooplankton. Eur J Biochem, 2004 Jul, 271(14), 2949 - 55 A new rice zinc-finger protein binds to the O2S box of the alpha-amylase gene promoter; Peng R et al.; A putative transcription factor, named RAMY, that binds to the 20-bp O2S sequences of the regulatory region of the Amy2 gene promoter has been identified using the yeast one-hybrid system from a rice library . The full length RAMY cDNA clone encodes a 218-amino acid protein and is homologous to the late embryogenesis-abundant protein (LEA5) . In vitro mutagenesis and electrophoretic mobility shift assays confirmed that RAMY can bind with O2S specifically through an unusual zinc finger with a CXCX(4)CX(2)H consensus sequence . Low levels of RAMY mRNAs were detected in rice leaves and roots by Northern blot hybridization . The plant hormone gibberellin (GA) induces expression of both RAMY and Amy2 genes, as performed by Northern blot hybridization, but the increase in RAMY mRNA level occurs prior to that of the Amy2 mRNA level in the GA-treated aleurone tissues . These data suggest that RAMY may act as a trans-acting protein and is probably involved in the GA-induced expression of the rice alpha-amylase gene. Biochem J, 2004 Sep 15, 382(Pt 3), 841 - 8 Membrane-bound carboxypeptidase E facilitates the entry of eosinophil cationic protein into neuroendocrine cells; Wu CM et al.; ECP (eosinophil cationic protein) is a major component of eosinophil granule proteins, and is used as a clinical biomarker for asthma and allergic inflammatory disease . ECP has been implicated in damage to the cell membrane of many tissue types, but the mechanism is not well known . In the present study, mECP-eGFP-6H, a recombinant fusion protein containing mature ECP (mECP), enhanced green fluorescence protein (eGFP) and a His(6) tag (6H), has been expressed, purified and added to GH3 neuroendocrine cells to study the internalization ability of ECP . We found that mECP-eGFP-6H entered into GH3 neuroendocrine cells and inhibited the growth of the cells with an IC(50) of 0.8 microM . By yeast two-hybrid screening and immunoprecipitation, we have identified a specific protein-protein interaction between mECP and CPE (carboxypeptidase E), a well characterized metalloprotease . Further in vivo yeast two-hybrid screening has also revealed that residues 318-387 located in a region of unknown function in mature CPE are indispensable for association with mECP . In addition, the uptake of mECP-eGFP-6H is suppressed by dominant-negative expression of the recycling defect mutant pre-pro-HA-CPE(S471A,E472A) in GH3 cells, suggesting that the entry of mECP-eGFP-6H is associated with the recycling of CPE in GH3 cells . Taken together, we have demonstrated that CPE possesses a novel function to facilitate the entry of ECP to neuroendocrine cells, and such an endocytotic process allows the cytotoxic ECP to inhibit growth of the target cells. J Biol Chem, 2004 Sep 10, 279(37), 38881 - 8 Epub 2004 Jul 01. Asb6, an adipocyte-specific ankyrin and SOCS box protein, interacts with APS to enable recruitment of elongins B and C to the insulin receptor signaling complex; Wilcox A et al.; The APS adapter protein plays a pivotal role in coupling the insulin receptor to CAP and c-Cbl in the phosphatidylinositol 3-kinase-independent pathway of insulin-stimulated glucose transport . Yeast two-hybrid screening of a 3T3-L1 adipocyte library using APS as a bait identified a 418-amino acid ankyrin and SOCS (suppressor of cytokine signaling) box protein Asb6 as an interactor . Asb6 is an orphan member of a larger family of Asb proteins that are ubiquitously expressed . However, Asb6 expression appears to be restricted to adipose tissue . Asb6 was specifically expressed in 3T3-L1 adipocytes as a 50-kDa protein but not in fibroblasts . In Chinese hamster ovary-insulin receptor (CHO-IR) cells Myc epitope-tagged APS interacted constitutively with FLAG-tagged Asb6 in the presence or absence of insulin stimulation and insulin stimulation did not alter the interaction . In 3T3-L1 adipocytes, insulin receptor activation was accompanied by the APS-dependent recruitment of Asb6 . Asb6 did not appear to undergo tyrosine phosphorylation . Immunofluorescence and confocal microscopy studies revealed that Asb6 colocalized with APS in CHO cells and in 3T3-L1 adipocytes . In immunoprecipitation studies in CHO cells or 3T3-L1 adipocytes, the Elongin BC complex was found to be bound to Asb6, and activation of the insulin receptor was required to facilitate Asb6 recruitment along with Elongins B/C . Prolonged insulin stimulation resulted in the degradation of APS when Asb6 was co-expressed but not in the absence of Asb6 . We conclude that Asb6 functions to regulate components of the insulin signaling pathway in adipocytes by facilitating degradation by the APS-dependent recruitment of Asb6 and Elongins BC. J Bacteriol, 2004 Jul, 186(14), 4796 - 801 Interaction between protein subunits of the type IV secretion system of Bartonella henselae; Shamaei-Tousi A et al.; In this study we used the yeast two-hybrid system to identify interactions between protein subunits of the virB type IV secretion system of Bartonella henselae . We report interactions between inner membrane and periplasmic proteins, the pilus polypeptide, and the core complex and a novel interaction between VirB3 and VirB5 . Genes Dev, 2004 Jul 1, 18(13), 1577 - 91 Conserved MYC transcription factors play a key role in jasmonate signaling both in tomato and Arabidopsis; Boter M et al.; Jasmonates (JA) are important regulators of plant defense responses that activate expression of many wound-induced genes including the tomato proteinase inhibitor II (pin2) and leucine aminopeptidase (LAP) genes . Elements required for JA induction of the LAP gene are all present in the -317 to -78 proximal promoter region . Using yeast one-hybrid screening, we have identified the bHLH-leu zipper JAMYC2 and JAMYC10 proteins, specifically recognizing a T/G-box AACGTG motif in this promoter fragment . Mutation of the G-box element decreases JA-responsive LAP promoter expression . Expression of JAMYC2 and JAMYC10 is induced by JA, with a kinetics that precedes that of the LAP or pin2 transcripts . JAMYC overexpression enhanced JA-induced expression of these defense genes in potato, but did not result in constitutive transcript accumulation . Using footprinting assays, an additional protected element was identified, located directly adjacent to the T/G-box motif . Mutation of this element abolishes JA response, showing that recognition of this duplicated element is also required for gene expression . Knockout mutants in the AtMYC2 homolog gene of Arabidopsis are insensitive to JA and exhibit a decreased activation of the JA-responsive genes AtVSP and JR1 . Activation of the PDF1.2 and b-CHI, ethylene/JA-responsive genes, is, however, increased in these mutants . These results show that the JAMYC/AtMYC2 transcription factors function as members of a MYC-based regulatory system conserved in dicotyledonous plants with a key role in JA-induced defense gene activation. Adv Exp Med Biol, 2004, 547, 21 - 30 A systems approach to discovering signaling and regulatory pathways--or, how to digest large interaction networks into relevant pieces; Ideker T; In the post-genomic era, the first step in any study of protein function is a homology search against the complete genome sequence of the organism of interest . By analogy, if we also wish to elucidate the cadre of signaling and regulatory pathways in the cell, an extremely powerful first step is to construct a complete network of protein-protein and transcriptional interactions and then search through this network to identify critical pathways in a top-down fashion . Like genomic sequence, the molecular interaction network provides a broad foundation for more directed studies to follow . We illustrate this strategy using a large network of 12,232 interactions in yeast . A variety of applications are discussed, including screening the network to identify pathways responsible for gene expression changes observed in galactose-induced cells, and identifying groups of interacting proteins that are essential (by phenotypic assay) for the cellular response to DNA damage. Mol Pharmacol, 2004 Oct, 66(4), 817 - 23 Epub 2004 Jun 30. The copper influx transporter human copper transport protein 1 regulates the uptake of cisplatin in human ovarian carcinoma cells; Holzer AK et al.; Cells selected for resistance to cisplatin are often cross-resistant to copper and vice versa, and the major copper influx transporter copper transport protein 1 (CTR1) has been shown to regulate the uptake of cisplatin, carboplatin, and oxaliplatin in yeast . To further define the role of hCTR1 in human tumor cells, the ovarian carcinoma cell line A2780 was molecularly engineered to increase expression of hCTR1 by a factor of 20-fold . Enhanced expression of hCTR1 in the A2780/hCTR1 cells was associated with a 6.5-fold increase in basal steady-state copper content and a 13.7-fold increase in initial copper influx, demonstrating that the exogenously expressed hCTR1 was functional in altering copper homeostasis . The A2780/hCTR1 cells accumulated 46% more platinum after a 1-h exposure to 2 microM cisplatin, and 55% more after a 24 h exposure, than the control A2780/empty vector cells . The initial uptake of cisplatin was 81% higher in the A2780/hCTR1 cells when measured at 5 min . Thus, increased expression of hCTR1 had a substantially larger effect on the cellular pharmacology of copper than cisplatin . Interestingly, the increased uptake of copper and cisplatin was accompanied by only a marginal increase in sensitivity to the cytotoxic effect of copper and cisplatin, and there was no increase in the extent of cisplatin-DNA adduct formation . Thus, although increased expression of hCTR1 mediates greater cellular accumulation of copper and cisplatin, hCTR1 delivers these compounds into intracellular compartments from which they do not have ready access to their key cytotoxic targets. J Neurosci, 2004 Jun 30, 24(26), 5966 - 73 YB-1 and CTCF differentially regulate the 5-HTT polymorphic intron 2 enhancer which predisposes to a variety of neurological disorders; Klenova E et al.; The serotonin transporter (5-HTT) gene contains a variable number tandem repeat (VNTR) domain within intron 2 that is often associated with a number of neurological conditions, including affective disorders . The implications of this polymorphism are not yet understood, however, we have previously demonstrated that the 5-HTT VNTR is a transcriptional regulatory domain, and the allelic variation supports differential reporter gene expression in vivo and in vitro . The aim of this study was to identify transcription factors responsible for the regulation of this VNTR . Using a yeast one-hybrid screen, we found the transcription factor Y box binding protein 1 (YB-1) interacts with the 5-HTT VNTR . Consistent with this, we demonstrate in a reporter gene assay that the polymorphic VNTR domains differentially respond to exogenous YB-1 and that YB-1 will bind to the VNTR in vitro in a sequence-specific manner . Interestingly, the transcription factor CCTC-binding factor (CTCF), previously shown to interact with YB-1, interferes with the ability of the VNTR to support YB-1-directed reporter gene expression . In addition, CTCF blocks the binding of YB-1 to its DNA recognition sequences in vitro, thus providing a possible mechanism of regulation of YB-1 activation of the VNTR by CTCF . Therefore, we have identified YB-1 and CTCF as transcription factors responsible, at least in part, for modulation of VNTR function as a transcriptional regulatory domain . Our data suggest a novel mechanism that explains, in part, the ability of the distinct VNTR copy numbers to support differential reporter gene expression based on YB-1 binding sites. J Neurosci, 2004 Jun 30, 24(26), 5881 - 91 The gamma2 subunit of GABA(A) receptors is a substrate for palmitoylation by GODZ; Keller CA et al.; The neurotransmitter GABA activates heteropentameric GABA(A) receptors, which are composed mostly of alpha, beta, and gamma2 subunits . Regulated membrane trafficking and subcellular targeting of GABA(A) receptors is important for determining the efficacy of GABAergic inhibitory function . Of special interest is the gamma2 subunit, which is mostly dispensable for assembly and membrane insertion of functional receptors but essential for accumulation of GABA(A) receptors at synapses . In a search for novel receptor trafficking proteins, we have used the SOS-recruitment system and isolated a Golgi-specific DHHC zinc finger protein (GODZ) as a novel gamma2 subunit-interacting protein . GODZ is a member of the superfamily of DHHC cysteine-rich domain (DHHC-CRD) polytopic membrane proteins shown recently in yeast to represent palmitoyltransferases . GODZ mRNA is found in many tissues; however, in brain the protein is detected in neurons only and highly concentrated and asymmetrically distributed in the Golgi complex . GODZ interacts with a cysteine-rich 14-amino acid domain conserved specifically in the large cytoplasmic loop of gamma1-3 subunits but not in other GABA(A) receptor subunits . Coexpression of GODZ and GABA(A) receptors in heterologous cells results in palmitoylation of the gamma2 subunit in a cytoplasmic loop domain-dependent manner . Neuronal GABA(A) receptors are similarly palmitoylated . Thus, GODZ-mediated palmitoylation represents a novel posttranslational modification that is selective for gamma subunit-containing GABA(A) receptor subtypes, a mechanism that is likely to be important for regulated trafficking of these receptors in the secretory pathway. J Biol Chem, 2004 Sep 10, 279(37), 38803 - 12 Epub 2004 Jun 30. Post-translational modification of Rta of Epstein-Barr virus by SUMO-1; Chang LK et al.; Epstein-Barr virus (EBV) expresses an immediate-early protein, Rta, to activate the transcription of EBV lytic genes and the lytic cycle . This work identifies Ubc9 and PIAS1 as binding partners of Rta in a yeast two-hybrid screen . These bindings are verified by glutathione S-transferase pull-down assay, coimmunoprecipitation, and confocal microscopy . The interactions appear to cause Rta sumoylation, because not only can Rta be sumoylated in vitro but also sumoylated Rta can be detected in P3HR1 cells following lytic induction and in 293T cells after transfecting plasmids that express Rta and SUMO-1 . Moreover, PIAS1 stimulates conjugation of SUMO-1 to Rta, thus acting as an E3 ligase . Furthermore, transfecting plasmids that express Ubc9, PIAS1, and SUMO-1 increases the capacity of Rta to transactivate the promoter that includes an Rta response element, indicating that the modification by SUMO-1 increases the transactivation activity of Rta . This study reveals that Rta is sumoylated at the Lys-19, Lys-213, and Lys-517 residues and that SUMO-1 conjugation at the Lys-19 residue is crucial for enhancing the transactivation activity of Rta . These results indicate that sumoylation of Rta may be important in EBV lytic activation. Development, 2004 Aug, 131(15), 3649 - 59 Epub 2004 Jun 30. Molecular and genetic interactions between STYLOSA and GRAMINIFOLIA in the control of Antirrhinum vegetative and reproductive development; Navarro C et al.; STYLOSA (STY) in Antirrhinum and LEUNIG (LUG) in Arabidopsis control the spatially correct expression of homeotic functions involved in the control of floral organ identity . We show here that the sty mutant also displays alteration in leaf venation patterns and hypersensitivity towards auxin and polar auxin transport inhibitors, demonstrating that STY has a more general role in plant development . STY and LUG are shown to be orthologues that encode proteins with structural relation to GRO/TUP1-like co-repressors . Using a yeast-based screen we found that STY interacts with several transcription factors, suggesting that STY, like GRO/TUP1, forms complexes in vivo . Proteins of the YABBY family, characterised by containing a partial HMG domain, represent a major group of such interactors . In vivo association of STY with one of the YABBY proteins, GRAMINIFOLIA (GRAM), is supported by enhanced phenotypic defects in sty gram double mutants, for instance in the control of phyllotaxis, floral homeotic functions and organ polarity . Accordingly, the STY and GRAM protein and mRNA expression patterns overlap in emerging lateral organ primordia . STY is expressed in all meristems and later becomes confined to the adaxial domain and (pro)vascular tissue . This pattern is similar to genes that promote adaxial identity, and, indeed, STY expression follows, although does not control, adaxial fate . We discuss the complex roles of STY and GRAM proteins in reproductive and vegetative development, performed in part in physical association but also independently. Mol Microbiol, 2004 Jul, 53(2), 365 - 72 Trypanosomatid histones; Alsford S et al.; The histones are responsible for packaging and regulating access to eukaryotic genomes . Trypanosomatids are flagellated protists that diverged early from the eukaryotic lineage and include parasites that cause disease in humans and other mammals . Here, we review the properties of histones in parasitic trypanosomatids, from gene organization and sequence to expression, post-translational modification and function within chromatin . Phylogenetic and experimental analysis indicates that certain specifically conserved histone sequence motifs, particularly within the N-terminal 'tail' domains, possibly represent functionally important modification substrates conserved throughout the eukaryotic lineage . For example, histone H3 contains a highly conserved methylation substrate . Trypanosomatids also possess at least three variant histones . Among these is an orthologue of H2A.Z, a histone involved in protecting 'active' chromatin from silencing in yeast . Histones provide docking platforms for a variety of regulatory factors . The presence of histone modification and variant histones in trypanosomatids therefore represents evidence for a network that provides the discrimination required to regulate transcription, recombination, repair and chromosome replication and segregation. Biochem J, 2004 Oct 1, 383(Pt 1), 19 - 26 Characterization of three novel members of the Arabidopsis thaliana equilibrative nucleoside transporter (ENT) family; Wormit A et al.; Research on metabolism of nucleotides and their derivatives has gained increasing interest in the recent past . This includes de novo synthesis, analysis of salvage pathways, breakdown and transport of nucleotides, nucleosides and nucleobases . To perform a further step towards the analysis of nucleoside transport in Arabidopsis, we incubated leaf discs with various radioactively labelled nucleosides . Leaf cells imported labelled nucleosides and incorporated these compounds into RNA, but not into DNA . Furthermore, we report on the biochemical properties of three so far uncharacterized members of the Arabidopsis ENT (equilibrative nucleoside transporter) family (AtENT4, AtENT6 and AtENT7) . After heterologous expression in yeast, all three proteins exhibited broad substrate specificity and transported the purine nucleosides adenosine and guanosine, as well as the pyrimidine nucleosides cytidine and uridine . The apparent K(m) values were in the range 3-94 microM, and transport was inhibited most strongly by deoxynucleosides, and to a smaller extent by nucleobases . Typical inhibitors of mammalian ENT proteins, such as dilazep and NBMPR (nitrobenzylmercaptopurine ribonucleoside, also known as nitrobenzylthioinosine) surprisingly exerted almost no effect on Arabidopsis ENT proteins . Transport mediated by the AtENT isoforms differed in pH-dependency, e.g . AtENT7 was not affected by changes in pH, AtENT3, 4 and 6 exhibited a less pronounced pH-dependency, and AtENT1 activity was clearly pH-dependent . Using a GFP (green fluorescent protein)-fusion protein transiently expressed in tobacco leaf protoplasts, a localization of AtENT6 in the plant plasma membrane has been revealed. Proc Natl Acad Sci U S A, 2004 Jul 6, 101(27), 9988 - 93 Epub 2004 Jun 28. Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity; Bugreev DV et al.; Human Rad51 (hRad51) protein plays a key role in homologous recombination and DNA repair . hRad51 protein forms a helical filament on single-stranded DNA (ssDNA), which performs the basic steps of homologous recombination: a search for homologous double-stranded DNA (dsDNA) and DNA strand exchange . hRad51 protein possesses DNA-dependent ATPase activity; however, the role of this activity has not been understood . Our current results show that Ca(2+) greatly stimulates DNA strand exchange activity of hRad51 protein . We found that Ca(2+) exerts its stimulatory effect by modulating the ATPase activity of hRad51 protein . Our data demonstrate that, in the presence of Mg(2+), the hRad51-ATP-ssDNA filament is quickly converted to an inactive hRad51-ADP-ssDNA form, due to relatively rapid ATP hydrolysis and slow dissociation of ADP . Ca(2+) maintains the active hRad51-ATP-ssDNA filament by reducing the ATP hydrolysis rate . These findings demonstrate a crucial role of the ATPase activity in regulation of DNA strand exchange activity of hRad51 protein . This mechanism of Rad51 protein regulation by modulating its ATPase activity is evolutionarily recent; we found no such mechanism for yeast Rad51 (yRad51) protein. J Cell Sci, 2004 Jul 15, 117(Pt 16), 3547 - 59 Epub 2004 Jun 29. Sister-chromatid cohesion mediated by the alternative RF-CCtf18/Dcc1/Ctf8, the helicase Chl1 and the polymerase-alpha-associated protein Ctf4 is essential for chromatid disjunction during meiosis II; Petronczki M et al.; Cohesion between sister chromatids mediated by a multisubunit complex called cohesin is established during DNA replication and is essential for the orderly segregation of chromatids during anaphase . In budding yeast, a specialized replication factor C called RF-C(Ctf18/Dcc1/Ctf8) and the DNA-polymerase-alpha-associated protein Ctf4 are required to maintain sister-chromatid cohesion in cells arrested for long periods in mitosis . We show here that CTF8, CTF4 and a helicase encoded by CHL1 are required for efficient sister chromatid cohesion in unperturbed mitotic cells, and provide evidence that Chl1 functions during S-phase . We also show that, in contrast to mitosis, RF-C(Ctf18/Dcc1/Cft8), Ctf4 and Chl1 are essential for chromosome segregation during meiosis and for the viability of meiotic products . Our finding that cells deleted for CTF8, CTF4 or CHL1 undergo massive meiosis II non-disjunction suggests that the second meiotic division is particularly sensitive to cohesion defects . Using a functional as well as a cytological assay, we demonstrate that CTF8, CHL1 and CTF4 are essential for cohesion between sister centromeres during meiosis but dispensable for cohesin's association with centromeric DNA . Our finding that mutants in fission yeast ctf18 and dcc1 have similar defects suggests that the involvement of the alternative RF-C(Ctf18/Dcc1/Ctf8) complex in sister chromatid cohesion might be highly conserved. J Biol Chem, 2004 Sep 17, 279(38), 39296 - 302 Epub 2004 Jun 29. Domain swapping localizes the structural determinants of regioselectivity in membrane-bound fatty acid desaturases of Caenorhabditis elegans; Sasata RJ et al.; Most fatty acid desaturases are members of a large superfamily of integral membrane, O(2)-dependent, iron-containing enzymes that catalyze a variety of oxidative modifications to lipids . Sharing a similar primary structure and membrane topology, these enzymes are broadly categorized according to their positional specificity or regioselectivity, which designates the preferred position for substrate modification . To investigate the structural basis of regioselectivity in membrane-bound desaturases, the Caenorhabditis elegans omega-3 (FAT-1) and "Delta12" (FAT-2) desaturases were used as a model system . With the use of unnatural substrates, the regioselectivity of C . elegans FAT-2 was clearly defined as nu+3, i.e . it "measures" three carbons from an existing double bond . The structural basis for nu+3 and omega-3 regioselectivities was examined through construction and expression of chimeric DNA sequences based on FAT-1 and FAT-2 . Each sequence was divided into seven domains, and chimeras were constructed in which specific domains were replaced with sequence from the other desaturase . When tested by expression in yeast using exogenously supplied substrates, chimeric sequences were found in which domain swapping resulted in a change of regioselectivity from nu+3 to omega-3 and vice versa . In this way, the structural determinants of regioselectivity in FAT-1 and FAT-2 have been localized to two interdependent regions: a relatively hydrophobic region between the first two histidine boxes and the carboxyl-terminal region. FEBS Lett, 2004 Jul 2, 569(1-3), 18 - 26 Novel Sm-like proteins with long C-terminal tails and associated methyltransferases; Albrecht M et al.; Sm and Sm-like proteins of the Lsm (like Sm) domain family are generally involved in essential RNA-processing tasks . While recent research has focused on the function and structure of small family members, little is known about Lsm domain proteins carrying additional domains . Using an integrative bioinformatics approach, we discovered five novel groups of Lsm domain proteins (Lsm12-16) with long C-terminal tails and investigated their functions . All of them are evolutionarily conserved in eukaryotes with an N-terminal Lsm domain to bind nucleic acids followed by as yet uncharacterized C-terminal domains and sequence motifs . Based on known yeast interaction partners, Lsm12-16 may play important roles in RNA metabolism . Particularly, Lsm12 is possibly involved in mRNA degradation or tRNA splicing, and Lsm13-16 in the regulation of the mitotic G2/M phase . Lsm16 proteins have an additional C-terminal YjeF_N domain of as yet unknown function . The identification of an additional methyltransferase domain at the C-terminus of one of the Lsm12 proteins also led to the recognition of three new groups of methyltransferases, presumably dependent on S-adenosyl-l-methionine . Further computational analyses revealed that some methyltransferases contain putative RNA-binding helix-turn-helix domains and zinc fingers. Mol Cell, 2004 Jul 2, 15(1), 141 - 52 The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis; Teige M et al.; The Arabidopsis mitogen-activated protein kinase (MAPK) kinase 2 (MKK2) and the downstream MAPKs MPK4 and MPK6 were isolated by functional complementation of osmosensitive yeast mutants . In Arabidopsis protoplasts, MKK2 was specifically activated by cold and salt stress and by the stress-induced MAPK kinase kinase MEKK1 . Yeast two-hybrid, in vitro, and in vivo protein kinase assays revealed that MKK2 directly targets MPK4 and MPK6 . Accordingly, plants overexpressing MKK2 exhibited constitutive MPK4 and MPK6 activity, constitutively upregulated expression of stress-induced marker genes, and increased freezing and salt tolerance . In contrast, mkk2 null plants were impaired in MPK4 and MPK6 activation and were hypersensitive to salt and cold stress . Full genome transcriptome analysis of MKK2-overexpressing plants demonstrated altered expression of 152 genes involved in transcriptional regulation, signal transduction, cellular defense, and stress metabolism . These data identify a MAP kinase signaling cascade mediating cold and salt stress tolerance in plants. Mol Cell, 2004 Jul 2, 15(1), 17 - 29 Physical and functional interaction between Pes1 and Bop1 in mammalian ribosome biogenesis; Lapik YR et al.; Molecular mechanisms of mammalian ribosome biogenesis remain largely unexplored . Here we develop a series of transposon-derived dominant mutants of Pes1, the mouse homolog of the zebrafish Pescadillo and yeast Nop7p implicated in ribosome biogenesis and cell proliferation control . Six Pes1 mutants selected by their ability to reversibly arrest the cell cycle also impair maturation of the 28S and 5.8S rRNAs in mouse cells . We show that Pes1 physically interacts with the nucleolar protein Bop1, and both proteins direct common pre-rRNA processing steps . Interaction with Bop1 is essential for the efficient incorporation of Pes1 into nucleolar preribosomal complexes . Pes1 mutants defective for the interaction with Bop1 lose the ability to affect rRNA maturation and the cell cycle . These data show that coordinated action of Pes1 and Bop1 is necessary for the biogenesis of 60S ribosomal subunits. J Mol Biol, 2004 Jul 16, 340(4), 641 - 53 STRA13 interacts with STAT3 and modulates transcription of STAT3-dependent targets; Ivanova AV et al.; STRA13 is a pVHL-dependent bHLH transcription factor up-regulated on the mRNA level in multiple cancer cell lines and implicated recently in the regulation of immune cell homeostasis and autoimmunity . In searching for STRA13-interacting proteins with oncogenic potential by the yeast two-hybrid screening, we identified STAT3 beta as a STRA13-binding partner . We showed that STRA13 binds predominantly to phosphorylated (active) STAT3 alpha and beta isoforms via its HLH and C-terminal regions . We also found that STRA13 was able to activate transcription from STAT-dependent cis-elements . Expression of endogenous STRA13 was shown to be cytokine-inducible, consistent with STRA13 involvement in STAT-dependent transcription regulation . We demonstrated that the STAT3-regulated promoter of the pro-apoptotic Fas gene was activated upon STRA13 over-expression and that co-expression of STRA13 with STAT3 beta or STAT3 alpha modulated the transcriptional outcome . Forced expression of STRA13 induced apoptosis, in agreement with the STRA13 activation effect on the Fas promoter . Simultaneous expression of STRA13 and STAT3 beta resulted in alleviation of the STRA13 pro-apoptotic effect . Thus, for the first time, we identify STRA13 as a STAT3 partner and provide a consistent line of evidence for STRA13 involvement into regulation of apoptosis via the STAT pathways. Bioessays, 2004 Jul, 26(7), 719 - 29 Ups and downs of tissue and planar polarity in plants; Grebe M; The polar orientation of cells within a tissue is an intensively studied research area in animal cells . The term planar polarity refers to the common polar arrangement of cells within the plane of an epithelium . In plants, the subcellular analysis of tissue polarity has been limited by the lack of appropriate markers . Recently, research on plant tissue polarity has come of age . Advances are based on studies of Arabidopsis patterning, cell polarity and auxin transport mutants employing the coordinated, polar localization of auxin transporters and the planar polarity of root epidermal hairs as markers . These approaches have revealed auxin transport and response, vesicular trafficking, membrane sterol and cytoskeletal requirements of tissue polarity . This review summarizes recent progress in research on vascular tissue and planar epidermal polarity in the Arabidopsis root and compares it to findings on planar polarity in animals and cell polarity in yeast . Proteomics, 2004 Jul, 4(7), 1939 - 49 Assessing factors for reliable quantitative proteomics based on two-dimensional gel electrophoresis; Fievet J et al.; We statistically analysed various factors to get accurate estimates of protein quantities from two-dimensional gels . Yeast proteins were labelled with (35)S or stained with Coomassie Brilliant Blue G-250, and spots were automatically quantified with software packages Kepler, ImageQuaNT, Melanie 3.0 and Progenesis . The different software packages proved to have very similar performances . With (35)S-labelled actin spot as a reference, we studied the staining efficiency of colloidal Coomassie blue as a function of amino acid composition of the protein, and derived an equation to estimate the number of molecules per cell from blue-stained proteins . Absolute quantification of most glycolytic enzymes was carried out in two yeast strains. Protoplasma, 2004 Jun, 223(2-4), 229 - 32 Epub 2004 Jun 22. Disposal of chloroplasts with abnormal function into the vacuole in Arabidopsis thaliana cotyledon cells; Niwa Y et al.; Autophagy is a process in which cell membrane rearrangement allows for the sequestration and degradation of part of the cytoplasm . Many protein components of the autophagic mechanism and their corresponding genes have been identified in yeast cells by molecular genetics, and this has enabled researchers to identify homologues of these genes in mammalian and plant systems . Autophagy is involved in the starvation response in which part of the cytoplasm is degraded in order to produce essential substrates to allow the cell to survive during extreme substrate-limiting conditions . However, autophagy may also be important as a quality control mechanism in normal cells . By screening Arabidopsis thaliana T-DNA insert mutants, we isolated an A . thaliana mutant that lacks the AtTIC40 gene and found that the cotyledon cells of this mutant contained undeveloped plastids . Moreover, many toluidine-stained particulate structures were found in the vacuoles of these mutant cells . The images from electron microscopy suggested that some of these particulate structures were partially degraded chloroplasts . Furthermore, oil bodies were found in the cotyledon cells of mutant and wild-type plants, which suggests that the mutant seedlings were not "starved" under the experimental conditions . These results may indicate that under nutrient-sufficient conditions, plant cells remove abnormal plastids by autophagy and that this mechanism is involved in the quality control of organelles. Planta, 2004 Aug, 219(4), 547 - 60 Epub 2004 Jun 22. The plant endosomal system--its structure and role in signal transduction and plant development; Geldner N; Endosomes are highly dynamic membrane systems that receive endocytosed plasma membrane proteins and sort them for either degradation or recycling back to the cell surface . In addition, they receive newly synthesised proteins destined for vacuolar/lysosomal compartments . Sorting in the endosomes is necessary for the establishment and maintenance of cell polarity and it is needed to control levels and function of receptors and transporters at the cellular surface . Both processes are crucial for correct cell behaviour during tissue and organ development and for intercellular communication in general . It has therefore become an imperative to investigate structure and function of the endosomal system if we want to obtain a deeper mechanistic understanding of signal transduction and development . This review will compare our current understanding of endosomal trafficking in animals and yeast with what is known in plants, and will highlight some important breakthroughs in our understanding of the role of endosomes in signal transduction and multicellular development in Drosophila, as well as in Arabidopsis. Plant Cell Rep, 2004 Aug, 23(1-2), 99 - 103 Epub 2004 Jun 24. Uncapped mRNA introduced into tobacco protoplasts can be imported into the nucleus and is trapped by leptomycin B; Stuger R et al.; The mechanism of nuclear export of RNAs in yeast and animal cells is rapidly being uncovered, but RNA export in plants has received little attention . We introduced capped and uncapped fluorescent mRNAs into tobacco (Nicotiana plumbaginifolia) protoplasts and studied their cellular localization . Following insertion, capped transcripts were found in the cytoplasm, while uncapped messengers transiently appeared in the nucleus in about one-quarter to one-third of the cells . These mRNAs were trapped by the nuclear export-inhibiting drug leptomycin B, pointing to an export mechanism in plants similar to Rev-NES-mediated RNP export in other organisms. Anal Bioanal Chem, 2004 Jul, 379(5-6), 842 - 8 Epub 2004 Jun 18. Solid-phase microextraction-capillary gas chromatography combined with microwave-induced plasma atomic-emission spectrometry for selenite determination; Dimitrakakis E et al.; The use of solid-phase microextraction (SPME) with gas chromatography coupled to microwave-induced plasma atomic-emission detection (GC-MIP-AED) is described for selenite {Se(IV)} speciation . Aqueous standards were derivatised with sodium tetraethyl- or tetrapropylborate and extracted by SPME . Headspace extraction of the ethyl and propyl derivatives was studied . Relevant experimental conditions were optimised, including conditions for derivatisation and extraction and those of gas chromatographic analysis . The limits of detection achieved for headspace sampling of derivatised Se(IV) were in the low ng mL(-1) range for both ethylation and propylation . When the method was applied to analysis of selenite in selenised yeast reference material results were in good agreement with the indicated values. Oncogene, 2004 Sep 9, 23(41), 6914 - 23 Negative regulation of transforming growth factor-beta (TGF-beta) signaling by WW domain-containing protein 1 (WWP1); Komuro A et al.; Smad7 negatively regulates transforming growth factor (TGF)-beta superfamily signaling by binding to activated type I receptors, thereby preventing the phosphorylation of receptor-regulated Smads (R-Smads), as well as by recruiting HECT-type E3 ubiquitin ligases to degrade type I receptors through a ubiquitin-dependent mechanism . To elucidate the regulatory mechanisms of TGF-beta signaling, we searched for novel members of proteins that interact with Smad7 using a yeast two-hybrid system . One of the proteins identified was the WW domain-containing protein 1 (WWP1) that is structurally related to Smad ubiquitin regulatory factors (Smurfs), E3 ubiquitin ligases for Smads and TGF-beta superfamily receptors . Using a TGF-beta-responsive reporter in mammalian cells, we found that WWP1 inhibited transcriptional activities induced by TGF-beta . Similar to Smurfs, WWP1 associated with Smad7 and induced its nuclear export, and enhanced binding of Smad7 to TGF-beta type I receptor to cause ubiquitination and degradation of the receptor . Consistent with these results, WWP1 inhibited phosphorylation of Smad2 induced by TGF-beta . WWP1 thus negatively regulates TGF-beta signaling in cooperation with Smad7 . However, unlike Smurfs, WWP1 failed to ubiquitinate R-Smads and SnoN . Importantly, WWP1 and Smurfs were expressed in distinct patterns in human tissues and carcinoma cell lines, suggesting unique pathophysiological roles of WWP1 and Smurfs. Nat Genet, 2004 Jul, 36(7), 714 - 9 Epub 2004 Jun 27. A new, tenth subunit of TFIIH is responsible for the DNA repair syndrome trichothiodystrophy group A; Giglia-Mari G et al.; DNA repair-deficient trichothiodystrophy (TTD) results from mutations in the XPD and XPB subunits of the DNA repair and transcription factor TFIIH . In a third form of DNA repair-deficient TTD, called group A, none of the nine subunits encoding TFIIH carried mutations; instead, the steady-state level of the entire complex was severely reduced . A new, tenth TFIIH subunit (TFB5) was recently identified in yeast . Here, we describe the identification of the human TFB5 ortholog and its association with human TFIIH . Microinjection of cDNA encoding TFB5 (GTF2H5, also called TTDA) corrected the DNA-repair defect of TTD-A cells, and we identified three functional inactivating mutations in this gene in three unrelated families with TTD-A . The GTF2H5 gene product has a role in regulating the level of TFIIH . The identification of a new evolutionarily conserved subunit of TFIIH implicated in TTD-A provides insight into TFIIH function in transcription, DNA repair and human disease. Cancer Lett, 2004 Aug 10, 211(2), 209 - 18 Expression of AIE-75 PDZ-domain protein induces G2/M cell cycle arrest in human colorectal adenocarcinoma SW480 cells; Hirai A et al.; AIE-75 has been known as a 75-kDa autoantigen detected in the serum of autoimmune enteropathy (AIE) and as a colon cancer-related antigen, and now designated as a gene causative of Usher syndrome type 1C hereditary syndromic hearing loss . It binds to a novel putative tumor suppressor MCC2 that is homologous to MCC (mutated in colon cancer) through a PSD-95/Dlg/ZO-1 (PDZ) domain . To clarify the functional role in colon cancer cells, we transfected AIE-75 gene into SW480 colon cancer cells which do not express AIE-75 . Expression of AIE-75 suppressed growth of SW480 cells in vitro in correlation with the expression levels . It was due mainly to G2/M phase cell cycle arrest associated with mitotic slippage, resulting in emergence of hyperploid giant-nucleated or multi-nucleated cells . Screening of proteins that bound to PDZ domains of AIE-75 by a yeast two hybrid system showed that three serine/threonine phosphatase catalytic subunits (PP2AC-alpha, PP2AC-beta, and PPP6C) could bind to AIE-75 . Since PP2AC is known to regulate G2/M checkpoint, we suggest that AIE-75 interacts with PP2AC and prevent cells to transit mitotic phase. Cell Immunol, 2004 Apr, 228(2), 81 - 90 Possible role of factor XIII subunit A in Fcgamma and complement receptor-mediated phagocytosis; Sarvary A et al.; Besides its traditional role in hemostasis, factor XIII subunit A (FXIII-A) is supposed to function as a cellular transglutaminase and to be involved in certain intracellular processes, including cytoskeletal remodeling . To investigate its intracellular role, the aim of the present study was to follow changes in FXIII-A production in combination with the receptor-mediated phagocytic activities of monocytes/macrophages and to examine the phagocytic functions of monocytes in patients with FXIII-A deficiency . Human blood monocytes were isolated from the buffy coats of healthy volunteers and cultured for 4 days . The FcgammaR-mediated phagocytosis of sensitized erythrocytes (EA) and the complement receptor (CR)-mediated phagocytosis of complement-coated yeast particles were studied during monocyte/macrophage differentiation . Changes in the gene expression of FXIII-A were detected by real-time quantitative RT-PCR . FXIII-A protein production was investigated with fluorescent image analysis at single cell level and Western immunoblot analysis . Both the FcgammaR and CR-mediated phagocytosis increased during culturing, which peaked on day 3 . The phagocytic activity of the cells could be markedly inhibited with monodansylcadaverine, an inhibitor of the transglutaminase-induced crosslinking of proteins . The phagocytosis of EA, complement-coated and uncoated yeast particles was found to be strongly diminished in monocytes of FXIII-A deficient patients . The phagocytic functions of cultured cells showed a change in parallel with the alterations in FXIII-A mRNA expression, as well as with that in FXIII-A in protein synthesis detected by image and Western immunoblot analyses in concert . Our results suggest that FXIII-A plays a role in the Fcgamma and complement receptor-mediated phagocytic activities of monocytes/macrophages. Science, 2004 Jun 25, 304(5679), 1971 - 6 RNAi-independent heterochromatin nucleation by the stress-activated ATF/CREB family proteins; Jia S et al.; At the silent mating-type interval of fission yeast, the RNA interference (RNAi) machinery cooperates with cenH, a DNA element homologous to centromeric repeats, to initiate heterochromatin formation . However, in RNAi mutants, heterochromatin assembly can still occur at low efficiency . Here, we report that Atf1 and Pcr1, two ATF/CREB family proteins, act in a parallel mechanism to the RNAi pathway for heterochromatin nucleation . Deletion of atf1 or pcr1 alone has little effect on silencing at the mating-type region, but when combined with RNAi mutants, double mutants fail to nucleate heterochromatin assembly . Moreover, deletion of atf1 or pcr1 in combination with cenH deletion causes loss of silencing and heterochromatin formation