Cell Cycle, 2003 Nov-Dec, 2(6), 553 - 4 Interdependence of the contractile ring and spindle midzone in cleavage plane maintenance; Finger FP; How segregation of the chromosomes is coordinated with the ensuing cell cleavage to complete the cell cycle is not well understood . A recent study of cytokinesis in fission yeast by Pardo and Nurse suggests that the contractile ring is required for assembly of the post-mitotic microtubule array (PAA) . In turn, the PAA is required to maintain the contractile ring at the cleavage plane, as well as to keep the nuclei separated at the poles of the cleaving cell . These functions may be particularly important for a cell cycle checkpoint ensuring that if cytokinesis is delayed, septation will occur between the two daughter nuclei. Genetics, 2003 Sep, 165(1), 159 - 69 The AF-6 homolog canoe acts as a Rap1 effector during dorsal closure of the Drosophila embryo; Boettner B et al.; Rap1 belongs to the highly conserved Ras subfamily of small GTPases . In Drosophila, Rap1 plays a critical role in many different morphogenetic processes, but the molecular mechanisms executing its function are unknown . Here, we demonstrate that Canoe (Cno), the Drosophila homolog of mammalian junctional protein AF-6, acts as an effector of Rap1 in vivo . Cno binds to the activated form of Rap1 in a yeast two-hybrid assay, the two molecules colocalize to the adherens junction, and they display very similar phenotypes in embryonic dorsal closure (DC), a process that relies on the elongation and migration of epithelial cell sheets . Genetic interaction experiments show that Rap1 and Cno act in the same molecular pathway during DC and that the function of both molecules in DC depends on their ability to interact . We further show that Rap1 acts upstream of Cno, but that Rap1, unlike Cno, is not involved in the stimulation of JNK pathway activity, indicating that Cno has both a Rap1-dependent and a Rap1-independent function in the DC process. Genetics, 2003 Sep, 165(1), 145 - 57 The Caenorhabditis elegans spe-39 gene is required for intracellular membrane reorganization during spermatogenesis; Zhu GD et al.; Caenorhabditis elegans spermatid formation involves asymmetric partitioning of cytoplasm during the second meiotic division . This process is mediated by specialized ER/Golgi-derived fibrous body-membranous organelles (FB-MOs), which have a fibrous body (FB) composed of bundled major sperm protein filaments and a vesicular membranous organelle (MO) . spe-39 mutant spermatocytes complete meiosis but do not usually form spermatids . Ultrastructural examination of spe-39 spermatocytes reveals that MOs are absent, while FBs are disorganized and not surrounded by the membrane envelope usually observed in wild type . Instead, spe-39 spermatocytes contain many small vesicles with internal membranes, suggesting they are related to MOs . The spe-39 gene was identified and it encodes a novel hydrophilic protein . Immunofluorescence with a specific SPE-39 antiserum reveals that it is distributed through much of the cytoplasm and not specifically associated with FB-MOs in spermatocytes and spermatids . The spe-39 gene has orthologs in Drosophila melanogaster and humans but no homolog was identified in the yeast genome . This suggests that the specialized membrane biogenesis steps that occur during C . elegans spermatogenesis are part of a conserved process that requires SPE-39 homologs in other metazoan cell types. Genetics, 2003 Sep, 165(1), 115 - 25 A targeted histone acetyltransferase can create a sizable region of hyperacetylated chromatin and counteract the propagation of transcriptionally silent chromatin; Chiu YH et al.; Transcriptionally silent chromatin is associated with reduced histone acetylation and its propagation depends on histone hypoacetylation promoted by histone deacetylases . We show that tethered histone acetyltransferase (HAT) Esa1p or Gcn5p creates a segment of hyperacetylated chromatin that is at least 2.6 kb in size and counteracts transcriptional silencing that emanates from a silencer in yeast . Esa1p and Gcn5p counteract URA3 silencing even when they are targeted 1.7 kb downstream of the promoter and >2.0 kb from the silencer . The anti-silencing effect of a targeted HAT is strengthened by increasing the number of targeting sites, but impaired by events that enhance silencing . A tethered HAT can also counteract telomeric silencing . The anti-silencing effect of Gcn5p is abolished by a mutation that eliminated its HAT activity or by deleting the ADA2 gene encoding a structural component of Gcn5p-containing HAT complexes . These results demonstrate that a tethered HAT complex can create a sizable region of histone hyperacetylation and serve as a barrier to encroaching repressive chromatin. BMC Genomics . 2003 Sep 22;4(1):39. The interrelationship between DRIM gene expression and cytogenetic and phenotypic characteristics in human breast tumor cell lines; Goodison S et al.; BACKGROUND: In order to facilitate the identification of genes involved in the metastatic phenotype we have previously developed a pair of cell lines from the human breast carcinoma cell line MDA-MB-435, which have diametrically opposite metastatic potential in athymic mice . Differential display analysis of this model previously identified a novel gene, DRIM (down regulated in metastasis), the decreased expression of which correlated with metastatic capability . DRIM encodes a protein comprising 2785 amino acids with significant homology to a protein in yeast and C . elegans, but little else is currently known about its function or pattern of expression . In a detailed analysis of the DRIM gene locus we quantitatively evaluated gene dosage and the expression of DRIM transcripts in a panel of breast cell lines of known metastatic phenotype . RESULTS: Fluorescent in situ hybridization (FISH) analyses mapped a single DRIM gene locus to human chromosome 12q23~24, a region of conserved synteny to mouse chromosome 10 . We confirmed higher expression of DRIM mRNA in the non-metastatic MDA-MB-435 clone NM2C5, relative to its metastatic counterpart M4A4, but this appeared to be due to the presence of an extra copy of the DRIM gene in the cell line's genome . The other non-metastatic cell lines in the series (T47D MCF-7, SK-BR-3 and ZR-75-1) contained either 3 or 4 chromosomal copies of DRIM gene . However, the expression level of DRIM mRNA in M4A4 was found to be 2-4 fold higher than in unrelated breast cells of non-metastatic phenotype . CONCLUSIONS: Whilst DRIM expression is decreased in metastatic M4A4 cells relative to its non-metastatic isogenic counterpart, neither DRIM gene dosage nor DRIM mRNA levels correlated with metastatic propensity in a series of human breast tumor cell lines examined . Collectively, these findings indicate that the expression pattern of the DRIM gene in relation to the pathogenesis of breast tumor metastasis is more complex than previously recognized. Mol Cells, 2003 Aug 31, 16(1), 34 - 9 Induction of phenylalanine ammonia-lyase gene expression by paraquat and stress-related hormones in Rehmannia glutinosa; Lee BK et al.; Rehmannia glutinosa is a medicinal herb that is tolerant to the non-selective herbicide paraquat . Acteoside, a phenolic compound present in the plant, has been shown to inhibit paraquat . To understand regulation of the phenylpropanoid pathway that produces the acteoside moiety, we isolated a phenylalanine ammonia-lyase (PAL) cDNA clone (RgPAL1) and used it to examine PAL expression . The deduced 712 amino acid sequence of the open reading frame contains the conserved active site and potential phosphorylation sites of other plant PALs . RgPAL1 mRNA was detected in the leaves, flowers and roots of healthy plants, and the level of the mRNA was higher in leaves than in flowers and roots . RgPAL1 mRNA was induced in leaves by paraquat, H2O2, UV light, wounding, yeast extract, jasmonic acid and ethephon . The transcript level and enzyme activity increased gradually from 6 to 24 h after exposure to paraquat or jasmonic acid . Induction of RgPAL1 by paraquat and stress-related phytohormones suggests that it is involved in the regulation of the phenylpropanoid pathway under oxidative stress. Neurol Res, 2003 Sep, 25(6), 665 - 74 Mitochondria and vascular lesions as a central target for the development of Alzheimer's disease and Alzheimer disease-like pathology in transgenic mice; Aliev G et al.; Accumulating evidence strongly suggests that the AD brain is characterized by impairments in energy metabolism, and vascular hypoperfusion, whereby oxidative stress appears to be an especially important contributor to neuronal death and development of AD pathology . We hypothesized that mitochondria play a key role in the generation of reactive oxygen species, resulting in oxidative damage to neuronal cell bodies, as well as other cellular compartments in the AD brain . All of these changes have been found to accompany AD pathology . In this review we have outlined recent evidence from the literature and our own original studies concerning the role of mitochondrial abnormalities and vascular damage in the pathogenesis of AD and AD-like pathology in transgenic mice (as a model for human AD) . We examined ultrastructural features of vascular lesions and mitochondria from vascular wall cells in human AD brain biopsies, in human short post-mortem brain tissues and in yeast artificial chromosome (YAC) and C57B6/SJL transgenic positive (Tg+) mice overexpressing amyloid beta precursor protein (A beta PP) . In situ hybridization using mitochondrial DNA (mtDNA) probes for human wild type, 5kb deleted and mouse mtDNA was performed along with immunocytochemistry using antibodies against amyloid beta precursor protein (A beta PP), 8-hydroxy-2'-guanosine (8OHG) and cytochrome C oxidase (COX) were studied at the electron microscopic levels . There was a higher degree of amyloid deposition in the vascular walls of the human AD, YAC and C57B6/SJL Tg(+) mice compared to aged-matched controls . In addition, vessels with more severe lesions showed immunopositive staining for APP and possessed large, lipid-laden vacuoles in the cytoplasm of endothelial cells (EC) . Significantly more mitochondrial abnormalities were seen in human AD, YAC and C57B6/SJL Tg(+) mouse microvessels where lesions occurred . In situ hybridization using wild and chimera (5 kB) mtDNA probes revealed positive signals in damaged mitochondria from the vascular endothelium and in perivascular cells of lesioned microvessels close to regions of large amyloid deposition . These features were absent in undamaged regions of human AD tissues, YAC and C57B6/SJL Tg(+) mouse tissues and in aged-matched control subjects . In addition, vessels with atherosclerotic lesions revealed endothelium and perivascular cells possessing clusters of wild and deleted mtDNA positive probes . These mtDNA deletions were accompanied by increased amounts of immunoreactive APP, 8OHG and COX in the same cellular compartment . Our observations first time demonstrate that vascular wall cells, especially their mitochondria, appear to be a central target for oxidative stress induced damage. Annu Rev Plant Biol, 2003, 54, 165 - 82 The COP9 signalosome: regulating plant development through the control of proteolysis; Serino G et al.; The COP9 signalosome (CSN) is a multiprotein complex that was initially identified in plants as a repressor of photomorphogenesis . It is now known to play major roles in several other developmental pathways, from auxin response to flower development . Furthermore, the COP9 signalosome shares homologies with the lid sibcomplex of the proteasome and is evolutionarily conserved from fission yeast to humans . It is important for the proper development of virtually all higher eukaryotes . In recent years, significant progress has been made in unraveling the molecular, cellular, and physiological mode of action of the COP9 signalosome . This review discusses our current understanding of the COP9 signalosome function with particular emphasis on its recently defined role in modulating a wide variety of cellular processes by regulating specific protein degradation events. J Cell Physiol, 2003 Nov, 197(2), 214 - 24 NF1 modulates the effects of Ras oncogenes: evidence of other NF1 function besides its GAP activity; Corral T et al.; Neurofibromin (NF1) (the product of Nf1 gene) is a large cytosolic protein known as a negative regulator of Ras . A fragment of some 400 residues located at the center of the NF1 GAP-Related Domain (NF1-GRD) has strong identity with other molecules of the GAP family, which comprises, among others, the mammalian proteins NF1 and p120GAP, and the yeast proteins IRA1 and IRA2 . GAP family members are known by their ability to promote the GTPase activity of Ras proteins, facilitating the transit of those proteins to their inactive state . Recent findings (Tong et al., 2002, Nat Neurosci 5:95-96) indicate that NF1 may be involved in the regulation of adenyl cyclase activity . Our results show that NF1-GRD cooperates with Ras in the anchorage-independent growth capacity of Ras-expressing fibroblasts, without affecting: (i) their ability to grow in low serum, (ii) their cellular adhesion capability, or (iii) the expression of key proteins involved in cell-cell and cell-matrix interactions . On the other hand, NF1 overexpression induces an increase in the expression levels of the focal adhesion kinase (FAK), and specific changes in the activation status of the mitogen-activated protein kinases (MAPKs) . These results suggest the existence of a Ras-independent NF1-dependent pathway able to modify the levels of expression of FAK and the levels of activation of MAPKs . Because FAK and many proteins recently found to bind NF1 have a role in the cytoskeleton, this pathway may involve rearrangement of cytoskeletal components that facilitate anchorage independence . Mol Vis, 2003 Sep 18, 9, 449 - 59 A proline-rich domain in the gamma subunit of phosphodiesterase 6 mediates interaction with SH3-containing proteins; Morin F et al.; PURPOSE: Phosphodiesterase 6 (PDE6) is the primary effector of phototransduction in vertebrate photoreceptors . Previous studies described the expression of the regulatory subunit of rod PDE6 (Pgamma-rod) in non-photosensitive tissues of the adult rat and the effects of this protein on MAP kinase pathways . Upon examination of the Pgamma-rod sequence, we detected a proline-rich domain that might reveal its ability to interact with SH3-containing proteins . Therefore, the present study was initiated to identify new protein partners of Pgamma-rod . METHODS: A yeast two-hybrid screen of a rat brain cDNA library was performed using Pgamma-rod as a bait . Pgamma-rod-SH3 interaction was confirmed by GST pull-down of in vitro-translated proteins . The aminoacids involved in the interaction were mapped by site-directed mutagenesis . Rnase protection assay, RT-PCR and western blot analysis were used to detect Pgamma-rod expression in various rat tissues . RESULTS: A clone was isolated twice, that consisted essentially of the SH3 domain of the formin-binding protein 17 (FBP17) . This interaction was confirmed by GST pull-down . Mutational analysis of the Pgamma-rod-FBP17 interaction confirmed it involved the proline-rich domain of Pgamma-rod and the SH3 domain of FBP17 . This proline-rich domain also allowed Pgamma-rod to interact with Cdc42-interacting protein 4 (CIP4), another SH3-containing protein . RT-PCR and Rnase protection assay detected different amounts of Pgamma-rod mRNA in adult and embryonic rat tissues . Western blots confirmed the presence of low levels of Pgamma-rod protein only in embryonic tissues . CONCLUSIONS: Our data suggest that Pgamma-rod participates in SH3-mediated cellular pathways and may therefore play a wider role than previously appreciated . One possibility is that FBP17 interaction with sorting nexin 2 might connect Pgamma-rod to receptor tyrosine kinase recycling . However, further studies are still required to identify the diversity of SH3-containing proteins that interact with Pgamma-rod . This effort should provide a rationale to understand how Pgamma-rod can affect receptor internalization-dependent MAP kinase activity. Anticancer Drugs, 2003 Sep, 14(8), 595 - 600 Selenium and inhibition of disease progression in men diagnosed with prostate carcinoma: study design and baseline characteristics of the 'Watchful Waiting' Study; Stratton MS et al.; Impediment of the promotion and progression stages of carcinogenesis of the prostate could have a profound impact on treatment choice and prognosis for prostate cancer . Efficacious chemopreventive agents that elicit their activity by slowing the processes of progression could make watchful waiting a viable alternative for a large population of men or could delay the necessity for surgery, radiation or other more invasive treatment modalities associated with frequent side effects . Reports from the Nutritional Prevention of Cancer (NPC) study reported that dietary supplementation with selenium significantly reduced the risk of developing prostate cancer . These data led to initiation of the Watchful Waiting Study, a phase II, multi-center, randomized, double-blind, placebo-controlled clinical intervention study testing the effects of two doses of selenized yeast on progression of prostate cancer . Participants are men with biopsy-proven prostate cancer who have elected to forgo therapy and be closely followed by 'watchful waiting' that includes quarterly prostate-specific antigen (PSA) screening . Subjects are randomized to receive 200 or 800 microg of selenized yeast or matched placebo daily . Endpoints include time to disease progression and PSA velocity . Secondary endpoints include time to initiation of therapy as well as biochemical markers of disease progression including chromagranin A and alkaline phosphatase . Immunohistochemical analyses for indicators of apoptosis, proliferation and differentiation will be performed on baseline and subsequent prostate biopsy specimens . This report summarizes the primary objectives, research methods and the randomized subjects in this important clinical trial. Anticancer Drugs, 2003 Sep, 14(8), 589 - 94 Selenium and prevention of prostate cancer in high-risk men: the Negative Biopsy Study; Stratton MS et al.; Epidemiological and clinical studies suggesting a significant inverse relationship between intake of dietary selenium and overall cancer risk have led to initiation of a randomized, placebo-controlled, phase III clinical trial testing the safety and efficacy of selenized yeast as a chemopreventive agent for prostate cancer . Participants eligible for the 'Negative Biopsy Study', which was initiated in August 1999, are men considered to be at high risk for prostate cancer because of at least one negative sextant prostate biopsy, which was clinically indicated within 1 year of enrollment to the study . After a 30-day run-in period to ensure protocol compliance, participants are randomized to receive either 200 or 400 microg selenized yeast or matched placebo once daily . Primary study endpoints include development of prostate cancer and prostate-specific antigen (PSA) velocity . Secondary biochemical endpoints include change in chromagranin A and alkaline phosphatase . As of 1 June 2003, 514 eligible participants had been enrolled . Randomization schema was effective for selected parameters including age, body mass index, smoking status, baseline PSA and baseline plasma selenium level . Various data, including medical history, family history, and urological symptoms and specimens (including blood and subsequent prostate biopsy samples) had been collected at baseline, and throughout both the intervention and follow-up stages of the protocol . The goal for accrual is 700 evaluable participants. Science, 2003 Sep 19, 301(5640), 1731 - 3 Demography of dietary restriction and death in Drosophila; Mair W et al.; Dietary restriction (DR) increases life-span in organisms from yeast to mammals, presumably by slowing the accumulation of aging-related damage . Here we show that in Drosophila, DR extends life-span entirely by reducing the short-term risk of death . Two days after the application of DR at any age for the first time, previously fully fed flies are no more likely to die than flies of the same age that have been subjected to long-term DR . DR of mammals may also reduce short-term risk of death, and hence DR instigated at any age could generate a full reversal of mortality. Science, 2003 Sep 19, 301(5640), 1679 - 81 Aging . It's never too late; Vaupel JW et al.; When organisms as diverse as yeast and rodents are subjected to a restricted diet, they live longer . The good news is, according to Vaupel, Carey, and Christensen in their Perspective, that switching to a restricted diet at any age can yield the benefit of increased longevity--at least in flies (Mair et al.). Proc Natl Acad Sci U S A, 2003 Sep 30, 100(20), 11237 - 42 Epub 2003 Sep 19. Probe selection for high-density oligonucleotide arrays; Mei R et al.; High-density oligonucleotide microarrays enable simultaneous monitoring of expression levels of tens of thousands of transcripts . For accurate detection and quantitation of transcripts in the presence of cellular mRNA, it is essential to design microarrays whose oligonucleotide probes produce hybridization intensities that accurately reflect the concentration of original mRNA . We present a model-based approach that predicts optimal probes by using sequence and empirical information . We constructed a thermodynamic model for hybridization behavior and determined the influence of empirical factors on the effective fitting parameters . We designed Affymetrix GeneChip probe arrays that contained all 25-mer probes for hundreds of human and yeast transcripts and collected data over a 4,000-fold concentration range . Multiple linear regression models were built to predict hybridization intensities of each probe at given target concentrations, and each intensity profile is summarized by a probe response metric . We selected probe sets to represent each transcript that were optimized with respect to responsiveness, independence (degree to which probe sequences are nonoverlapping), and uniqueness (lack of similarity to sequences in the expressed genomic background) . We show that this approach is capable of selecting probes with high sensitivity and specificity for high-density oligonucleotide arrays. Proc Natl Acad Sci U S A, 2003 Sep 30, 100(20), 11261 - 6 Epub 2003 Sep 18. NMR structure of the forkhead-associated domain from the Arabidopsis receptor kinase-associated protein phosphatase; Lee GI et al.; Forkhead-associated (FHA) domains are phosphoprotein-binding modules found in diverse signaling proteins that bind partners phosphorylated on threonine or serine . Kinase-associated protein phosphatase from Arabidopsis employs its FHA domain for negative regulation of receptor-like kinase signaling pathways, which are important in plant development . The solution structure of the free state of kinase-interacting FHA domain (KI-FHA) of kinase-associated protein phosphatase has been determined with high precision and accuracy using residual dipolar couplings . KI-FHA is a sandwich of a five-stranded mixed beta-sheet with a six-stranded antiparallel beta-sheet . Despite homology only in the recognition loops, this fold is shared with FHA domains from checkpoint proteins from yeast and humans, as well as with nonhomologous MH2 domains of Smad tumor suppressors . A shared pattern of hydrophobicity throughout FHA domains and Smad MH2 domains may stabilize the core of the beta-sandwich . Evolutionary trace analysis of FHA domains suggests class-specific residues in the recognition loops that could tune their phosphoprotein-binding specificity . This surface agrees with that of KI-FHA in contact with a phosphothreonine peptide ligand . Evolutionary trace analysis also predicts an unexpected swath of class-specific residues on another face of FHA domains . Protein interactions with these faces may affect assembly of transmembrane signaling complexes in plants, and in other FHA domain-containing assemblies. J Biol Chem, 2003 Dec 5, 278(49), 49573 - 81 Epub 2003 Sep 18. Serine/threonine kinase Mirk/Dyrk1B is an inhibitor of epithelial cell migration and is negatively regulated by the Met adaptor Ran-binding protein M; Zou Y et al.; Minibrain-related kinase (Mirk)/Dyrk1B is an arginine-directed serine/threonine kinase that is active in skeletal muscle development but is also expressed in various carcinomas . In the current study, the Met adaptor protein Ran-binding protein M (RanBPM) was identified as a Mirk-binding protein by yeast two-hybrid analysis . The Mirk-RanBPM association was confirmed by glutathione S-transferase pull-down assays, co-immunoprecipitation studies, and in vivo cross-linking . Met plays an important role in tumor cell invasion and cell migration . RanBPM has been reported to bind to the tyrosine kinase domain of the hepatocyte growth factor (HGF) receptor Met, enhance Met downstream signaling, and enhance HGF-induced A704 kidney carcinoma cell invasion (Wang, D., Li, Z., Messing, E . M., and Wu, G . (2002) J . Biol . Chem . 277, 36216-36222) . We made a stable Mirk-inducible subline from nontransformed Mv1Lu lung epithelial cells and now demonstrate that induction of Mirk inhibited the migration of these cells in wounding experiments and inhibited their invasion through polycarbonate Transwell filters . Furthermore the ability of Mirk to inhibit Mv1Lu cell migration was attenuated when cells were exposed to HGF or to elevated levels of transiently expressed RanBPM . RanBPM inhibited the kinase activity of Mirk/Dyrk1B and Dyrk1A . In addition, RanBPM and HGF inhibited the function of Mirk as a transcriptional coactivator . Our findings suggest that Mirk plays a role in modulating cell migration through opposing the action of the Met signaling cascade adaptor protein RanBPM. J Biol Chem, 2003 Nov 28, 278(48), 47434 - 40 Epub 2003 Sep 18. PIAS1-mediated sumoylation of focal adhesion kinase activates its autophosphorylation; Kadare G et al.; Focal adhesion kinase (FAK) is a protein tyrosine kinase enriched in focal adhesions, which plays a critical role in integrin-dependent cell motility and survival . The crucial step in its activation is autophosphorylation on Tyr-397, which promotes the recruitment of several enzymes including Src family kinases and the activation of multiple signaling pathways . We found in a yeast two-hybrid screen that the N-terminal domain of FAK interacted with protein inhibitor of activated STAT1 (PIAS1) . This interaction was confirmed and shown to be direct using in vitro assays . PIAS1 was co-immunoprecipitated with FAK from transfected cells and brain extracts . PIAS1 has recently been recognized as a small ubiquitin-like modifier (SUMO) ligase . In the presence of PIAS1 and SUMO-1, FAK was sumoylated in intact cells, whereas PYK2, a closely related enzyme, was not . Sumoylation occurred on Lys-152, a residue conserved in FAK during evolution . Sumoylated FAK, like PIAS1, was recovered predominantly from the nuclear fraction . Sumoylation did not require the catalytic activity or autophosphorylation of FAK . In contrast, sumoylation increased dramatically the ability of FAK to autophosphorylate in intact cells and in immune precipitate kinase assays . Endogenous FAK was sumoylated in the presence of PIAS1 and SUMO-1 independently of cell adhesion, and autophosphorylation of sumoylated FAK was persistently increased in suspended cells . These observations show that sumoylation controls the activity of a protein kinase and suggest that FAK may play a novel role in signaling between the plasma membrane and the nucleus. Cancer Res, 2003 Sep 1, 63(17), 5178 - 87 Alternative splicing of the human proto-oncogene c-H-ras renders a new Ras family protein that trafficks to cytoplasm and nucleus; Guil S et al.; We characterized a novel protein of the Ras family, p19 (H-RasIDX) . The c-H-ras proto-oncogene undergoes alternative splicing of the exon termed IDX . We show that the alternative p19 mRNA is stable and as abundant as p21 (p21 H-Ras4A) mRNA in all of the human tissues and cell lines tested . IDX is spliced into stable mRNA in different mammalian species, which present a high degree of nucleotide conservation . Both the endogenous and the transiently expressed p19 protein are detected in COS-1 and HeLa cells and show nuclear diffuse and speckled patterns as well as cytoplasmic localization . In yeast two-hybrid assays, p19 did not interact with two known p21 effectors, Raf1 and Rin1, but was shown to interact with RACK1, a scaffolding protein that promotes multiprotein complexes in different signaling pathways . This observation suggests that p19 and p21 play differential and complementary roles in the cell. J Mol Biol, 2003 Oct 3, 332(5), 1059 - 69 Structural insights into the lipase/esterase behavior in the Candida rugosa lipases family: crystal structure of the lipase 2 isoenzyme at 1.97A resolution; Mancheno JM et al.; The yeast Candida rugosa produces several closely related extracellular lipases that differ in their substrate specificity . Here, we report the crystal structure of the isoenzyme lipase 2 at 1.97A resolution in its closed conformation . Lipase 2 shows a 79.4% amino acid sequence identity with lipase 1 and 82.2% with lipase 3, which makes it relevant to compare these three isoenzymes . Despite this high level of sequence identity, structural comparisons reveal several amino acid changes affecting the flap (residue 69), the substrate-binding pocket (residues 127, 132 and 450) and the mouth of the hydrophobic tunnel (residues 296 and 344), which may be responsible for the different substrate specificity and catalytic properties of this group of enzymes . Also, these comparisons reveal two distinct regions in the hydrophobic tunnel: a phenylalanyl-rich region and an aliphatic-rich region . Whereas this last region is essentially identical in the three isoenzymes, the phenylalanyl content in the first one is specific for each lipase, resulting in a different environment of the catalytic triad residues, which probably tunes finely their lipase/esterase character . The greater structural similarity observed between the monomeric form of lipase 3 and lipase 2 concerning the above-mentioned key residues led us to propose a significant esterase activity for this last protein . This enzymatic activity has been confirmed with biochemical experiments using cholesteryl {1-14C}oleate as substrate . Surprisingly, lipase 2 is a more efficient esterase than lipase 3, showing a twofold specific activity against cholesteryl {1-14C}oleate in our experimental conditions . These results show that subtle amino acid changes within a highly conserved protein fold may produce protein variants endowed with new enzymatic properties. Biochim Biophys Acta, 2003 Sep 23, 1651(1-2), 50 - 9 CHRK1, a chitinase-related receptor-like kinase, interacts with NtPUB4, an armadillo repeat protein, in tobacco; Kim M et al.; CHRK1 is a receptor-like kinase containing a chitinase-related sequence in the extracellular domain in Nicotiana tabacum . The previous study indicated that CHRK1 plays a role in a signaling pathway regulating plant development and the endogenous cytokinin levels . In this study, we identified NtPUB4 as a CHRK1-interacting protein using yeast two-hybrid screening . NtPUB4 contains the U-box and five arm repeats, and is homologous to Arabidopsis AtPUB4 with unknown function and to Brassica arm repeat containing 1 (ARC1) that interacts with SRK receptor-like kinases during self-incompatibility response . The arm repeats of NtPUB4 are important for the interaction with CHRK1 . CHRK1-NtPUB4 interaction was confirmed by in vitro binding assay using the recombinant proteins . NtPUB4 exhibited spatial and temporal expression patterns that are very similar to those of CHRK1 . Finally, GFP and RFP fusion experiments demonstrated that both CHRK1 and NtPUB4 are localized at the plasma membrane in vivo . These results strongly indicate that NtPUB4 is an interacting partner of CHRK1 receptor-like kinase, and is likely involved in modulating the plant developmental signaling pathway mediated by CHRK1. Peptides, 2003 Jul, 24(7), 987 - 98 A peptide from the extension of Lys-tRNA synthetase binds to transfer RNA and DNA; Yiadom KP et al.; Eukaryotic aminoacyl-tRNA synthetases have dispensable extensions appended at the amino- or carboxyl-terminus as compared to their bacterial counterparts . While a synthetic peptide corresponding to the basic amino-terminal extension in yeast Asp-tRNA synthetase binds to DNA, the extension in the intact protein evidently binds to tRNA and enhances the tRNA specificity of Asp-tRNA synthetase . On the other hand, the amino-terminal extension in human Asp-tRNA synthetase, both within the intact protein and as a synthetic peptide, binds to tRNA . Here, the tRNA binding of a synthetic peptide, hKRS(Arg(25)-Glu(42)), corresponding to the amino-terminal extension of human Lys-tRNA synthetase (hKRS) was analyzed . This basic peptide bound to tRNA(Phe) and the apparent-binding constant increased with increasing concentrations of Mg(2+) . The hKRS peptide also bound to DNA and polyphosphate; however, the apparent DNA-binding constants decreased at increasing concentrations of Mg(2+) . The ability of the hKRS peptide to adopt alpha-helical conformation was demonstrated by NMR and circular dichroism . A Lys-rich peptide derived from the elongation factor 1alpha was also examined and bound to DNA but not to tRNA. BioDrugs, 2003, 17(5), 375 - 9 Somatomedin-1 binding protein-3: insulin-like growth factor-1 binding protein-3, insulin-like growth factor-1 carrier protein; Characterization and expression of the Neurospora crassa nmt-1 gene; Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, K1S 5B6, CanadaThe Neurospora crassa homologue of the yeast no message in thiamine ( nmt-1) gene was characterized . The deduced 342-amino-acid gene product has more than 60% identity with other fungal homologues and 42% similarity to a putative bacterial permease . In addition to three introns disrupting the coding sequence, a differentially spliced intron in the 5' untranslated region was also detected . Unlike other fungi, the N . crassa nmt-1 gene is repressed only 6- to 8-fold by exogenous thiamine concentrations above 0.5 microM; and a high basal level of nmt-1 mRNA persists even at 5 microM thiamine . Immuno-blotting with purified antibodies detected two variants of NMT-1 which differ in size and charge . The more abundant 39-kDa form is more strongly repressed by thiamine than the 37-kDa protein . NMT-1 abundance modulates slowly in response to changes in the concentration of exogenous thiamine, suggesting that N . crassa maintains thiamine reserves in excess of immediate needs . Disruption of the nmt-1 gene demonstrated that it is essential for growth in the absence of exogenous thiamine . NMT-1-deficient strains had a growth rate and colony density which was about 70% of the wild type, despite supplementation with a wide range of exogenous thiamine . These results suggest that the nmt-1 gene plays some other role in addition to thiamine biosynthesis. Oncogene, 2003 Sep 18, 22(40), 6151 - 9 Low molecular weight inhibitors of Myc-Max interaction and function; Yin X et al.; c-Myc is helix-loop-helix-leucine zipper (HLH-ZIP) oncoprotein that is frequently deregulated in human cancers . In order to bind DNA, regulate target gene expression, and function in a biological context, c-Myc must dimerize with another HLH-ZIP protein, Max . A large number of c-Myc target genes have been identified, and many of the encoded proteins are transforming . Such functional redundancy, however, complicates therapeutic strategies aimed at inhibiting any single target gene product . Given this consideration, we have instead attempted to identify ways by which c-Myc itself could be effectively disabled . We have used a yeast two-hybrid approach to identify low-molecular-weight compounds that inhibit c-Myc-Max association . All of the compounds prevented transactivation by c-Myc-Max heterodimers, inhibited cell cycle progression, and prevented the in vitro growth of fibroblasts in a c-Myc-dependent manner . Several of the compounds also inhibited tumor growth in vivo . These results show that the yeast two-hybrid screen is useful for identifying compounds that can be exploited in mammalian cells . More specifically, they provide a means by which structural analogs, based upon these first-generation Myc-Max inhibitors, can be developed to enhance antitumor efficacy. J Gen Virol, 2003 Oct, 84(Pt 10), 2861 - 9 The central and C-terminal domains of VPg of Clover yellow vein virus are important for VPg-HCPro and VPg-VPg interactions; Yambao ML et al.; Interactions between the major proteins of Clover yellow vein virus (ClYVV) were investigated using a GAL4 transcription activator-based yeast two-hybrid system (YTHS) . Self-interactions manifested by VPg and HCPro and an interaction between NIb and NIaPro were observed in ClYVV . In addition, a strong HCPro-VPg interaction was detected by both YTHS and by in vitro far-Western blot analysis in ClYVV . A potyvirus HCPro-VPg interaction has not been reported previously . Using YTHS, domains in ClYVV for the VPg self-interaction and the HCPro-VPg interaction were mapped . The VPg C-terminal region (38 amino acids) was important for the VPg-VPg interaction and the central 19 amino acids were needed for the HCPro-VPg interaction. J Biol Chem, 2003 Nov 28, 278(48), 48491 - 7 Epub 2003 Sep 17. Xenopus cold-inducible RNA-binding protein 2 interacts with ElrA, the Xenopus homolog of HuR, and inhibits deadenylation of specific mRNAs; Aoki K et al.; Xenopus cold-inducible RNA-binding protein 2 (xCIRP2) is a major cytoplasmic RNA-binding protein in oocytes . In this study, we identify another RNA-binding protein ElrA, the Xenopus homolog of HuR, as an interacting protein of xCIRP2 by yeast two-hybrid screening . As ElrA stabilizes the RNA body in the in vitro mRNA stability system, we examine the role of xCIRP2 in the stabilization of mRNA and find that xCIRP2 inhibits deadenylation of AU-rich element-containing mRNA . These results suggest that xCIRP2 and ElrA may be involved in the regulation of mRNA stability at different steps . By immunoprecipitation with anti-xCIRP2 antibody, we find that xCIRP2 interacts with several mRNAs including mRNA encoding the centrosomal kinase Nek2B in oocytes . xCIRP2 also inhibits deadenylation of the mRNA substrate containing the 3'-untranslated region of Nek2B mRNA in the in vitro system . Our results suggest that xCIRP2 associates with specific mRNAs and can regulate the length of poly(A) tail in Xenopus oocytes. Biochem Biophys Res Commun, 2003 Oct 3, 309(4), 1005 - 10 In vitro characterization of four novel non-functional variants of the thiopurine S-methyltransferase; Hamdan-Khalil R et al.; Human thiopurine S-methyltransferase (TPMT) is an enzyme responsible for the detoxification of widely used thiopurine drugs such as azathioprine (Aza) . Its activity is inversely related to the risk of developing severe hematopoietic toxicity in certain patients treated with standard doses of thiopurines . DNA samples from four leucopenic patients treated with Aza were screened by PCR-SSCP analysis for mutations in the 10 exons of the TPMT gene . Four missense mutations comprising two novel mutations, A83T (TPMT*13, Glu(28)Val) and C374T (TPMT*12, Ser(125)Leu), and two previously described mutations, G430C (TPMT*10, Gly(144)Arg) and T681G (TPMT*7, His(227)Gln) were identified . Using a recombinant yeast expression system, kinetic parameters (K(m) and V(max)) of 6-thioguanine S-methylation of the four TPMT variants were determined and compared to those obtained with wild-type TPMT . This functional analysis suggests that these rare allelic variants are defective TPMT alleles . The His(227)Gln variant retained only 10% of the intrinsic clearance value (V(max)/K(m) ratio) of the wild-type enzyme . The Ser(125)Leu and Gly(144)Arg variants were associated with a significant decrease in intrinsic clearance values, retaining about 30% of the wild-type enzyme, whereas the Glu(28)Val variant produced a more modest decrease (57% of the wild-type enzyme) . The data suggest that the sporadic contribution of the rare Glu(28)Val, Ser(125)Leu, Gly(144)Arg, and His(227)Gln variants may account for the occurrence of altered metabolism of TPMT substrates . These findings improve our knowledge of the genetic basis of interindividual variability in TPMT activity and would enhance the efficiency of genotyping methods to predict patients at risk of inadequate responses to thiopurine therapy. Biochem Biophys Res Commun, 2003 Oct 3, 309(4), 980 - 5 Casper/c-FLIP is physically and functionally associated with NF-kappaB1 p105; Li Z et al.; Casper/c-FLIP is a caspase-8-related molecule critically involved in regulation of death receptor-induced apoptosis . It has been shown that Casper can either promote or antagonize apoptosis and can activate the transcription factor NF-kappaB . The exact functions of Casper are controversial . To further understand how Casper signals, we searched Casper-interacting proteins by yeast two-hybrid screening . This effort identified NF-kappaB1 (p105), an atypical IkappaB molecule and the precursor of NF-kappaB subunit p50 . Co-immunoprecipitation experiments indicated that Casper interacted with p105 in 293 cells and this interaction was mediated through the C-terminal IkappaB-like domain (IkappaBgamma) . Overexpression of p105 and IkappaBgamma inhibited Casper-induced NF-kappaB activation and potentiated Casper-induced apoptosis . Furthermore, Casper and its C-terminal caspase-like domain inhibited p105 processing into p50 . Our findings suggest that p105 is involved in Casper-mediated regulation of apoptosis and NF-kappaB activation. Biochem Biophys Res Commun, 2003 Oct 3, 309(4), 848 - 56 Effects of deficiencies of STAMs and Hrs, mammalian class E Vps proteins, on receptor downregulation; Kanazawa C et al.; The STAM family proteins, STAM1 and STAM2/EAST/Hbp, are phosphotyrosine proteins that contain SH3 domains and ubiquitin-interacting motifs . Their yeast homologue, Hse1, and its binding protein, Vps27, are involved in the vacuolar membrane transport machinery . Here we show that STAM1 and STAM2 are localized to the endosomal membrane . Some of these complexes contain Eps15, an endocytic protein, which accumulates in clumps upon expression of a dominant-negative form of Vps4-A, an AAA-type ATPase, that is required for normal endosome function . These results support the idea that the STAMs are mammalian vacuolar protein sorting (Vps) proteins . We also demonstrate that ligand-mediated epidermal growth factor receptor (EGFR) degradation is partially but not completely impaired in both Hrs(-/-) and STAM1(-/-)STAM2(-/-) mouse embryonic fibroblasts . Furthermore, endosome swelling is seen in both Hrs(-/-) and STAM1(-/-)STAM2(-/-) cells . These results suggest that the STAMs and Hrs play important roles in the mammalian endosomal/vacuolar protein sorting pathway. Curr Biol, 2003 Sep 16, 13(18), R711 - 3 Cell polarity: a new mod(e) of anchoring; Martin SG et al.; Microtubules play a central role in the establishment of cell polarity by directing the transport of polarity determinants to their site of action . Recent work has revealed a novel membrane-anchoring mechanism which complements the microtubule transport of the fission yeast polarity determinant tea1p to ensure its retention at the cell tip. J Chromatogr A, 2003 Aug 15, 1009(1-2), 111 - 7 Characterization of recombinant human serum albumin using matrix-assisted laser desorption ionization time-of-flight mass spectrometry; Flensburg J et al.; Chromatographically purified recombinant human serum albumin (rHSA), produced in genetically transformed yeast cells, was characterized using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS techniques . The molecular mass of the intact protein was determined to be 66671, in good agreement with that of purified HSA which was used as a standard . The identity of rHSA to its natural counterpart was established with high precision using peptide mass fingerprinting of tryptic peptides . Partial amino acid sequence data for rHSA were obtained using Ettan CAF MALDI Sequencing Kit and post-source decay on the tryptic peptides . The results achieved provide strong evidence that MALDI-TOF-MS is an important analytical technique for characterising gene products and for establishing the identity and bio-compatibility of recombinant proteins relative to their natural counterparts. Plant Mol Biol, 2003 Jul, 52(4), 715 - 27 The Arabidopsis SKP1-like genes present a spectrum of expression profiles; Marrocco K et al.; The yeast Skp1 protein is a component of the SCF complex, an E3 enzyme involved in the specific protein degradation pathway via ubiquitination . Skp1 binds to F-box proteins to trigger specific recognition of proteins targeted for degradation . SKP1-like genes have been found in a variety of eukaryotes including yeast, man, Caenorhabditis elegans and Arabidopsis thaliana . The Arabidopsis genome contains 20 SKP1-like genes called ASK (for Arabidopsis SKP1-like), among which only ASK1 has been characterized in detail . The analysis of the expression pattern of the ASK genes in Arabidopsis should provide key information for the understanding of the biological role of this family in protein degradation and in different cellular mechanisms . In this paper, we describe the expression profiles of 19 ASK promoter-GUS fusions in stable transformants of Arabidopsis, with a special emphasis on floral organ development . Four ASK promoters did not show any detectable expression in either inflorescences or seedlings . Our results on the ASK1 expression profile are consistent with previous reports . Several ASK promoters show clear tissue-specific expression (for instance in the connective of anthers or in the embryo) . We also found that almost half (9/19) of ASK promoters direct a post-meiotic expression in the male gametophyte . Tight regulation of the expression of this gene family indicates a crucial role of the ubiquitin degradation pathway during development, particularly during male gametophyte development. RNA, 2003 Oct, 9(10), 1171 - 3 The GW182 protein colocalizes with mRNA degradation associated proteins hDcp1 and hLSm4 in cytoplasmic GW bodies; Eystathioy T et al.; A novel cytoplasmic compartment referred to as GW bodies (GWBs) was initially identified using antibodies specific to a 182-kD protein termed GW182 . GW182 was characterized by multiple glycine(G)-tryptophan(W) repeats and an RNA recognition motif (RRM) that bound a subset of HeLa cell messenger RNAs (mRNAs) . The function of GWBs was not known; however, more recent evidence suggested similarities between GWBs and cytoplasmic structures that contain hLSm proteins and hDcp1, the human homolog to a yeast decapping enzyme subunit . In this study, we used antibodies to hLSm4 and hDcp1 to show that both of these markers of an mRNA degradation pathway colocalize to the same structures as GW182 . Our studies demonstrate that GW182, hLSm4, and hDcp1 are found in the same cytoplasmic structures and suggest that GW182 is involved in the same mRNA processing pathway as hLSm4 and hDcp1. J Lipid Res, 2004 Jan, 45(1), 63 - 70 Epub 2003 Sep 16. Mutation of lysine residues in apolipoprotein B-100 causes defective lipoprotein{a} formation; Liu CY et al.; Lipoprotein{a} (Lp{a}) is assembled by a two-step process involving an initial lysine-dependent binding between apolipoprotein B-100 (apoB-100) and apolipoprotein{a} (apo{a}) that facilitates the formation of a disulphide bond between apoB-100Cys4,326 and apo{a}Cys4,057 . Previous studies of transgenic mice expressing apoB-95 (4,330 amino acids) and apoB-97 (4,397 amino acids) have shown that apoB-100 amino acids 4,330-4,397 are important for the initial binding to apo{a} . Furthermore, a lysine-rich peptide spanning apoB-100 amino acids 4,372-4,392 has recently been shown to bind apo{a} and inhibit Lp{a} assembly in vitro . This suggests that a putative apo{a} binding site exists in the apoB-4,372-4,392 region . The aim of our study was to establish whether the apoB-4,372-4,392 sequence was important for Lp{a} assembly in the context of the full-length apoB-100 . Transgenic mice were created that expressed a mutant human apoB-100, apoB-100K4-->S4, in which all four lysine residues in the 4,372-4,392 sequence were mutated to serines . The apoB-100K4-->S4 mutant showed a reduced capacity to form Lp{a} in vitro compared with wild-type human apoB-100 . Double transgenic mice expressing both apoB-100K4-->S4 and apo{a} contained significant amounts of free apo{a} in the plasma, indicating a less-efficient assembly of Lp{a} in vivo . Taken together, these results clearly show that the apoB-4,372-4,392 sequence plays a role in Lp{a} assembly. Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1655 - 64 Novel anamorphic mite-associated fungi belonging to the Ustilaginomycetes: Meira geulakonigii gen . nov., sp . nov., Meira argovae sp . nov . and Acaromyces ingoldii gen . nov., sp . nov; Boekhout T et al.; Three novel mite-associated basidiomycetous species are described in two new anamorph genera as Meira geulakonigii gen . nov., sp . nov . (type CBS 110052(T)=NRRL Y-27483(T)=AS 004(T)), Meira argovae sp . nov . (type CBS 110053(T)=NRRL Y-27482(T)=AS 005(T)) and Acaromyces ingoldii gen . nov., sp . nov . (type CBS 110050(T)=NRRL Y-27484(T)=AS 001(T)) . Morphologically, these fungi are similar to the yeast-like fungi classified in the Ustilaginales, such as Pseudozyma species . However, analysis of the D1/D2 domain of the LSU rDNA suggests that they belong to two different lineages within the Exobasidiomycetidae of the Ustilaginomycetes (Basidiomycota) . Furthermore, these fungi may be of interest for the biocontrol of mites, as they reduced mite numbers by approximately 80 % after inoculation. J Biol Chem, 2003 Nov 28, 278(48), 47644 - 53 Epub 2003 Sep 16. Expression profiles of Arabidopsis thaliana in mineral deficiencies reveal novel transporters involved in metal homeostasis; Wintz H et al.; Plants directly assimilate minerals from the environment and thus are key for acquisition of metals by all subsequent consumers . Limited bio-availability of copper, zinc and iron in soil decreases both the agronomic productivity and the nutrient quality of crops . Understanding the molecular mechanisms underlying metal homeostasis in plants is a prerequisite to optimizing plant yield and metal nutrient content . To absorb and maintain a balance of potentially toxic metal ions, plants utilize poorly understood mechanisms involving a large number of membrane transporters and metal binding proteins with overlapping substrate specificities and complex regulation . To better understand the function and the integrated regulation, we analyzed in Arabidopsis the expression patterns in roots and in leaves of 53 genes coding for known or potential metal transporters, in response to copper, zinc, and iron deficiencies in Arabidopsis . Comparative analysis of gene expression profiles revealed specific transcriptional regulation by metals of the genes contrasting with the known wide substrate specificities of the encoded transporters . Our analysis suggested novel transport roles for several gene products and we used functional complementation of yeast mutants to correlate specific regulation by metals with transport activity . We demonstrate that two ZIP genes, ZIP2 and ZIP4, are involved in copper transport . We also present evidence that AtOPT3, a member of the oligopeptide transporter gene family with significant similarities to the maize iron-phytosiderophore transporter YS1, is regulated by metals and heterologous expression AtOPT3 can rescue yeast mutants deficient in metal transport. J Biol Chem, 2003 Dec 12, 278(50), 50203 - 11 Epub 2003 Sep 15. Critical determinants of the G protein gamma subunits in the Gbetagamma stimulation of G protein-activated inwardly rectifying potassium (GIRK) channel activity; Peng L et al.; The betagamma subunits of G proteins modulate inwardly rectifying potassium (GIRK) channels through direct interactions . Although GIRK currents are stimulated by mammalian Gbetagamma subunits, we show that they were inhibited by the yeast Gbetagamma (Ste4/Ste18) subunits . A chimera between the yeast and the mammalian Gbeta1 subunits (ymbeta) stimulated or inhibited GIRK currents, depending on whether it was co-expressed with mammalian or yeast Ggamma subunits, respectively . This result underscores the critical functional influence of the Ggamma subunits on the effectiveness of the Gbetagamma complex . A series of chimeras between Ggamma2 and the yeast Ggamma revealed that the C-terminal half of the Ggamma2 subunit is required for channel activation by the Gbetagamma complex . Point mutations of Ggamma2 to the corresponding yeast Ggamma residues identified several amino acids that reduced significantly the ability of Gbetagamma to stimulate channel activity, an effect that was not due to improper association with Gbeta . Most of the identified critical Ggamma residues clustered together, forming an intricate network of interactions with the Gbeta subunit, defining an interaction surface of the Gbetagamma complex with GIRK channels . These results show for the first time a functional role for Ggamma in the effector role of Gbetagamma. Genome Res, 2003 Oct, 13(10), 2265 - 70 Epub 2003 Sep 15. The secreted protein discovery initiative (SPDI), a large-scale effort to identify novel human secreted and transmembrane proteins: a bioinformatics assessment; Clark HF et al.; A large-scale effort, termed the Secreted Protein Discovery Initiative (SPDI), was undertaken to identify novel secreted and transmembrane proteins . In the first of several approaches, a biological signal sequence trap in yeast cells was utilized to identify cDNA clones encoding putative secreted proteins . A second strategy utilized various algorithms that recognize features such as the hydrophobic properties of signal sequences to identify putative proteins encoded by expressed sequence tags (ESTs) from human cDNA libraries . A third approach surveyed ESTs for protein sequence similarity to a set of known receptors and their ligands with the BLAST algorithm . Finally, both signal-sequence prediction algorithms and BLAST were used to identify single exons of potential genes from within human genomic sequence . The isolation of full-length cDNA clones for each of these candidate genes resulted in the identification of >1000 novel proteins . A total of 256 of these cDNAs are still novel, including variants and novel genes, per the most recent GenBank release version . The success of this large-scale effort was assessed by a bioinformatics analysis of the proteins through predictions of protein domains, subcellular localizations, and possible functional roles . The SPDI collection should facilitate efforts to better understand intercellular communication, may lead to new understandings of human diseases, and provides potential opportunities for the development of therapeutics. Insect Mol Biol, 2003 Oct, 12(5), 427 - 32 Functional dissection of the hexamerin receptor and its ligand arylphorin in the blowfly Calliphora vicina; Hansen IA et al.; The process of receptor-mediated uptake of hexamerin storage proteins from insect haemolymph by fat body cells is a unique feature of the class Insecta . We identified the binding domains of the hexamerin receptor and the hexamerin ligand arylphorin in the blowfly, by means of the yeast-two-hybrid-system . The receptor-binding domain of arylphorin was located within domain 3 of the arylphorin monomer . The ligand-binding domain of the hexamerin receptor was mapped to the extreme N-terminus of the receptor . The binding domains identified exhibit no similarity to any functional protein domains known to date . Additionally, we identified two previously unknown protein-interactors of the hexamerin receptor . The results of this study provide further insights regarding the mechanism of the receptor-mediated endocytosis of storage proteins in insects. Biochem J, 2004 Jan 1, 377(Pt 1), 51 - 9 Cytoskeletal protein 4.1G binds to the third intracellular loop of the A1 adenosine receptor and inhibits receptor action; Lu D et al.; To identify binding partners of the A1AR (A1 adenosine receptor), yeast two-hybrid screening of a rat embryonic cDNA library was performed . This procedure led to the identification of erythrocyte membrane cytoskeletal protein (represented as 4.1G) as an A1AR-binding partner . Truncation studies revealed that the C-terminal domain of 4.1G was essential for binding to A1ARs and that the C-terminal domain of 4.1G and the third intracellular loop of A1ARs interacted . A1AR-4.1G interaction was also confirmed in studies using brain tissue . Studies in HEK-293 (human embryonic kidney 293) cells and Chinese-hamster ovary cells showed that 4.1G interfered with A1AR signal transduction, as 4.1G reduced A1AR-mediated inhibition of cAMP accumulation and intracellular calcium release . 4.1G also altered cell-surface A1AR expression . These observations identify 4.1G as a novel A1AR-binding partner that can regulate adenosine action. Biochemistry, 2003 Sep 23, 42(37), 11032 - 9 A specific segment of the transmembrane domain of Wbp1p is essential for its incorporation into the oligosaccharyl transferase complex; Li G et al.; Wbp1p, a type I transmembrane protein, is an essential component of oligosaccharyl transferase (OT), which consists of nine different subunits in yeast . It has been proposed that three subunits, Wbp1p, Ost2p, and Swp1p, physically interact with each other, but the mechanism of these interactions is unknown . To explore the mode of interaction, we have focused on the single-transmembrane protein, Wbp1p, and made several deletions and mutations within the short cytosolic domain and the transmembrane domain . Our results show that the deletion of the cytosolic domain has no effect on cell growth, but mutation of all 17 amino acids in the transmembrane domain to 17 Leu residues or replacement of the transmembrane and cytosolic domains with the counterparts of Ost1p results in lethality . Immunoprecipitation experiments show that Wbp1p mutated in these two ways is not incorporated into the OT complex . This finding suggests that the transmembrane domain of Wbplp may mediate its association with the other subunits . A series of mutations of the transmembrane domain have revealed that block alterations in the half of the transmembrane domain facing the lumen of the endoplasmic reticulum (ER) impaired cell viability . Seven single-Lys mutants in the same domain were temperature sensitive for growth at 37 degrees C . In contrast, block mutations in the other half of the transmembrane domain facing the cytosol did not result in lethality and indicated that this portion of the transmembrane domain was not involved in stable incorporation of Wbp1p into the OT complex. Mol Cell Biol, 2003 Oct, 23(19), 6944 - 57 A novel human Ada2 homologue functions with Gcn5 or Brg1 to coactivate transcription; Barlev NA et al.; In yeast, the transcriptional adaptor yeast Ada2 (yAda2) is a part of the multicomponent SAGA complex, which possesses histone acetyltransferase activity through action of the yGcn5 catalytic enzyme . yAda2, among several SAGA proteins, serves to recruit SAGA to genes via interactions with promoter-bound transcription factors . Here we report identification of a new human Ada2 homologue, hAda2beta . Ada2beta differs both biochemically and functionally from the previously characterized hAda2alpha, which is a stable component of the human PCAF (human Gcn5 homologue) acetylase complex . Ada2beta, relative to Ada2alpha, interacted selectively, although not stably, with the Gcn5-containing histone acetylation complex TFTC/STAGA . In addition, Ada2beta interacted with Baf57 (a component of the human Swi/Snf complex) in a yeast two-hybrid screen and associated with human Swi/Snf in vitro . In functional assays, hAda2beta (but not Ada2alpha), working in concert with Gcn5 (but not PCAF) or Brg1 (the catalytic component of hSwi/Snf complex), increased transcription via the B-cell-specific transcription factor Pax5/BSAP . These findings support the view that Gcn5 and PCAF have distinct roles in vivo and suggest a new mechanism of coactivator function, in which a single adaptor protein (Ada2beta) can coordinate targeting of both histone acetylation and chromatin remodeling activities. Mol Biol Cell, 2003 Sep, 14(9), 3664 - 74 Epub 2003 Jul 11. Spy1 interacts with p27Kip1 to allow G1/S progression; Porter LA et al.; Progression through the G1/S transition commits cells to synthesize DNA . Cyclin dependent kinase 2 (CDK2) is the major kinase that allows progression through G1/S phase and subsequent replication events . p27 is a CDK inhibitor (CKI) that binds to CDK2 to prevent premature activation of this kinase . Speedy (Spy1), a novel cell cycle regulatory protein, has been found to prematurely activate CDK2 when microinjected into Xenopus oocytes and when expressed in mammalian cells . To determine the mechanism underlying Spy1-induced proliferation in mammalian cell cycle regulation, we used human Spy1 as bait in a yeast two-hybrid screen to identify interacting proteins . One of the proteins isolated was p27; this novel interaction was confirmed both in vitro, using bacterially expressed and in vitro translated proteins, and in vivo, through the examination of endogenous and transfected proteins in mammalian cells . We demonstrate that Spy1 expression can overcome a p27-induced cell cycle arrest to allow for DNA synthesis and CDK2 histone H1 kinase activity . In addition, we utilized p27-null cells to demonstrate that the proliferative effect of Spy1 depends on the presence of endogenous p27 . Our data suggest that Spy1 associates with p27 to promote cell cycle progression through the G1/S transition. J Mol Biol, 2003 Sep 26, 332(4), 877 - 87 Muscle-type creatine kinase interacts with central domains of the M-band proteins myomesin and M-protein; Hornemann T et al.; Muscle-type creatine kinase (MM-CK) is a member of the CK isoenzyme family with key functions in cellular energetics . MM-CK interacts in an isoform-specific manner with the M-band of sarcomeric muscle, where it serves as an efficient intramyofibrillar ATP-regenerating system for the actin-activated myosin ATPase located nearby on both sides of the M-band . Four MM-CK-specific and highly conserved lysine residues are thought to be responsible for the interaction of MM-CK with the M-band . A yeast two-hybrid screen led to the identification of MM-CK as a binding partner of a central portion of myomesin (My7-8) . An interaction was observed with domains six to eight of the closely related M-protein but not with several other Ig-like domains, including an M-band domain, of titin . The observed interactions were corroborated and characterised in detail by surface plasmon resonance spectroscopy (BiaCore) . In both cases, they were CK isoform-specific and the MM-CK-specific lysine residues (K8 . K24, K104 and K115) are involved in this interaction . At pH 6.8, the dissociation constants for the myomesin/MM-CK and the M-protein/MM-CK binding were in the range of 50-100 nM and around 1 microM, respectively . The binding showed pronounced pH-dependence and indicates a dynamic association/dissociation behaviour, which most likely depends on the energy state of the muscle . Our data propose a simple model for the regulation of this dynamic interaction. Mol Cell Endocrinol, 2003 Sep 30, 207(1-2), 31 - 8 Cloning of a protein binding to the most proximal Pit-1 binding element of prolactin gene from human pituitary cDNA library; Fumoto M et al.; A human pituitary cDNA library was screened using a yeast one-hybrid system to find a factor binding Pit-1 binding elements in the PRL gene other than Pit-1 . Beside colonies containing Pit-1 or Oct-1 cDNA, three colonies contained mPOU cDNA, a member of the POU protein family . Immunohistochemical analysis showed mPOU-like immunoreactivity was present in human PRL-producing pituitary tumors but not in non-functioning pituitary tumors . Mobility shift analysis revealed that mPOU bound to Pit-1 binding elements of the PRL gene, 1P and 3P . mPOU activated the expression of 0.6 k PRL and 7x1P reporter genes in the presence of Pit-1 and cAMP, although it did not enhance Pit-1-induced expression of 7x3P reporter gene . These findings suggest that mPOU is involved in the activation of the PRL gene by cAMP through 1P in the presence of Pit-1. Chromosome Res, 2003, 11(5), 471 - 84 Comparative analysis of the functional genome architecture of animal and plant cell nuclei; Mayr C et al.; Many studies have shown that the functional architecture of eukaryotic genomes displays striking similarities in evolutionarily distant organisms . For example, late-replicating and transcriptionally inactive chromatin is associated with the nuclear periphery in organisms as different as budding yeast and man . These findings suggest that eukaryotic genomes are organized in cell nuclei according to conserved principles . In order to investigate this, we examined nuclei of different animal and plant species by comparing replicational pulse-labelling patterns and their topological relationship to markers for heterochromatin and euchromatin . The data show great similarities in the nuclear genome organization of the investigated animal and plant species, supporting the idea that eukaryotic genomes are organized according to conserved principles . There are, however, differences between animals and plants with regard to histone acetylation patterns and the nuclear distribution of late-replicating chromatin. Oncogene, 2003 Sep 11, 22(39), 7905 - 12 Multiple molecular mechanisms contribute to radiation sensitivity in mantle cell lymphoma; M'kacher R et al.; Mantle cell lymphomas (MCL) are characterized by their aggressive behavior and poor response to chemotherapy regimens . We report here evidence of increased in vitro radiation sensitivity in two cell lines that we have generated from two MCL patients (UPN1 and UPN2) . However, despite their increased radiation sensitivity, UPN2 cells were totally resistant to apoptotic cell death, whereas UPN1 cells underwent massive apoptosis 6 h after irradiation . The frequency of induced chromosomal abnormalities was higher in UPN1 as compared to UPN2 . Distinct mechanisms have been found to contribute to this phenotype: a major telomere shortening (UPN1 and UPN2), deletion of one ATM allele and a point mutation in the remaining allele in UPN2, mutation of p53 gene (UPN1 and UPN2) with absence of functional p53 as revealed by functional yeast assays . After irradiation, Ku70 levels in UPN1 increased and decreased in UPN2, whereas in the same conditions, DNA-PKcs protein levels decreased in UPN1 and remained unchanged in UPN2 . Thus, irradiation-induced apoptotic cell death can occur despite the nonfunctional status of p53 (UPN1), suggesting activation of a unique pathway in MCL cells for the induction of this event . Overall, our study demonstrates that MCL cells show increased radiation sensitivity, which can be the result of distinct molecular events . These findings could clinically be exploited to increase the dismal response rates of MCL patients to the current chemotherapy regimens. Plant Physiol, 2003 Sep, 133(1), 328 - 38 Reduction of stability of arabidopsis genomic and transgenic DNA-repeat sequences (microsatellites) by inactivation of AtMSH2 mismatch-repair function; Leonard JM et al.; Highly conserved mismatch repair (MMR) systems promote genomic stability by correcting DNA replication errors, antagonizing homeologous recombination, and responding to various DNA lesions . Arabidopsis and other plants encode a suite of MMR protein orthologs, including MSH2, the constant component of various specialized eukaryotic mismatch recognition heterodimers . To study MMR roles in plant genomic stability, we used Arabidopsis AtMSH2::TDNA mutant SALK_002708 and AtMSH2 RNA-interference (RNAi) lines . AtMSH2::TDNA and RNAi lines show normal growth, development, and fertility . To analyze AtMSH2 effects on germ line DNA fidelity, we measured insertion-deletion mutation of dinucleotide-repeat sequences (microsatellite instability) at nine loci in 16 or more progeny of two to four different wild-type or AtMSH2-deficient plants . Scoring 992 total alleles revealed 23 (2.3%) unique and 51 (5.1%) total repeat length shifts ({+2}, {-2}, {+4}, or {-4} bp) . For the six longest repeat loci, the corresponding frequencies were 22/608 and 50/608 . Two of four AtMSH2-RNAi plants showed similar microsatellite instability . In wild-type progeny, only one unique repeat length allele was found in 576 alleles tested . This endogenous microsatellite instability, shown for the first time in MMR-defective plants, is similar to that seen in MMR-defective yeast and mice, indicating that plants also use MMR to promote germ line fidelity . We used a frameshifted reporter transgene, (G)(7)GUS, to measure insertion-deletion reversion as blue-staining beta-glucuronidase-positive leaf spots . Reversion rates increased only 5-fold in AtMSH2::TDNA plants, considerably less than increases in MSH2-deficient yeast or mammalian cells for similar mononucleotide repeats . Thus, MMR-dependent error correction may be less stringent in differentiated leaf cells than in plant equivalents of germ line tissue. Plant Physiol, 2003 Sep, 133(1), 182 - 90 Functional characterization and expression analyses of the glucose-specific AtSTP9 monosaccharide transporter in pollen of Arabidopsis; Schneidereit A et al.; A genomic clone and the corresponding cDNA of a new Arabidopsis monosaccharide transporter AtSTP9 were isolated . Transport analysis of the expressed protein in yeast showed that AtSTP9 is an energy-dependent, uncoupler-sensitive, high-affinity monosaccharide transporter with a K(m) for glucose in the micromolar range . In contrast to all previously characterized monosaccharide transporters, AtSTP9 shows an unusual specificity for glucose . Reverse transcriptase-polymerase chain reaction analyses revealed that AtSTP9 is exclusively expressed in flowers, and a more detailed approach using AtSTP9 promoter/reporter plants clearly showed that AtSTP9 expression is restricted to the male gametophyte . AtSTP9 expression is not found in other floral organs or vegetative tissues . Further localization on the cellular level using a specific antibody revealed that in contrast to the early accumulation of AtSTP9 transcripts in young pollen, the AtSTP9 protein is only found weakly in mature pollen but is most prominent in germinating pollen tubes . This preloading of pollen with mRNAs has been described for genes that are essential for pollen germination and/or pollen tube growth . The pollen-specific expression found for AtSTP9 is also observed for other sugar transporters and indicates that pollen development and germination require a highly regulated supply of sugars. J Virol, 2003 Oct, 77(19), 10237 - 49 Proteasome activator PA28gamma-dependent nuclear retention and degradation of hepatitis C virus core protein; Moriishi K et al.; Hepatitis C virus (HCV) core protein plays an important role in the formation of the viral nucleocapsid and a regulatory protein involved in hepatocarcinogenesis . In this study, we have identified proteasome activator PA28gamma (11S regulator gamma) as an HCV core binding protein by using yeast two-hybrid system . This interaction was demonstrated not only in cell culture but also in the livers of HCV core transgenic mice . These findings are extended to human HCV infection by the observation of this interaction in liver specimens from a patient with chronic HCV infection . Neither the interaction of HCV core protein with other PA28 subtypes nor that of PA28gamma with other Flavivirus core proteins was detected . Deletion of the PA28gamma-binding region from the HCV core protein or knockout of the PA28gamma gene led to the export of the HCV core protein from the nucleus to the cytoplasm . Overexpression of PA28gamma enhanced the proteolysis of the HCV core protein . Thus, the nuclear retention and stability of the HCV core protein is regulated via a PA28gamma-dependent pathway through which HCV pathogenesis may be exerted. Bioinformatics, 2003 Sep 1, 19(13), 1612 - 9 Diametrical clustering for identifying anti-correlated gene clusters; Dhillon IS et al.; MOTIVATION: Clustering genes based upon their expression patterns allows us to predict gene function . Most existing clustering algorithms cluster genes together when their expression patterns show high positive correlation . However, it has been observed that genes whose expression patterns are strongly anti-correlated can also be functionally similar . Biologically, this is not unintuitive-genes responding to the same stimuli, regardless of the nature of the response, are more likely to operate in the same pathways . RESULTS: We present a new diametrical clustering algorithm that explicitly identifies anti-correlated clusters of genes . Our algorithm proceeds by iteratively (i) . re-partitioning the genes and (ii) . computing the dominant singular vector of each gene cluster; each singular vector serving as the prototype of a 'diametric' cluster . We empirically show the effectiveness of the algorithm in identifying diametrical or anti-correlated clusters . Testing the algorithm on yeast cell cycle data, fibroblast gene expression data, and DNA microarray data from yeast mutants reveals that opposed cellular pathways can be discovered with this method . We present systems whose mRNA expression patterns, and likely their functions, oppose the yeast ribosome and proteosome, along with evidence for the inverse transcriptional regulation of a number of cellular systems. Am J Physiol Cell Physiol, 2004 Jan, 286(1), C164 - 9 Epub 2003 Sep 10. Characterization of Cos-7 cells overexpressing the rat secretory pathway Ca2+-ATPase; Reinhardt TA et al.; On the basis of sequence similarities to the yeast PMR1 and hSPCA gene, the rat alternatively spliced mRNA has been suggested to be a Golgi secretory pathway Ca2+-ATPase (SPCA) . Data in this report lend further support for this hypothesis in that sucrose gradient fractionation of rat liver microsomes resulted in SPCA comigrating with the Golgi calcium binding protein CALNUC, which was well resolved from the endoplasmic reticulum marker calreticulin . Also, in PC-12 cells, antibody to SPCA colocalized with an antibody to the Golgi marker alpha-mannosidase II . To study the biological effects of SPCA expression, we performed stable overexpression of SPCA in COS-7 cells . Seven clones were selected for further comparison with COS-7 cells containing an empty expression vector . Overexpression of SPCA resulted in a significant reduction of plasma membrane Ca2+-ATPase, sarco(endo)plasmic reticulum Ca2+-ATPase, and calreticulin expression in these clones . In contrast, the expression of the Golgi calcium-binding protein CALNUC increased significantly . The phosphoenzyme intermediate formed using membranes from clone G11/5 was calcium dependent, significantly more intense than in COS-7 cells, and not affected by La3+ treatment . Calcium uptake by G11/5 microsomes was ATP dependent and significantly greater than in microsomes from parent COS-7 cells . The overexpression of SPCA significantly increased the growth rate of these cells compared with COS-7 cells containing only the empty vector . These data demonstrate that overexpression of the rat SPCA results in significant changes in the expression of calcium transport and storage proteins in COS-7 cells. Trends Pharmacol Sci, 2003 Sep, 24(9), 444 - 6 A chemical genomics approach to understanding drug action; Giaever G; The complete collection of yeast deletion strains represents a unique, living biological computer for understanding gene function . The molecular 'barcodes' present in each of the deletion strains allow a quantitative ranking of the importance of any gene under any experimental condition of choice . In this article, some of the recent results generated from experiments that exploit the yeast deletion collection to understand mechanisms of drug action are discussed. Mol Biochem Parasitol, 2003 Sep, 131(1), 45 - 54 Cloning of Schistosoma mansoni Seven in Absentia (SmSINA)(+) homologue cDNA, a gene involved in ubiquitination of SmRXR1 and SmRXR2; Fantappie MR et al.; Drosophila (SINA) and human Seven in Absentia (SIAH-1 and SIAH-2) have been implicated in ubiquitin-mediated proteolysis of different target proteins . Using the Schistosoma mansoni nuclear receptor SmRXR2 as bait in a yeast two-hybrid system, we identified a DNA fragment that encodes part of the schistosome homologue of the Seven in Absentia protein (SmSINA) . Screening of S . mansoni cDNA expression library resulted in the isolation of a cDNA containing the full-length coding region of SmSINA . SmSINA contains the characteristic structural features of other SINA proteins including a conserved N-terminal RING finger domain and a cysteine-rich C-terminus . We demonstrate that SmSINA associates with SmRXR2 and SmRXR1 both in vivo and in vitro, and define the binding domains in SmRXR2 and SmRXR1 that mediate their interaction . Schistosome SINA co-localizes with SmRXR2 and SmRXR1 in vitelline cells . In addition, we show that SmSINA stimulates the ubiquination of both SmRXR2 and SmRXR1 in vitro . Our findings suggest that SmSINA regulates ubiquitination and ubiquitin-induced degradation of schistosome nuclear receptors (RXR1 and RXR2) via the ubiquitin-proteasome pathway. Mol Biochem Parasitol, 2003 Sep, 131(1), 11 - 23 Characterization of a lysosomal serine carboxypeptidase from Trypanosoma cruzi; Parussini F et al.; Trypanosoma cruzi, the flagellate protozoan which is the causative agent of the American trypanosomiasis, Chagas disease has carboxypeptidase activity . The enzyme has been purified to protein homogeneity, and shown to be a lysosomal monomeric glycoprotein with a molecular mass of about 54kDa . The enzyme has an optimum acidic pH (4.5 with furyl acryloyl-Phe-Phe as substrate), is highly specific for hydrophobic C-terminal amino acid residues, and is strongly inhibited by 3,4-dichloroisocoumarin (IC(50) value 0.3 microM) . The enzyme is encoded by a number of genes arrayed in head-to-tail tandems; one of these genes has been cloned and sequenced . Sequence comparisons indicate that the enzyme belongs to the C group of serine carboxypeptidases, within the S10 serine peptidase family, and shows the higher similarity to plant and yeast enzymes . The residues involved in catalysis and most of those involved in substrate binding are conserved in the T . cruzi enzyme as well as 8 out of 10 Cys residues known to be involved in disulfide bridges in the yeast enzyme . This is the first report of an S10 family enzyme in trypanosomatids . The presence of serine carboxypeptidases is not restricted to T . cruzi, being possibly a general character of trypanosomatids. Dev Cell, 2003 Sep, 5(3), 499 - 511 Ent3p Is a PtdIns(3,5)P2 effector required for protein sorting to the multivesicular body; Friant S et al.; PtdIns(3,5)P(2) is required for cargo-selective sorting to the vacuolar lumen via the multivesicular body (MVB) . Here we show that Ent3p, a yeast epsin N-terminal homology (ENTH) domain-containing protein, is a specific PtdIns(3,5)P(2) effector localized to endosomes . The ENTH domain of Ent3p is essential for its PtdIns(3,5)P(2) binding activity and for its membrane interaction in vitro and in vivo . Ent3p is required for protein sorting into the MVB but not for the internalization step of endocytosis . Ent3p is associated with clathrin and is necessary for normal actin cytoskeleton organization . Our results show that Ent3p is required for protein sorting into intralumenal vesicles of the MVB through PtdIns(3,5)P(2) binding via its ENTH domain. Dev Cell, 2003 Sep, 5(3), 363 - 4 PtdIns(3,5)P2 finds a partner; Hicke L; Phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P(2)) is required for the sorting of a subset of membrane proteins at the late endosome . Unlike other phosphoinositides, binding partners for PtdIns(3,5)P(2) and its mechanism of action have not been characterized . New work by in this issue of Developmental Cell describes the identification of a yeast epsin-like protein that binds PtdIns(3,5)P(2) and functions in the transport of proteins through late endosomes to the lysosome-like vacuole. Org Lett, 2003 Sep 18, 5(19), 3435 - 7 Synthesis of a TMC-95A ketomethylene analogue by cyclization via intramolecular Suzuki coupling; Kaiser M et al.; {structure: see text} A TMC-95A analogue extended at the C-terminus with NlePsi{COCH(2)}Gly-Ala-Ala-NH(2) was synthesized via side-chain cyclization of the linear precursor by a Suzuki cross-coupling reaction in solution to analyze the effect of additional P' residues on the inhibitory potency against yeast proteasome. J Biol Chem, 2003 Nov 28, 278(48), 48105 - 11 Epub 2003 Sep 09. The direct activation of MIK, a germinal center kinase (GCK)-like kinase, by MARK, a maize atypical receptor kinase, suggests a new mechanism for signaling through kinase-dead receptors; Llompart B et al.; Signaling by receptor protein kinases (RPKs) involves their dimerization and transphosphorylation . However, atypical RPKs with kinase-defective domains have been described recently . Some of them are essential for proper signaling in animal systems, although the precise mechanism involved is unknown in most cases . Here we describe the cloning and characterization of an atypical plant receptor kinase from maize, MARK, which does not phosphorylate in vitro . A yeast two-hybrid approach has allowed us to identify a new germinal center kinase (GCK)-related protein, MIK, that interacts with MARK . Interestingly, the interaction of the intracellular domain of MARK with the regulator domain of MIK strongly induces MIK kinase activity . As some GCK-related proteins connect cell-surface receptors to the intracellular MAPK cascades, the activation of MIK by direct interaction with MARK could illustrate a new mechanism for signaling through atypical RPKs. J Biol Chem, 2003 Nov 14, 278(46), 45485 - 91 Epub 2003 Sep 09. Convergence of peroxisome proliferator-activated receptor gamma and Foxo1 signaling pathways; Dowell P et al.; The forkhead factor Foxo1 (or FKHR) was identified in a yeast two-hybrid screen as a peroxisome proliferator-activated receptor (PPAR) gamma-interacting protein . Foxo1 antagonized PPARgamma activity and vice versa indicating that these transcription factors functionally interact in a reciprocal antagonistic manner . One mechanism by which Foxo1 antagonizes PPARgamma activity is through disruption of DNA binding as Foxo1 inhibited the DNA binding activity of a PPARgamma/retinoid X receptor alpha heterodimeric complex . The Caenorhabditis elegans nuclear hormone receptor, DAF-12, interacted with the C . elegans forkhead factor, DAF-16, paralleling the interaction between PPARgamma and Foxo1 . daf-12 and daf-16 have been implicated in C . elegans insulin-like signaling pathways, and PPARgamma and Foxo1 likewise have been linked to mammalian insulin signaling pathways . These results suggest a convergence of PPARgamma and Foxo1 signaling that may play a role in insulin action and the insulinomimetic properties of PPARgamma ligands . A more general convergence of nuclear hormone receptor and forkhead factor pathways may be important for multiple biological processes and this convergence may be evolutionarily conserved. J Biochem (Tokyo), 2003 Aug, 134(2), 239 - 44 Crystal structure of chloroplastic ascorbate peroxidase from tobacco plants and structural insights into its instability; Wada K et al.; Ascorbate peroxidase (APX) is a heme-containing protein that plays a central role in scavenging H(2)O(2) in higher plants . The structure of stromal APX (sAPX) was determined at 1.6 A to an R-factor of 19.1% and an R-free-factor of 22.3% . The electrostatic potential of the gamma-channel that connects the molecular surface of sAPX to the gamma-edge of heme was more positive than that of cytosolic APX (cAPX) from pea, so sAPX might bind more easily with ascorbate than cAPX . The overall structure of sAPX was similar to those of cAPX from pea and cytochrome c peroxidase (CCP) from yeast, with a substantial difference in a loop structure located in the vicinity of the heme . The side chain of Arg169 in sAPX corresponding to His169 in cAPX and His181 in CCP extended in the opposite direction from the heme, forming two hydrogen bonds with carbonyl groups in the loop structure . The rapid inactivation of sAPX might be due to the characteristic conformation of Arg169 owing to the loop structure of sAPX. Proc R Soc Lond B Biol Sci, 2003 Jul 7, 270(1522), 1373 - 8 Killer-sensitive coexistence in metapopulations of micro-organisms; Czaran TL et al.; Many micro-organisms are known to produce efficient toxic substances against conspecifics and closely related species . The widespread coexistence of killer (toxin producer) and sensitive (non-producer) strains is a puzzle calling for a theoretical explanation . Based on stochastic cellular automaton simulations and the corresponding semi-analytical configuration-field approximation models, we suggest that metapopulation dynamics offers a plausible rationale for the maintenance of polymorphism in killer-sensitive systems . A slight trade-off between toxin production and population growth rate is sufficient to maintain the regional coexistence of toxic and sensitive strains, if toxic killing is a local phenomenon restricted to small habitat patches and local populations regularly go extinct and are renewed via recolonizations from neighbouring patches . Pattern formation on the regional scale does not play a decisive part in this mechanism, but the local manner of interactions is essential. J Plant Physiol, 2003 Aug, 160(8), 903 - 11 Isolation and characterisation of an invertase cDNA from perennial ryegrass (Lolium perenne); Johnson X et al.; An invertase (LpFT2) cDNA from perennial ryegrass was isolated and sequenced . Nucleotide sequence analysis revealed an ORF of 2016 bp encoding a protein of 671 amino acids . LpFT2 is 76% identical to sugarcane soluble acid invertase, and contains invertase and fructosyltransferase functional domains . LpFT2 is present as a single copy gene and maps to the distal region of LG6 in perennial ryegrass . The expression pattern analysis of LpFT2 revealed transcript accumulation in seedlings and in mature leaf sheaths . The LpFT2 recombinant protein expressed in yeast showed invertase and fructan exohydrolase-like activities with complete breakdown of sucrose, 1-kestose (DP3), 1,1-kestotetraose (DP4) and 1,1,1-kestopentaose (DP5) into glucose and fructose. Anal Chem, 2003 Jul 1, 75(13), 3263 - 6 Electron capture dissociation and 13C,15N depletion for deuterium localization in intact proteins after solution-phase exchange; Charlebois JP et al.; For localization of deuterium atoms after solution-phase exchange with D2O, intact proteins are often digested prior to analysis by mass spectrometry (MS) and tandem MS (MS/MS) . Amelioration of limitations associated with this approach (e.g., <70% sequence coverage and some D atom scrambling during MS/MS) were sought using intact proteins and two newer methods applied to tracking H/D exchange dynamics for the first time . Using 2-4-fold signal enhancements through depletion of 13C and 15N isotopes and implementing the new MS/MS technique of electron capture dissociation (ECD) yielded an increased number of c and z* ions observed (43 vs 25) for recombinant yeast ubiquitin (9.3 kDa) . Initial determination of D atom content in consecutive c ion series (c4-c7, c28, c31, c32, and c33) was demonstrated . The improved ion signal and experiment speed combined with narrower isotopic distributions markedly increases the degree of localization and feasibility of ECD-based MS/MS after solution-phase H/D exchange. Cell Cycle, 2003 Sep-Oct, 2(5), 424 - 5 Polo-like kinase 1 in the life and death of cancer cells; Liu X et al.; The polo-like kinase family plays a vital role in many cell cycle related events . The family includes mammalian Plkl, Snk (Plk2), and Fnk/Prk (Plk3), Xenopus laevis Plxl,Drosophila polo, fission yeast Plol, and budding yeast Cdc5 . These enzymes, in addition to a conserved kinase domain at the N-terminus, have highly conserved sequences called polo-box(s) in the non-catalytic C-terminal domain . Genetic and biochemical experiments with several different organisms have documented that polo-like kinases are involved in many aspects of the cell cycle, such as activation of Cdc2, centrosome assembly and maturation, activation of the anaphase-promoting complex (APC) during the metaphase-anaphase transition, and cytokinesis. Proc Natl Acad Sci U S A, 2003 Sep 16, 100(19), 10629 - 34 Epub 2003 Sep 08. Structure of the topoisomerase II ATPase region and its mechanism of inhibition by the chemotherapeutic agent ICRF-187; Classen S et al.; Type IIA topoisomerases both manage the topological state of chromosomal DNA and are the targets of a variety of clinical agents . Bisdioxopiperazines are anticancer agents that associate with ATP-bound eukaryotic topoisomerase II (topo II) and convert the enzyme into an inactive, salt-stable clamp around DNA . To better understand both topo II and bisdioxopiperazine function, we determined the structures of the adenosine 5'-{beta,gamma-imino}-triphosphate-bound yeast topo II ATPase region (ScT2-ATPase) alone and complexed with the bisdioxopiperazine ICRF-187 . The drug-free form of the protein is similar in overall fold to the equivalent region of bacterial gyrase but unexpectedly displays significant conformational differences . The ternary drug-bound complex reveals that ICRF-187 acts by an unusual mechanism of inhibition in which the drug does not compete for the ATP-binding pocket, but bridges and stabilizes a transient dimer interface between two ATPase protomers . Our data explain why bisdioxopiperazines target ATP-bound topo II, provide a structural rationale for the effects of certain drug-resistance mutations, and point to regions of bisdioxopiperazines that might be modified to improve or alter drug specificity. Mol Cell Proteomics, 2003 Nov, 2(11), 1225 - 33 Epub 2003 Sep 08. Identification of novel protein-protein interactions using a versatile Mammalian tandem affinity purification expression system; Knuesel M et al.; Identification of protein-protein interactions is essential for elucidating the biochemical mechanism of signal transduction . Purification and identification of individual proteins in mammalian cells have been difficult, however, due to the sheer complexity of protein mixtures obtained from cellular extracts . Recently, a tandem affinity purification (TAP) method has been developed as a tool that allows rapid purification of native protein complexes expressed at their natural level in engineered yeast cells . To adapt this method to mammalian cells, we have created a TAP tag retroviral expression vector to allow stable expression of the TAP-tagged protein at close to physiological levels . To demonstrate the utility of this vector, we have fused a TAP tag, consisting of a protein A tag, a cleavage site for the tobacco etch virus (TEV) protease, and the FLAG epitope, to the N terminus of human SMAD3 and SMAD4 . We have stably expressed these proteins in mammalian cells at desirable levels by retroviral gene transfer and purified native SMAD3 protein complexes from cell lysates . The combination of two different affinity tags greatly reduced the number of nonspecific proteins in the mixture . We have identified HSP70 as a specific interacting protein of SMAD3 . We demonstrated that SMAD3, but not SMAD1, binds HSP70 in vivo, validating the TAP purification approach . This method is applicable to virtually any protein and provides an efficient way to purify unknown proteins to homogeneity from the complex mixtures found in mammalian cell lysates in preparation for identification by mass spectrometry. J Biol Chem, 2003 Nov 21, 278(47), 46452 - 60 Epub 2003 Sep 08. Sphingosine kinase type 1 induces G12/13-mediated stress fiber formation, yet promotes growth and survival independent of G protein-coupled receptors; Olivera A et al.; Sphingosine 1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors (GPCRs) that regulate a wide variety of important cellular functions, including growth, survival, cytoskeletal rearrangements, and cell motility . However, whether it also has an intracellular function is still a matter of great debate . Overexpression of sphingosine kinase type 1, which generated S1P, induced extensive stress fibers and impaired formation of the Src-focal adhesion kinase signaling complex, with consequent aberrant focal adhesion turnover, leading to inhibition of cell locomotion . We have dissected biological responses dependent on intracellular S1P from those that are receptor-mediated by specifically blocking signaling of Galphaq, Galphai, Galpha12/13, and Gbetagamma subunits, the G proteins that S1P receptors (S1PRs) couple to and signal through . We found that intracellular S1P signaled "inside out" through its cell-surface receptors linked to G12/13-mediated stress fiber formation, important for cell motility . Remarkably, cell growth stimulation and suppression of apoptosis by endogenous S1P were independent of GPCRs and inside-out signaling . Using fibroblasts from embryonic mice devoid of functional S1PRs, we also demonstrated that, in contrast to exogenous S1P, intracellular S1P formed by overexpression of sphingosine kinase type 1 promoted growth and survival independent of its GPCRs . Hence, exogenous and intracellularly generated S1Ps affect cell growth and survival by divergent pathways . Our results demonstrate a receptor-independent intracellular function of S1P, reminiscent of its action in yeast cells that lack S1PRs. J Mol Biol, 2003 Sep 19, 332(3), 675 - 87 Alternative splicing as a mechanism for regulating 14-3-3 binding: interactions between hD53 (TPD52L1) and 14-3-3 proteins; Boutros R et al.; D52 (TPD52)-like proteins are coiled-coil motif-bearing proteins first identified through their expression in human breast carcinoma, which have been proposed to represent signalling intermediates and regulators of vesicle trafficking . D52-like gene transcripts are subject to alternative splicing, with sequences encoding a region termed insert 3 being affected in all three D52-like genes . We have now identified a 14-3-3 binding motif within one of two alternatively spliced exons encoding insert 3 . As predicted from the distribution of 14-3-3 binding motifs in four hD52-like bait proteins tested, only a hD53 isoform encoding a 14-3-3 binding motif bound both 14-3-3beta and 14-3-3zeta preys in the yeast two-hybrid system . Since D53 proteins carrying 14-3-3 binding motifs are predicted to be widely expressed, polyclonal antisera were derived to specifically detect these isoforms . Using soluble protein extracts from breast carcinoma cell lines, pull-down assays replicated interactions between recombinant 14-3-3beta and 14-3-3zeta isoforms and exogenously expressed hD53, and co-immunoprecipitation analyses demonstrated interactions between endogenous 14-3-3 and both endogenously and exogenously-expressed hD53 protein . Co-expressed hD53 and 14-3-3 proteins were similarly demonstrated to co-localise within the cytoplasm of breast carcinoma cell lines . These results identify 14-3-3 proteins as partners for hD53, and alternative splicing as a mechanism for regulating 14-3-3 binding. J Ethnopharmacol, 2003 Oct, 88(2-3), 261 - 7 Studies on the use of Cassia singueana in malaria ethnopharmacy; Adzu B et al.; Cassia singueana (Family: Fabaceae) is used in northern Nigeria for the treatment of acute malaria attack . We investigated the activities of the methanol extract of the root bark of this plant against rodent plasmodia infection, nociception, pyrexia and inflammation in mice and rats . The studies were carried out using acetic acid-induced writhing, hot plate algesia, rodent plasmodia (Plasmodium berghei) in mice; formalin test, yeast-induced pyrexia and egg-albumin-induced inflammation in rats . The results showed that the extract exhibited significant antinociceptive, antipyretic and antiplasmodial activity in all the models used . Phytochemical screening of the extract revealed the presence of phenols, saponins, tannins and some traces of anthraquinones . The LD50 of the extract was established to be 847+/-30 mg/kg, i.p . in mice . The observed pharmacological activities might be the scientific basis for the folkloric use of the plant in treating acute malaria attack . The study also paves way for the possible development of it, as a phytodrug against malaria. Biochem Biophys Res Commun, 2003 Sep 26, 309(3), 552 - 7 RanBPM is a novel binding protein for p75NTR; Bai D et al.; The neurotrophin receptor p75NTR can induce signal transduction both in vivo and in vitro . The mechanisms by which p75NTR transduces signals have remained mostly unknown . Using yeast two-hybrid system, we identified the Ran-binding protein (RanBPM) as an interactor with the intracytoplasmic domain of p75NTR (p75ICD) . The interaction was then validated by immunoprecipitation in mammalian cells and immunoblotting analysis . The domain in p75ICD interacting with RanBPM was mapped to the death domain. Biochem Biophys Res Commun, 2003 Sep 26, 309(3), 520 - 7 Identification of a Bcl-XL binding region within the ATPase domain of Apaf-1; Yajima H et al.; CED-4, a pro-apoptotic factor in Caenorhabditis elegans, activates the cell death protease CED-3 . CED-9 directly binds to CED-4 and represses this . However, it has remained unclear whether a mammalian CED-9 homologue, Bcl-XL, inhibits the function of the mammalian CED-4 homologue, Apaf-1, by direct binding . To analyze the interaction, we adopted a yeast two-hybrid system . Since Bcl-XL and the CED-4-like portion of Apaf-1 failed to exhibit a positive result in the assay, we prepared "fragment libraries" of bcl-XL or apaf-1 cDNA . By screening of the apaf-1 "fragment library," we obtained nine clones interacting with Bcl-XL, all containing the same region within the ATPase domain, designated BBR: the Bcl-XL binding region . Binding of BBR to Bcl-XL was also confirmed by immunoprecipitation assays . Bcl-2, Bcl-w, A1/Bfl-1, and Boo/Diva failed to show the same capacity for binding to BBR as Bcl-XL . These results indicate that Bcl-XL directly binds to a specific region in Apaf-1. Mol Cell Biochem, 2003 Aug, 250(1-2), 189 - 95 Interaction of insulin-like growth factor binding protein-3 with latent transforming growth factor-beta binding protein-1; Gui Y et al.; Insulin-like growth factor binding protein-3 (IGFBP-3) inhibits the replication and promotes apoptosis in various cell lines in an IGF-independent manner . We utilized a yeast two-hybrid system to identify binding partners for IGFBP-3 in a mouse embryo cDNA library . A partial cDNA encoding mouse latent transforming growth factor beta (TGF-beta) binding protein-1 (LTBP-1) was identified . This cDNA encoded a mouse LTBP-1 mRNA fragment corresponding to amino acid residues 1160-1712 . Analysis of C-terminal deleted mutants indicated that the IGFBP-3 interacting domain resides in the 552 residue C-terminal fragment encoding amino acids 831-1383 . The interaction of IGFBP-3 with recombinant human LTBP-1 immobilized on nitrocellulose was also demonstrated . Neither binding of IGF-1 to IGFBP-3 nor binding of latency associated protein (LAP) with LTBP-1 inhibited the interaction of IGFBP-3 with LTBP-1 . Furthermore the large latent complex, 125I-TGF-beta/LAP/LTBP-1 was able to bind to immobilized IGFBP-3 . These data demonstrate that IGFBP-3 can bind to LTBP-1 and provide a potential mechanism whereby IGFBP-3 can interact with the TGF-beta system. Mol Biol Cell, 2003 Nov, 14(11), 4448 - 57 Epub 2003 Sep 05. Recycling of Raft-associated prohormone sorting receptor carboxypeptidase E requires interaction with ARF6; Arnaoutova I et al.; Little is known about the molecular mechanism of recycling of intracellular receptors and lipid raft-associated proteins . Here, we have investigated the recycling pathway and internalization mechanism of a transmembrane, lipid raft-associated intracellular prohormone sorting receptor, carboxypeptidase E (CPE) . CPE is found in the trans-Golgi network (TGN) and secretory granules of (neuro)endocrine cells . An extracellular domain of the IL2 receptor alpha-subunit (Tac) fused to the transmembrane domain and cytoplasmic tail of CPE (Tac-CPE25) was used as a marker to track recycling of CPE . We show in (neuro)endocrine cells, that upon stimulated secretory granule exocytosis, raft-associated Tac-CPE25 was rapidly internalized from the plasma membrane in a clathrin-independent manner into early endosomes and then transported through the endocytic recycling compartment to the TGN . A yeast two-hybrid screen and in vitro binding assay identified the CPE cytoplasmic tail sequence S472ETLNF477 as an interactor with active small GTPase ADP-ribosylation factor (ARF) 6, but not ARF1 . Expression of a dominant negative, inactive ARF6 mutant blocked this recycling . Mutation of residues S472 or E473 to A in the cytoplasmic tail of CPE obliterated its binding to ARF6, and internalization from the plasma membrane of Tac-CPE25 mutated at S472 or E473 was significantly reduced . Thus, CPE recycles back to the TGN by a novel mechanism requiring ARF6 interaction and activity. Mol Biol Cell, 2003 Dec, 14(12), 5038 - 50 Epub 2003 Sep 05. The angiotensin II type I receptor-associated protein, ATRAP, is a transmembrane protein and a modulator of angiotensin II signaling; Lopez-Ilasaca M et al.; Our group identified angiotensin II type 1 (AT1) receptor-associated protein (ATRAP) in a yeast two-hybrid screen for proteins that bind to the carboxyl-terminal cytoplasmic domain of the AT1 . In this work, we characterize ATRAP as a transmembrane protein localized in intracellular trafficking vesicles and plasma membrane that functions as a modulator of angiotensin II-induced signal transduction . ATRAP contains three hydrophobic domains at the amino-terminal end of the protein, encompassing the amino acid residues 14-36, 55-77, and 88-108 and a hydrophilic cytoplasmic carboxyl-terminal tail from residues 109-161 . Endogenous and transfected ATRAP cDNA shows a particulate distribution; electron microscopy reveals the presence of ATRAP in prominent perinuclear vesicular membranes; and colocalization analysis by immunofluorescence shows that ATRAP colocalizes in an intracellular vesicular compartment corresponding to endoplasmic reticulum, Golgi, and endocytic vesicles . Real-time tracking of ATRAP vesicles shows constitutive translocation toward the plasma membrane . Using epitope-tagged forms of ATRAP at either the amino or carboxyl end of the molecule, we determined the orientation of the amino end as being outside the cell . Mutant forms of ATRAP lacking the carboxyl end are unable to bind to the AT1 receptor, leading to the formation of prominent perinuclear vesicle clusters . Functional analysis of the effects of ATRAP on angiotensin II-induced AT1 receptor signaling reveals a moderate decrease in the generation of inositol lipids, a marked decrease in the angiotensin II-stimulated transcriptional activity of the c-fos promoter luciferase reporter gene, and a decrease in cell proliferation. Endocrinology, 2003 Oct, 144(10), 4393 - 402 Epub 2003 Jul 03. Regulation of follitropin receptor cell surface residency by the ubiquitin-proteasome pathway; Cohen BD et al.; Little is known of the normal physiological processes that govern the cell surface residency of the human follitropin receptor (hFSHR), a G protein-coupled receptor expressed in the ovary and testis . In the hFSHR, the third intracellular (3i) loop is considered to be pivotal in attenuation of ligand activation, particularly internalization . To gain a better understanding of these processes, we used a yeast-based interaction trap to identify cytoplasmic proteins in a human ovarian cDNA library that interacted with the hFSHR 3i loop . Among the cDNA identified, four encoded isoforms of ubiquitin . Immunoprecipitated hFSHR probed with an antiubiquitin antibody revealed that the receptor is ubiquitinated, although not exclusively on the 3i loop . Cell-surface hFSHR levels increased when expressed at nonpermissive temperature in a temperature-sensitive, ubiquitination-defective cell line . Similarly, after treatment with proteasome inhibitors, HEK293 cells stably transfected with an hFSHR expression plasmid showed an increase in follitropin binding . Proteasome inhibitors did not affect the rate of FSH internalization when receptors were saturated before internalization was measured . In contrast, internalization decreased when binding experiments were performed under nonequilibrium conditions . A mutant hFSHR-K555R, which removes the only lysine in the 3i loop available for ubiquitination, was still ubiquitinated, illustrating that, although the third loop enables and interaction with ubiquitin, it is not the sole site of ubiquitination . These observations are consistent with a role for ubiquitination in the regulation of hFSHR cell surface residency . Additionally, it can be inferred that a sequence in the 3i loop is involved in regulating receptor ubiquitination and internalization. Biochem J, 2003 Dec 15, 376(Pt 3), 795 - 9 Oligomerization of the cardiac ryanodine receptor C-terminal tail; Stewart R et al.; The C-terminal 100 amino acids of the RyR (ryanodine receptor), referred to as the C-terminal tail, is a highly conserved sequence that is present in all known RyR isoforms and which has been implicated in channel function . Deleting the final 15 amino acids from the full-length skeletal muscle RyR resulted in an inactive channel, attributed to impaired assembly of a tetrameric RyR complex {Gao, Tripathy, Lu and Meissner (1997) FEBS Lett . 412, 223-226} . To account for these observations, the C-terminal tail itself may be an important molecular determinant of oligomerization . Alternatively, the large N-terminal cytoplasmic domain may fold back upon itself to interact with the C-terminal tail to provide a correctly folded tetrameric structure . We explored these possibilities for RyR2 (cardiac RyR) using the yeast two-hybrid interaction assay and in vitro translation followed by immunoprecipitation and chemical cross-linking . The data indicate that the C-terminal tail of RyR2 is capable of self-tetramerization . Moreover, a truncated C-terminal tail, lacking the final 15 amino acids, failed to self-associate . These observations suggest that the intrinsic ability of the RyR C-terminal tail to self-tetramerize may be vitally important for the oligomeric assembly of the native RyR channel. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Sep, 35(9), 816 - 22 {Researching a novel NPC-related candidate suppressor gene BRD7 by two-dimensional gel electrophoresis and MALDI-TOF-MS}; Peng C et al.; BRD7 was isolated through cDNA representational difference analysis (RDA) (GenBank accession No . AF152604) . Previous studies showed that BRD7 gene was down-regulated in nasopharyngeal carcinoma (NPC) cells and tissues, and three cSNPs (coding-region single nucleotide polymorphisms) were found on BRD7 . In addition, six BRD7-interacting proteins were identified by yeast two-hybrid system . To study the function of this gene on carcinogenesis in NPC, BRD7 gene was transfected into HNE1 cells low-expressed BRD7 by using liposomes and a stable cell line over-expressing BRD7 was established . After two-dimensional gel electrophoresis(2-DE), twenty differentially expressed proteins were identified by MALDI-TOF-MS including argininosuccinate lyase, metalloproteinase inhibitor-2 precursor, proteaseome activator28 beta subunit, thyroid transcriptional factor-1, cyclinH (MO15-associated protein), and so on . These differentially expressed proteins are related to cell cycling, transcription regulation, signaling pathway etc . Therefore, BRD7 may exert its functions by mediating differential expression of these proteins. J Biol Chem, 2003 Nov 14, 278(46), 46087 - 93 Epub 2003 Sep 04. The scaffolding protein RACK1 interacts with androgen receptor and promotes cross-talk through a protein kinase C signaling pathway; Rigas AC et al.; The androgen receptor (AR), a member of the nuclear hormone receptor superfamily, functions as a ligand-dependent transcription factor that regulates genes involved in cell proliferation and differentiation . Using a C-terminal region of the human AR in a yeast two-hybrid screen, we have identified RACK1 (receptor for activated C kinase-1) as an AR-interacting protein . In this report we found that RACK1, which was previously shown to be a protein kinase C (PKC)-anchoring protein that determines the localization of activated PKCbetaII isoform, facilitates ligand-independent AR nuclear translocation upon PKC activation by indolactam V . We also observed RACK1 to suppress ligand-dependent and -independent AR transactivation through PKC activation . In chromatin immunoprecipitation assays, we demonstrate a decrease in AR recruitment to the AR-responsive prostate-specific antigen (PSA) promoter following stimulation of PKC . Furthermore, prolonged exposure to indolactam V, a PKC activator, caused a reduction in PSA mRNA expression in prostate cancer LNCaP cells . Finally, we found PKC activation to have a repressive effect on AR and PSA protein expression in androgen-treated LNCaP cells . Our data suggest that RACK1 may function as a scaffold for the association and modification of AR by PKC enabling translocation of AR to the nucleus but rendering AR unable to activate transcription of its target genes. Front Biosci, 2003 Sep 01, 8, d1128 - 33 Polo-like kinases in cell cycle checkpoint control; Dai W et al.; Recent studies from various eukaryotic model systems indicate that polo-like kinases (Plks) play an ever-increasing role in the regulation of cell cycle progression . Early genetic studies have demonstrated that Cdc5, a budding yeast counterpart of vertebrate Plks, is essential for mitosis . Mammalian Plks primarily localize to the microtubule organization center during interphase and undergo dramatic subcellular relocation during mitotic progression . Many key cell cycle regulators such as p53, Cdc25C, cyclin B, and components of the anaphase promoting complex are directly targeted by Plks . Although the exact mechanisms of action of these protein kinases in vivo remain to be elucidated, Plks appear to orchestrate various cell cycle checkpoints (intra-S phase, G2/M transition, spindle assembly, and cytokinesis checkpoints) that protect cells against genetic instability during cell division. Proc Natl Acad Sci U S A, 2003 Sep 16, 100(19), 10694 - 9 Epub 2003 Sep 03. The first external loop of the metal ion transporter DCT1 is involved in metal ion binding and specificity; Cohen A et al.; The yeast null mutant smf1Delta cannot grow on medium containing EGTA . Expression of Smf1p or the mammalian transporter DCT1 (Slc11a2) suppresses the above-mentioned phenotype . Both can also be expressed in Xenopus oocytes, and the uptake activity and their electrophysiological properties can be studied . We used these systems to analyze the properties of mutations in the predicted external loop I of DCT1 . The sensitivity of the transporter to amino acid substitutions in this region is manifested by the mutation G119A, which resulted in almost complete inhibition of the metal ion uptake activity and marked changes in the pre-steady-state currents in Xenopus oocytes . The mutation Q126D abolished the uptake and the electrophysiology, but the double mutant D124A/Q126D partially restored it and changed the metal ion specificity in favor of Fe2+ . The maximal pre-steady-state currents at negatively imposed potentials shifted to a lower pH of approximately 5 . The triple mutant G119A/D