Yeasts

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/D124A/Q126D, which has no apparent transport activity, exhibited remarkable pre-steady-state currents at pH 7.5 . Moreover, Zn2+ had a dual effect on this mutant; at pH 7.5 it eliminated the pre-steady state without generating steady-state currents, and at pH 5.5 it induced large pre-steady-state currents . The mutant D124A retained appreciable Fe2+ uptake activity but exhibited very little Mn2+ uptake at pH 5.5 and was abolished at pH 6.5 . The properties of the various mutants suggest that loop I is involved in the metal ion binding and its coupling to the proton-driving force.

Biol Reprod, 2004 Jan, 70(1), 106 - 13 Epub 2003 Sep 03.
Tsp57: a novel gene induced during a specific stage of spermatogenesis; Kim YS et al.; Recently, we described the identification of a novel protein, nuclear receptor-associated protein 80 (RAP80), which is highly expressed in spermatocytes and appears to have a role in regulating gene expression . To identify proteins interacting with this protein, we performed yeast two-hybrid screening using full-length RAP80 as bait . This screen identified one in-frame clone encoding a novel testis-specific protein (Tsp), referred to as Tsp57 . Tsp57 encodes a basic protein with a mass of 56.8 kDa . The amino acid sequence of Tsp57 is highly conserved (87%) between mouse and human . The mouse and human Tsp57 genes map to chromosomes 9A1 and 11q21, respectively . Northern blot analysis showed that the expression of Tsp57 mRNA was highly restricted to the testis and temporally regulated during testicular development . Tsp57 mRNA was greatly induced between Day 21 and Day 25 of postnatal testicular development . In situ hybridization analysis demonstrated that the hybridization signal for Tsp57 mRNA was strongest in sections of seminiferous tubules at stages VI-VIII of spermatogenesis, consistent with the conclusion that Tsp57 is most highly expressed in round spermatids . Study of Tsp57 expression in several purified subpopulations of spermatogenic cells confirmed maximum levels of expression in round spermatids . Consistently, Tsp57 expression was absent in testes from vitamin A-deficient mice, which do not have any round spermatids, and was reduced in RARalpha null mice, which have lowered numbers of round spermatids in their testes . These results indicate the possibility that Tsp57 protein plays a role in the postmeiotic phase of germ cell differentiation . Tsp57 contains two putative nuclear localization signals: NLS1 and NLS2 . Examination of the cellular localization showed that the green fluorescent protein-Tsp57 fusion protein localized to both cytoplasm and nucleus . After deletion of NLS1 but not NLS2, Tsp57 localized solely to the cytoplasm, indicating a role for NLS1 in the nuclear localization of Tsp57 . The localization suggests a nuclear function for Tsp57 . Pull-down analysis demonstrated that Tsp57 and RAP80 form a complex in intact cells.

J Biol Chem, 2003 Nov 7, 278(45), 44320 - 5 Epub 2003 Sep 03.
Substrate specificity of the Arabidopsis thaliana sucrose transporter AtSUC2; Chandran D et al.; The Arabidopsis sucrose transporter AtSUC2 is expressed in the companion cells of the phloem (specialized vascular tissue) and is essential for the long distance transport of carbohydrates within the plant . A variety of glucosides are known to inhibit sucrose uptake into yeast expressing AtSUC2; however, it remains unknown whether glucosides other than sucrose could serve as transported substrates . By expression of AtSUC2 in Xenopus oocytes and two-electrode voltage clamping, we have tested the ability of AtSUC2 to transport a range of physiological and synthetic glucosides . Sucrose induced inward currents with a K0.5 of 1.44 mM at pH 5 and a membrane potential of -137 mV . Of the 24 additional sugars tested, 8 glucosides induced large inward currents allowing kinetic analysis . These glucosides were maltose, arbutin (hydroquinone-beta-D-glucoside), salicin (2-(hydroxymethyl)phenyl-beta-D-glucoside), alpha-phenylglucoside, beta-phenylglucoside, alpha-paranitrophenylglucoside, beta-paranitrophenylglucoside, and paranitrophenyl-beta-thioglucoside . In addition, turanose and alpha-methylglucoside induced small but significant inward currents indicating that they were transported by At-SUC2 . The results indicate that AtSUC2 is not highly selective for alpha-over beta-glucosides and may function in transporting glucosides besides sucrose into the phloem, and the results provide insight into the structural requirements for transport by AtSUC2.

Plant Cell, 2003 Sep, 15(9), 2165 - 80
A grape ASR protein involved in sugar and abscisic acid signaling; Cakir B et al.; The function of ASR (ABA {abscisic acid}-, stress-, and ripening-induced) proteins remains unknown . A grape ASR, VvMSA, was isolated by means of a yeast one-hybrid approach using as a target the proximal promoter of a grape putative monosaccharide transporter (VvHT1) . This promoter contains two sugar boxes, and its activity is induced by sucrose and glucose . VvMSA and VvHT1 share similar patterns of expression during the ripening of grape . Both genes are inducible by sucrose in grape berry cell culture, and sugar induction of VvMSA is enhanced strongly by ABA . These data suggest that VvMSA is involved in a common transduction pathway of sugar and ABA signaling . Gel-shift assays demonstrate a specific binding of VvMSA to the 160-bp fragment of the VvHT1 promoter and more precisely to two sugar-responsive elements present in this target . The positive regulation of VvHT1 promoter activity by VvMSA also is shown in planta by coexpression experiments . The nuclear localization of the yellow fluorescent protein-VvMSA fusion protein and the functionality of the VvMSA nuclear localization signal are demonstrated . Thus, a biological function is ascribed to an ASR protein . VvMSA acts as part of a transcription-regulating complex involved in sugar and ABA signaling.

J Cell Sci, 2003 Oct 1, 116(Pt 19), 4021 - 34
Epigenetic assembly of centromeric chromatin at ectopic alpha-satellite sites on human chromosomes; Nakano M et al.; To investigate the mechanism of chromatin assembly at human centromeres, we isolated cultured human cell lines in which a transfected alpha-satellite (alphoid) YAC was integrated ectopically into the terminal region of host chromosome 16, where it was stably maintained . Centromere activity of the alphoid YAC was suppressed at ectopic locations on the host chromosome, as indicated by the absent or reduced assembly of CENP-A and -C . However, long-term culture in selective medium, or short-term treatment with the histone deacetylase inhibitor Trichostatin A (TSA), promoted the re-assembly of CENPA, -B and -C at the YAC site and the release of minichromosomes containing the YAC integration site . Chromatin immunoprecipitation analyses of the re-formed minichromosome and the alphoid YAC-based stable human artificial chromosome both indicated that CENP-A and CENP-B assembled only on the inserted alphoid array but not on the YAC arms . On the YAC arms at the alphoid YAC integration sites, TSA treatment increased both the acetylation level of histone H3 and the transcriptional level of a marker gene . An increase in the level of transcription was also observed after long-term culture in selective medium . These activities, which are associated with changes in chromatin structure, might reverse the suppressed chromatin state of the YAC at ectopic loci, and thus might be involved in the epigenetic change of silent centromeres on ectopic alphoid loci.

J Biol Chem, 2003 Nov 14, 278(46), 46029 - 34 Epub 2003 Sep 02.
PACSIN3 binds ADAM12/meltrin alpha and up-regulates ectodomain shedding of heparin-binding epidermal growth factor-like growth factor; Mori S et al.; A disintegrin and metalloprotease 12 (ADAM12/meltrin alpha) is a key enzyme implicated in the ectodomain shedding of membrane-anchored heparin-binding epidermal growth factor (EGF)-like growth factor (proHB-EGF)-dependent epidermal growth factor receptor (EGFR) transactivation . However, the activation mechanisms of ADAM12 are obscure . To determine how ADAM12 is activated, we screened proteins that bind to the cytoplasmic domain of ADAM12 using a yeast two-hybrid system and identified a protein called PACSIN3 that contains a Src homology 3 domain . An analysis of interactions between ADAM12 and PACSIN3 using glutathione S-transferase fusion protein revealed that a proline-rich region (amino acid residues 829-840) of ADAM12 was required to bind PACSIN3 . Furthermore, co-immunoprecipitation and co-localization analyses of ADAM12 and PACSIN3 proteins also revealed their interaction in mammalian cells expressing both of them . The overexpression of PACSIN3 in HT1080 cells enhanced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced proHB-EGF shedding . Furthermore, knockdown of endogenous PACSIN3 by small interfering RNA in HT1080 cells significantly attenuated the shedding of proHB-EGF induced by TPA and angiotensin II . Our data indicate that PACSIN3 has a novel function as an up-regulator in the signaling of proHB-EGF shedding induced by TPA and angiotensin II.

J Biol Chem, 2003 Nov 14, 278(46), 45603 - 10 Epub 2003 Sep 02.
Homo- and hetero-oligomerization of ammonium transporter-1 NH4 uniporters; Ludewig U et al.; In most organisms, high affinity ammonium uptake is catalyzed by members of the ammonium transporter family (AMT/MEP/Rh) . A single point mutation (G458D) in the cytosolic C terminus of the plasma membrane transporter LeAMT1;1 from tomato leads to loss of function, although mutant and wild type proteins show similar localization when expressed in yeast or plant protoplasts . Co-expression of LeAMT1;1 and mutant in Xenopus oocytes inhibited ammonium transport in a dominant negative manner, suggesting homo-oligomerization . In vivo interaction between LeAMT1;1 proteins was confirmed by the split ubiquitin yeast two-hybrid system . LeAMT1;1 is isolated from root membranes as a high molecular mass oligomer, converted to a approximately 35-kDa polypeptide by denaturation . To investigate interactions with the LeAMT1;2 paralog, co-localizing with LeAMT1;1 in root hairs, LeAMT1;2 was characterized as a lower affinity NH4+ uniporter . Co-expression of wild types with the respective G458D/G465D mutants inhibited ammonium transport in a dominant negative manner, supporting the formation of heteromeric complexes in oocytes . Thus, in yeast, oocytes, and plants, ammonium transporters are able to oligomerize, which may be relevant for regulation of ammonium uptake.

J Cell Biol, 2003 Sep 1, 162(5), 765 - 72
Dynamic phosphoregulation of the cortical actin cytoskeleton and endocytic machinery revealed by real-time chemical genetic analysis; Sekiya-Kawasaki M et al.; We used chemical genetics to control the activity of budding yeast Prk1p, which is a protein kinase that is related to mammalian GAK and AAK1, and which targets several actin regulatory proteins implicated in endocytosis . In vivo Prk1p inhibition blocked pheromone receptor endocytosis, and caused cortical actin patches to rapidly aggregate into large clumps that contained Abp1p, Sla2p, Pan1p, Sla1p, and Ent1p . Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated Arp2/3-stimulated actin assembly . Electron microscopy/immunoelectron microscopy analysis and tracking of the endocytic membrane marker FM4-64 revealed vesicles of likely endocytic origin within the actin clumps . Upon inhibitor washout, the actin clumps rapidly disassembled, and properly polarized actin patches reappeared . Our results suggest that actin clumps result from blockage at a normally transient step during which actin assembly is stimulated by endocytic proteins . Thus, we revealed tight phosphoregulation of an intrinsically dynamic, actin patch-related process, and propose that Prk1p negatively regulates the actin assembly-stimulating activity of endocytic proteins.

Genes Dev, 2003 Sep 15, 17(18), 2271 - 82 Epub 2003 Sep 02.
Stable inheritance of telomere chromatin structure and function in the absence of telomeric repeats; Sadaie M et al.; It is generally believed that telomeric repeats are a necessary and sufficient cis-element for telomere function . Here we show that telomere structure and meiotic function are stably inherited in fission yeast circular chromosomes that have lost all telomeric repeats . We found that the telomeric repeat binding protein, Taz1, and the heterochromatin protein, Swi6, remain associated with subtelomeres in the absence of telomeric repeats . We also found that the fusion point of circular chromosomes that lack telomeric repeats associates with SPB (the yeast counterpart of the centrosome) in the premeiotic horsetail stage, similarly to wild-type telomeres . However, a taz1+ deletion/reintroduction experiment revealed that the maintenance of Taz1 binding and premeiotic function is achieved via different strategies . Taz1 is recruited to subtelomeres by an autonomous element present in subtelomeric DNA, thus in a genetic mechanism . In contrast, the premeiotic subtelomere-SPB association is maintained in an epigenetic manner . These results shed light on the previously unrecognized role played by the subtelomere and underscore the robust nature of the functional telomere complex that is maintained by both genetic and epigenetic mechanisms . Furthermore, we suggest that the establishment and the maintenance of the functional telomere complex are mechanistically distinguishable.

Genes Dev, 2003 Sep 1, 17(17), 2138 - 50
A novel and essential mechanism determining specificity and activity of protein phosphatase 2A (PP2A) in vivo; Fellner T et al.; Protein phosphatase 2A (PP2A) is an essential intracellular serine/threonine phosphatase containing a catalytic subunit that possesses the potential to dephosphorylate promiscuously tyrosine-phosphorylated substrates in vitro . How PP2A acquires its intracellular specificity and activity for serine/threonine-phosphorylated substrates is unknown . Here we report a novel and phylogenetically conserved mechanism to generate active phospho-serine/threonine-specific PP2A in vivo . Phosphotyrosyl phosphatase activator (PTPA), a protein of so far unknown intracellular function, is required for the biogenesis of active and specific PP2A . Deletion of the yeast PTPA homologs generated a PP2A catalytic subunit with a conformation different from the wild-type enzyme, as indicated by its altered substrate specificity, reduced protein stability, and metal dependence . Complementation and RNA-interference experiments showed that PTPA fulfills an essential function conserved from yeast to man.

Dermatol Online J . 2003 Aug;9(3):13.
Topical pimecrolimus in the treatment of seborrheic dermatitis; Brownell I et al.; Seborrheic dermatitis is a chronic inflammatory disease that mainly affects seborrheic areas of skin . An inflammatory response to the yeast Pityrosporum ovale has been thought to be important in the etiology of the condition . Therefore, topical antifungals and corticosteroids have been the mainstay of treatment . The recent development of topical macrolactam immunomodulators has offered a useful, safe alternative to corticosteroids in the treatment of various inflammatory skin disorders . We report successful treatment of seborrheic dermatitis with pimecrolimus.

Genome Biol . 2003;4(9):117 . Epub 2003 Aug 29.
Comparing protein abundance and mRNA expression levels on a genomic scale; Greenbaum D et al.; Attempts to correlate protein abundance with mRNA expression levels have had variable success . We review the results of these comparisons, focusing on yeast . In the process, we survey experimental techniques for determining protein abundance, principally two-dimensional gel electrophoresis and mass-spectrometry . We also merge many of the available yeast protein-abundance datasets, using the resulting larger 'meta-dataset' to find correlations between protein and mRNA expression, both globally and within smaller categories.

Connect Tissue Res, 2003, 44 Suppl 1, 149 - 53
Temporal and spatial parameters of skeletal gene expression: targeting RUNX factors and their coregulatory proteins to subnuclear domains; Stein GS et al.; Key components of the basal transcription machinery and several tissue-specific transcription factor complexes are functionally compartmentalized as specialized subnuclear domains . We have identified a unique 31-38 amino acid targeting signal (NMTS) that directs the Runx (Cbfa/AML) transcription factors to distinct nuclear matrix-(NM) associated sites within the nucleus that support gene expression . Our determination of the NMTS crystal structure, yeast 2 hybrid screens to identify NM interacting proteins, and in situ colocalization studies with Runx interacting factors (YAP, Smad, TLE) suggest that localization of Runx transcription factors at intranuclear sites facilitates the assembly and activity of regulatory complexes that mediate activation and suppression of target genes . Mice homozygous for the deletion of the intranuclear Runx2 targeting signal in a homologous recombination (Runx2 deltaC) do not form bone due to maturational arrest of osteoblasts, demonstrating the importance of fidelity of subnuclear localization for tissue-differentiating activity . These results provide evidence that Runx2 subnuclear targeting and the associated regulatory functions are essential for a spatiotemporal placement that facilitates activation of Runx-dependent genes involved in tissue differentiation during embryonic development.

Connect Tissue Res, 2003, 44 Suppl 1, 52 - 7
Amelogenin self-assembly and the role of the proline located within the carboxyl-teleopeptide; Paine ML et al.; A hallmark of biological systems is a reliance on protein assemblies to perform complex functions . We have focused attention on mammalian enamel formation because it relies on a self-assembling protein complex to direct mineral habit . The principle protein of enamel is amelogenin that self-assembles to form nanospheres . In mice, the principal amelogenin product is a 180 amino acid hydrophobic protein . The yeast two-hybrid assay has been used to demonstrate the importance of amelogenin self-assembly domains . We have generated specific variants of amelogenin to analyze contributions of individual amino acids to the self-assembly process . These amelogenin variants have been produced either by deleting carboxyl-terminal amino acids (to generate proteins that relate to the documented proteolytic products of mouse amelogenin) or by a site-directed mutagenesis approach . Assessment of variant amelogenins truncated at the carboxyl-terminal imply that the proline at position 169 of mouse amelogenin (M180) plays a significant role in amelogenin self-assembly . Site-directed mutagenesis of this particular proline, however, failed to disrupt the amelogenin self-assembly property . These conflicting data add to the complexity of protein-protein assembly mechanisms as they relate to the enamel matrix . Available data suggest a robustness of this enamel protein (amelogenin) that ensures a functional, even though mechanically less than optimal, enamel results despite either minor or major genetic errors to the amelogenin gene locus.

J Muscle Res Cell Motil, 2002, 23(7-8), 829 - 38
Mitochondrial membrane dynamics are altered in cluA- mutants of Dictyostelium; Fields SD et al.; In cluA- mutants of Dictyostelium, mitochondria are clustered near the cell center rather than being dispersed throughout the cytoplasm . We have examined two possible mechanisms that could account for this phenotype . First, we sought evidence that the cytoskeleton or a presumptive mitochondrion-cytoskeleton linkage was altered in mutant cells . We found that cytoskeletal structures in cluA- cells appeared normal by immunostaining, and that the distribution of peroxisomes in mutant cells was indistinguishable from that in wild type cells . Treatment of wild type cells with drugs that disrupted microtubules or actin filaments did not mimic the cluA- phenotype . Thus, cytoskeletal defects seemed unlikely to account for the mitochondrial clustering in cluA- cells . Observation of the movement of GFP-tagged mitochondria in wild type cells suggested that mitochondria are transported along microtubules, as in mammalian cells, rather than along actin filaments, as in budding yeast . Therefore, the similar phenotypes of cluA- Dictyostelium cells and clu1delta yeast cells argued against CluA/Clu1p acting as a mitochondrion-cytoskeleton linker . We next examined the ultrastructure of mitochondria in freeze-substituted, thin-sectioned cells . We found that the clustered mitochondria in cluA- cells are interconnected . Often, adjacent mitochondria are linked by narrow membranous strands, although sometimes the mitochondria are partially merged . The presence of narrow constrictions at presumptive division sites argues that the constriction step of division proceeds normally . Our data suggest that cluA- cells may be blocked at a very late step in fission of the outer mitochondrial membrane.

Biosci Biotechnol Biochem, 2003 Aug, 67(8), 1667 - 74
MpFAE3, a beta-ketoacyl-CoA synthase gene in the liverwort Marchantia polymorpha L., is preferentially involved in elongation of palmitic acid to stearic acid; Kajikawa M et al.; Fatty acid chain elongation is a crucial step in the biosynthesis of long chain fatty acids . An essential reaction in the elongation process is condensation of malonyl-CoA with acyl-CoA, which is catalyzed by beta-ketoacyl-CoA synthase (KCS) in plants . We have isolated and characterized the MpFAE3 gene, one of the KCS gene family in the liverwort Marchantia polymorpha . Transgenic M . polymorpha plants overexpressing MpFAE3 accumulate fatty acids 18:0, 20:0, and 22:0 . In these plants, the amount of 16:0 is reduced to 50% of wild type . In a heterologous assay, transgenic methylotrophic yeast expressing the MpFAE3 gene accumulates fatty acid 18:0 and generates several longer fatty acids which are not detectable in the control, accompanied by a decrease of 16:0 . These observations indicate that the MpFAE3 protein is preferentially involved in the elongation of 16:0 to 18:0 and also in the subsequent steps of 18:0 to 20:0 and 20:0 to 22:0 in M . polymorpha.

Biochem Biophys Res Commun, 2003 Sep 19, 309(2), 414 - 8
Imidocarb, a potent anti-protozoan drug, up-regulates interleukin-10 production by murine macrophages; Katayama T et al.; Interleukin-10 (IL-10), a potent antiinflammatory and immunosuppressive cytokine, plays an important role in the regulation of immune responses . To discover small molecules that stimulate IL-10 production, a cell-based screening assay was performed using a murine macrophage cell line, RAW264.7 . Imidocarb, (3,3'-bis-2-imidazolin-2-yl)-carbanilide, which has been used as an anti-protozoan drug for the prevention and treatment of babesiosis in cattle, was thus identified . Imidocarb markedly enhanced LPS-induced IL-10 production not only by RAW264.7 cells but also by murine peritoneal macrophages in a concentration-dependent manner . It also augmented IL-10 production in the presence of zymosan, a yeast cell wall component . In contrast, imidocarb inhibited LPS-induced tumor necrosis factor (TNF)-alpha production by peritoneal macrophages . In mice, intraperitoneal administration of imidocarb significantly increased serum IL-10 levels and lowered TNF-alpha levels . Our results suggest that a novel anti-inflammatory property of imidocarb could lead to new therapeutic approaches in inflammatory conditions.

Virology, 2003 Aug 15, 313(1), 224 - 34
Adenovirus ADP protein (E3-11.6K), which is required for efficient cell lysis and virus release, interacts with human MAD2B; Ying B et al.; The human subgroup C adenovirus (Ad) protein named adenovirus death protein (ADP) (previously named E3-11.6K) is synthesized at very late stages of infection when it mediates efficient lysis of cells and release of adenovirus to infect other cells . ADP is an integral membrane N-linked, O-linked palmitoylated glycoprotein of 101 amino acids (aa) that localizes to the nuclear membrane, endoplasmic reticulum (ER), and Golgi . It has a single membrane spanning region (roughly aa 40-60) and is oriented with aa 1-40 in the lumen and aa 61-101 in the nucleoplasm and cytoplasm . Using aa 61-101 of Ad2 ADP as bait in a yeast two-hybrid screen, we isolated a cDNA for a 211-aa protein that initially was not in the database but has now been published by others with the names human MAD2B, MAD2L2, and REV7 . ADP binds strongly to human MAD2B not only in yeast but also in GST pull-down experiments and in coimmunoprecipitations of ADP and MAD2B synthesized in vitro or in vivo . ADP mutants with deletions throughout the bait region do not interact with human MAD2B, whereas a Pro69Pro70 to Ala69Ala70 mutant in the "basic-proline" domain of ADP does interact . Northern blot analyses indicate that human MAD2B is expressed ubiquitously . Human MAD2B is about 25% identical to human MAD2, a spindle assembly checkpoint protein . Two human A549 cell lines were made that constitutively overexpress MAD2B . Wild-type adenovirus lyses these cells significantly more slowly than it lyses parental A549 cells, raising the possibility that ADP and MAD2B act in opposition and suggesting that the ADP-MAD2B interaction is biologically relevant.

J Microsc, 2003 Sep, 211(Pt 3), 230 - 48
Automatic fluorescent tag localization II: Improvement in super-resolution by relative tracking; Thomann D et al.; We present an algorithm for the three-dimensional (3D) tracking of multiple fluorescent subresolution tags with super-resolution in images of living cells . Recently, we described an algorithm for the automatic detection of such tags in single frames and demonstrated its potential in a biological system . The algorithm presented here adds to the tag detector a module for relative tracking of the signals between frames . As with tag detection, the main problem in relative tracking arises when signals of multiple tags interfere . We propose a novel multitemplate matching framework that exploits knowledge of the microscope point spread function to separate the intensity contribution of each tag in image regions with signal interferences . We use this intensity splitting to reconstruct a template for each tag in the source frame and a patch in the target frame, which are both free of intensity contributions from other tag signals . Tag movements between frames are then tracked by seeking, for each template-patch pair, the displacement vector providing the best signal match in terms of the sum of squared intensity differences . Because template and patch generation of tags with overlapping signals are interdependent, the matching is carried out simultaneously for all tags, and in an iterative manner . We have examined the performance of our approach using synthetic 3D data and observed a significant increase in resolution and robustness as compared with our previously described detector . It is now possible to localize and track tags separated by a distance three times smaller than the Rayleigh limit with a relative positional accuracy of better than 50 nm . We have applied the new tracking system to extract metaphase trajectories of fluorescently tagged chromosomes relative to the spindle poles in budding yeast.

Biochem J, 2003 Dec 1, 376(Pt 2), 465 - 72
Dual action of oestrogens on the mouse constitutive androstane receptor; Makinen J et al.; mCAR (mouse constitutive androstane receptor; NR1I3) controls the expression of cytochrome P450 as well as other enzymes involved in drug and steroid metabolism . The high basal activity of mCAR can be modulated by inhibitory steroids related to androstenol and by activating xenobiotic chemicals such as 1,4-bis-{2-(3,5-dichloropyridyloxy)}benzene and chlorpromazine . The ability of oestrogens and some other xenobiotics to activate mCAR is not clear . In the present study, co-transfection assays in HEK-293 cells indicated that oestrogens varied in their efficacy to activate mCAR, depending on variation at the steroid D-ring and position of hydroxy groups . In general, oestrogens were weaker activators of mCAR than 1,4-bis-{2-(3,5-dichloropyridyloxy)}benzene and chlorpromazine . Also, the induction of CYP2B10 mRNA by oestrogens was less pronounced in mouse primary hepatocytes . Yeast two-hybrid assays indicated that, unlike androstenol and the established activators, oestrogens attracted both nuclear receptor co-repressors and co-activators to the mCAR ligand-binding domain, thus limiting the extent of mCAR activation . This novel dual action is not limited to oestrogens, but is shared by some xenobiotic CYP2B inducers such as clotrimazole and methoxychlor . These findings offer an alternative explanation for the recently suggested nuclear activation step of mCAR.

EMBO Rep, 2003 Sep, 4(9), 889 - 93 Epub 2003 Aug 29.
An exosome-like complex in Sulfolobus solfataricus; Evguenieva-Hackenburg E et al.; We present the first experimental evidence for the existence of an exosome-like protein complex in Archaea . In Eukarya, the exosome is essential for many pathways of RNA processing and degradation . Co-immunoprecipitation with antibodies directed against the previously predicted Sulfolobus solfataricus orthologue of the exosome subunit ribosomal-RNA-processing protein 41 (Rrp41) led to the purification of a 250-kDa protein complex from S . solfataricus . Approximately half of the complex cosediments with ribosomal subunits . It comprises four previously predicted orthologues of the core exosome subunits from yeast (Rrp41, Rrp42, Rrp4 and Csl4 (cepl synthetic lethality 4; an RNA-binding protein and exosome sub-unit)), whereas other predicted subunits were not found . Surprisingly, the archaeal homologue of the bacterial DNA primase DnaG was tightly associated with the complex . This suggests an RNA-related function for the archaeal DnaG-like proteins.Comparison of experimental data from different organisms shows that the minimal core of the exosome consists of at least one phosphate-dependent ribonuclease PH homologue, and of Rrp4 and Csl4 . Such a protein complex was probably present in the last common ancestor of Archaea and Eukarya.

J Biol Chem, 2003 Nov 7, 278(45), 44843 - 51 Epub 2003 Aug 28.
Mouse proton pump ATPase C subunit isoforms (C2-a and C2-b) specifically expressed in kidney and lung; Sun-Wada GH et al.; The vacuolar-type H+-ATPases (V-ATPases) are multimeric proton pumps involved in a wide variety of physiological processes . We have identified two alternative splicing variants of C2 subunit isoforms: C2-a, a lung-specific isoform containing a 46-amino acid insertion, and C2-b, a kidney-specific isoform without the insert . Immunohistochemistry with isoform-specific antibodies revealed that V-ATPase with C2-a is localized specifically in lamellar bodies of type II alveolar cells, whereas the C2-b isoform is found in the plasma membranes of renal alpha and beta intercalated cells . Immunoprecipitation combined with immunohistological analysis revealed that C2-b together with other kidney-specific isoforms was selectively assembled to form a unique proton pump in intercalated cells . Furthermore, a chimeric yeast V-ATPase with mouse the C2-a or C2-b isoform showed a lower Km(ATP) and lower proton transport activity than that with C1 or Vma5p (yeast C subunit) . These results suggest that V-ATPases with the C2-a and C2-b isoform are involved in luminal acidification of lamellar bodies and regulation of the renal acid-base balance, respectively.

Can J Neurol Sci, 2003 Aug, 30(3), 244 - 51
HnRNP A1 and A/B interaction with PABPN1 in oculopharyngeal muscular dystrophy; Fan X et al.; BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive ptosis, dysphagia and proximal limb weakness . The autosomal dominant form of this disease is caused by short expansions of a (GCG)6 repeat to (GCG) in the PABPN1 gene . The mutations lead to the expansion of a polyalanine stretch from 10 to 12-17 alanines in the N-terminus of PABPN1 . The mutated PABPN1 (mPABPN1) induces the formation of intranuclear filamentous inclusions that sequester poly(A) RNA and are associated with cell death . METHODS: Human fetal brain cDNA library was used to look for PABPNI binding proteins using yeast two-hybrid screen . The protein interaction was confirmed by GST pull-down and co-immunoprecipitation assays . Oculopharyngeal muscular dystrophy cellular model and OPMD patient muscle tissue were used to check whether the PABPN1 binding proteins were involved in the formation of OPMD intranuclear inclusions . RESULTS: We identify two PABPNI interacting proteins, hnRNP A1 and hnRNP A/B . When co-expressed with mPABPN1 in COS-7 cells, predominantly nuclear protein hnRNP A1 and A/B co-localize with mPABPN1 in the insoluble intranuclear aggregates . Patient studies showed that hnRNP A1 is sequestered in OPMD nuclear inclusions . CONCLUSIONS: The hnRNP proteins are involved in mRNA processing and mRNA nucleocytoplasmic export, sequestering of hnRNPs in OPMD intranuclear aggregates supports the view that OPMD intranuclear inclusions are "poly(A) RNA traps", which would interfere with RNA export, and cause muscle cell death.

J Clin Pathol, 2003 Sep, 56(9), 687 - 9
Routine use of a one minute trehalase and maltase test for the identification of Candida glabrata in four laboratories; Piens MA et al.; AIMS: To evaluate the rapid identification of Candida glabrata using a one minute trehalase and maltase test in four clinical laboratories . METHOD: The test was evaluated with 944 freshly isolated yeasts comprising 572 C glabrata and 372 non-C glabrata strains . These strains were isolated on one of three differential media-Candida ID, CHROMagar Candida, or Albicans ID2 medium-and all strains were fully identified using standard methods . RESULTS: The trehalase and maltase test allowed the overall identification of 550 of 572 C glabrata strains (sensitivity, 96.2%) and only 11 of 372 isolates of other yeast species yielded a false positive result (specificity, 96.8 %) . Sensitivity and specificity were consistent from one laboratory to another . Using Candida ID medium, the rapid trehalase and maltase test showed a sensitivity of 95% and specificity of 96.2% . Using CHROMagar Candida, sensitivity and specificity were 95.6% and 98.1%, respectively . Using Albicans ID2 medium (tested by two laboratories), the sensitivity was 100% and 98.5% and specificity was 98.1% and 98.2% . In 60% of cases, the test could be performed directly from the primary isolation medium, thus reducing the time for identification . CONCLUSION: The rapid trehalase and maltase test was highly reliable for the presumptive identification of C glabrata on primary isolation using three different chromogenic media . Direct recognition of C albicans by means of their characteristic colour on chromogenic media coupled with one minute trehalase maltase testing performed only on suspect colonies of C glabrata allowed for rapid presumptive identification of the two yeast species most commonly encountered in clinical samples.

Mol Cell Biol, 2003 Sep, 23(18), 6373 - 84
The human I-mfa domain-containing protein, HIC, interacts with cyclin T1 and modulates P-TEFb-dependent transcription; Young TM et al.; Positive transcription elongation factor b (P-TEFb) hyperphosphorylates the carboxy-terminal domain of RNA polymerase II, permitting productive transcriptional elongation . The cyclin T1 subunit of P-TEFb engages cellular transcription factors as well as the human immunodeficiency virus type 1 (HIV-1) transactivator Tat . To identify potential P-TEFb regulators, we conducted a yeast two-hybrid screen with cyclin T1 as bait . Among the proteins isolated was the human I-mfa domain-containing protein (HIC) . HIC has been reported to modulate expression from both cellular and viral promoters via its C-terminal cysteine-rich domain, which is similar to the inhibitor of MyoD family a (I-mfa) protein . We show that HIC binds cyclin T1 in yeast and mammalian cells and that it interacts with intact P-TEFb in mammalian cell extracts . The interaction involves the I-mfa domain of HIC and the regulatory histidine-rich region of cyclin T1 . HIC also binds Tat via its I-mfa domain, although the sequence requirements are different . HIC colocalizes with cyclin T1 in nuclear speckle regions and with Tat in the nucleolus . Expression of the HIC cDNA modulates Tat transactivation of the HIV-1 long terminal repeat (LTR) in a cell type-specific fashion . It is mildly inhibitory in CEM cells but stimulates gene expression in HeLa, COS, and NIH 3T3 cells . The isolated I-mfa domain acts as a dominant negative inhibitor . Activation of the HIV-1 LTR by HIC in NIH 3T3 cells occurs at the RNA level and is mediated by direct interactions with P-TEFb.

Hum Mol Genet, 2003 Nov 1, 12(21), 2845 - 52 Epub 2003 Aug 27.
Elucidation of ataxin-3 and ataxin-7 function by integrative bioinformatics; Scheel H et al.; The spinocerebellar ataxias (SCAs) are a class of hereditary neurodegenerative diseases, which are caused by the pathological expansion of unstable CAG triplet repeats found in a number of apparently unrelated genes . The proteins encoded by the SCA genes typically translate this expanded (CAG)n repeat into an expanded poly(Q) stretch . Several pathological features are common to all SCAs, irrespective of the gene harbouring the expansion . The specific contributions of the mutated genes are currently hard to assess, as the physiological role of most of the so-called ataxins is not known . By combining the results of profile-based sequence analysis with genome-wide functional data available for model organisms, we have derived detailed predictions of the physiological function of two SCA gene products . Ataxin-3, the protein mutated in Machado Joseph Disease (SCA3), belongs to a novel group of cysteine-proteases and is predicted to be active against ubiquitin chains or related substrates . The catalytic site of this enzyme class is similar to that found in UBP and UCH type ubiquitin proteases . For ataxin-7, the gene product of the SCA7 gene, we have identified an orthology relationship to the yeast open reading frame Ygl066c . Recently published evidence from genome-wide studies suggests that Ygl066c is a component of the SAGA histone acetyltransferase complex . By analogy, a similar role for the mammalian ataxin-7 can be expected . The functional predictions reported here are sufficiently precise to allow a direct experimental verification . Moreover, both findings have implications for the general pathogenesis of spinocerebellar ataxias by providing a direct connection of these diseases with ubiquitin metabolism and histone acetylation.

J Biochem (Tokyo), 2003 Jul, 134(1), 143 - 50
Germ cell-specific expression of microphthalmia-associated transcription factor mRNA in mouse testis; Saito H et al.; The gene coding for microphthalmia-associated transcription factor (Mitf) contains many promoters that could generate multiple Mitf isoforms with distinct amino-termini, such as ubiquitously expressed Mitf-A and Mitf-H . To gain further insight into Mitf isoform multiplicity and the regulation of the promoter usage of the Mitf gene, we have analyzed the function of the amino-terminal domains of Mitf isoforms and the expression of Mitf mRNA in mouse postnatal testis, which is characterized by spermatogenesis and by a cool temperature because of its unique location . Here we show that the amino-terminal domain of Mitf-A possesses a transactivation activity, as judged by yeast expression analysis . We also show the expression of Mitf-A and Mitf-D mRNAs in testis by PCR-based methods . Moreover, in situ hybridization analysis revealed that an Mitf mRNA, probably representing Mitf-A and/or Mitf-D, is expressed in germ cells, including spermatogonia, spermatocytes that undergo meiosis, and round spermatids with the haploid genome, but is undetectable in elongated spermatids with remodeled and condensed chromatin . Notably, Mitf mRNA is undetectable in somatic Leydig cells and peritubular cells . Therefore, multiple promoters may direct differential expression of the Mitf gene in the testis and contribute to functional diversity of Mitf isoforms.

J Biochem (Tokyo), 2003 Jul, 134(1), 71 - 82
A temperature-sensitive mutant of the mammalian RNA helicase, DEAD-BOX X isoform, DBX, defective in the transition from G1 to S phase; Fukumura J et al.; ts ET24 cells are a novel temperature-sensitive (ts) mutant for cell proliferation of hamster BHK21 cells . The human genomic DNA which rescued the temperature-sensitive lethality of ts ET24 cells was isolated and screened for an open reading frame in the deposited human genomic library . X chromosomal DBX gene encoding the RNA helicase, DEAD-BOX X isoform, which is homologous to yeast Ded1p, was found to be defective in this mutant . The single point mutation (P267S) was localized between the Motifs I and Ia of the hamster DBX of ts ET24 cells . At the nonpermissive temperature of 39.5 degrees C, ts ET24 cells were arrested in the G1-phase and survived for more than 3 days . In ts ET24 cells, total protein synthesis was not reduced at 39.5 degrees C for 24 h, while mRNA accumulated in the nucleus after incubation at 39.5 degrees C for 17 h . The amount of cyclin A mRNA decreased in ts ET24 cells within 4 h after the temperature shift to 39.5 degrees C, consistent with the fact that the entry into the S-phase was delayed by the temperature shift.

J Biochem (Tokyo), 2003 Jul, 134(1), 1 - 8
Ubiquitylation as a quality control system for intracellular proteins; Hatakeyama S et al.; Quality control of intracellular proteins is essential for cellular homeostasis . Molecular chaperones recognize and contribute to the refolding of misfolded or unfolded proteins, whereas the ubiquitin-proteasome system mediates the degradation of such abnormal proteins . Ubiquitin-protein ligases (E3s) determine the substrate specificity for ubiquitylation and have been classified into HECT and RING-finger families . More recently, however, U-box proteins, which contain a domain (the U box) of about 70 amino acids that is conserved from yeast to humans, have been identified as a new type of E3 . The prototype U-box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor (E4) that cooperates with a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and an E3 to catalyze the formation of a ubiquitin chain on artificial substrates . Yeast Ufd2 is functionally implicated in cell survival under stressful conditions . This review addresses recent progress in characterization of the role of E3 enzymes, especially that of U-box proteins, in quality control of intracellular proteins.

Mol Cell Endocrinol, 2003 Aug 29, 206(1-2), 93 - 111
The gonadotropin releasing hormone (GnRH) receptor activating sequence (GRAS) is a composite regulatory element that interacts with multiple classes of transcription factors including Smads, AP-1 and a forkhead DNA binding protein; Ellsworth BS et al.; Activin responsiveness of the murine GnRH receptor gene promoter is mediated at a regulatory element we termed the GnRH receptor activating sequence (GRAS) . Here, we have sought to define the complex of transcription factors that interact at this element . Consistent with activin regulation at GRAS, gel shift analyses and yeast one-hybrid assays reveal Smad4 interaction at the 5' end of GRAS . While overexpression of Smad3 activates a GRAS reporter, Smad3 binding at GRAS was not detectable . A functional interaction of Smad3 at GRAS was, however, detectable in yeast expressing Smad4 . Thus, Smad3 interaction at GRAS appears to be dependent on the presence of Smad4 . Mutations located at the 3' end of GRAS do not affect Smad binding but eliminate functional activity . Thus, Smad binding alone cannot account for the functional attributes of GRAS . Consistent with this notion, we find that AP-1 binding is immediately juxtaposed to and, in fact, partially overlaps the Smad binding site . Finally, a recently identified member of the forkhead family of transcription factors, FoxL2, is also capable of interacting at GRAS . Furthermore, FoxL2 activation at GRAS is lost with mutation of either the 5' Smad binding site or a putative forkhead binding site located at the 3' end of the element . We suggest that GRAS is a composite regulatory element whose functional activity is dependent on the organization of a multi-protein complex consisting of Smads, AP-1 and a member of the forkhead family of DNA binding proteins.

J Steroid Biochem Mol Biol, 2003 Jun, 85(2-5), 449 - 56
Regulation of differential COUP-TF-coregulator interactions in adrenal cortical steroidogenesis; Shibata H et al.; Hyperfunctioning adrenocortical adenomas produce excessive amounts of various corticosteroids due to dysregulated expression of steroidogenic enzymes . Since no genetic mutations in steroidogenic enzyme genes have been identified as yet, the dysregulated expression at the transcription level may be crucial . Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) and steroidogenic factor-1 (SF-1) play key roles in the transcriptional regulation of steroidogenic P450 genes . Transfection studies showed that SF-1 activated and COUP-TFs repressed the transcription of bovine CYP17 gene promoter from the CRS2 element in a mutually exclusive manner in Y-1 cells . The results indicate that COUP-TFs negatively regulate the transcriptional activity of SF-1, a steroidogenic cell-specific activator of various steroidogenic P450 genes . Expression of both COUP-TFI and COUP-TFII was significantly decreased in the cortisol-producing adenomas, in which CYP17 was drastically overexpressed, indicating that decreased expression of COUP-TFs play a key role in overexpression of CYP17 in this type of tumors . We then screened for COUP-TFI-interacting proteins from a cortisol-producing adenoma cDNA library using a yeast two-hybrid system and identified a novel RING finger-containing protein which can function as a coregulator for COUP-TFI . Notably, COUP-TFI activated rather than repressed several target genes including the human CYP11B2 gene promoter, the results of which were opposite to those of the CYP17 promoter . The bifunctional activities of COUP-TFI may be derived from the promoter context and our newly identified COUP-TFI coregulator.

J Steroid Biochem Mol Biol, 2003 Jun, 85(2-5), 133 - 8
Nuclear receptor modifications and endocrine cell proliferation; Fu M et al.; Heritable and reversible changes in gene expression can occur without alterations in DNA sequence largely dependent upon the position of a gene within an accessible (euchromatic) chromatin environment . This position effect variegation in Drosophila and S . pombe, and higher order chromatin structure regulation in yeast, is orchestrated by modifier genes of the Su(var) group (e.g . histone deacetylases (HDACs), protein phosphatases) and enhancer E(var) group (e.g . ATP-dependent nucleosome remodeling proteins) . Higher order chromatin structure is regulated in part by covalent modification of the N-terminal histone tails of chromatin and histone tails in turn serve as platforms for recruitment of signaling modules that include non-histone proteins such as HP1 and NuRD . As the enzymes governing chromatin structure through covalent modifications of histones (acetylation, methylation, phosphorylation, ubiquitination) can also target non-histone substrates, a mechanism is in place by which epigenetic regulatory processes can affect the function of these alternate substrates . The nuclear receptor (NR) superfamily consists of conserved modular transcriptional regulators . Herein, we review the functional properties of nuclear receptors regulated by their direct acetylation including ligand-dependent activation, cellular growth and apoptosis.

J Steroid Biochem Mol Biol, 2003 Jun, 85(2-5), 101 - 4
Estrogen-responsive RING finger protein controls breast cancer growth; Horie K et al.; Most of the breast cancers initially respond to endocrine therapy that reduces the levels of estrogens or competes with estrogen for binding to its receptor . Most of the patients, however, acquire resistance to endocrine therapy with tamoxifen and aromatase inhibitors later . We assumed that identification of estrogen-responsive genes those regulate the growth of breast cancer is indispensable to develop new strategies targeting the genes and overcome the resistance to current endocrine therapy . Estrogen-responsive finger protein (Efp) is one of the estrogen receptor (ER)-target genes we have cloned using genomic binding site cloning . Efp features a structure of the RING-finger B-box coiled-coil (RBCC) motif . We postulated that Efp is a critical factor in proliferation of breast tumors . In a model system using MCF7 cells grown in xenografts, we showed that inhibition of Efp expression by antisense oligonucleotide reduced the tumor growth . MCF7 cells overexpressing Efp formed tumors in xenografts even in estrogen deprivation environment . By yeast two-hybrid screen, we identified that Efp interacts with 14-3-3sigma, which is known as a cell cycle brake that causes G2 arrest and expressed in normal mammary glands . In vitro studies have revealed that Efp functions as a ubiquitin-protein ligase (E3) that targets 14-3-3sigma . These data suggest that Efp controls breast cancer growth through ubiquitin-dependent proteolysis of 14-3-3sigma . Future studies may provide a new therapy to block breast tumor proliferation by targeting Efp.

Biochem Biophys Res Commun, 2003 Sep 12, 309(1), 189 - 95
Identification of EloA-BP1, a novel Elongin A binding protein with an exonuclease homology domain; Tamura K et al.; The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II by suppressing the transient pausing of the polymerase at many sites along the DNA template . Elongin is composed of a transcriptionally active A subunit, and two positive regulatory B and C subunits . Although the NH(2)-terminal approximately 120 amino acid region of Elongin A is dispensable for its transcriptional activity in vitro, it shares significant sequence similarity with the NH(2)-terminus of other class of transcription factors SII and CRSP70, suggesting that the NH(2)-terminus mediates interactions important for the regulation of transcription in vivo . To identify proteins that can bind to these conserved sequences, a human B cell cDNA library was screened using the NH(2)-terminus of Elongin A as bait in a yeast two-hybrid system . Here, we report on the cloning and characterization of a novel human exonuclease domain-containing protein, Elongin A-binding protein 1 (EloA-BP1) . EloA-BP1 is composed of 1221 amino acids and its mRNA is ubiquitously expressed . Double immunofluorescence labeling in COS7 cells suggested that EloA-BP1 and Elongin A are colocalized to the cell nucleus . By using an in vitro binding assay, we show that EloA-BP1 is capable of binding not only the NH(2)-terminal approximately 120 amino acid region of Elongin A, but also that of SII . Although the purified EloA-BP1 had no detectable effect on the rate of transcription elongation in vitro, it may play some role in the regulation of elongation in vivo.

Biochem Biophys Res Commun, 2003 Sep 12, 309(1), 44 - 51
Different isoforms of PRIP-interacting protein with methyltransferase domain/trimethylguanosine synthase localizes to the cytoplasm and nucleus; Enunlu I et al.; A protein family including the recently identified PIMT/Tgs1 (PRIP-interacting protein with methyltransferase domain/trimethylguanosine synthase) was identified by searching databases for homologues of a newly identified Drosophila protein with RNA-binding activity and methyltransferase domain . Antibodies raised against a short peptide of the mammalian homologue show a 90-kDa isoform expressed specifically in rat brain and testis and a 55-kDa form expressed ubiquitously . In HeLa cells, the larger isoform of the protein is nuclear and associated with a 600-kDa complex, while the smaller isoform is mainly cytoplasmic and co-localizes to the tubulin network . Inhibition of PIMT/Tgs1 expression by siRNA in HeLa cells resulted in an increase in the percentage of cells in G2/M phases . In yeast two-hybrid and in vitro GST pull down experiments, the conserved C-terminal region of PIMT/Tgs1 interacted with the WD domain containing EED/WAIT-1 that acts as a polycomb-type repressor in the nucleus and also binds to integrins in the cytoplasm . Our experiments, together with earlier data, indicate that isoforms of the PIMT/Tgs1 protein with an RNA methyltransferase domain function both in the nucleus and in the cytoplasm and associate with both elements of the cytoskeletal network and nuclear factors known to be involved in gene regulation.

Plant J, 2003 Jan, 33(1), 177 - 88
pistillata-5, an Arabidopsis B class mutant with strong defects in petal but not in stamen development; Yang Y et al.; The Arabidopsis floral organ identity genes APETALA3 (AP3) and PISTILLATA (PI) encode related DNA-binding proteins of the MADS family . Considerable evidence supports the hypothesis that a heterodimer of AP3 and PI is an essential component of B class activity . All ap3 and pi alleles characterized to date exhibit equivalent phenotypic defects in both whorls 2 and 3 . In strong ap3 and pi mutants, petals and stamens are missing and sepals and carpels develop in their place . Weak ap3 and pi mutants exhibit partial conversions of petals to sepals and stamens to carpels . In this report, we describe the isolation and characterization of pi-5, an unusual B class mutant that exhibits defects in whorl 2 where sepals develop in place of petals, but third whorl stamens are most often normal . pi-5 flowers resemble those from 35S::SEP3 antisense plants . pi-5 contains missense mutation in the K domain (PIE125K) . PIE125K exhibits defects in heterodimerization with its partner protein AP3 . Via a reverse yeast two-hybrid screen, AP3K139E was isolated as a compensatory mutant of PIE125K . The compensatory interaction between PIE125K and AP3K139E is observed both in yeast two-hybrid assays and in planta . On its own, AP3K139E exhibits defects in specifying both petal and stamen identity . In addition, PIE125K is defective in interaction with SEPALLATA proteins in both two- and three-hybrid assays suggesting that PIE125K is defective in forming higher order complexes of MADS proteins . The decreased concentration of PI/AP3/SEP complexes offers an explanation for the petal defects observed in both pi-5 and 35S::SEP3 antisense plants.

Plant J, 2003 Jan, 33(1), 139 - 47
Antisense repression of StubGAL83 affects root and tuber development in potato; Lovas A et al.; StubGAL83 is a potato gene that encodes the beta-subunit of a protein kinase complex similar to the yeast SNF1, and the mammalian AMPK complexes that are modulated by changes in the cellular AMP/ATP ratio and are important regulators of metabolic and stress responses . Here we show that the expression of StubGAL83 in potato foliage is much higher in the dark than in the light and can be repressed by metabolisable sugars in the dark . The amounts of StubGAL83 mRNA are higher in sink than in source leaves . To unravel the role of StubGAL83, transgenic potato plants expressing a part of the StubGAL83 cDNA in antisense orientation under the control of the constitutive CaMV35S promoter were generated . Northern analysis revealed a reduction up to 90-95% in StubGAL83 mRNA accumulation in leaves of seven lines . Five out of these seven lines exhibited a reduction of StubGAL83 mRNA levels also in root and tuber tissues . Independent on the type of repression, the transgenic lines showed a delay in rooting and an increased sensitivity to salt stress . The roots were stunted and possessed less pronounced tap roots than the controls albeit with different severity in the different transgenic lines . The root cells were smaller and some of them had irregular shape . Tuberisation of the antisense-StubGAL83 lines was delayed, the size of the tubers was reduced while the number of tubers per plant was increased . These results together suggest that StubGAL83 affects root and tuber development probably by altering the metabolic status of the leaves.

Plant J, 2003 Jan, 33(1), 47 - 59
The K domain mediates heterodimerization of the Arabidopsis floral organ identity proteins, APETALA3 and PISTILLATA; Yang Y et al.; MADS genes in plants encode key developmental regulators of vegetative and reproductive development . The majority of well-characterized plant MADS proteins contain two conserved domains, the DNA-binding MADS domain and the K domain . The K domain is predicted to form three amphipathic alpha-helices referred to as K1, K2, and K3 . In this report, we define amino acids and subdomains important for heterodimerization between the two Arabidopsis floral organ identity MADS proteins APETALA3 (AP3) and PISTILLATA (PI) . Analysis of mutants defective in dimerization demonstrates that K1, K2 and the region between K1 and K2 are critical for the strength of AP3/PI dimerization . The majority of the critical amino acids are hydrophobic indicating that the K domain mediates AP3/PI interaction primarily through hydrophobic interactions . Specially, K1 of AP3 and PI resembles a leucine zipper motif . Most mutants defective in AP3/PI heterodimerization in yeast exhibit partial floral organ identity function in transgenic Arabidopsis . Our results also indicate that the motif containing Asn-98 and specific charged residues in K1 (Glu-97 in PI and Arg-102 in AP3) are important for both the strength and specificity of AP3/PI heterodimer formation.

Med Sci (Paris), 2003 Jun-Jul, 19(6-7), 717 - 23
{Molecular mechanisms of meiosis in plants}; Horlow C et al.; Meiosis is a key step in diploid sexual reproduction . Apart from its cytological description, the molecular mechanisms involved in this specialized cell division are being deciphered in plants thanks to the model plant Arabidopsis thaliana . While some meiotic mutants of Arabidopsis confirm the central role of functions that have been described either in yeast or in mice, others led to the identification of previously unknown genes . Numerous plants also exist as polyploids, which represent a special case with regard to meiosis.

J Biol Chem, 2003 Nov 7, 278(45), 44439 - 47 Epub 2003 Aug 26.
A novel type of chloroplast stromal hexokinase is the major glucose-phosphorylating enzyme in the moss Physcomitrella patens; Olsson T et al.; Hexokinase catalyzes the first step in the metabolism of glucose but has also been proposed to be involved in sugar sensing and signaling both in yeast and in plants . We have cloned a hexokinase gene, PpHXK1, in the moss Physcomitrella patens where gene function can be studied directly by gene targeting . PpHxk1 is a novel type of chloroplast stromal hexokinase that differs from previously studied membrane-bound plant hexokinases . Enzyme assays on a knock-out mutant revealed that PpHxk1 is the major glucose-phosphorylating enzyme in Physcomitrella, accounting for 80% of the total activity in protonemal tissue . The mutant is deficient in the response to glucose, which in wild type moss induces the formation of caulonemal filaments that protrude from the edge of the colony . Growth on glucose in the dark is strongly reduced in the mutant . Sequence data suggest that most plants including Physcomitrella and Arabidopsis have both chloroplast-imported hexokinases similar to PpHxk1 and traditional membrane-bound hexokinases . We propose that the two types of plant hexokinases have distinct physiological roles.

J Virol, 2003 Sep, 77(18), 9758 - 68
Identification of a novel cellular transcriptional repressor interacting with the latent nuclear antigen of Kaposi's sarcoma-associated herpesvirus; Pan HY et al.; The latent nuclear antigen (LNA) of Kaposi's sarcoma-associated herpesvirus (KSHV) has an essential role in viral latent infection . LNA maintains the stability of KSHV episomes and modulates the expression of cellular genes . A novel cellular protein KLIP1 was identified to interact with LNA through yeast two-hybrid screening, and confirmed by a glutathione S-transferase pull down assay . Domain mapping showed that KLIP1 interacted with the N-terminal domain of LNA . Northern blot hybridization with a KLIP1 probe identified a major transcript of 1.8 kb and a minor transcript of 2.8 kb . cDNA library screening and 5'-RACE revealed that the major transcript encoded an open-reading-frame of 1,257 bp and had a 5'-untranslated region of 73 nucleotides . The major KLIP1 transcript was ubiquitously present in different cell types examined . A KLIP1 synthetic peptide antibody detected a doublet of 58-kDa and 63-kDa proteins in a Western blot assay . KLIP1 had two putative nuclear localization signals and showed punctate nuclear localization when expressed as a GFP-fusion protein . KLIP1 interacted with LNA in vivo, as demonstrated by coimmunoprecipitation using KSHV-infected cells and colocalization when they were expressed as GFP- and DsRed-fusion proteins, respectively . Consistent with its interaction with LNA, nuclear localization, and possession of two leucine zipper motifs, KLIP1 behaved like a transcriptional factor and repressed herpes simplex virus thymidine kinase (TK) promoter activity in a mammalian one-hybrid assay . In addition, cotransfection with LNA alleviated the transcriptional repression effect of KLIP1 on TK promoter activity . These results suggest that KLIP1 is a new member of cellular transcriptional repressors, and that LNA is involved in deregulating cellular transcription process.

Plant Cell Physiol, 2003 Aug, 44(8), 856 - 60
Point mutation is responsible for Arabidopsis tz-201 mutant phenotype affecting thiamin biosynthesis; Papini-Terzi FS et al.; A point mutation in the thi1 gene, involved in the synthesis of thiamin, has been identified in a tz-201 mutant line of Arabidopsis thaliana . The mutation occurs in a conserved protein domain and prevents the mutant plants from synthesizing thiamin . Complementation assays in yeast thi4 mutant confirm that this mutation hinders thiamin synthesis and, thus, is responsible for the tz phenotype . Northern blot analyses indicate that, in contrast to the yeast homologue, thi1 expression is not influenced by the presence of thiamin; however, reduced transcription of the gene is observed in roots and dark grown plants.

Plant Cell Physiol, 2003 Aug, 44(8), 783 - 94
Ectopic expression of an orchid (Oncidium Gower Ramsey) AGL6-like gene promotes flowering by activating flowering time genes in Arabidopsis thaliana; Hsu HF et al.; An AP1/AGL9 group of MADS box gene, OMADS1, with extensive homology to the Arabidopsis AGAMOUS-like 6 gene (AGL6) was characterized from orchid (Oncidium Gower Ramsey) . OMADS1 mRNA was detected in apical meristem and in the lip and carpel of flower . Yeast two-hybrid analysis indicated that OMADS1 is able to strongly interact with OMADS3, a TM6-like protein that was involved in flower formation and floral initiation in orchid . Transgenic Arabidopsis and tobacco ectopically expressed OMADS1 showed similar novel phenotypes by significantly reducing plant size, flowering extremely early, and losing inflorescence indeterminacy . In addition, homeotic conversion of sepals into carpel-like structures and petals into staminoid structures were also observed in flowers of 35S::OMADS1 Arabidopsis . This result indicated that OMADS1 was involved in floral formation and initiation in transgenic plants . Further analysis indicated that the expression of flowering time genes FT, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) and flower meristem identity genes LEAFY (LFY), APETALA1 (AP1) was significantly up-regulated in 35S::OMADS1 transgenic Arabidopsis plants . Furthermore, ectopic expression of OMADS1 rescued late-flowering phenotype in gi-1, co-3 but not for ft-1 and fwa-1 mutants . These results supported that ectopic expression of OMADS1 influenced flower transition and formation by acting as an activator for FT and SOC1 in Arabidopsis.

Cancer Res, 2003 Aug 15, 63(16), 5151 - 8
Identification of a novel inhibitor of breast cell growth that is down-regulated by estrogens and decreased in breast tumors; Wittmann BM et al.; Lifetime exposure to estrogens is a major risk factor in breast cancer, but the mechanism for this action is not fully defined . To better determine this mechanism, the activation domain of estrogen receptor (ER) alpha was used in yeast two-hybrid screenings . These screenings resulted in the identification of a novel antiproliferative protein, estrogen down-regulated gene 1 (EDG1), of which the mRNA and protein were shown to be down-regulated directly by estrogens . Our studies additionally suggested an important role for EDG1 in ER alpha-mediated breast cancer development . Analysis of 43 invasive breast cancer samples and 40 adjacent normal breast samples demonstrated EDG1 protein levels to be significantly higher in normal breast epithelial tissue as compared with breast epithelial tumor tissue . EDG1 expression levels were also correlated with the proliferation activity and ER alpha status of the tumors to examine the prognostic value of EDG1 in invasive breast tumors . EDG1 expression was more disassociated from proliferative activity as compared with ER alpha expression in tumor cells . A growth regulatory function for EDG1 is additionally indicated by studies wherein overexpression of EDG1 protein in breast cells resulted in decreased cell proliferation and decreased anchorage-independent growth . Conversely, inhibiting EDG1 expression in breast cells resulted in increased breast cell growth . Thus, we have identified a novel growth inhibitor that is down-regulated by estrogens and colocalizes with ER alpha in breast tissue . These studies support a role for EDG1 in breast cancer.

Cancer Res, 2003 Aug 15, 63(16), 4792 - 5
BP75, bromodomain-containing M(r) 75,000 protein, binds dishevelled-1 and enhances Wnt signaling by inactivating glycogen synthase kinase-3 beta; Kim S et al.; To identify novel regulators of Wnt signaling, we performed yeast two-hybrid analyses with Dvl-1 and identified BP75 as a candidate . Here, we demonstrated that BP75 directly interacts with Dvl-1 in mammalian cells and enhances TCF-dependent gene expression induced by Dvl-1 . In support of these results, BP75 in cooperation with Dvl-1 was found to facilitate dephosphorylation at Tyr216 of glycogen synthase kinase-3beta and consequently inhibit its kinase activity . Furthermore, the nuclear translocation and formation of vesicular structures of beta-catenin were induced by BP75 and Dvl-1 in a synergistic manner . Collectively, these results provided us a novel mechanism in Wnt signaling where BP75 plays important regulatory roles between glycogen synthase kinase-3beta and Dvl.

EMBO J, 2003 Sep 1, 22(17), 4455 - 64
JNK-interacting protein 3 associates with Toll-like receptor 4 and is involved in LPS-mediated JNK activation; Matsuguchi T et al.; Lipopolysaccharide (LPS) is recognized by Toll-like receptor (TLR) 4 and activates NF-kappaB and a set of MAP kinases . Here we have investigated proteins associated with the cytoplasmic domain of mouse TLR4 by yeast two-hybrid screening and identified JNK-interacting protein 3 (JIP3), a scaffold protein for JNK, as a TLR4-associated protein . In mammalian cells, JIP3, through its N-terminal region, constitutively associates with TLR4 . The association is specific to JIP3, as the two other JIPs, JIP1 and JIP2, failed to bind TLR4 . In HEK 293 cells exogenously expressing TLR4, MD2 and CD14, co-expression of JIP3 significantly increased the complex formation of TLR4-JNK and LPS-mediated JNK activation . In contrast, expression of C-terminally truncated forms of JIP3 impaired LPS-induced JNK activation in a mouse macrophage cell line, RAW264.7 . Moreover, RNA interference of JIP3 inhibited LPS-mediated JNK activation . In RAW264.7 cells, JIP3 associates MEKK-1, but not with TAK-1 . Finally, JIP3 also associates with TLR2 and TLR9, but not with TLR1 or TLR6 . Altogether, our data indicate the involvement of JIP3 in JNK activation in downstream signals of some TLRs.

Comp Biochem Physiol B Biochem Mol Biol, 2003 Sep, 136(1), 99 - 106
Spontaneous haemolytic activity of Atlantic halibut (Hippoglossus hippoglossus L.) and sea bass (Dicentrarchus labrax) serum; Lange S et al.; The spontaneous haemolytic (SH) activity of sera was compared in groups of cultured halibut and sea bass . The optimum assay temperature was determined for each species and different red blood cell donors were tested . The effects of heat inactivation, storage temperature and of different agents like EDTA, EGTA, yeast cell components and bacterial LPS were compared . Halibut sera gave optimum lysis with sheep red blood cells (RBC) at 16 degrees C whereas sea bass sera showed optimum lysis with rabbit RBC at 37 degrees C . The haemolytic activity of halibut sera was inactivated at 45 degrees C while sea bass sera were inactivated at 56 degrees C . The haemolytic activity of halibut sera was significantly reduced during short-term storage at -80 degrees C, whereas the sea bass sera maintained fairly good activity after 1-year storage at -80 degrees C . EGTA and EDTA inhibited the spontaneous haemolytic activity of sera from both the species . Zymosan and MacroGard from yeast cells also inhibited the haemolytic activity of the sera of both species, whereas LPS had a very slight effect . Considerable variation in haemolytic activity was observed within both the halibut and sea bass groups studied.

Microbes Infect, 2003 Sep, 5(11), 933 - 8
Variants of Sporothrix schenckii with attenuated virulence for mice; de Lima RF et al.; Strains of Sporothrix schenckii preserved under mineral oil were examined for virulence in BALB/c mice . The mice were inoculated with S . schenckii conidia and development of cutaneous lesions, signs of inactivity, weight loss, survival rates, number of viable yeast cells in lung and spleen, splenomegaly and organ lesions were evaluated . After intravenous injection of 7.5 x 10(6) conidia, two of five S . schenckii strains were unable to induce systemic disease and to kill the mice, only producing cord-like lesions on the tail that regressed with mouse maturation . Very small numbers of viable cells isolated from the spleen confirmed the lower invasive ability of these strains when compared with other strains studied here . These results suggest a relationship between the attenuation of virulence and the storage method under mineral oil after long periods of time.

Arch Biochem Biophys, 2003 Sep 15, 417(2), 219 - 26
Complex sphingolipid synthesis in plants: characterization of inositolphosphorylceramide synthase activity in bean microsomes; Bromley PE et al.; Complex glycophosphosphingolipids present in plants are composed of ceramide, inositolphosphate, and diverse polar oligosaccharide substituents . The activity of inositolphosphorylceramide (IPC) synthase (phosphatidylinositol:ceramide inositolphosphate transferase), the enzyme proposed to catalyze the initial committed step in the formation of these complex sphingolipids, was characterized in wax bean hypocotyl microsomes . Enzyme activity was assayed by monitoring the incorporation of fluorescent NBD-C(6) ceramide or {3H}inositolphosphate from radiolabeled phosphatidylinositol (PI) into product identified by TLC . IPC synthase was found to utilize nonhydroxy fatty acid-containing ceramide, hydroxy fatty acid-containing ceramide, and NBD-C(6) ceramide as substrate . Maximum product formation was observed at PI concentrations in excess of 600 microM (with half-maximum activity at approximately 200 microM) . Both endogenous PI and ceramide appeared to serve as substrates . Aureobasidin A and rustmicin, two potent inhibitors of fungal IPC synthase, inhibited enzyme activity in bean microsomes with values for IC(50) of 0.4-0.8 and 16-20 nM, respectively . IPC synthase activity appeared most closely associated with the Golgi based on results using selected marker enzymes . Enzyme activity was detected in a variety of plant tissues . This report, the first to characterize IPC synthase in plant tissues, demonstrates the similarities between the plant enzyme and its yeast counterpart, and provides insight into plant glycophosphosphingolipid biology.

Invest Ophthalmol Vis Sci, 2003 Sep, 44(9), 3920 - 6
Studies on the mechanism of the UVA light-dependent loss of glutathione reductase activity in human lenses; Linetsky M et al.; PURPOSE: To determine the mechanism that leads to the UVA light-dependent loss of glutathione reductase (GR) activity in human lens (HL) . METHODS: Both the HL water-soluble (WS) fraction and yeast GR were irradiated with UVA light (200 mW/(cm(2) . h) for 1 hour at +20 degrees C, and the specific activity (SA) was observed . GR apoenzyme (apo-GR) was prepared from either HL-WS fractions or yeast GR by treatment with a cold solution of acidic ammonium sulfate . Reconstitution of apo-GR was conducted by mixing enzyme with an excess of flavine adenine dinucleotide (FAD) and purification of GR on a size-exclusion separation column . RESULTS: One hour of UVA photolysis of an HL-WS fraction resulted in a 96% decrease in the SA of GR (6.32 +/- 0.22 vs . 0.39 +/- 0.01 mU/mg lens protein) . Action spectra of GR SA in the WS fraction from HL within the range 320 to 500 nm showed that the enzyme was most vulnerable to the wavelengths in the UVA region with the highest decrease in the SA at 320 to 350 nm ( approximately 23%-28% activity loss within 1 hour of irradiation), and lowest with the wavelengths beyond 400 nm (7%-8% SA loss) . UVA irradiation of apo-GR in the crude HL-WS fraction, followed by reconstitution with FAD, showed that 90% of the original SA was recovered . The original GR activity either in HL or yeast GR, however, was not recovered by (NH(4))(2)SO(4) (pH 2.25) treatment followed by reconstitution with FAD after UVA photolysis . Experiments with UVA-photolyzed yeast GR revealed that UVA photolysis caused the formation of additional SH groups within the enzyme, as shown by the incorporation of an SH-specific fluorescent probe, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) . Similar results were obtained on the photolyzed iodoacetamide-alkylated yeast GR, which was evaluated by matrix-assisted desorption ionization-time of flight (MALDI-TOF) mass spectrometry . CONCLUSIONS: The results show that the reduction of HL GR activity by UVA light was directly linked to the presence of FAD within the enzyme . That the irradiated GR showed de novo formed SH groups argues that UVA photolysis of GR leads to the reduction of the redox-active disulfide within the reaction center of the enzyme, making it inactive.

J Med Virol, 2003 Oct, 71(2), 188 - 94
Soluble CD14 levels are increased and inversely correlated with the levels of hepatitis B surface antigen in chronic hepatitis B patients; Steyaert S et al.; Because it was observed recently that yeast-derived recombinant HBsAg interacts in a lipopolysaccharide binding protein-dependent manner with CD14 expressed on human monocytes, we investigated whether HBsAg influences the serum levels of sCD14, lipopolysaccharide binding protein and C-reactive protein in hepatitis B patients . Samples from acute and chronic hepatitis B and chronic hepatitis C patients were tested . All analytes were measured using commercial assays . HBsAg was quantified using an NIBSC titrated standard . sCD14 levels were higher in chronic hepatitis B and C patients than in healthy controls (P = 0.0006 and P < 0.0001, respectively) . In chronic hepatitis B patients an inverse correlation was found between sCD14 and HBsAg (P = 0.0309) . Lipopolysaccharide binding protein and C-reactive protein levels were higher in acute hepatitis B patients than in control subjects (P = 0.0217 and P = 0.0034, respectively) . In chronic hepatitis B and C, sCD14 and C-reactive protein levels were higher in cirrhotic than in non-cirrhotic patients (P = 0.0072 and P = 0.0223, respectively) .

Bioessays, 2003 Sep, 25(9), 847 - 55
Intrinsically unstructured proteins evolve by repeat expansion; Tompa P; The proportion of the genome encoding intrinsically unstructured proteins increases with the complexity of organisms, which demands specific mechanism(s) for generating novel genetic material of this sort . Here it is suggested that one such mechanism is the expansion of internal repeat regions, i.e., coding micro- and minisatellites . An analysis of 126 known unstructured sequences shows the preponderance of repeats: the percentage of proteins with tandemly repeated short segments is much higher in this class (39%) than earlier reported for all Swiss-Prot (14%), yeast (18%) or human (28%) proteins . Furthermore, prime examples, such as salivary proline-rich proteins, titin, eukaryotic RNA polymerase II, the prion protein and several others, demonstrate that the repetitive segments carry fundamental function in these proteins . In addition, their repeat numbers show functionally significant interspecies variation and polymorphism, which underlines that these regions have been shaped by intense evolutionary activity . In all, the major point of this paper is that the genetic instability of repetitive regions combined with the structurally and functionally permissive nature of unstructured proteins has powered the extension and possible functional expansion of this newly recognized protein class .

Am J Clin Nutr, 2003 Sep, 78(3), 361 - 9
Calorie restriction and aging: review of the literature and implications for studies in humans; Heilbronn LK et al.; Calorie restriction (CR) extends life span and retards age-related chronic diseases in a variety of species, including rats, mice, fish, flies, worms, and yeast . The mechanism or mechanisms through which this occurs are unclear . CR reduces metabolic rate and oxidative stress, improves insulin sensitivity, and alters neuroendocrine and sympathetic nervous system function in animals . Whether prolonged CR increases life span (or improves biomarkers of aging) in humans is unknown . In experiments of nature, humans have been subjected to periods of nonvolitional partial starvation . However, the diets in almost all of these cases have been of poor quality . The absence of adequate information on the effects of good-quality, calorie-restricted diets in nonobese humans reflects the difficulties involved in conducting long-term studies in an environment so conducive to overfeeding . Such studies in free-living persons also raise ethical and methodologic issues . Future studies in nonobese humans should focus on the effects of prolonged CR on metabolic rate, on neuroendocrine adaptations, on diverse biomarkers of aging, and on predictors of chronic age-related diseases.

FEBS Lett, 2003 Aug 28, 550(1-3), 139 - 43
MIP-T3 associates with IL-13Ralpha1 and suppresses STAT6 activation in response to IL-13 stimulation; Niu Y et al.; To unravel the mechanism of interleukin-13 (IL-13)-specific functions, we sought to identify IL-13 receptor (IL-13R) binding molecules . A novel human IL-13Ralpha1 binding protein (IL13RBP1) has been identified using yeast tri-hybrid system, which was found to encode the same protein as MIP-T3 (microtubule interacting protein that associates with tumor necrosis factor (TNF) receptor associating factor-3 (TRAF3)) . It constitutively associates with IL-13Ralpha1 and suppresses IL-4/13-induced signal transducer and activator of transcription-6 (STAT6) phosphorylation . IL-13-induced STAT6 activation was also inhibited as determined by dual luciferase assay and electrophoretic mobility shift assay (EMSA) . These results suggest that MIP-T3 is a novel inhibitor of IL-13 signaling and may be a useful molecule in ameliorating various conditions in which IL-13 plays a central role.

FEBS Lett, 2003 Aug 28, 550(1-3), 119 - 23
The Cbl proteins are binding partners for the Cool/Pix family of p21-activated kinase-binding proteins; Flanders JA et al.; Members of the Cool protein family contain SH3, Dbl, and pleckstrin homology domains and are binding partners for the p21-activated kinase (PAK) . Using the yeast two-hybrid screen, we identified Cbl-b as a Cool family binding partner . We co-immunoprecipitated endogenous Cool and Cbl-b from a variety of breast cancer cell lines . The Cool-Cbl-b interaction requires the SH3 domain of Cool and competes with the binding of PAK to Cool proteins . Expression of Cbl-b effectively blocks the ability of Cool-2 to stimulate PAK, thus providing an additional mechanism, aside from catalyzing receptor ubiquitination, by which Cbl-b acts as a negative regulator for signaling activities requiring PAK activation.

FEBS Lett, 2003 Aug 28, 550(1-3), 1 - 4
Membrane traffic fuses with cartilage development; Sacher M; The ability of cells to synthesize and secrete proteins is essential for numerous cellular functions . Therefore, when mutations in one component of the secretory pathway result in a tissue-specific defect, a unique opportunity arises to examine the molecular mechanisms at play . The recent finding that a defect in the protein sedlin, whose yeast counterpart is involved in the first step of the secretory pathway, leads to a cartilage-specific disorder in humans raises numerous questions and interesting possibilities for understanding both the pathobiology involved and the role of membrane traffic in normal cartilage development.

J Comput Biol, 2003, 10(3-4), 599 - 615
Discriminative motifs; Sinha S; This paper takes a new view of motif discovery, addressing a common problem in existing motif finders . A motif is treated as a feature of the input promoter regions that leads to a good classifier between these promoters and a set of background promoters . This perspective allows us to adapt existing methods of feature selection, a well-studied topic in machine learning, to motif discovery . We develop a general algorithmic framework that can be specialized to work with a wide variety of motif models, including consensus models with degenerate symbols or mismatches, and composite motifs . A key feature of our algorithm is that it measures overrepresentation while maintaining information about the distribution of motif instances in individual promoters . The assessment of a motif's discriminative power is normalized against chance behaviour by a probabilistic analysis . We apply our framework to two popular motif models and are able to detect several known binding sites in sets of co-regulated genes in yeast.

J Comput Biol, 2003, 10(3-4), 433 - 52
Bayesian estimation of transcript levels using a general model of array measurement noise; Dror RO et al.; Gene arrays demonstrate a promising ability to characterize expression levels across the entire genome but suffer from significant levels of measurement noise . We present a rigorous new approach to estimate transcript levels and ratios from one or more gene array experiments, given a model of measurement noise and available prior information . The Bayesian estimation of array measurements (BEAM) technique provides a principled method to identify changes in expression level, combine repeated measurements, or deal with negative expression level measurements . BEAM is more flexible than existing techniques, because it does not assume a specific functional form for noise and prior models . Instead, it relies on computational techniques that apply to a broad range of models . We use Affymetrix yeast chip data to illustrate the process of developing accurate noise and prior models from existing experimental data . The resulting noise model includes novel features such as heavy-tailed additive noise and a gene-specific bias term . We also verify that the resulting noise and prior models fit data from an Affymetrix human chip set.

Oncogene, 2003 Aug 21, 22(35), 5408 - 14
Functional interaction between the small GTP-binding protein Rin and the N-terminal of Brn-3a transcription factor; Calissano M et al.; Brn-3a is a transcription factor belonging to the class IV of POU domain transcription factors . It is expressed throughout the peripheral nervous system but especially in postmitotic sensory neurons of dorsal root ganglia . Brn-3a is known to regulate different genes involved in neuronal differentiation and survival . It has been shown that some of these genes require the N-terminal domain of Brn-3a in order to be activated and this effect is observed only in neurons suggesting that it may require a neuronal-specific cofactor . In order to identify this putative factor(s) we screened a cDNA library via a variant of the original yeast two-hybrid system . By using the N-terminal of Brn-3a as the bait, we have repeatedly isolated a protein named Rin, an incompletely characterized small GTP-binding protein expressed only in neurons . In this work, we describe the evidence for a functional interaction between Brn-3a and Rin and demonstrate the role of Rin in modulating the activation of the Brn-3a regulated egr-1 promoter by the N-terminal domain of Brn-3a.

Intervirology, 2003, 46(4), 239 - 44
The Tat protein of the caprine arthritis encephalitis virus interacts with the Notch2 EGF-like repeats and the epithelin/granulin precursor; Shoham N et al.; Using the yeast two-hybrid system, we screened a human placenta cDNA library and identified two proteins that interacted with the Tat protein of the caprine arthritis encephalitis virus (CAEV): the EGF-like repeats 1-6 of the extracellular domain of the human Notch2 receptor and the epithelin/granulin growth factor precursor . This interaction was also confirmed in mammalian cells . Using in vitro mutagenesis assays, we showed that each one of the three cysteine residues located within the cysteine-rich domain of the CAEV Tat protein is essential for the binding of Tat to both the Notch2 and the epithelin/granulin protein . It is thus suggested that the cysteine-rich domain of Tat plays a role in the interaction between the Tat and either Notch2 or the epithelin/granulin domains, both of which exhibit EGF-like-repeat-imposed spatial conformation . It is assumed that such interactions might modulate the physiological functions of Notch2 and epithelin/granulin, thereby affecting various pathologies associated with CAEV .

Protein Sci, 2003 Sep, 12(9), 1925 - 33
Solution structure of the C-terminal domain from poly(A)-binding protein in Trypanosoma cruzi: a vegetal PABC domain; Siddiqui N et al.; PABC is a phylogenetically conserved peptide-binding domain primarily found within the C terminus of poly(A)-binding proteins (PABPs) . This domain recruits a series of translation factors including poly(A)-interacting proteins (Paip1 and Paip2) and release factor 3 (RF3/GSPT) to the initiation complex on mRNA . Here, we determine the solution structure of the Trypanosoma cruzi PABC domain (TcPABC), a representative of the vegetal class of PABP proteins . TcPABC is similar to human PABC (hPABC) and consists of five alpha-helices, in contrast to the four helices observed in PABC domains from yeast (yPABC) and hyper plastic disk proteins (hHYD) . A mobile N-terminal helix is observed in TcPABC that does not pack against the core of the protein, as found in hPABC . Characteristic to all PABC domains, the last four helices of TcPABC fold into a right-handed super coil . TcPABC demonstrates high-affinity binding to PABP interacting motif-2 (PAM-2) and reveals a peptide-binding surface homologous to that of hPABC . Our results demonstrate the last four helices in TcPABC are sufficient for peptide recognition and we predict a similar binding mode in PABC domains . Furthermore, these results point to the presence of putative PAM-2 site-containing proteins in trypanosomes.

Nucleic Acids Res, 2003 Sep 1, 31(17), 5090 - 100
Analysis of the DNA joining repertoire of Chlorella virus DNA ligase and a new crystal structure of the ligase-adenylate intermediate; Odell M et al.; Chlorella virus DNA ligase is the smallest eukaryotic ATP-dependent DNA ligase known; it suffices for yeast cell growth in lieu of the essential yeast DNA ligase Cdc9 . The Chlorella virus ligase-adenylate intermediate has an intrinsic nick sensing function and its DNA footprint extends 8-9 nt on the 3'-hydroxyl (3'-OH) side of the nick and 11-12 nt on the 5'-phosphate (5'-PO4) side . Here we establish the minimal length requirements for ligatable 3'-OH and 5'-PO4 strands at the nick (6 nt) and describe a new crystal structure of the ligase-adenylate in a state construed to reflect the configuration of the active site prior to nick recognition . Comparison with a previous structure of the ligase-adenylate bound to sulfate (a mimetic of the nick 5'-PO4) suggests how the positions and contacts of the active site components and the bound adenylate are remodeled by DNA binding . We find that the minimal Chlorella virus ligase is capable of catalyzing non-homologous end-joining reactions in vivo in yeast, a process normally executed by the structurally more complex cellular Lig4 enzyme . Our results suggest a model of ligase evolution in which: (i) a small 'pluripotent' ligase is the progenitor of the much larger ligases found presently in eukaryotic cells and (ii) gene duplications, variations within the core ligase structure and the fusion of new domains to the core structure (affording new protein-protein interactions) led to the compartmentalization of eukaryotic ligase function, i.e . by enhancing some components of the functional repertoire of the ancestral ligase while disabling others.

Nucleic Acids Res, 2003 Sep 1, 31(17), 5016 - 24
Elongation by RNA polymerase II on chromatin templates requires topoisomerase activity; Mondal N et al.; Transcription on chromatin by RNA polymerase II (pol II) is repressed as compared with transcription on histone-free DNA . In this study, we show that human topoisomerase I (topo I) and yeast topoisomerase II (topo II), each of which relax both positive and negative superhelical tension, reverse the transcriptional repression by chromatin . In the presence of bacterial topo I, which can relax only negative superhelical tension, the transcription is repressed on chromatin templates . The data together show that the relaxation of positive superhelical tension by these enzymes was the key property required for RNA synthesis from chromatin templates . In the absence of topoisomerase, transcriptional repression on chromatin depended on RNA length . The synthesis of transcripts of 100 nt or shorter was unaffected by chromatin, but repression was apparent when the RNA transcript was 200 nt or longer . These findings suggest that transcription on chromatin templates results in the accumulation of positive superhelical tension by the elongating polymerase, which in turn inhibits further elongation in the absence of topoisomerase activity.

Proc Natl Acad Sci U S A, 2003 Sep 2, 100(18), 10237 - 42 Epub 2003 Aug 20.
The alternative Ctf18-Dcc1-Ctf8-replication factor C complex required for sister chromatid cohesion loads proliferating cell nuclear antigen onto DNA; Bermudez VP et al.; The linkage of sister chromatids after DNA replication ensures the faithful inheritance of chromosomes by daughter cells . In budding yeast, the establishment of sister chromatid cohesion requires Ctf8, Dcc1, and Ctf18, a homologue of the p140 subunit of the replication factor C (RFC) . In this report we demonstrate that in 293T cells, Flag-tagged Ctf18 forms a seven-subunit cohesion-RFC complex comprised of Ctf18, Dcc1, Ctf8, RFCp40, RFCp38, RFCp37, and RFCp36 (Ctf18-RFC) . We demonstrate that a stoichiometric heteroheptameric Ctf18-RFC complex can be assembled by coexpressing the seven proteins in baculovirus-infected insect cells . In addition, the two other stable subcomplexes were formed, which include a pentameric complex comprised of Ctf18, RFCp40, RFCp38, RFCp37, and RFCp36 and a dimeric Dcc1-Ctf8 . Both the five- and seven-subunit Ctf18-RFC complexes bind to single-stranded and primed DNAs and possess weak ATPase activity that is stimulated by the addition of primed DNA and proliferating cell nuclear antigen (PCNA) . These complexes catalyzed the ATP-dependent loading of PCNA onto primed and gapped DNA but not onto double-stranded nicked or single-stranded circular DNAs . Consistent with these observations, both Ctf18-RFC complexes substituted for the replicative RFC in the PCNA-dependent DNA polymerase delta-catalyzed DNA replication reaction . These results support a model in which sister chromatid cohesion is linked to DNA replication.

Mol Cell Proteomics, 2003 Oct, 2(10), 1104 - 19 Epub 2003 Aug 19.
ERp19 and ERp46, New Members of the Thioredoxin Family of Endoplasmic Reticulum Proteins; Knoblach B et al.; Using a proteomic analysis of the luminal environment of the endoplasmic reticulum (ER), we have identified 141 proteins, of which 6 were previously unknown . Two newly discovered ER luminal proteins, designated ERp19 and ERp46, are related to protein disulphide isomerase . Western and Northern blot analyses revealed that both ERp19 and ERp46 and their respective mRNAs are highly expressed in the liver as compared with other tissues . Both proteins were enriched in purified liver ER vesicles and were localized specifically to the ER in McA-RH7777 hepatocytes . Functional analysis with yeast complementation studies showed that ERp46 but not ERp19 can substitute for protein disulphide isomerase function in vivo.

J Biol Chem, 2003 Oct 31, 278(44), 43041 - 50 Epub 2003 Aug 19.
Involvement of the histone deacetylase SIRT1 in chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2-mediated transcriptional repression; Senawong T et al.; Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting proteins 1 and 2 (CTIP1 and CTIP2) enhance transcriptional repression mediated by COUP-TF II and have been implicated in hematopoietic cell development and malignancies . CTIP1 and CTIP2 are also sequence-specific DNA-binding proteins that repress transcription through direct, COUP-TF-in-dependent binding to a GC-rich response element . CTIP1- and CTIP2-mediated transcriptional repression is insensitive to trichostatin A, an inhibitor of known class I and II histone deacetylases . However, chromatin immunoprecipitation assays revealed that expression of CTIP2 in mammalian cells resulted in deacetylation of histones H3 and/or H4 that were associated with the promoter region of a reporter gene . CTIP2-mediated transcriptional repression, as well as deacetylation of promoter-associated histones H3/H4 in CTIP2-transfected cells, was reversed by nicotinamide, an inhibitor of class III histone deacetylases such as the mammalian homologs of yeast Silent Information Regulator 2 (Sir2) . The human homolog of yeast Sir2, SIRT1, was found to interact directly with CTIP2 and was recruited to the promoter template in a CTIP2-dependent manner . Moreover, SIRT1 enhanced the deacetylation of template-associated histones H3/H4 in CTIP2-transfected cells, and stimulated CTIP2-dependent transcriptional repression . Finally, endogenous SIRT1 and CTIP2 co-purified from Jurkat cell nuclear extracts in the context of a large (1-2 mDa) complex . These findings implicate SIRT1 as a histone H3/H4 deacetylase in mammalian cells and in transcriptional repression mediated by CTIP2.

Zhonghua Yi Xue Za Zhi, 2003 Jul 25, 83(14), 1270 - 3
{Wnt/Frizzled signaling pathway in renal carcinoma}; Gong K et al.; OBJECTIVE: To investigate the transduction of Wnt/Frizzled signaling pathway, especially the function of T cell factor 4 (TCF(4)), in renal carcinoma . METHODS: A renal carcinoma yeast two hybrid library and a human TCF(4) yeast two hybrid expression vector were constructed . Proteins interacting with the bait protein human TCF(4) were obtained from the renal carcinoma yeast two hybrid library by reverse yeast two hybrid system . RESULTS: 67 positive clones interacting with the bait protein TCF(4) were obtained by reverse yeast two hybrid system, including 18 beta-catenin clones, 24 TCF(4) clones and 25 unknown clones . CONCLUSION: Wnt/Frizzled signaling pathway exists in renal carcinoma . TCF(4), its important signal factor, interacts with beta-catenin and forms homodimer or homocopolymer by itself, thus displaying its constitutive transcriptional activity.

J Bone Miner Res, 2003 Aug, 18(8), 1419 - 29
P300/CBP acts as a coactivator to cartilage homeoprotein-1 (Cart1), paired-like homeoprotein, through acetylation of the conserved lysine residue adjacent to the homeodomain; Iioka T et al.; The paired-like homeoprotein, Cart1, is involved in skeletal development . We describe here that the general coactivator p300/CBP controls the transcription activity of Cart1 through acetylation of a lysine residue that is highly conserved in other homeoproteins . Acetylation of this residue increases the interaction between p300/CBP and Cart1 and enhances its transcriptional activation . INTRODUCTION: Cart1 encodes a paired-like homeoprotein expressed selectively in chondrocyte lineage during embryonic development . Although its target gene remains unknown, gene disruption studies have revealed that Cart1 plays an important role for craniofacial bone formation as well as limb development by cooperating with another homeoprotein, Alx4 . In this report, we study the functional involvement of p300/CBP, coactivators with intrinsic histone acetyltransferase (HAT) activity, in the transcriptional control of Cart1 . METHODS: To study the transcription activity of Cart1, a reporter construct containing a putative Cart1 binding site was transiently transfected with the expression vectors of each protein . The interaction between p300/CBP and Cart1 was investigated by glutathione S-transferase (GST) pull-down, yeast two-hybrid, and immunoprecipitation assays . In vitro acetylation assay was performed with the recombinant p300-HAT domain and Cart1 in the presence of acetyl-CoA . RESULTS AND CONCLUSIONS: p300 and CBP stimulate Cart1-dependent transcription activity, and this transactivation is inhibited by E1A and Tax, oncoproteins that suppress the activity of p300/CBP . Cart1 binds to p300 in vivo and in vitro, and this requires the homeodomain of Cart 1 and N-terminal 139 amino acids of p300 . Confocal microscopy analysis shows that Cart1 recruits overexpressed and endogenous p300 to a Cart1-specific subnuclear compartment . Cart1 is acetylated in vivo and sodium butyrate and trichostatin A, histone deacetylase inhibitors, markedly enhance the transcription activity of Cart1 . Deletion and mutagenesis analysis identifies the 131st lysine that locates immediately adjacent to the homeodomain as a target of p300-HAT, and a point mutation to this residue attenuates the binding affinity to p300 as well as p300-dependent transcription activity . Together, these results indicate that p300/CBP acts as a cotransactivator to Cart1 through a direct interaction and specific lysine acetylation . In addition, because 131st lysine is highly conserved in other types of homeoprotein, this lysine may be a common target for HAT of p300/CBP for these proteins.

Hum Mol Genet, 2003 Oct 15, 12(20), 2711 - 21 Epub 2003 Aug 19.
Genomic microarray analysis reveals distinct locations for the CENP-A binding domains in three human chromosome 13q32 neocentromeres; Alonso A et al.; Human neocentromeres are fully functional centromeres that provide mitotic stability to rearranged chromosomes that have separated from endogenous centromeres . A disproportionate number of neocentromeres has been observed in certain regions such as chromosome 3q (n=6), 15q (n=9) and 13q32 (n=7), suggesting that these regions contain DNA sequences with a high propensity for neocentromere formation . Therefore, we have addressed the role of primary DNA sequence in neocentromere formation by asking whether multiple independent neocentromeres that were cytologically localized to chromosome 13q32 are in fact localized to the same underlying genomic DNA . Analysis of four independent 13q32 neocentromeres using simultaneous FISH with ordered YAC probes and immunofluorescence with antibodies to CENP-C have localized three neocentromeres to a distal approximately 7 Mb domain in chromosome 13q32, and one to an overlapping proximal domain of approximately 7 Mb . DNA was obtained from three of these neocentromeres by CENP-A chromatin immunoprecipitation (ChIP) and used to screen ordered BACs using both a slot-blotted BAC pool approach and a genomic microarray that contiguously spans 13q31.3-13q33.1 . The CENP-A binding domains from each of these neocentromeres was identified to distinct genomic locations of approximately 130, 215 and 275 kb within an approximately 6.5 Mb region . Thus, the lack of coincidence of these neocentromeres to the same underlying DNA sequence refutes the idea of a DNA sequence based neocentromere 'hotspot' in 13q32 and further supports the sequence-independent epigenetic formation of human neocentromeres . The screening of genomic microarrays with ChIP DNA provides a powerful method to identify mammalian DNA sequences associated with particular functional chromatin states.

Brain Res Brain Res Protoc, 2003 Aug, 12(1), 35 - 40
An abbreviated procedure for the cloning and identification of Ets transcription factors regulating the expression of the human presenilin 1 gene; Pastorcic M et al.; We have previously defined a crucial DNA element controlling 90% of the expression of the presenilin 1 gene at (-35 to +6) . This region contains an Ets transcription factor binding motif, and a two-basepair alteration within the core sequence (GGAA to TTAA) of the Ets consensus also reduced transcription by over 90% . We have shown that Ets1/2 transcription factors bind specifically to the -10 Ets element and activate PS1 transcription . The identification of other transcription factors recognizing specifically this promoter area should provide insights into the regulation of PS1 . We have used the -10 Ets element as a bait in yeast one hybrid screening of a human brain cDNA library using a His3 reporter construct . We describe an abbreviated one-hybrid protocol to screen cDNA libraries . This assay selected four factors from the Ets family: Ets2, ER81, ERM and Elk1 . We have also shown that specific DNA binding activity to the -10 Ets element of PS1 could easily be detected in yeast clones by EMSAs including protein extracts from yeast cells, thus confirming specific DNA binding activity without further sequencing and subcloning into suitable expression vectors . Ultimately the identity of putative clones was confirmed by DNA sequencing . We also confirmed the specific DNA binding properties of the factors identified by showing that the proteins produced by in vitro translation of the entire cDNAs from Elk1 and ER81 indeed binds specifically to the -10 region of the PS1 promoter.

Biochem Biophys Res Commun, 2003 Sep 5, 308(4), 793 - 801
hLodestar/HuF2 interacts with CDC5L and is involved in pre-mRNA splicing; Leonard D et al.; hLodestar/HuF2 belongs to the SNF2 family of proteins . This family of proteins has been shown to play a critical role in altering protein-DNA interactions in a variety of cellular contexts . We have identified an unexpected interaction between hLodestar/HuF2 and CDC5L in both the yeast two-hybrid system and HeLa nuclear extract . CDC5L is a well-characterized pre-mRNA splicing factor in yeast and humans . Our findings demonstrate that hLodestar/HuF2 associates with human splicing complexes . We also found that a truncated hLodestar/HuF2 polypeptide that overlaps with the CDC5L-binding region can inhibit pre-mRNA splicing by disrupting spliceosome assembly . These findings indicate that hLodestar/HuF2 may have a role in pre-mRNA splicing . These data are consistent with a close co-ordination of the transcription and splicing pathways in eukaryotes . Although many members of the DExH/D helicase superfamily have been linked to pre-mRNA splicing, this is the first SNF2 family member to be implicated in this pathway.

Cancer, 2003 Aug 25, 99(4), 223 - 32
ThinPrep-processed fine-needle samples of breast are effective material for RNA- and DNA-based molecular diagnosis: application to p53 mutation analysis; Tisserand P et al.; BACKGROUND: Fine-needle sampling is the least invasive method of in vivo breast carcinoma sampling and can provide material for breast carcinoma diagnosis . The aim of the current study was to assess the accuracy of molecular diagnosis techniques using fine-needle sample (FNS) material stored in PreservCyt (Cytyc Corp., Boxborough, MA) . METHODS: The p53 tumor suppressor gene was chosen as a model because it can be used for DNA, RNA, and protein analysis . Molecular analysis was performed using a yeast functional assay and DNA sequencing . p53 accumulation was evaluated by immunocytochemistry . RESULTS: DNA and protein analysis indicated that samples stored for periods of several months, either at room temperature, 4 degrees C, or -20 degrees C, can be processed reliably . For RNA-based diagnosis, samples were still intact after 5 months of storage in PreservCyt at 4 degrees C . In addition, using FNS material that was stored for 16 months at 4 degrees C, the authors detected p53 mutations with either the functional assay for separating alleles in yeast (an RNA-based functional assay) or direct cDNA sequencing . CONCLUSIONS: Fine-needle samples stored in PreservCyt at 4 degrees C are very good material for molecular diagnosis techniques . In addition, it is feasible to adopt a strategy of storing excess FNS material to create cellular banks that will be invaluable for future gene studies .

Mol Biol Cell, 2003 Aug, 14(8), 3437 - 48 Epub 2003 Apr 17.
Dependence of endoplasmic reticulum-associated degradation on the peptide binding domain and concentration of BiP; Kabani M et al.; ER-associated degradation (ERAD) removes defective and mis-folded proteins from the eukaryotic secretory pathway, but mutations in the ER lumenal Hsp70, BiP/Kar2p, compromise ERAD efficiency in yeast . Because attenuation of ERAD activates the UPR, we screened for kar2 mutants in which the unfolded protein response (UPR) was induced in order to better define how BiP facilitates ERAD . Among the kar2 mutants isolated we identified the ERAD-specific kar2-1 allele (Brodsky et al . J . Biol . Chem . 274, 3453-3460) . The kar2-1 mutation resides in the peptide-binding domain of BiP and decreases BiP's affinity for a peptide substrate . Peptide-stimulated ATPase activity was also reduced, suggesting that the interdomain coupling in Kar2-1p is partially compromised . In contrast, Hsp40 cochaperone-activation of Kar2-1p's ATPase activity was unaffected . Consistent with UPR induction in kar2-1 yeast, an ERAD substrate aggregated in microsomes prepared from this strain but not from wild-type yeast . Overexpression of wild-type BiP increased substrate solubility in microsomes obtained from the mutant, but the ERAD defect was exacerbated, suggesting that simply retaining ERAD substrates in a soluble, retro-translocation-competent conformation is insufficient to support polypeptide transit to the cytoplasm.

Mol Biol Cell, 2003 Aug, 14(8), 3427 - 36 Epub 2003 May 18.
Schizosacchromyces pombe Dpb2 binds to origin DNA early in S phase and is required for chromosomal DNA replication; Feng W et al.; Genetic evidence suggests that DNA polymerase epsilon (Pol epsilon) has a noncatalytic essential role during the early stages of DNA replication initiation . Herein, we report the cloning and characterization of the second largest subunit of Pol epsilon in fission yeast, called Dpb2 . We demonstrate that Dpb2 is essential for cell viability and that a temperature-sensitive mutant of dpb2 arrests with a 1C DNA content, suggesting that Dpb2 is required for initiation of DNA replication . Using a chromatin immunoprecipitation assay, we show that Dpb2, binds preferentially to origin DNA at the beginning of S phase . We also show that the C terminus of Pol epsilon associates with origin DNA at the same time as Dpb2 . We conclude that Dpb2 is an essential protein required for an early step in DNA replication . We propose that the primary function of Dpb2 is to facilitate assembly of the replicative complex at the start of S phase . These conclusions are based on the novel cell cycle arrest phenotype of the dpb2 mutant, on the previously uncharacterized binding of Dpb2 to replication origins, and on the observation that the essential function of Pol epsilon is not dependent on its DNA synthesis activity.

Hum Mol Genet, 2003 Oct 15, 12(20), 2587 - 97 Epub 2003 Aug 12.
The autosomal recessive juvenile Parkinson disease gene product, parkin, interacts with and ubiquitinates synaptotagmin XI; Huynh DP et al.; Inactivating mutations of the gene encoding parkin are responsible for some forms of autosomal recessive juvenile Parkinson disease . Parkin is a ubiquitin ligase that ubiquitinates misfolded proteins targeted for the proteasome-dependent protein degradation pathway . Using the yeast two-hybrid system and co-immunoprecipitation methods, we identified synaptotagmin XI as a protein that interacts with parkin . Parkin binds to the C2A and C2B domains of synaptotagmin XI resulting in the polyubiquitination of synaptotagmin XI . Truncated and missense mutated parkins reduce parkin-sytXI binding affinity and ubiquitination . Parkin-mediated ubiquitination also enhances the turnover of sytXI . In sporadic PD brain sections, sytXI was found in the core of the Lewy bodies . Since synaptotagmin XI is a member of the synaptotagmin family that is well characterized in their importance for vesicle formation and docking, the interaction with this protein suggests a role for parkin in the regulation of the synaptic vesicle pool and in vesicle release . Loss of parkin could thus affect multiple proteins controlling vesicle pools, docking and release and explain the deficits in dopaminergic function seen in patients with parkin mutations.

J Biol Chem, 2003 Nov 14, 278(46), 45611 - 9 Epub 2003 Aug 17.
Scapinin, a putative protein phosphatase-1 regulatory subunit associated with the nuclear nonchromatin structure; Sagara J et al.; It is thought that the nuclear nonchromatin structures, such as the nuclear matrix and lamina, play regulatory roles in gene expression . In this study, we identified an insoluble protein that was associated with the chromatin-depleted nuclear structure of proliferating human leukemia HL-60 cells . Preparation of the chromatin-depleted nuclear structure, referred to as the nuclear matrix-intermediate filament scaffold (Fey, E., Krochmalnic, G., and Penman, S . (1986) J . Cell . Biol . 102, 1654-1665), involved cell extraction using a series of buffers containing Triton X-100, DNase I, and 2 M NaCl . A yeast two-hybrid assay revealed that this protein bound to the catalytic subunit of protein phosphatase-1 (PP1) . Furthermore, it inhibited PP1 activity in vitro . We therefore named it scapinin (scaffold-associated PP1 inhibiting protein) . cDNA cloning revealed that scapinin had two splicing variants of 448 amino acids (scapinin-S) and 518 amino acids (scapinin-L) . Scapinin was down-regulated by differentiation in HL-60 cells . These results suggest that scapinin is a putative regulatory subunit of PP1 and is involved in transformed or immature phenotypes of HL-60 cells . We also describe the presence of scapinin family proteins from worm to human.

Nutr Cancer, 2003, 46(1), 73 - 81
Effect of in utero-administered coumestrol, equol, and organic selenium on biomarkers for phase 2 enzyme capacity and redox status; Kramer F et al.; The aim of the present study was to investigate the effect of in utero administration of coumestrol, equol, and selenium-enriched yeast on selected hepatic phase 2 enzymes, plasma hormone levels, and markers for redox status in plasma and red blood cells (RBCs) . The test compounds were administered via the diet to pregnant Sprague-Dawley rats throughout gestation . Within 24 h following delivery dams and offspring were sacrificed, and blood, liver, and reproductive organs were sampled . Coumestrol, equol, and selenium-enriched yeast did not significantly affect hepatic glutathione S-transferase (GST), quinone reductase (QR), or RBC glutathione peroxidase (GPx) in the offspring, whereas significant increases in GST, QR, and GPx activities in dams were observed following administration of selenium-enriched yeast . The level of 17beta-estradiol in offspring from coumestrol-exposed dams was significantly increased compared with the control . The present results indicate that selenium-enriched yeast, coumestrol, and equol affect selected hepatic phase 2 enzymes and GPx in RBC in dams, whereas the offspring in general were refractive to the employed treatments . Further studies are warranted to investigate whether the observed in utero effects imposed by the selected plant compounds confer permanent alterations on the health status of the animal resulting in an altered resistance to cancer.

J Biol Chem, 2003 Oct 24, 278(43), 41988 - 97 Epub 2003 Aug 14.
Direct interaction between the actin-binding protein filamin-A and the inwardly rectifying potassium channel, Kir2.1; Sampson LJ et al.; The role of filamins in actin cross-linking and membrane stabilization is well established, but recently their ability to interact with a variety of transmembrane receptors and signaling proteins has led to speculation of additional roles in scaffolding and signal transduction . Here we report a direct interaction between filamin-A and Kir2.1, an isoform of inwardly rectifying potassium channel expressed in vascular smooth muscle and an important regulator of vascular tone . Yeast two-hybrid screening of a porcine coronary artery cDNA library using the carboxyl terminus of Kir2.1 as bait yielded cDNA encoding a fragment of filamin-A (residues 2481-2647) . Interaction between filamin-A and Kir2.1 was confirmed by in vitro overlay assay of membrane-bound Kir2.1 with glutathione S-transferase fusion protein of the isolated filamin clone . Additionally, antibodies directed against Kir2.1 coimmunoprecipitated filamin-A from arterial smooth muscle cell lysates, and immunocytochemical analysis of individual arterial smooth muscle cells showed that Kir2.1 and filamin co-localize in "hotspots" at the cell membrane . Interaction with filamin-A was found to have no effect on Kir2.1 channel behavior but, rather, increased the number of functional channels resident within the membrane . We conclude that filamin-A is potentially an important regulator of Kir2.1 surface expression and location within vascular smooth muscle.

J Biol Chem, 2003 Oct 24, 278(43), 42190 - 9 Epub 2003 Aug 14.
PDZ Domain-mediated interaction of interleukin-16 precursor proteins with myosin phosphatase targeting subunits; Bannert N et al.; The cytokine interleukin-16 is generated by posttranscriptional cleavage by caspase-3 of two large precursor isoforms . The smaller protein of 67 kDa (pro-IL-16) is expressed in cells of the immune system and contains three PDZ (postsynaptic density/disc large/zona occludens-1) domains, whereas the larger 141-kDa neuronal variant (npro-IL-16) has two additional PDZ domains in its N-terminal extension that interact with neuronal ion channels . Using the yeast two-hybrid approach we have identified three closely related myosin phosphatase targeting subunits, MYPT1, MYPT2, and MBS85, as binding partners of the IL-16 precursor proteins . These interactions were verified using pull-down assays, coimmunoprecipitations, and plasmon resonance experiments . Binding requires the intact PDZ2 domain of pro-IL-16 and highly related C-terminal regions in the ligands consisting of a short leucine zipper and an indispensable serine at the -1 position, suggesting a novel unconventional PDZ binding mode . Pro-IL-16 and the myosin phosphatase targeting subunits colocalize along actomyosin filaments and stress fibers in transfected COS-7 cells . By modulating and targeting the catalytic phosphatase subunit to its substrates, MYPT1, MYPT2, and MBS85 regulate various contractile processes in muscle and non-muscle cells . Our findings indicate an involvement of the IL-16 precursor molecules in myosin-based contractile processes, most likely in cell motility, providing a functional link to the chemotactic activity of the mature cytokine . Alternatively, an intracellular complex of npro-IL-16, ion channels, and components of myosin motors in neurons suggests a role in protein targeting.

Genes Dev, 2003 Sep 1, 17(17), 2083 - 7 Epub 2003 Aug 15.
Heritable activity: a prion that propagates by covalent autoactivation; Roberts BT et al.; Known prions (infectious proteins) are self-propagating amyloids or conformationally altered proteins, but in theory an enzyme necessary for its own activation could also be a prion (or a gene composed of protein).We show that yeast protease B is such a prion, called {beta}.{beta} is infectious, reversibly curable, and its de novo generation is induced by overexpression of the pro-protease . Present in normal cells but masked by the functionally redundant protease A, {beta} is advantageous during starvation and necessary for sporulation.We propose that other enzymes whose active, modified, form is necessary for their maturation might also be prions.

Virus Res, 2003 Sep, 95(1-2), 59 - 73
Encephalomyocarditis virus (EMCV) proteins 2A and 3BCD localize to nuclei and inhibit cellular mRNA transcription but not rRNA transcription; Aminev AG et al.; We have followed the viral processing cascade and polyprotein precursor fates during encephalomyocarditis virus (EMCV) infection of HeLa cells using a panel of monoclonal antibodies (mAbs) . Within the first 2-4 h of infection, signals of antibodies specific for the 2A, 3B(VPg), 3C(pro) and 3D(pol) proteins were found to co-localize in nucleoli at the rRNA synthesis and cellular protein B23 (nucleophosmin) sites . Cellular fractionation identified viral protein precursor 3BCD as the common source of the P3-region antibody signals . Previously thought to be a minor product of the polymerase region cleavage pathways, the nuclear targeting of this precursor was localized with engineered mutations to five P2 and P3 region polyprotein processing sites . A nuclear localization motif (NLS), similar to that in many yeast ribosomal proteins, was identified near the N-terminus of the 3D(pol) sequence . Point mutations within this motif prevented nuclear and nucleolar localization by all forms of 3B(VPg), 3C(pro) and 3D(pol), and were lethal to the virus because they also prevented genome replication . However, viral RNA synthesis was not required for nucleolar transport and 3BCD was found in nuclei, even when the 3D(pol) was inactivated . Co-immunoprecipitation experiments showed a tight association between 3BCD and B23 (nucleophosmin), suggesting a possible ribosomal protein-like mechanism for nuclear transport . Infected cell extracts analyzed with microarrays, quantitative slot-blots and pulse-labeling experiments confirmed a nearly complete shutoff of host pol-II-dependent mRNA synthesis during EMCV infection, in reactions that depended on wild-type 2A protein . In contrast to human rhinovirus-16 infection, rRNA synthesis by pol-I and pol-III were not turned off by EMCV, although the cellular concentration of rRNA decreased during infection, relative to control samples . The data suggest that nuclear targeting by 2A and 3BCD may be responsible for regulating cellular mRNA and rRNA transcription during infection, perhaps via a proteolytic mechanism catalyzed by the endogenous 3C(pro) sequence.

Virus Res, 2003 Sep, 95(1-2), 45 - 57
Encephalomyocarditis viral protein 2A localizes to nucleoli and inhibits cap-dependent mRNA translation; Aminev AG et al.; Panels of monoclonal antibodies were raised against viral non-structural proteins of encephalomyocarditis virus (EMCV) and used to probe infected cells in laser confocal microscopy experiments and Western analyses . Surprisingly, all Mengovirus and EMCV-infected cells showed strong targeting of protein 2A, 3B(VPg), 3C(pro), and 3D(pol) signals to cellular nuclei, in particular to nucleoli, from the earliest times of infection . Viral capsid proteins (1AB, 1C, and 1D) and other non-structural proteins (2B, 2C, and 3A) did not target nuclei and remained cytoplasmic throughout the infection . The cardioviral 2A protein (subject of this article) has a novel 143 amino acid sequence, terminating in a 19 amino acid COOH-terminal processing cassette (PCC) that participates in autocatalytic, co-translational primary cleavage of the viral polyprotein . The remainder of the 2A protein shares only limited similarity with other viral or cellular sequences, except for a short motif (KRvRPFRLP) near PCC resembling the nuclear localization signals (NLS) common to many yeast ribosomal proteins . Deletions within the EMCV 2A protein that impinge on this region have been reported to diminish the ability of virus to inhibit cap-dependent translation of cellular mRNAs . We have now observed that these same deletions prevented nuclear localization . Cellular expression of 2A protein from RNA transcripts or cDNAs confirmed that it does not require other viral proteins or activities for nuclear transport; even when expressed as a single protein, 2A protein effectively shuts off translation from capped reporter mRNAs . Within infected, transfected, or DNA vector-transformed cells, the 2A protein was always found in close association with the nucleolar ribosomal chaperone protein B23, which may help the traffic 2A into nucleoli like a surrogate ribosomal protein, by virtue of the putative nucleolar localization signal (NoLS) . The data are consistent with a novel mechanism for virus-induced host protein shut off in cardioviruses, whereby 2A helps to upregulate the synthesis of new and modified ribosomes that have an inherent preference for internal ribosomal entry site (IRES)-dependent viral genome translation over cap-dependent host mRNA translation.

Prog Neuropsychopharmacol Biol Psychiatry, 2003 Aug, 27(5), 723 - 7
Possible role of 3'(2')-phosphoadenosine-5'-phosphate phosphatase in the etiology and therapy of bipolar disorder; Agam G et al.; Bipolar affective disorder (BPD) is a multifactorial, severe, chronic and disabling illness with 50% heritability that affects 1-2% of the population . Lithium ions (Li) are the drug of choice for BPD . Yet, 20-40% of patients fail to respond to Li . Although numerous biochemical and cellular effects have been attributed to Li, its therapeutic mechanism of action has not been elucidated . This review presents the possible involvement of 3'(2')-phosphoadenosine-5'-phosphate (PAP) phosphatase in the etiology of bipolar disorder and the mechanism of action of Li . Of the enzymes inhibited by Li, PAP phosphatase is inhibited with the lowest Ki (0.3 mM) . At therapeutic concentrations of Li (0.5-1.5 mM), inhibition is greater than 80% . Therefore, PAP phosphatase is a strong candidate for Li's therapeutic mechanism of action . In yeast, a PAP phosphatase knockout mutation leads to the accumulation of PAP, which affects ribosomal-, transfer- and small nucleolar-RNA processing . PAP accumulation in the mammalian brain following Li inhibition of PAP phosphatase may very well account for the observed effects of Li on gene expression and behavior . Furthermore, we have reported significant changes in PAP phosphatase levels in postmortem frontal cortex of bipolar patients.

Ying Yong Sheng Tai Xue Bao, 2003 Apr, 14(4), 627 - 31
{Physiological and molecular biological mechanisms of heavy metal absorption and accumulation in hyperaccumulators}; Li W et al.; In comparison with normal plants, hyperaccumulators have the ability to accumulate heavy metals in their shoots far exceeding those observed in soil, without suffering from detrimental effects . With the help of molecular technologies, the research on the mechanisms of heavy metal accumulation in hyperaccumulators has been made a great progress . A number of trace element transporters have been cloned by functional complementation with yeast mutants defective in metal absorption . The relations between glutathione, phytochelatins metallothioneins, organic acids and heavy metals have been studied by molecular technologies . This review concentrated on the physiological and molecular mechanisms of heavy metal absorption and sequestration in hyperaccumulators.

Pflugers Arch, 2004 Feb, 447(5), 807 - 12 Epub 2003 Aug 15.
Non-erythroid Rh glycoproteins: a putative new family of mammalian ammonium transporters; Nakhoul NL et al.; The Rhesus (Rh) glycoproteins, originally described in human blood cells, are mostly recognized for their immunogenic characteristics and importance in pregnancy . The Rh proteins in the red blood cell are expressed as an "Rh complex" made up of one D-subunit, one CE-subunit and two Rh-associated glycoprotein (RhAG) subunits . In addition to its antigenic property, the Rh complex is thought to contribute to membrane stability and structure of red blood cells . The exact function is yet to be determined . Recently, two non-erythroid Rh glycoproteins were cloned from mice (Rhcg and Rhbg) and humans (RhCG and RhBG) . RhCG is expressed at the membrane surface alone with no apparent need for heteromeric interaction with other glycoproteins . It is more similar to RhAG than to Rh CE/D, occurs late in development and is expressed abundantly and broadly in kidney and testis . In the kidney RhCG is localized to the apical cell membrane of the collecting duct . Rhbg and its human analog (RhBG) are expressed mainly in liver, skin and the kidney tubules . In the kidney collecting duct, Rhbg is localized to the basolateral membrane . Based on structural similarities to the methylammonium and ammonium permease/ammonium (MEP/Amt) transporters in yeast and their sequence homology, these proteins probably function as NH(4)(+) transporters . An initial study has indicated that RhAG or RhCG promote efflux of NH(4)(+), whereas another study has suggested that RhAG functions as an NH(4)(+)-H(+) exchanger . Evidence for such a function is still circumstantial and data indicating that Rh proteins function as NH(4)(+) transporters are indirect.

Theor Appl Genet, 2003 Nov, 107(8), 1402 - 9 Epub 2003 Aug 13.
A rice transcription factor OsbHLH1 is involved in cold stress response; Wang YJ et al.; Cold stress adversely affects plant growth and crop production . Some plants express a series of cold-responsive genes during cold acclimation to reduce the damage of cold stress . Among them, transcription factors play important roles in enhancing plant cold tolerance . A bHLH-type gene OsbHLH1 was isolated from rice . The predicted OsbHLH1 protein has a putative nuclear-localization signal and a putative DNA binding-domain bHLH-ZIP . The genomic sequence of the OsbHLH1 gene is unique in rice genome and has four introns . The transcription of the OsbHLH1 gene was specifically induced in roots of rice seedlings by cold but not by NaCl, PEG and ABA treatments . The OsbHLH1 protein was located in the nucleus of plant cells and had the ability to activate the transcription of the reporter gene in yeast . In addition, OsbHLH1 had the ability to dimerize . These results indicate that the OsbHLH1 may function as a transcription factor in a cold signal-transduction pathway.

J Biol Chem, 2003 Oct 31, 278(44), 43460 - 9 Epub 2003 Aug 14.
Interaction protein for cytohesin exchange factors 1 (IPCEF1) binds cytohesin 2 and modifies its activity; Venkateswarlu K; The ADP-ribosylation factor 6 (ARF6) small GTPase functions as a GDP/GTP-regulated switch in the pathways that stimulate actin reorganization and membrane ruffling . The formation of active ARF6GTP is stimulated by guanine nucleotide exchange factors (GEFs) such as cytohesins, which translocate to the plasma membrane in agonist-stimulated cells by binding the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate through the pleckstrin homology domain with subsequent ARF6 activation . Using cytohesin 2 as bait in yeast two-hybrid screening, we have isolated a cDNA encoding a protein termed interaction protein for cytohesin exchange factors 1 (IPCEF1) . Using yeast two-hybrid and glutathione S-transferase pull-down assays coupled with deletion mutational analysis, the specific domains required for the cytohesin 2-IPCEF1 interaction were mapped to the coiled-coil domain of cytohesin 2 and the C-terminal 121 amino acids of IPCEF1 . IPCEF1 also interacts with the other members of the cytohesin family of ARF GEFs, suggesting that the interaction with IPCEF1 is highly conserved among the cytohesin family of ARF GEFs . The interaction of cytohesin 2 and IPCEF1 in mammalian cells was demonstrated by immunoprecipitation . Immunofluorescence analysis revealed that IPCEF1 co-localizes with cytohesin 2 to the cytosol in unstimulated cells and translocates to the plasma membrane via binding to cytohesin 2 in epidermal growth factor-stimulated cells . However, a deletion mutant of IPCEF1 that lacks the cytohesin 2 binding site failed to co-migrate with cytohesin 2 to the membrane in stimulated cells . The functional significance of the IPCEF1-cytohesin 2 interaction is demonstrated by showing that IPCEF1 increases the in vitro and in vivo stimulation of ARFGTP formation by cytohesin 2.

J Biol Chem, 2003 Oct 24, 278(43), 42596 - 603 Epub 2003 Aug 14.
An SH2 domain-dependent, phosphotyrosine-independent interaction between Vav1 and the Mer receptor tyrosine kinase: a mechanism for localizing guanine nucleotide-exchange factor action; Mahajan NP et al.; Mer belongs to the Mer/Axl/Tyro3 receptor tyrosine kinase family, which regulates immune homeostasis in part by triggering monocyte ingestion of apoptotic cells . Mutations in Mer can also cause retinitis pigmentosa, again due to defective phagocytosis of apoptotic material . Although, some functional aspects of Mer have been deciphered, how receptor activation lead to the physiological consequences is not understood . By using yeast two-hybrid assays, we identified the carboxyl-terminal region of the guanine nucleotide-exchange factor (GEF) Vav1 as a Mer-binding partner . Unlike similar (related) receptors, Mer interacted with Vav1 constitutively and independently of phosphotyrosine, yet the site of binding localized to the Vav1 SH2 domain . Mer activation resulted in tyrosine phosphorylation of Vav1 and release from Mer, whereas Vav1 was neither phosphorylated nor released from kinase-dead Mer . Mutation of the Vav1 SH2 domain phosphotyrosine coordinating Arg-696 did not alter Mer/Vav1 constitutive binding or Vav1 tyrosine phosphorylation but did retard Vav1 release from autophosphorylated Mer . Ligand-dependent activation of Mer in human monocytes led to Vav1 release and stimulated GDP replacement by GTP on RhoA family members . This unusual constitutive, SH2 domain-dependent, but phosphotyrosine-independent, interaction and its regulated local release and subsequent activation of Rac1, Cdc42, and RhoA may explain how Mer coordinates precise cytoskeletal changes governing the ingestion of apoptotic material by macrophages and pigmented retinal epithelial cells.

Virology, 2003 Aug 1, 312(2), 381 - 94
Dual interaction of plant PCNA with geminivirus replication accessory protein (Ren) and viral replication protein (Rep); Castillo AG et al.; Geminiviruses replicate their small, single-stranded DNA genomes in plant nuclei using host replication machinery . Similar to most dicotyledonous plant-infecting geminiviruses, Tomato yellow leaf curl Sardinia virus (TYLCSV) encodes a protein, REn, that enhances viral DNA accumulation through an unknown mechanism . Earlier studies showed that REn protein from another geminivirus, Tomato golden mosaic virus (TGMV), forms oligomers and interacts with Rep protein, the only viral protein essential for replication . It has been shown that both proteins from TGMV also interact with a plant homolog of the mammalian tumor suppressor retinoblastoma protein (RBR) . By using yeast two-hybrid technology and the TYLCSV REn protein as bait, we have isolated three clones of the proliferating cell nuclear antigen (PCNA) of Arabidopsis thaliana, a ring-shaped protein that encircles DNA and plays an essential role in eukaryotic chromosomal DNA replication . We also demonstrate by the two-hybrid system and a pull-down assay that REn interacts with tomato PCNA (LePCNA) . Analysis of truncated proteins has located the REn-binding domain of LePCNA between amino acids 132 and 187, whereas all REn deletions used abolished or decreased dramatically its ability to interact with PCNA . Tomato PCNA also interacts with TYLCSV Rep . We propose that the interaction between PCNA and REn/Rep takes place during virus infection, inducing the assembly of the plant replication complex (replisome) close to the virus origin of replication.

Virology, 2003 Aug 1, 312(2), 306 - 19
Homotypic interactions of the infectious bursal disease virus proteins VP3, pVP2, VP4, and VP5: mapping of the interacting domains; Tacken MG et al.; Infectious bursal disease virus (IBDV), a nonenveloped double-stranded RNA virus of chicken, encodes five proteins . Of these, the RNA-dependent RNA polymerase (VP1) is specified by the smaller genome segment, while the large segment directs synthesis of a nonstructural protein (VP5) and a structural protein precursor from which the capsid proteins pVP2 and VP3 as well as the viral protease VP4 are derived . Using the recently redefined processing sites of the precursor, we have reevaluated the homotypic interactions of the viral proteins using the yeast two-hybrid system . Except for VP1, which interacted weakly, all proteins appeared to self-associate strongly . Using a deletion mutagenesis approach, we subsequently mapped the interacting domains in these polypeptides, where possible confirming the observations made in the two-hybrid system by performing coimmunoprecipitation analyses of tagged protein constructs coexpressed in avian culture cells . The results revealed that pVP2 possesses multiple interaction domains, consistent with available structural information about this external capsid protein . VP3-VP3 interactions were mapped to the amino-terminal part of the polypeptide . Interestingly, this domain is distinct from two other interaction domains occurring in this internal capsid protein: while binding to VP1 has been mapped to the carboxy-terminal end of the protein, interaction with the genomic dsRNA segments has been suggested to occur just upstream thereof . No interaction sites could be assigned to the VP4 protein; any deletion applied abolished its self-association . Finally, one interaction domain was detected in the central, most hydrophobic region of VP5, supporting the idea that this virulence determinant may function as a membrane pore-forming protein in infected cells.

Anal Chem, 2003 May 15, 75(10), 2309 - 15
An automated noncontact deposition interface for liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry; Ericson C et al.; A new multichannel deposition system was developed for off-line liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry (LC/MALDI-MS) . This system employs a pulsed electric field to transfer the eluents from multiple parallel columns directly onto MALDI targets without the column outlets touching the target surface . The deposition device performs well with a wide variety of solvents that have different viscosities, vapor pressures, polarities, and ionic strengths . Surface-modified targets were used to facilitate concentration and precise positioning of samples, allowing for efficient automation of high-throughput MALDI analysis . The operational properties of this system allow the user to prepare samples using MALDI matrixes whose properties range from hydrophilic to hydrophobic . The latter, exemplified by alpha-cyano-4-hydroxycinnamic acid, were typically processed with a multistep deposition method consisting of precoating of individual spots on the target plate, sample deposition, and sample recrystallization steps . Using this method, 50 amol of angiotensin II was detected reproducibly with high signal-to-noise ratio after LC separation . Experimental results show that there is no significant decrease in chromatographic resolution using this device . To assess the behavior of the apparatus for complex mixtures, 5 microg of a tryptic digest of the cytosolic proteins of yeast was analyzed by LC/MALDI-MS and more than 13,500 unique analytes were detected in a single LC/MS analysis.

Oncogene, 2003 Aug 14, 22(34), 5362 - 6
Grap-2, a novel RET binding protein, is involved in RET mitogenic signaling; Ludwig L et al.; Signal transduction of the RET receptor tyrosine kinase is involved in developmental processes as well as in neoplastic transformation . Activation of RET initiates receptor autophosphorylation on specific tyrosines that act as docking sites for downstream signaling molecules . Using the cytoplasmatic part of RET as bait in a yeast two-hybrid screen, we identified a novel SH2 and SH3 domain containing adaptor protein previously termed Grap-2/Grf40/GrpL/GRID and its murine homologue as Gads/Mona, respectively . This protein, predominantly expressed in cells of hematopoietic origin, is involved in signaling downstream of the T-cell receptor and the receptor for monocyte colony-stimulating factor . Here, we show that Grap-2 is also expressed in neuroendocrine tumors and cell lines known to bear mutated forms of RET . Endogenously expressed RET and Grap-2 coimmunoprecipitate from lysates of a medullary thyroid carcinoma cell line . Grap-2 directly associates with RET in pull-down experiments using in vitro translated proteins . Overexpression of Grap-2 inhibits RET-induced NF-kappaB activation, and cotransfection of Grap-2 significantly reduces focus formation induced by oncogenic RET in NIH 3T3 cells . Taken together, these results suggest that besides being involved in tyrosine kinase signaling in hematopoietic cells, Grap-2 plays a tissue-specific role as an inhibitor of RET mitogenic signaling.

Oncogene, 2003 Aug 14, 22(34), 5221 - 8
A porphobilinogen deaminase (PBGD) Ran-binding protein interaction is implicated in nuclear trafficking of PBGD in differentiating glioma cells; Greenbaum L et al.; Porphobilinogen deaminase (PBGD) is a rate-limiting enzyme of the heme biosynthesis pathway, whose level is elevated in various human tumors . PBGD was observed in both nuclear and cytoplasmic fractions of C6 glioma cells by immunostaining . During mitosis, chromatids were intensely stained for PBGD in comparison to the interphase chromatin . Using the yeast two-hybrid system, we identified RanBPM, the nuclear Ran-binding protein, as an interacting partner of PBGD . During butyrate-induced differentiation of C6, both nuclear and cytoplasmic PBGD levels declined as did Ran protein and its nucleotide exchange factor RCC1 . N,N'-hexamethylene bis-acetamide-dependent differentiation resulted in an increase of the cytoplasmic PBGD, whereas nuclear PBGD, Ran protein and RCC1 remained unchanged . mRNA levels of PBGD remained unchanged during stimulation with both butyrate and N,N'-hexamethylene bis-acetamide . The enzymatic activity of PBGD and protoporphyrin IX synthesis in C6 cells were dependent on the differentiation induction agent . We conclude that PBGD possibly has a nuclear role in addition to its cytosolic enzymatic activity required for heme synthesis, which is related to cell transformation and differentiation.

J Gen Virol, 2003 Sep, 84(Pt 9), 2317 - 22
Equine arteritis virus non-structural protein 1, an essential factor for viral subgenomic mRNA synthesis, interacts with the cellular transcription co-factor p100; Tijms MA et al.; Non-structural protein 1 (nsp1), the N-terminal subunit of the replicase polyprotein of the arterivirus Equine arteritis virus (EAV), is essential for viral subgenomic mRNA synthesis, but fully dispensable for genome replication . However, at the molecular level, the role of nsp1 in EAV subgenomic mRNA synthesis is poorly understood . A yeast two-hybrid screen did not reveal interactions between EAV nsp1 and other viral non-structural proteins or the nucleocapsid protein, although both nsp1 and the nucleocapsid protein were found to form homomers . Subsequently, a yeast two-hybrid screen of a HeLa cell cDNA library was performed using nsp1 as bait . Remarkably, this analysis revealed (potential) interactions between EAV nsp1 and factors that are involved in host cell transcriptional regulation . The interaction of nsp1 with one of these proteins, p100, a transcription co-activator that also interacts with regulatory proteins of other viruses, was confirmed by mutual co-immunoprecipitation from lysates of EAV-susceptible mammalian cells.

J Biol Chem, 2003 Oct 24, 278(43), 42064 - 71 Epub 2003 Aug 13.
Signal-anchor domains of proteins of the outer membrane of mitochondria: structural and functional characteristics; Waizenegger T et al.; We have studied the topogenesis of a class of mitochondrial outer membrane proteins that expose a hydrophilic domain to the cytosol and are anchored to the membrane by a single transmembrane domain in the N-terminal region . To determine the role of these latter sequences in the targeting and insertion of such proteins we took two approaches . First, a functional complementation assay was used to define the structural elements that together with the anchor domain make up the topogenic signal . Moderate hydrophobicity of the transmembrane domain was found to be the most important requirement . Variants with a scrambled sequence of the membrane-spanning segment were only partially functional suggesting that specificity in the amino acid sequence is also of considerable importance . A net positive charge at both flanking regions of the transmembrane domain contributes to the efficiency of targeting and membrane integration but is not an essential structural feature of this signal . Second, chimeras of Tom20, Tom70, and OM45 were generated that contained the cytosolic domain of Tom20 or Tom70 and the anchor domain of one of the other members of the class . These hybrid proteins were able to rescue the growth of cells lacking Tom20 or Tom70 . Thus, anchor domains of outer membrane proteins are functionally interchangeable . They play only a minor role in the specific function of these proteins, but have a decisive role in topogenic signaling.

Mol Cell Biol, 2003 Sep, 23(17), 6150 - 8
Retention but not recruitment of Crb2 at double-strand breaks requires Rad1 and Rad3 complexes; Du LL et al.; The fission yeast checkpoint protein Crb2, related to budding yeast Rad9 and human 53BP1 and BRCA1, has been suggested to act as an adapter protein facilitating the phosphorylation of specific substrates by Rad3-Rad26 kinase . To further understand its role in checkpoint signaling, we examined its localization in live cells by using fluorescence microscopy . In response to DNA damage, Crb2 localizes to distinct nuclear foci, which represent sites of DNA double-strand breaks (DSBs) . Crb2 colocalizes with Rad22 at persistent foci, suggesting that Crb2 is retained at sites of DNA damage during repair . Damage-induced Crb2 foci still form in cells defective in Rad1, Rad3, and Rad17 complexes, but these foci do not persist as long as in wild-type cells . Our results suggest that Crb2 functions at the sites of DNA damage, and its regulated persistent localization at damage sites may be involved in facilitating DNA repair and/or maintaining the checkpoint arrest while DNA repair is under way.

Mol Cell Biol, 2003 Sep, 23(17), 6000 - 12
Expression of MIS in the testis is downregulated by tumor necrosis factor alpha through the negative regulation of SF-1 transactivation by NF-kappa B; Hong CY et al.; The expression of Mullerian inhibiting substance (MIS), a key molecule in sex differentiation and reproduction, is tightly regulated . It has been suggested that meiotic germ cells repress MIS expression in testicular Sertoli cells, although the substance responsible for this cell-cell communication remains unknown . Here, we present the cytokine tumor necrosis factor alpha (TNF-alpha) as a strong candidate for such a substance and its downstream molecular events . TNF-alpha inhibited MIS expression in testis organ cultures, and TNF-alpha(-/-) testes showed high and prolonged MIS expression . Furthermore, in transient-transfection assays TNF-alpha suppressed the MIS promoter that was activated by steroidogenic factor 1 (SF-1), one of the major transcription factors that regulate MIS expression . The modulation of SF-1 transactivation by TNF-alpha is through the activation of NF-kappa B, which subsequently interacts with SF-1 and represses its transactivation . The physical association of NF-kappa B with SF-1 was shown by yeast two-hybrid protein interaction, glutathione S-transferase pull-down, and coimmunoprecipitation (ChIP) analyses . ChIP assays also revealed that endogenous NF-kappa B, as well as SF-1, is recruited to the MIS promoter upon TNF-alpha signaling . SF-1-bound NF-kappa B subsequently recruits histone deacetylases to inhibit the SF-1-activated gene expression . These results may identify, for the first time, the responsible substance and its action mechanism underlying the repression of MIS expression by meiotic germ cells in the testis.

Development, 2003 Oct, 130(20), 4859 - 69 Epub 2003 Aug 13.
A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis; Zhang F et al.; GLABRA3 (GL3) encodes a bHLH protein that interacts with the WD repeat protein, TTG1 . GL3 overexpression suppresses the trichome defect of the pleiotropic ttg1 mutations . However, single gl3 mutations only affect the trichome pathway with a modest trichome number reduction . A novel unlinked bHLH-encoding locus is described here, ENHANCER OF GLABRA3 (EGL3) . When mutated, egl3 gives totally glabrous plants only in the gl3 mutant background . The double bHLH mutant, gl3 egl3, has a pleiotropic phenotype like ttg1 having defective anthocyanin production, seed coat mucilage production, and position-dependent root hair spacing . Furthermore, the triple bHLH mutant, gl3 egl3 tt8, phenocopies the ttg1 mutation . Yeast two-hybrid and plant overexpression studies show that EGL3, like GL3, interacts with TTG1, the myb proteins GL1, PAP1 and 2, CPC and TRY, and it will form heterodimers with GL3 . These results suggest a combinatorial model for TTG1-dependent pathway regulation by this trio of partially functionally redundant bHLH proteins.

Cancer Epidemiol Biomarkers Prev, 2003 Aug, 12(8), 809 - 12
Common polymorphisms in checkpoint kinase 2 are not associated with breast cancer risk; Kuschel B et al.; A substantial proportion of the familial risk of breast cancer may be attributable to genetic variants each contributing a small effect . Polymorphisms in DNA repair genes are good candidates for such low penetrance breast cancer susceptibility alleles . Checkpoint kinase 2 (CHEK2) is a kinase in which the yeast counterpart regulates a cell cycle checkpoint and causes cells to arrest proliferation after DNA damage . A rare, protein truncating mutation in the CHEK2 gene has recently been shown to confer a modest risk of breast cancer . The aim of this study was to determine whether common polymorphic variants in CHEK2 are associated with an increase in breast cancer risk . We assessed two variants in CHEK2 using a case control study design (n = 1786 cases and 1828 controls) . No differences in genotype frequencies were found between cases and control for either the IVS1 + 38insa or the a1013g polymorphisms (P = 0.3 and 0.2 respectively), and no genotype-specific risk was significantly different from unity . The haplotype frequency distribution in cases and controls were also similar (P = 0.3) . We conclude that the CHEK2 polymorphisms IVS + 1a and a1013g do not confer an increased risk of breast cancer . It is also unlikely that other, as yet unidentified, common polymorphisms that affect risk are present in the gene in the British population.

Am J Physiol Cell Physiol, 2003 Dec, 285(6), C1494 - 503 Epub 2003 Aug 13.
Localization and interaction of NHERF isoforms in the renal proximal tubule of the mouse; Wade JB et al.; In expression systems and in yeast, Na/H exchanger regulatory factor (NHERF)-1 and NHERF-2 have been demonstrated to interact with the renal brush border membrane proteins NHE3 and Npt2 . In renal tissue of mice, however, NHERF-1 is required for cAMP regulation of NHE3 and for the apical targeting of Npt2 despite the presence of NHERF-2, suggesting another order of specificity . The present studies examine the subcellular location of NHERF-1 and NHERF-2 and their interactions with target proteins including NHE3, Npt2, and ezrin . The wild-type mouse proximal tubule expresses both NHERF-1 and NHERF-2 in a distinct pattern . NHERF-1 is strongly expressed in microvilli in association with NHE3, Npt2, and ezrin . Although NHERF-2 can be detected weakly in the microvilli, it is expressed predominantly at the base of the microvilli in the vesicle-rich domain . NHERF-2 appears to associate directly with ezrin and NHE3 but not Npt2 . NHERF-1 is involved in the apical expression of Npt2 and the presence of other Npt2-binding proteins does not compensate totally for the absence of NHERF-1 in NHERF-1-null mice . Although NHERF-1 links NHE3 to the actin cytoskeleton through ezrin, the absence of NHERF-1 does not result in a generalized disruption of the architecture of the cell . Thus the mistargeting of Npt2 seen in NHERF-1-null mice likely represents a specific disruption of pathways mediated by NHERF-1 to achieve targeting of Npt2 . These findings suggest that the organized subcellular distribution of the NHERF isoforms may play a role in the specific interactions mediating physiological control of transporter function.

Chronobiol Int, 2003 Jul, 20(4), 543 - 58
Surface plasmon resonance spectroscopy (SPR) interaction studies of the circadian-controlled tomato LHCa4*1 (CAB 11) protein with its promoter; Hoffrogge R et al.; Feedback regulation is an important biochemical mechanism which is also able to direct the circadian timing at the transcriptional level . Independent investigations highlighted a conserved ca . 10 nucleotide motif present in many circadian regulated Lhc genes . Two of such nucleotide motifs exist within 119 nucleotides of the Lhca4*1 promoter from tomato . This promoter fragment was used as a bait in a yeast one hybrid screen and interestingly a clone encoding with sequence identity to the LHCa4*1 protein was isolated as an interaction partner . The LHCa4*1 protein was heterologous expressed and binding to the 119bp promoter fragment was demonstrated by surface plasmon resonance spectroscopy (SPR, Biacore) . This result allows to postulate an autoregulatory feedback loop involved in expression of the Lhca4*1 gene.

Phytother Res, 2003 Aug, 17(7), 804 - 6
Evaluation of antipyretic potential of Vernonia cinerea extract in rats; Gupta M et al.; The methanol extract of the whole plant of Vernonia cinerea (MEVC) was evaluated for its antipyretic potential on normal body temperature and yeast-induced pyrexia in rats . MEVC significantly reduced the normal body temperature at doses of 250 and 500 mg/kg body weight p.o . MEVC also lowered the elevated body temperature in the case of yeast-induced pyrexia in a dose dependent manner . The antipyretic effect of the extract at a dose of 500 mg/kg was identical to that of the standard drug paracetamol .

Hum Mol Genet, 2003 Oct 1, 12(19), 2503 - 10 Epub 2003 Aug 05.
Direct interaction of the Fanconi anaemia protein FANCG with BRCA2/FANCD1; Hussain S et al.; Fanconi anaemia (FA) is an autosomal recessive genetic disorder characterized by progressive bone marrow failure, multiple congenital abnormalities, and an increased risk of cancer . FA cells are characterized by chromosomal instability and hypersensitivity to DNA interstrand crosslinking agents . At least eight complementation groups exist (FA-A to G), and the genes for all of these except FA-B have been cloned . Functional linkage between the FA pathway and genes involved in susceptibility to breast cancer has been demonstrated by the interaction of the FANCA and FANCD2 proteins with BRCA1, and the discovery that the FANCD1 gene is identical to BRCA2 . Here we have used the yeast two-hybrid system to test for direct interaction between BRCA2 or its effector RAD51 and the FANCA, FANCC and FANCG proteins . We found that FANCG was capable of binding to two separate sites in the BRCA2 protein, located either side of the BRC repeats . Furthermore, FANCG could be co-immunoprecipitated with BRCA2 from human cells, and FANCG co-localized in nuclear foci with both BRCA2 and RAD51 following DNA damage with mitomycin C . These results demonstrate that BRCA2 is directly connected to a pathway that is deficient in interstrand crosslink repair, and that at least one other FA protein is closely associated with the homologous recombination DNA repair machinery.

Hum Mol Genet, 2003 Sep 15, 12(18), 2359 - 68 Epub 2003 Jul 29.
The Lafora disease gene product laforin interacts with HIRIP5, a phylogenetically conserved protein containing a NifU-like domain; Ganesh S et al.; Lafora disease is an autosomal recessive type of progressive myoclonus epilepsy caused by mutations in the EPM2A gene . The EPM2A gene-encoded protein laforin is a dual-specificity phosphatase that associates with polyribosomes . Because the cellular functions of laforin are largely unknown, we used the yeast-two hybrid system to screen for protein(s) that interact with laforin . We found that laforin interacts with a phylogenetically conserved protein HIRIP5 that harbors a NifU-like domain . Both in vitro and in vivo assay have shown that the interaction is specific and that laforin probably uses its N-terminal CBD-4 domain to interact with the C-terminal NifU-like domain of the HIRIP5 protein . HIRIP5 encodes a cytosolic protein and is expressed ubiquitously, perhaps reflecting a house-keeping function . The presence of a NifU-like domain in the HIRIP5 protein raises an interesting possibility that it may be involved in iron homeostasis . Although the significance of the interaction between HIRIP5 and laforin proteins is not yet fully known, because laforin dephosphorylated HIRIP5 in vitro, HIRIP5 promises to be an interesting laforin-binding partner and would contribute to the understanding of the molecular pathology of Lafora disease.

Biochim Biophys Acta, 2003 Aug 18, 1641(2-3), 195 - 202
Mitochondrial membrane fusion; Westermann B; Mitochondrial fusion has been observed in a great variety of organisms from yeast to man . It serves to mix and unify the mitochondrial compartment and plays roles in cellular aging, cell development, energy dissipation and mitochondrial DNA inheritance . Large GTPases in the mitochondrial outer membrane, termed Fzo or mitofusins, have been identified as key components of the mitochondrial fusion machinery in yeast, flies and mammalian cells . Recent studies in yeast suggest an involvement of a dynamin-related protein in the intermembrane space . Additional components have been identified by genetic screens . These findings suggest a unique and evolutionarily conserved mechanism for mitochondrial membrane fusion.

Biochim Biophys Acta, 2003 Aug 18, 1641(2-3), 137 - 43
Calcium and calmodulin in membrane fusion; Burgoyne RD et al.; Regulated exocytosis was the first intracellular membrane fusion step that was suggested to involve both Ca(2+) and calmodulin . In recent years, it has become clear that calmodulin is not an essential Ca(2+) sensor for exocytosis but that it is likely to have a more regulatory role . A requirement for cytosolic Ca(2+) in other vesicle fusion events within cells has become apparent and in certain cases, such as homotypic fusion of early endosomes and yeast vacuoles, calmodulin may be the primary Ca(2+) sensor . A number of distinct targets for calmodulin have been identified including SNARE proteins and subunits of the vacuolar ATPase . The extent to which calmodulin regulates different intracellular fusion events through conserved SNARE-dependent or other mechanisms remains to be resolved.

Biochim Biophys Acta, 2003 Aug 18, 1641(2-3), 111 - 9
Control of eukaryotic membrane fusion by N-terminal domains of SNARE proteins; Dietrich LE et al.; SNARE proteins function at the center of membrane fusion reactions by forming complexes with each other via their coiled-coil domains . Several SNAREs have N-terminal domains (NTDs) that precede the coiled-coil domain and have critical functions in regulating the fusion cascade . This review will highlight recent findings on NTDs of syntaxins, the longin domain of VAMP proteins and SNAP-23/25 homologues in yeast . Biochemical and genetic experiments as well as the resolution of several NMR and crystal structures of SNARE NTDs shed light on their diverse function.

FEBS Lett, 2003 Aug 14, 549(1-3), 1 - 6
Macropexophagy in Hansenula polymorpha: facts and views; Kiel JA et al.; The hallmark of eukaryotic cells is compartmentalization of distinct cellular functions into specific organelles . This necessitates the cells to run energetically costly mechanisms to precisely control maintenance and function of these compartments . One of these continuously controls organelle activity and abundance, a process termed homeostasis . Yeast peroxisomes are favorable model systems for studies of organelle homeostasis because both the proliferation and degradation of these organelles can be readily manipulated . Here, we highlight recent achievements in regulation of peroxisome turnover in yeast, in particular Hansenula polymorpha, with a focus on directions of future research.

Cell, 2003 Aug 8, 114(3), 299 - 310
Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi; Wang YJ et al.; Phosphatidylinositol 4 phosphate {PI(4)P} is essential for secretion in yeast, but its role in mammalian cells is unclear . Current paradigms propose that PI(4)P acts primarily as a precursor to phosphatidylinositol 4,5 bisphosphate (PIP2), an important plasma membrane regulator . We found that PI(4)P is enriched in the mammalian Golgi, and used RNA interference (RNAi) of PI4KIIalpha, a Golgi resident phosphatidylinositol 4 kinase, to determine whether PI(4)P directly regulates the Golgi . PI4KIIalpha RNAi decreases Golgi PI(4)P, blocks the recruitment of clathrin adaptor AP-1 complexes to the Golgi, and inhibits AP-1-dependent functions . This AP-1 binding defect is rescued by adding back PI(4)P . In addition, purified AP-1 binds PI(4)P, and anti-PI(4)P inhibits the in vitro recruitment of cytosolic AP-1 to normal cellular membranes . We propose that PI4KIIalpha establishes the Golgi's unique lipid-defined organelle identity by generating PI(4)P-rich domains that specify the docking of the AP-1 coat machinery.

Curr Neurol Neurosci Rep, 2003 Sep, 3(5), 423 - 32
Mitochondrial disorders; Zeviani M et al.; We present here a discussion on the most relevant recent publications on mitochondrial disease . In addition to many papers concerning the description of the genotype-to-phenotype correlations in mitochondrial DNA-related disorders, this very broad area of neurogenetics includes a number of novel observations on the basic aspects of mitochondrial biogenesis that can be relevant in explaining the molecular mechanisms of mitochondrial abnormalities . The completion of the human genome project and the wealth of knowledge gained on the genetics of oxidative phosphorylation in yeast have promoted a substantial acceleration in the discovery of a remarkable number of nuclear genes associated with specific mitochondrial disorders . A further development of these contributions has been the generation of several cellular and animal models of disease that can now be exploited for testing both pathogenetic hypotheses and therapeutic strategies . Most of the latter are based on the use of chemical compounds aimed at reducing the negative impact of mitochondrial defects on both energy production and generation of reactive oxygen species . The first successful attempts for gene therapy of some mitochondrial diseases have recently been achieved and will hopefully increase in the near future.

Biosci Biotechnol Biochem, 2003 Jul, 67(7), 1465 - 71
Production of galactinol from sucrose by plant enzymes; Wakiuchi N et al.; Galactinol, 1-O-(alpha-D-galactopyranosyl)-myo-inositol, was produced from sucrose as a starting material . UDP-Glc was prepared with sucrose and UDP using sucrose synthase partially purified from sweet potato roots . Then, the UDP-Glc was converted to UDP-Gal using yeast UDP-Gal 4-epimerase from a commercial source . Finally, galactinol was produced from the UDP-Gal and myo-inositol using galactinol synthase partially purified from cucumber leaves . The product was identified as galactinol by the retention times of HPLC, alpha-galactosidase digestion, and NMR spectrometry.

Plant Physiol, 2003 Aug, 132(4), 1950 - 60
Peptide and amino acid transporters are differentially regulated during seed development and germination in faba bean; Miranda M et al.; Two peptide transporter (PTR) homologs have been isolated from developing seeds of faba bean (Vicia faba) . VfPTR1 was shown to be a functional peptide transporter through complementation of a yeast mutant . Expression patterns of VfPTR1 and VfPTR2 as well as of the amino acid permease VfAAP1 (Miranda et al., 2001) were compared throughout seed development and germination . In developing seeds, the highest levels of VfPTR1 transcripts were reached during midcotyledon development, whereas VfAAP1 transcripts were most abundant during early cotyledon development, before the appearance of storage protein gene transcripts, and were detectable until late cotyledon development . During early germination, VfPTR1 mRNA appeared first in cotyledons and later, during seedling growth, also in axes and roots . Expression of VfPTR2 and VfAAP1 was delayed compared with VfPTR1, and was restricted to the nascent organs of the seedlings . Localization of VfPTR1 transcripts showed that this PTR is temporally and spatially regulated during cotyledon development . In germinating seeds, VfPTR1 mRNA was localized in root hairs and root epidermal cells, suggesting a role in nutrient uptake from the soil . In seedling roots, VfPTR1 was repressed by a dipeptide and by an amino acid, whereas nitrate was without influence.

Plant Physiol, 2003 Aug, 132(4), 1861 - 9
Hyperphosphorylation of a mitochondrial protein, prohibitin, is induced by calyculin A in a rice lesion-mimic mutant cdr1; Takahashi A et al.; The rice (Oryza sativa) lesion-mimic mutants, cell death and resistance (cdr), show spontaneous cell death on the entire leaf and exhibited significant resistance to the rice blast fungus . Our previous studies showed that CDR1 and CDR2 genes negatively regulated the phosphorylation steps leading to the activation of NADPH oxidase, which is associated with oxidative burst . To identify novel factors involved in the phosphorylation steps, the phosphorylation level of total proteins was compared between cdr mutants and wild type using two-dimensional gel electrophoresis . Here, we show that the phosphorylation level of four proteins in cdr1 was increased as compared with the wild type after calyculin A treatment . Partial amino acid sequences revealed that one of the four proteins is homologous to prohibitin (PHB), which has been shown to be associated with senescence and cell death and to function as a chaperone in the assembly of mitochondrial respiratory chain complex in yeast and mammals . Analysis of green fluorescent protein fusions indicated that rice PHB (OsPHB1) was targeted to mitochondria as found in yeast and mammals, suggesting a possibility that PHB is involved in defense response and/or programmed cell death through the mitochondrial function.

Proc Natl Acad Sci U S A, 2003 Aug 19, 100(17), 10008 - 13 Epub 2003 Aug 11.
A nuclear target for interleukin-1alpha: interaction with the growth suppressor necdin modulates proliferation and collagen expression; Hu B et al.; There is growing evidence for the intracellular role of cytokines and growth factors, but the pathways by which these activities occur remain largely obscure . Previous work from our laboratory identified the constitutive, aberrant expression of the 31-kDa IL-1 alpha precursor (pre-IL-1 alpha) in the nuclei of fibroblasts from the lesional skin of patients with systemic sclerosis (SSc) . We established that pre-IL-1 alpha expression was associated with increased fibroblast proliferation and collagen production . Further investigation has led to the identification of a mechanism by which nuclear expression of pre-IL-1 alpha affects fibroblast growth and matrix production . By using a yeast two-hybrid method, we found that pre-IL-1 alpha binds necdin, a nuclear protein with growth suppressor activity . We mapped the region of pre-IL-1 alpha responsible for necdin binding and found it to be localized near the N terminus, a region that is present on pre-IL-1 alpha, but not the mature 17-kDa cytokine . Expression studies demonstrated that pre-IL-1 alpha associates with necdin in the nuclei of mammalian cell lines and regulates cell growth and collagen expression . Our results provide the first evidence, to our knowledge, of a nuclear target for pre-IL-1 alpha . Based on these findings, we propose that the constitutively up-regulated expression of pre-IL-1 alpha in the nuclei of SSc fibroblasts up-regulates proliferation and matrix production of SSc fibroblasts through binding necdin, and by counteracting its effects on cell growth and collagen production.

Hum Mol Genet, 2003 Aug 15, 12(16), 1981 - 93
Fgd1, the Cdc42 GEF responsible for Faciogenital Dysplasia, directly interacts with cortactin and mAbp1 to modulate cell shape; Hou P et al.; FGD1 mutations result in Faciogenital Dysplasia (FGDY), an X-linked human disease that affects skeletal formation and embryonic morphogenesis . FGD1 and Fgd1, the mouse FGD1 ortholog, encode guanine nucleotide exchange factors (GEF) that specifically activate Cdc42, a Rho GTPase that controls the organization of the actin cytoskeleton . To further understand FGD1/Fgd1 signaling and begin to elucidate the molecular pathophysiology of FGDY, we demonstrate that Fgd1 directly interacts with cortactin and mouse actin-binding protein 1 (mAbp1), actin-binding proteins that regulate actin polymerization through the Arp2/3 complex . In yeast two-hybrid studies, cortactin and mAbp1 Src homology 3 (SH3) domains interact with a single Fgd1 SH3-binding domain (SH3-BD), and biochemical studies show that the Fgd1 SH3-BD directly binds to cortactin and mAbp1 in vitro . Immunoprecipitation studies show that Fgd1 interacts with cortactin and mAbp1 in vivo and that Fgd1 SH3-BD mutations disrupt binding . Immunocytochemical studies show that Fgd1 colocalizes with cortactin and mAbp1 in lamellipodia and membrane ruffles, and that Fgd1 subcellular targeting is dynamic . By using truncated cortactin proteins, immunocytochemical studies show that the cortactin SH3 domain targets Fgd1 to the subcortical actin cytoskeleton, and that abnormal Fgd1 localization results in actin cytoskeletal abnormalities and significant changes in cell shape and viability . Thus, this study provides novel in vitro and in vivo evidence that Fgd1 specifically and directly interacts with cortactin and mAbp1, and that these interactions play an important role in regulating the actin cytoskeleton and, subsequently, cell shape.

J Biol Chem, 2003 Oct 31, 278(44), 43452 - 9 Epub 2003 Aug 11.
Two mammalian longevity assurance gene (LAG1) family members, trh1 and trh4, regulate dihydroceramide synthesis using different fatty acyl-CoA donors; Riebeling C et al.; Overexpression of upstream of growth and differentiation factor 1 (uog1), a mammalian homolog of the yeast longevity assurance gene (LAG1), selectively induces the synthesis of stearoyl-containing sphingolipids in mammalian cells (Venkataraman, K., Riebeling, C., Bodennec, J., Riezman, H., Allegood, J . C., Sullards, M . C., Merrill, A . H . Jr., and Futerman, A . H . (2002) J . Biol . Chem . 277, 35642-35649) . Gene data base analysis subsequently revealed a new subfamily of proteins containing the Lag1p motif, previously characterized as translocating chain-associating membrane (TRAM) protein homologs (TRH) . We now report that two additional members of this family regulate the synthesis of (dihydro)ceramides with specific fatty acid(s) when overexpressed in human embryonic kidney 293T cells . TRH1 or TRH4-overexpression elevated {3H}(dihydro)ceramide synthesis from l-{3-3H}serine and the increase was not blocked by the (dihydro)ceramide synthase inhibitor, fumonisin B1 (FB1) . Analysis of sphingolipids by liquid chromatography-electrospray tandem mass spectrometry revealed that TRH4 overexpression elevated mainly palmitic acid-containing sphingolipids whereas TRH1 overexpression increased mainly stearic acid and arachidic acid, which in both cases were further elevated upon incubation with FB1 . A similar fatty acid specificity was obtained upon analysis of (dihydro)ceramide synthase activity in vitro using various fatty acyl-CoA substrates, although in a FB1-sensitive manner . Moreover, in homogenates from TRH4-overexpressing cells, sphinganine, rather than sphingosine was the preferred substrate, whereas no preference was seen in homogenates from TRH1-overexpressing cells . These findings lend support to our hypothesis (Venkataraman, K., and Futerman, A . H . (2002) FEBS Lett . 528, 3-4) that Lag1p family members regulate (dihydro)ceramide synthases responsible for production of sphingolipids containing different fatty acids.

J Appl Microbiol, 2003, 95(3), 457 - 62
Flocculation and cell surface characterization of Kloeckera apiculata from wine; Farias ME et al.; AIMS: To characterize and analyze the flocculation phenomenon of Kloeckera apiculata mc1 from Argentinian wine to understand the cell-cell interaction pattern . METHODS AND RESULTS: Kloeckera apiculata mc1 possess intense cell-cell interactions in MYPG medium (0.5% malt extract, 1% yeast extract, 2% glucose, 2% peptone), pH 5.5 by shaking at 25 degrees C . Optimum flocculation is observed at pH 4.5 in the presence of 3 mmol l-1 Ca2+ . The flocculation is induced by peptone and malt extract and not by yeast extract and is reversed by 50 mmol l-1 galactose or lactose . The flocculation is highly susceptible to pronase, chymotrypsine and proteases types IV and XXVII and is partially resistant to trypsin . The electronic microscopy shows that the cells are attached to each other along their sides by fine hair-like threads . CONCLUSIONS: The mechanism of flocculation of K . apiculata mc1 is mediated by protein-carbohydrate interaction, stabilized by Ca2+ . SIGNIFICANCE AND IMPACT OF THE STUDY: The use of selected pure yeast inocula of known ability is preferred to wine elaboration, therefore the indigenous flora must be avoided and the flocculation of K . apiculata could be an economic method to do it.

Cell Biochem Funct, 2003 Sep, 21(3), 263 - 7
Analysis of intracellular distribution and apoptosis involvement of the Ufd1l gene product by over-expression studies; Amati F et al.; UFD1L is the human homologue of the yeast ubiquitin fusion degradation 1 (Ufd1) gene and maps on chromosome 22q11.2 in the typically deleted region (TDR) for DiGeorge/velocardiofacial syndromes (DGS/VCFS) . In yeast, Ufd1 protein is involved in a degradation pathway for ubiquitin fused products (UFD pathway) . Several studies have demonstrated that Ufd1 is a component of the Cdc48-Ufd1-Npl4 multiprotein complex which is active in the recognition of several polyubiquitin-tagged proteins and facilitates their presentation to the 26S proteasome for protein degradation or even more specific processing . The multiprotein complex Cdc48-Ufd-Npl4 is also active in mammalian cells . The biochemical role of UFD1L protein in human cells is unknown, even though the interaction between UFD1L and NPL4 proteins has been maintained . In order to clarify this issue, we examined the intracellular distribution of the protein in different mammalian cells and studied its involvement in the Fas and ceramide factors-mediated apoptotic pathways . We established that in mammalian cells, Ufd1l is localized around the nucleus and that it does not interfere with Fas-and ceramide-mediated apoptosis .

Support Care Cancer, 2003 Nov, 11(11), 717 - 21 Epub 2003 Aug 09.
Long-term oral Candida colonization, mucositis and salivary function after head and neck radiotherapy; Grotz KA et al.; The aim of this study was to follow the long-term effects of radiation therapy of head and neck malignancies on oral yeast colonization, mucositis and salivary function . Included in this prospective study were 32 patients with intended radiation therapy of a malignancy of the head and neck . In all patients the salivary glands lay within the radiation field and the patients had at least five teeth . The first examination was performed after oral hygiene instruction and removal of questionable teeth before the start of radiotherapy . The following examinations were conducted after 3, 6, 9 and 12 months . Together with the quantitative determination of Candida colonization, three "mucositis" variables were assessed: (1) examiner-rated mucositis score (LENT/SOMA), (2) patient-rated mucositis symptoms, and (3) scintigraphic salivary excretion fraction . The maximum Candida colonization was found 6 months after radiation therapy and this declined to above normal values after 12 months . Salivary flow was at a minimum 6 months after radiation therapy and had slightly recovered by 12 months . Examiner-rated mucositis and patient-rated xerostomia showed no significant recovery after 6 or 12 months . The results of this study show slight recovery of the oral ecological system . Although the causal role of a single parameter is not clear, persistently elevated Candida colonization should be taken into account therapeutically.

J Exp Bot, 2003 Oct, 54(391), 2231 - 7 Epub 2003 Aug 08.
Salt tolerance-related protein STO binds to a Myb transcription factor homologue and confers salt tolerance in Arabidopsis; Nagaoka S et al.; Regulating the intracellular Na+/K+ ratio is an essential process for salinity tolerance . The yeast mutant, can, which is deficient in calcineurin, can not grow on medium containing Na+ because it is unable to regulate the intracellular Na+/K+ ratio . Expression of the STO gene of Arabidopsis thaliana in the can mutant complements the salt-sensitive phenotype . A protein of Arabidopsis, an H-protein promoter binding factor (HPPBF-1), that binds to STO protein was isolated . HPPBF-1 cDNA has a sequence encoding a Myb DNA binding-motif and its gene expression is induced by salt stress . Furthermore, HPPBF-1 protein is localized in the nucleus . Although, the expression level of STO is not induced under salt-stress conditions, overexpression of STO in a transgenic Arabidopsis plant gave it a higher salt tolerance than was observed in the wild type . When STO transgenic plants and wild-type plants were subjected to salt stress, root growth was increased by 33-70% in the transgenic plants under salt stress . These results suggest that STO is involved in salt-stress responses in Arabidopsis.

J Biol Chem, 2003 Oct 24, 278(43), 42346 - 51 Epub 2003 Aug 09.
Karyopherin-alpha2 protein interacts with Chk2 and contributes to its nuclear import; Zannini L et al.; Chk2 is a nuclear protein kinase involved in the DNA damage-induced ataxia telangiectasia mutated-dependent checkpoint arrest at multiple cell cycle phases . Searching for Chk2-binding proteins by a yeast two-hybrid system, we identified a strong interaction with karyopherin-alpha2 (KPNA-2), a gene product involved in active nuclear import of proteins bearing a nuclear localization signal (NLS) . This finding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays . Of the three predicted Chk2 NLSs, located at amino acids 179-182 (NLS-1), 240-256 (NLS-2), and 515-522 (NLS-3), only the latter mediated the interaction with KPNA-2 in the yeast two-hybrid system, and in particular with its C terminus . Unlike mutations in NLS-1 or NLS-2, which left the nuclear localization of Chk2 unaffected, mutations in NLS-3 caused a cytoplasmic relocalization, indicating that the NLS-3 motif acts indeed as NLS for Chk2 in vivo . Finally, co-transfection experiments with green fluorescent protein (GFP)-Chk2 and wild type or mutant KPNA-2 confirmed the role of KPNA-2 in nuclear import of Chk2.

Gene, 2003 Jul 17, 312, 313 - 20
Isolation of new splice isoforms, characterization and expression analysis of the human septin SEPT8 (KIAA0202); Blaser S et al.; SEPT8 (KIAA0202) is a member of the highly conserved septin family . Septins are membrane-associated GTPases which are involved in cytokinesis and cellular morphogenesis . Using the yeast two-hybrid system and the glutathione-S-transferase pull-down assay we previously had identified the SEPT8 (KIAA0202) as interaction partner of the human septin SEPT5 (cell division cycle related-1, CDCrel-1) . Since the complete cDNA sequence of the human septin SEPT8 (KIAA0202) was not known at that time, we isolated new 5' and 3' cDNA sequence of SEPT8 (KIAA0202) by screening three different cDNA libraries . In addition, we performed the characterization of SEPT8 (KIAA0202) and identified new splice variants of SEPT8 (KIAA0202) . The expression pattern of SEPT8 (KIAA0202) and its interaction partner SEPT5 (CDCrel-1) is illustrated.

Mol Endocrinol, 2003 Nov, 17(11), 2240 - 50 Epub 2003 Aug 07.
Epitope map for a growth hormone receptor agonist monoclonal antibody, MAb 263; Wan Y et al.; Monoclonal antibody (MAb) 263 is a widely used monoclonal antibody that recognizes the extracellular domain (ECD) of the GH receptor . It has been shown to act as a GH agonist both in vitro and in vivo, and we report here that it must be divalent to exert its effect on the full-length receptor . To understand the mechanism of its agonist action, we have determined the precise epitope for this antibody using a novel random PCR mutagenesis approach together with expression screening in yeast . A library of 5200 clones of rabbit GH receptor ECD mutants were screened both with MAb 263 and with an anticarboxy-tag antibody to verify complete ECD expression . Sequencing for clones that expressed complete ECD but were not MAb 263 positive identified 20 epitope residues distributed in a discontinuous manner throughout the ECD . The major part of the epitope, as revealed after mapping onto the crystal structure model of the ECD molecule, was located on the side and upper portion of domain 1, particularly within the D-E strand disulfide loop 79-96 . Molecular dynamics docking of an antibody of the same isotype as MAb 263 was used to dock the bivalent antibody to the 1528-A2 epitope and to visualize the likely consequences of MAb binding . The minimized model enables the antibody to grasp two receptors in a pincer-like movement from opposite sides, facilitating alignment of the receptor dimerization domains in a manner similar to, but not identical with, GH.

Biochem Pharmacol, 2003 Aug 1, 66(3), 447 - 58
The anti-neoplastic and novel topoisomerase II-mediated cytotoxicity of neoamphimedine, a marine pyridoacridine; Marshall KM et al.; Topoisomerase IIalpha (top2) is a target of some of the most useful anticancer drugs . All clinically approved top2 drugs act to stabilize a drug-enzyme-DNA cleavable complex . Here we report the novel top2 activity of neoamphimedine, an isomer of the marine pyridoacridine amphimedine . Neoamphimedine was cytotoxic in yeast and mammalian cell lines . Neoamphimedine exhibited enhanced toxicity in top2 over-expressing yeast cells and was toxic in every mammalian cell line tested . However, neoamphimedine did not possess enhanced toxicity in a mammalian cell line sensitive to stabilized cleavable complexes . Therefore, we hypothesized that neoamphimedine is a top2-dependent drug, whose primary mechanism of action is not the stabilization of cleavable complexes . Top2-directed activity was determined in purified enzyme systems . Neoamphimedine-induced catenation of plasmid DNA only in the presence of active top2 . This catenation correlated with the ability of neoamphimedine to aggregate DNA . Catenation was also observed using a filter-binding assay and transmission electron microscopy . Catenation was confirmed when only restriction enzyme digestion could resolve the catenated plasmid complex to monomer length plasmid DNA . Neoamphimedine also showed potent anti-neoplastic activity in human xenograft tumors in athymic mice . Neoamphimedine was as effective as etoposide in mice bearing KB tumors and as effective as 9-aminocamptothecin in mice bearing HCT-116 tumors . Amphimedine did not induce DNA aggregation or catenation in vitro, nor did it display any significant anti-neoplastic activity . These results suggest that neoamphimedine has a novel top2-mediated mechanism of cytotoxicity and anticancer potential.

Curr Biol, 2003 Aug 5, 13(15), 1335 - 40
The mouse Formin mDia1 is a potent actin nucleation factor regulated by autoinhibition; Li F et al.; Formin proteins are widely expressed in eukaryotes and play essential roles in assembling specific cellular actin-based structures . Formins are defined by a Formin Homology 2 (FH2) domain, as well as a proline-rich FH1 domain that binds the actin monomer binding protein, profilin, and other ligands . Constructs including FH2 of budding yeast Bni1 or fission yeast Cdc12 formins nucleate actin filaments in vitro . In this study, we demonstrate that FH2-containing constructs of murine mDia1 (also called p140 mDia or Drf1) are much more potent actin nucleators than the yeast formins . FH1 is necessary for nucleation when actin monomers are profilin bound . mDia1 is a member of the Diaphanous formin subfamily (Dia), whose members contain an N-terminal Rho GTPase binding domain (GBD) and a C-terminal Diaphanous autoinhibitory domain (DAD, ) . Based on cellular and in vitro binding studies, an autoinhibitory model for Dia formin regulation proposes that GBD binding to DAD inhibits Dia-induced actin remodeling, whereas Rho binding activates by releasing GBD from DAD . Supporting this model, our results show that an N-terminal mDia1 construct strongly inhibits actin nucleation by the C terminus . RhoA partially relieves inhibition but does so when bound to either GDP or GTP analogs . Both N- and C-terminal mDia1 constructs appear to be multimeric.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2001 Apr, 23(2), 115 - 8
{Roles of G-protein beta and gamma subunits in the interaction of G beta gamma with adenylyl cyclases II}; Li JJ et al.; OBJECTIVE: To explore the individual roles of G protein beta and gamma subunit in the interactions with effectors . METHODS: We investigated the interactions of G beta 1 and G beta 1 gamma 2 with adenylyl cyclase II(AC II) using the yeast two-hybrid and three-hybrid systems . RESULTS: When assayed for the abilities to activate the reporter gene, the interactions among AD-beta 1, gamma 2 and BD-AC IIQ in the three-hybrid system were more potent than the interactions between AD-beta 1 and BD-AC II Q in the two-hybrid system . The expressions of BD-AC IIQ and AD-beta 1 in transformants coexpressed AD-beta 1 and BD-AC IIQ, and transformants coexpressed AD-beta 1, gamma 2 and BD-AC IIQ were respectively detected . The comparisons between the reporter activity and the expression levels of BD-AC IIQ and AD-beta 1 in the yeast cells show there was no correlations, i.e . The difference in the reporter activity was not a reflection of differential expression level of the hybrid proteins . CONCLUSIONS: All these results suggest that G protein beta 1 subunit is sufficient to maintain the basic interaction between G beta 1 gamma 2 and AC IIQ, and gamma 2 subunit plays an important role in the high affinity interaction of G beta 1 gamma 2 with AC IIQ.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2001 Oct, 23(5), 462 - 6
{Determination of the binding site of testis-specific nucleoporin BS-63 to transportin (karopherin beta 2) and the proof of their combination in vitro}; Cai Y et al.; OBJECTIVE: To locate the binding site of testis-specific nucleoporin BS-63 to transportin (karopherin beta 2) and confirm their combination in vitro . METHODS: Constructed different fragments of C terminal BS-63 was employed to localize the binding site of the testis-specific nucleoporin BS-63 to transportin by yeast two-hybrid system technique pull-down test was used to identify the interaction between the purified expressed fragments of BS-63 0.6 K and transportin in vitro . RESULTS: BS-63 binding site to transportin was shortened from 1.6 kb to 0.6 kb which included a Ran binding domain (RanBD) . SDS-PAGE and Western blot tests confirmed the recombinant purified protein coded by 0.6 kb fragment of BS-63 cDNA could interact with transportin in vitro . CONCLUSIONS: In germ cells, the function of the testis-specific nucleoporin BS-63 localized at cytoplasmic side of NPC importing cargoes into nuclear may be accomplished by transportin cooperation.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2001 Feb, 23(1), 27 - 31
{Cloning of human B lymphocyte activation-related novel gene}; Lu XW et al.; OBJECTIVE: To clone the novel activation-related gene of B lymphocyte . METHODS: The differential display reversal transcription PCR (DDRT-PCR) technique was applied to analyse the expression difference of mRNA between resting and activated B lymphocyte from human tonsil . The positive differential display cDNA fragment identified by Northern-blotting was chosen as probe to filtrate human activated B lymphocyte cDNA library . RESULTS: Sixty two differential display cDNA fragments (expressed sequence tag, EST) were obtained . Thirty-two of them were mainly expressed in resting B lymphocyte and thirty were expressed in activated cells . Twenty-five were positive ones after identification by Northern blot analysis . A novel cDNA clone was obtained after using EST30 as a probe to filtrate the human activated B cell cDNA library . The whole cDNA clone was 2,048 bp in length and contains a 630 bp open reading frame . The N end of the deduced amino acid sequence was homologous with KAR3 protein which is a member of kinesins superfamily in yeast . CONCLUSIONS: A novel possible activation-related gene in human B lymphocyte was obtained.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2002 Jun, 24(3), 310 - 4
{The primary study on a novel protein binding to the death domain of the death receptor 4}; Li XL et al.; OBJECTIVE: To clone and identify novel proteins binding to the death domain of the death receptor 4 (DR4) . METHODS: The yeast two-hybrid system was used for this study . Automatic sequencing was carried out for DNA sequencing . The sequence homology and the functional domains were analyzed by BLAST and the ScanProsite Tool softwares, respectively . Co-immunoprecipitate method was used to confirm human formyl peptide receptor-like 1 (FPRL1) binding specifically with DR4CD (the cytoplasmic domain of DR4) in HEK293T cells . RESULTS: Two positive clones, named as pADB1 and pADB2, were obtained . BLAST searching showed that the homology of the insert sequence of pADB1 with the mRNA of FPRL1 was 97% . The insert of pADB2 shared no homology with any known peptides in GeneBank . Co-immunoprecipitate analysis further confirmed that FPRL1 could bind to DR4CD in vivo specifically . CONCLUSIONS: FPRL1 may associate with DR4CD in vivo specifically . The functional studies of FPRL1 in signaling pathway mediated by TNF-related apoptosis inducing ligand (TRAIL) are in active progress in our laboratory.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2002 Jun, 24(3), 269 - 71
{Cloning cDNA encoding for GAGA-like element binding proteins in human Jurkat cells}; Mo ZC et al.; OBJECTIVE: To explore GAGA-like element binding protein in human cells . METHODS: Yeast one-hybrid system was used to screen the GAGA-like element binding proteins in HTLV-1 transformed Jurkat cell cDNA fusion library . Total RNA extracted from Jurkat cells was first labeled by reverse transcription, and was taken as cDNA probe to hybridize with the candidate positive clones . RESULTS: 9 positive clones were obtained, and 6 out of the 9 clones were positively hybridized with the cDNA probe . CONCLUSIONS: 6 candidate clones encoding for GAGA-like element binding proteins were obtained from Jurkat cells for further investigation.

Planta, 2003 Oct, 217(6), 904 - 11 Epub 2003 Aug 02.
Alteration of floral organ identity in rice through ectopic expression of OsMADS16; Lee S et al.; We used a transgenic approach and yeast two-hybrid experiments to study the role of the rice ( Oryza sativa L.) B-function MADS-box gene, OsMADS16 . Transgenic rice plants were generated that ectopically expressed OsMADS16 under the control of the maize ( Zea mays L.) ubiquitin1 promoter . Microscopic observations revealed that the innermost-whorl carpels had been replaced by stamen-like organs, which resembled the flowers of the previously described Arabidopsis thaliana (L.) Heynh . mutation superman as well as those ectopically expressing the AP3 gene . These results indicate that expression of OsMADS16 in the innermost whorl induces stamen development . Occasionally, carpels had completely disappeared . In addition, ectopic expression of OsMADS16 enhanced expression of OsMADS4, another B-function gene, causing superman phenotypes . In the yeast two-hybrid system, OsMADS16 did not form a homodimer but, rather, the protein interacted with OsMADS4 . OsMADS16 also interacted with OsMADS6 and OSMADS8, both of which are homologous to SEPALLATA proteins required for the proper function of class-B and class-C genes in Arabidopsis . Based on the gene expression pattern and our yeast two-hybrid data, we discuss a quartet model of MADS-domain protein interactions in the lodicule and stamen whorls of rice florets.

Proc Natl Acad Sci U S A, 2003 Aug 19, 100(17), 9803 - 7 Epub 2003 Aug 06.
Replication-initiator protein (UL9) of the herpes simplex virus 1 binds NFB42 and is degraded via the ubiquitin-proteasome pathway; Eom CY et al.; The ubiquitin-proteasome pathway plays a critical role in the degradation of short-lived and regulatory proteins in a variety of cellular processes . The F-box proteins are part of the ubiquitin-ligase complexes, which mediate ubiquitination and proteasome-dependent degradation of phosphorylated proteins . We previously identified NFB42, an F-box protein that is highly enriched in the nervous system, as a binding partner for the herpes simplex virus 1 UL9 protein, the viral replication-initiator protein, in a yeast two-hybrid screen . In the present work, we find that coexpression of NFB42 and UL9 genes in 293T cells leads to a significant decrease in the level of UL9 protein . Treatment with the 26S-proteasome inhibitor MG132 restores the UL9 protein to normal levels . We have observed also that the UL9 protein is polyubiquitinated in vivo and that the interaction between NFB42 and the UL9 protein is dependent upon phosphorylation of the UL9 protein . These results suggest that the interaction of the UL9 protein with NFB42 results in its polyubiquitination and subsequent degradation by the 26S proteasome . They suggest further a mechanism by which latency of herpes simplex virus 1 can be established in neuronal cells.

Proc Natl Acad Sci U S A, 2003 Aug 19, 100(17), 9791 - 6 Epub 2003 Aug 06.
Repression of Smad transcriptional activity by PIASy, an inhibitor of activated STAT; Long J et al.; Smad proteins mediate transforming growth factor beta (TGF-beta)-inducible transcriptional responses . Protein inhibitor of activated signal transducer and activator of transcription (PIAS) represents a family of proteins that inhibits signal transducer and activator of transcription and also regulates other transcriptional responses . In an effort to identify Smad-interacting proteins by a yeast three-hybrid screen with Smad3 and Smad4 as baits, we identified PIASy, a member of the PIAS family . In yeast, PIASy interacts strongly with Smad4 and also with receptor-regulated Smads . In mammalian cells, PIASy binds most strongly with Smad3 and also associates with other receptor-regulated Smads and Smad4 . The interaction between Smad3 and PIASy is increased in the presence of TGF-beta and occurs through the C-terminal domain of Smad3 . Moreover, Smad3, Smad4, and PIASy can form a ternary complex . PIASy does not inhibit Smad complex binding to DNA, but it represses Smad transcriptional activity . Interestingly, conditional overexpression of PIASy selectively inhibits a subset of endogenous TGF-beta-responsive genes, which includes the cyclin-dependent kinase inhibitor p15, and the plasminogen activator inhibitor 1 . We further show that PIASy can interact constitutively with histone deacetylase 1 (HDAC1) and that addition of HDAC inhibitor trichostatin A (TSA) can prevent the inhibitory function of PIASy . Taken together, our studies indicate that PIASy can inhibit TGF-beta/Smad transcriptional responses through interactions with Smad proteins and HDAC.

J Biol Chem, 2003 Oct 10, 278(41), 40169 - 76 Epub 2003 Aug 06.
Cofilin interacts with ClC-5 and regulates albumin uptake in proximal tubule cell lines; Hryciw DH et al.; Receptor-mediated endocytosis is a constitutive high capacity pathway for the reabsorption of proteins from the glomerular filtrate by the renal proximal tubule . ClC-5 is a voltage-gated chloride channel found in the proximal tubule where it has been shown to be essential for protein uptake, based on evidence from patients with Dent's disease and studies in ClC-5 knockout mice . To further delineate the role of ClC-5 in albumin uptake, we performed a yeast two-hybrid screen with the C-terminal tail of ClC-5 to identify any interactions of the channel with proteins involved in endocytosis . We found that the C-terminal tail of ClC-5 bound the actin depolymerizing protein, cofilin, a result that was confirmed by GST-fusion pulldown assays . In cultured proximal tubule cells, cofilin was distributed in nuclear, cytoplasmic, and microsomal fractions and co-localized with ClC-5 . Phosphorylation of cofilin by overexpressing LIM kinase 1 resulted in a stabilization of the actin cytoskeleton . Phosphorylation of cofilin in two proximal tubule cell models (porcine renal proximal tubule and opossum kidney) was also accompanied by a pronounced inhibition of albumin uptake . This study identifies a novel interaction between the C-terminal tail of ClC-5 and cofilin, an actin-associated protein that is crucial in the regulation of albumin uptake by the proximal tubule.

Expert Opin Drug Saf, 2003 Mar, 2(2), 113 - 22
A review of hepatitis B vaccination; Geier MR et al.; Hepatitis B is one of the most important infectious causes of acute and chronic liver disease both in the US and worldwide . In order to combat the life-threatening effects of hepatitis B infection, recombinant hepatitis B vaccines have been developed . The medical and scientific communities have generally accepted that recombinant hepatitis B vaccine - a highly purified, genetically engineered, single antigen vaccine - is a safe vaccine . Information is presented showing that hepatitis B vaccine contains yeast, aluminium, thimerosal and hepatitis B surface antigen epitopes, which may result in hepatitis B vaccine being associated with autoimmune diseases among susceptible adult vaccine recipients . There is little doubt that the benefits of this vaccine overall far outweigh its risks . Physicians and patients should evaluate the risks and benefits of hepatitis B vaccination and, together, make an informed consent decision as to whether to undergo vaccination . Individuals who experience an adverse reaction to hepatitis B vaccination should report it to the Vaccine Adverse Event Reporting System database and be advised that they may be eligible for compensation from the no-fault National Vaccine Injury Compensation Program, administered by the US Court of Claims . The authors strongly urge that additional research be conducted into the molecular basis of adverse events following hepatitis B vaccine administration, so that further recommendations may be made on how to improve their safety profiles.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2000 Dec, 22(6), 536 - 9
{Interactions between domains within the NH2- and COOH-terminal fragments of presenilins}; Mei P et al.; OBJECTIVE: To analyze the interactions between domains within the NH2- and COOH-terminal regions of presenilins . METHODS: The various constructions corresponding to NH2-terminal fragment (NTF) and COOH-terminal fragment (CTF) derivatives of presenilin 1 (PS1) and presenilin 2 (PS2) were generated by RT-PCR, and their interactions were assayed by yeast two-hybrid system . RESULTS: Domains within the NH- and COOH-terminal fragments of presenilins could directly interact with each other, and therefore form high molecular weight complex . The interaction site between domains within PS1 located at amino acid 361-447 of PS1 CTF, without the involvement of other partners . Similar interaction was not observed between PS11-360 and PS2341-448, PS2(1)-340 and PS1(361)-467 . CONCLUSIONS: Intramolecular interaction between domains within the NH2- and COOH-terminal regions of presenilins may be critical to the folding and assembly of mature PS molecules.

J Cell Sci, 2003 Sep 15, 116(Pt 18), 3803 - 10 Epub 2003 Aug 05.
Loss of responsiveness to chemotactic factors by deletion of the C-terminal protein interaction site of angiomotin; Levchenko T et al.; We have recently identified a novel protein, named angiomotin, by its ability to bind the angiogenesis inhibitor angiostatin in the yeast two-hybrid system . Angiomotin belongs to a family with two other members, AmotL-1 and -2 characterized by coiled-coil and C-terminal PDZ binding domains . Here we show that the putative PDZ binding motif of angiomotin serves as a protein recognition site and that deletion of three amino acids in this site results in inhibition of chemotaxis . Furthermore, endothelial cells expressing mutant angiomotin failed to migrate and form tubes in an in vitro tube formation assay . To study the effect of angiomotin on embryonic angiogenesis, we generated transgenic mice expressing wild-type angiomotin and the C-terminal deletion mutant driven by the endothelial cell-specific receptor tyrosine kinase (TIE) promoter . Expression of mutant angiomotin in endothelial cells inhibited migration into the neuroectoderm and intersomitic regions resulting in death at embryonic day 9.5 . In contrast, mice expressing wild-type angiomotin developed normally and were fertile . These results suggest that the putative PDZ binding motif of angiomotin plays a critical role in regulating the responsiveness of endothelial cells to chemotactic cues.

Appl Environ Microbiol, 2003 Aug, 69(8), 4689 - 96
Growth of Rhodosporidium toruloides strain DBVPG 6662 on dibenzothiophene crystals and orimulsion; Baldi F et al.; Strains DBVPG 6662 and DBVPG 6739 of Rhodosporidium toruloides, a basidiomycete yeast, grew on thiosulfate as a sulfur source and glucose (2 g liter(-1) or 10.75 mM) as a carbon source . DBVPG 6662 has a defective sulfate transport system, whereas DBVPG 6739 barely grew on sulfate . They were compared for the ability to use dibenzothiophene (DBT) and related organic sulfur compounds as sulfur sources . In the presence of glucose as a carbon source and DBT as a sulfur source, strain DBVPG 6662 grew better than DBVPG 6739 . In the presence of thiosulfate as a sulfur source, the two yeast strains did not use DBT, DBT-sulfone, benzenesulfonic acid, biphenyl, and fluorene . When the two strains were grown in the presence of glucose, strain DBVPG 6662 transformed 27% of the DBT present (10 micro M) at a rate of 0.023 micro mol liter(-1) h(-1) in 36 h . Traces of 2,2'-dihydroxylated biphenyl were transiently accumulated under these conditions . When the same strain was grown on glucose in the presence of a higher concentration of DBT (0.5 g liter(-1)), mainly in an insoluble form, the whole surface of the DBT crystals was colonized by a thick mycelium . This adherent structure was imaged by confocal microscopy with fluorescent concanavalin A, a lectin that specifically binds glucose and mannose residues . When DBVPG 6662 was grown on glucose in the presence of a commercial emulsion of bitumen, i.e., orimulsion, 68% of the benzo- and dibenzothiophenes and DBTs was removed after 15 days of incubation . The fungus adhered by hyphae to orimulsion droplets . When cultivated in the presence of commercial emulsifier-free fuel oil containing alkylated benzothiophenes and DBTs and having a composition similar to that of orimulsion, strain DBVPG 6662 removed only 11% of the total organic sulfur that occurs in the medium and did not adhere to the oil droplets . These results indicate that strain DBVPG 6662 is able to utilize the organic sulfur of DBT and a large variety of thiophenic compounds that occur extensively in commercial fuel oils by physically adhering to the organic sulfur source.

Appl Environ Microbiol, 2003 Aug, 69(8), 4438 - 47
Purification and characterization of a novel mannitol dehydrogenase from a newly isolated strain of Candida magnoliae; Lee JK et al.; Mannitol biosynthesis in Candida magnoliae HH-01 (KCCM-10252), a yeast strain that is currently used for the industrial production of mannitol, is catalyzed by mannitol dehydrogenase (MDH) (EC 1.1.1.138) . In this study, NAD(P)H-dependent MDH was purified to homogeneity from C . magnoliae HH-01 by ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography . The relative molecular masses of C . magnoliae MDH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, were 35 and 142 kDa, respectively, indicating that the enzyme is a tetramer . This enzyme catalyzed both fructose reduction and mannitol oxidation . The pH and temperature optima for fructose reduction and mannitol oxidation were 7.5 and 37 degrees C and 10.0 and 40 degrees C, respectively . C . magnoliae MDH showed high substrate specificity and high catalytic efficiency (k(cat) = 823 s(-1), K(m) = 28.0 mM, and k(cat)/K(m) = 29.4 mM(-1) s(-1)) for fructose, which may explain the high mannitol production observed in this strain . Initial velocity and product inhibition studies suggest that the reaction proceeds via a sequential ordered Bi Bi mechanism, and C . magnoliae MDH is specific for transferring the 4-pro-S hydrogen of NADPH, which is typical of a short-chain dehydrogenase reductase (SDR) . The internal amino acid sequences of C . magnoliae MDH showed a significant homology with SDRs from various sources, indicating that the C . magnoliae MDH is an NAD(P)H-dependent tetrameric SDR . Although MDHs have been purified and characterized from several other sources, C . magnoliae MDH is distinguished from other MDHs by its high substrate specificity and catalytic efficiency for fructose only, which makes C . magnoliae MDH the ideal choice for industrial applications, including enzymatic synthesis of mannitol and salt-tolerant plants.

J Ethnopharmacol, 2003 Sep, 88(1), 85 - 91
Antipyretic properties of the aqueous and ethanol extracts of the leaves of Ocimum suave and Ocimum lamiifolium in mice; Makonnen E et al.; Numerous plant species are used to treat ailments associated with pyrexia in the indigenous health care delivery system of Ethiopia . Notable among these are Ocimum suave and Ocimum lamiifolium . The objective of the present study was thus to evaluate the antipyretic effects of the aqueous and ethanol extracts of the leaves of Ocimum suave and Ocimum lamiifolium in mice . Rectal temperatures were recorded before and after inducing pyrexia as well as after administration of the respective extracts every half an hour for 3h . Parallel experiments were run with a standard antipyretic (acetylsalicylic acid) and the vehicle (distilled water) . All the plant extracts showed antipyretic property with reasonable onset and duration of action . Both ethanol and aqueous extracts of Ocimum suave were observed to be more potent than those of Ocimum lamiifolium . Aqueous extract of Ocimum suave and ethanol extract of Ocimum lamiifolium were more potent than their other counterpart extracts . Time dependent antipyretic effect was also observed with some extracts; reduced with time with aqueous extract of Ocimum suave and increased with time with both extracts of Ocimum lamiifolium.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2001 Dec, 23(6), 580 - 4
{Fragile X mental retardation protein interacts with human NDK/Nm23-H2}; Lou R et al.; OBJECTIVE: To investigate the physiological role of fragile X mental retardation protein (FMRP) and screen the proteins interacting with FMRP in human fetal hippocampus cDNA library . METHODS: Human fetal hippocampus cDNA library was constructed in yeast two-hybrid DAD vector pGAD10 . Quality of the library was measured by picking up random colonies as templates for PCR testing . Proteins interacting with FMRP were screened by yeast two-hybrid system . Furthermore, the interaction site of FMRP was mapped in yeast . RESULTS: The average length of inserts of the two-hybrid library was 1.5 kb, and the ratio of recombinant colonies was about 90% . Human NDK/Nm23-H2 was found interacting with FMRP . NDK/Nm23-H2 interacted with FMRP exon 1-12, as well as FMRP isoforms without exon 12, and exons 14-17 . NDK/Nm23-H2 couldn't interact with FMRP exon 1-6 and exon 2-7 fragments . CONCLUSIONS: Human NDK/Nm23-H2 can bind FMRP directly . The interaction site of FMRP is located at its exon 1-11 . This interaction in vitro might alter the intracellular distribution of NDK/Nm23-H2, and even regulates the transcription and expression of FMRP.

J Biol Chem, 2003 Oct 3, 278(40), 38113 - 6 Epub 2003 Aug 04.
A novel interaction between perlecan protein core and progranulin: potential effects on tumor growth; Gonzalez EM et al.; In an in vivo search of novel partners for perlecan, a major heparan sulfate proteoglycan of basement membranes and cell surfaces, we identified progranulin, a secreted growth factor, as a strong interacting protein . Unambiguous interaction, first observed with the yeast two-hybrid system, was corroborated by co-immunoprecipitation studies using cell-free transcription/translation and transient cell transfection assays . The interaction of progranulin with perlecan domain V involved the first two laminin- and epidermal growth factor-like repeats . Within progranulin, the subdomains interacting most with perlecan harbored granulins F and B . Kinetics analysis of the interaction using surface plasmon resonance showed a saturable binding of relative low affinity (KD approximately 1 microM) . These results were supported by significant expression overlap of these two proteins in a series of ovarian tumor tissue microarrays . Progranulin was present within proliferating blood vessels of ovarian carcinomas and perivascular matrices, with a distribution similar to perlecan . Notably, both progranulin and domain V stimulated the growth of adrenal carcinoma cells . However, when used together in equimolar amounts, the two proteins counteracted each other's activity . Thus, progranulin/perlecan interaction could contribute to a fine regulation of tumor angiogenesis and could ultimately affect cancer growth.

Biochem Cell Biol, 2003 Jun, 81(3), 123 - 9
Characteristics of gamma-H2AX foci at DNA double-strand breaks sites; Pilch DR et al.; Phosphorylated H2AX (gamma-H2AX) is essential to the efficient recognition and (or) repair of DNA double strand breaks (DSBs), and many molecules, often thousands, of H2AX become rapidly phosphorylated at the site of each nascent DSB . An antibody to gamma-H2AX reveals that this highly amplified process generates nuclear foci . The phosphorylation site is a serine four residues from the C-terminus which has been evolutionarily conserved in organisms from giardia intestinalis to humans . Mice and yeast lacking the conserved serine residue demonstrate a variety of defects in DNA DSB processing . H2AX Delta/Delta mice are smaller, sensitive to ionizing radiation, defective in class switch recombination and spermatogenesis while cells from the mice demonstrate substantially increased numbers of genomic defects . gamma-H2AX foci formation is a sensitive biological dosimeter and presents new and exciting opportunities to understand important biological processes, human diseases, and individual variations in radiation sensitivity . These potentialities demonstrate the importance of understanding the parameters and functions of gamma-H2AX formation.

Can J Microbiol, 2003 Apr, 49(4), 263 - 8
Release and regeneration of protoplasts from the fungus Trichothecium roseum; Balasubramanian N et al.; A protocol for isolating and regenerating protoplasts from Trichothecium roseum has been described . Protoplasts from T . roseum were isolated using (i) a lytic enzyme combination composed of Novozym 234, chitinase, cellulase, and pectinase at a 5-mg/mL concentration and (ii) 0.6 M KCl as an osmotic stabilizer . A maximum number of 28 x 10(4) protoplasts/mL were obtained at pH 5.5 . Experiments on the regeneration and reversion of protoplasts revealed a maximum regeneration (60.8%) in complete medium (potato dextrose--yeast extract agar) amended with 0.6 M KCl . The regenerated protoplasts were similar to the original parent strain in morphology, pigmentation, growth, and sporulation.

Biofactors, 2003, 17(1-4), 249 - 57
Molecular chaperones, stress proteins and redox homeostasis; Papp E et al.; Protection against oxidative stress is highly interrelated with the function of the most ancient cellular defense system, the network of molecular chaperones, heat shock, or stress-proteins . These ubiquitous, conserved proteins help other proteins and macromolecules to fold or re-fold and reach their final, native conformation . Redox regulation of protein folding becomes especially important during the preparation of extracellular proteins to the outside oxidative milieu, which should take place in a gradual and step-by-step controlled manner in the endoplasmic reticulum or in the periplasm . Several chaperones, such as members of the Hsp33 family in yeast and the plethora of small heat shock proteins as well as one of the major chaperones, Hsp70 are able to act against cytoplasmic oxidative damage . Abrupt changes of cellular redox status lead to chaperone induction . The function of several chaperones is tightly regulated by the surrounding redox conditions . Moreover, our recent data suggest that chaperones may act as a central switchboard for the transmission of redox changes in the life of the cell.

Plant Cell, 2003 Aug, 15(8), 1904 - 17
Regulation and processing of maize histone deacetylase Hda1 by limited proteolysis; Pipal A et al.; A maize histone deacetylase gene was identified as a homolog of yeast Hda1 . The predicted protein corresponds to a previously purified maize deacetylase that is active as a protein monomer with a molecular weight of 48,000 and is expressed in all tissues of germinating embryos . Hda1 is synthesized as an enzymatically inactive protein with an apparent molecular weight of 84,000 that is processed to the active 48-kD form by proteolytic removal of the C-terminal part, presumably via a 65-kD intermediate . The enzymatically inactive 84-kD protein also is part of a 300-kD protein complex of unknown function . The proteolytic cleavage of ZmHda1 is regulated during maize embryo germination in vivo . Expression of the recombinant full-length protein and the 48-kD form confirmed that only the smaller enzyme form is active as a histone deacetylase . In line with this finding, we show that the 48-kD protein is able to repress transcription efficiently in a reporter gene assay, whereas the full-length protein, including the C-terminal part, lacks full repression activity . This report on the processing of Hda1-p84 to enzymatically active Hda1-p48 demonstrates that proteolytic cleavage is a mechanism to regulate the function of Rpd3/Hda1-type histone deacetylases.

Plant Cell, 2003 Aug, 15(8), 1872 - 87
Arabidopsis AtGPAT1, a member of the membrane-bound glycerol-3-phosphate acyltransferase gene family, is essential for tapetum differentiation and male fertility; Zheng Z et al.; Membrane-bound glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) mediates the initial step of glycerolipid biosynthesis in the extraplastidic compartments of plant cells . Here, we report the molecular characterization of a novel GPAT gene family from Arabidopsis, designated AtGPAT . The corresponding polypeptides possess transmembrane domains and GPAT activity when expressed heterologously in a yeast lipid mutant . The functional significance of one isoform, AtGPAT1, is the focus of the present study . Disruption of the AtGPAT1 gene causes a massive pollen development arrest, and subsequent introduction of the gene into the mutant plant rescues the phenotype, illustrating a pivotal role for AtGPAT1 in pollen development . Microscopic examinations revealed that the gene lesion results in a perturbed degeneration of the tapetum, which is associated with altered endoplasmic reticulum profiles and reduced secretion . In addition to the sporophytic effect, AtGPAT1 also exerts a gametophytic effect on pollen performance, as the competitive ability of a pollen grain to pollinate is dependent on the presence of an AtGPAT1 gene . Deficiency in AtGPAT1 correlates with several fatty acid composition changes in flower tissues and seeds . Unexpectedly, however, a loss of AtGPAT1 causes no significant change in seed oil content.

Plant Cell, 2003 Aug, 15(8), 1749 - 70
The Arabidopsis basic/helix-loop-helix transcription factor family; Toledo-Ortiz G et al.; The basic/helix-loop-helix (bHLH) proteins are a superfamily of transcription factors that bind as dimers to specific DNA target sites and that have been well characterized in nonplant eukaryotes as important regulatory components in diverse biological processes . Based on evidence that the bHLH protein PIF3 is a direct phytochrome reaction partner in the photoreceptor's signaling network, we have undertaken a comprehensive computational analysis of the Arabidopsis genome sequence databases to define the scope and features of the bHLH family . Using a set of criteria derived from a previously defined consensus motif, we identified 147 bHLH protein-encoding genes, making this one of the largest transcription factor families in Arabidopsis . Phylogenetic analysis of the bHLH domain sequences permits classification of these genes into 21 subfamilies . The evolutionary and potential functional relationships implied by this analysis are supported by other criteria, including the chromosomal distribution of these genes relative to duplicated genome segments, the conservation of variant exon/intron structural patterns, and the predicted DNA binding activities within subfamilies . Considerable diversity in DNA binding site specificity among family members is predicted, and marked divergence in protein sequence outside of the conserved bHLH domain is observed . Together with the established propensity of bHLH factors to engage in varying degrees of homodimerization and heterodimerization, these observations suggest that the Arabidopsis bHLH proteins have the potential to participate in an extensive set of combinatorial interactions, endowing them with the capacity to be involved in the regulation of a multiplicity of transcriptional programs . We provide evidence from yeast two-hybrid and in vitro binding assays that two related phytochrome-interacting members in the Arabidopsis family, PIF3 and PIF4, can form both homodimers and heterodimers and that all three dimeric configurations can bind specifically to the G-box DNA sequence motif CACGTG . These data are consistent, in principle, with the operation of this combinatorial mechanism in Arabidopsis.

Mol Cell Biol, 2003 Aug, 23(16), 5680 - 91
HoxB5 is an upstream transcriptional switch for differentiation of the vascular endothelium from precursor cells; Wu Y et al.; Endothelial cells differentiate from mesoderm-derived precursors to initiate the earliest events in vascular development . Although the signaling events that regulate the successive steps of vascular development are known in some detail, the transcriptional processes that regulate the first steps in vasculogenesis are not well defined . We have studied the regulatory mechanisms of flk1 expression as a model to understand the upstream events in endothelial cell differentiation, since flk1 is the earliest marker of endothelial precursors . Using a variety of biochemical approaches, we identified a cis-acting element in the first intron of the flk1 gene that is required for endothelium-dependent expression in transgenic reporter gene assays . Using the yeast one-hybrid system, we identified HoxB5 as the transcription factor that binds this cis-acting element, the HoxB5-binding element (HBE) . HoxB5 mRNA colocalized with flk1 expression in differentiating embryoid bodies, and HoxB5 potently transactivated the flk1 promoter in an HBE-dependent fashion in transient-transfection assays . Overexpression of HoxB5 led to expansion of flk1(+) angioblasts in differentiating embryoid bodies and increased the number of PECAM (platelet-endothelial cell adhesion molecule)-positive primitive blood vessels . HoxB5 is necessary and sufficient to activate the cell-intrinsic events that regulate the differentiation of angioblasts and mature endothelial cells from their mesoderm-derived precursors.

J Biol Chem, 2003 Oct 17, 278(42), 41367 - 79 Epub 2003 Aug 03.
A region directly following the second transmembrane domain in gamma ENaC is required for normal channel gating; Booth RE et al.; We used a yeast one-hybrid complementation screen to identify regions within the cytosolic tails of the mouse alpha, beta, and gamma epithelial Na+ channel (ENaC) important to protein-protein and/or protein-lipid interactions at the plasma membrane . The cytosolic COOH terminus of alphaENaC contained a strongly interactive domain just distal to the second transmembrane region (TM2) between Met610 and Val632 . Likewise, gammaENaC contained such a domain just distal to TM2 spanning Gln573-Pro600 . Interactive domains were also localized within Met1-Gln54 and the last 17 residues of alpha- and betaENaC, respectively . Confocal images of Chinese hamster ovary cells transfected with enhanced green fluorescent fusion proteins of the cytosolic tails of mENaC subunits were consistent with results in yeast . Fusion proteins of the NH2 terminus of alphaENaC and the COOH termini of all three subunits co-localized with a plasma membrane marker . The functional importance of the membrane interactive domain in the COOH terminus of gammaENaC was established with whole-cell patch clamp experiments of wild type (alpha, beta, and gamma) and mutant (alpha, beta, and gammadeltaQ573-P600) mENaC reconstituted in Chinese hamster ovary cells . Mutant channels had about 13% of the activity of wild type channels with 0.33 +/- 0.14 versus 2.5 +/- 0.80 nA of amiloridesensitive inward current at -80 mV . Single channel analysis of recombinant channels demonstrated that mutant channels had a decrease in Po with 0.16 +/- 0.03 versus 0.67 +/- 0.07 for wild type . Mutant gammaENaC associated normally with the other two subunits in co-immunoprecipitation studies and localized to the plasma membrane in membrane labeling experiments and when visualized with evanescent-field fluorescence microscopy . Similar to deletion of Gln573-Pro600, deletion of Gln573-Arg583 but not Thr584-Pro600 decreased ENaC activity . The current results demonstrate that residues within Gln573-Arg583 of gammaENaC are necessary for normal channel gating.

Int J Dermatol, 2003 Sep, 42 Suppl 1, 19 - 22
Ciclopirox gel for seborrheic dermatitis of the scalp; Aly R et al.; BACKGROUND: Seborrheic dermatitis is a common inflammatory skin disorder that usually occurs in patients with pre-existing seborrhea . The etiology of seborrheic dermatitis is uncertain . Typically, sites dense with sebaceous glands support growth of the lipophilic yeast Malassezia furfur . Ciclopirox (Loprox) gel is a hydroxypyridone, broad-spectrum antifungal agent proven effective against the yeast M . furfur . OBJECTIVE: A multicenter, randomized, double-blind, vehicle controlled study of 178 subjects evaluated the efficacy of ciclopirox gel in treating seborrheic dermatitis of the scalp . METHODS: One hundred and seventy-eight subjects were randomized to apply either ciclopirox gel 0.77% twice daily, or vehicle twice daily for 28 days . Subjects' signs and symptoms of severity (erythema, scaling, pruritus and burning) were rated on a scale of 0-3 (none to severe); for inclusion, a minimum score of 4, for the sum of the individual ratings was required . Efficacy evaluations were performed at baseline, days 4, 8, 15, 22, 29, and at end-point (final visit, up to day 33) . The primary efficacy variable was clinical response assessed by a global improvement, based on a scale of 0-5 (100% clearance to flare of treatment area) . Changes in signs/symptoms severity scores within the target lesion were also evaluated . RESULTS: Global evaluation scores demonstrated that significantly more ciclopirox-treated subjects achieved over 75% improvement compared with vehicle at days 22, 29, and endpoint (P < 0.01) . Change-from-baseline mean score for total signs and symptoms was significantly greater in ciclopirox subjects compared with vehicle subjects at the same time points as above (P < 0.001), as well as day 15 (P < 0.01) . Twenty-nine percent of subjects rated ciclopirox as having excellent cosmetic acceptability . There were only mild adverse events, with the most common being burning sensation in 13% of ciclopirox subjects and 9% of vehicle subjects . CONCLUSION: Ciclopirox gel is effective and safe in the treatment of seborrheic dermatitis of the scalp.

Anticancer Res, 2003 May-Jun, 23(3A), 2173 - 8
Ets-2 interacts with co-repressor BS69 to repress target gene expression; Wei G et al.; BACKGROUND: The ETS-family of proteins consists of over 30 members that regulate the growth, differentiation and survival of both normal and tumor cells . How specificity is achieved within this family remains largely unresolved . One mechanism for attaining specificity is through the action of signaling pathways on specific family members . For example, Ets-2 is an activator modulated by ras-dependent phosphorylation of a single residue in the conserved pointed domain of this factor . We hypothesized that phosphorylation of the pointed domain regulates the proteins that can interact with ets-2 in the cell nucleus, resulting in regulation of target genes . MATERIALS AND METHODS: We used a combination of biochemical assays, yeast two-hybrid screens and transfection assays to identify and characterize proteins interacting with the pointed domain . RESULTS: BS69, a known co-repressor, was identified in a yeast two hybrid screen as an ets-2 interacting partner . BS69 can interact with ets-2 in vivo and phosphorylation of the ets-2 pointed domain decreased the interaction with BS69 in vitro . In transfection assays, co-expression of ets-2 and BS69 resulted in repression of defined ets-2 target genes . CONCLUSION: These results support a role for ets-2 as a repressor and indicate that BS69 is required as co-repressor . Phosphorylation of ets-2 may switch its activity from repressor to activator by interfering with formation of the BS69 complex.

Biochem Biophys Res Commun, 2003 Aug 1, 307(3), 569 - 77
Molecular characterization of a novel nucleolar protein, pNO40; Chang WL et al.; We report the discovery and characterization of a novel nucleolar protein . This protein, referred to as pNO40 based on its molecular weight on SDS-PAGE, was identified through yeast two hybrid interaction screen of a human kidney cDNA library using pinin (pnn) protein as the bait . The deduced amino acids of pNO40 derived from cDNA cloning of diverse species display high conservation; 95% identify between human and mouse and 57.3% identity for human and zebrafish . Several distinct domains are discernable in the ORF of pNO40, including a ribosomal protein S1 RNA binding region, a CCHC type zinc finger, and clusters of basic amino acid representing potential nucleolar targeting signal . Immunostaining of endogenous or transfected pNO40 indicated that it is localized to nucleoli of diverse cultured cells, with some concentration in the granular component of nucleoli . Northern blot analysis demonstrated that pNO40 message is expressed ubiquitously across all tissues examined . Characterization of human and mouse pNO40 gene revealed that mouse gene spans 44 kb in length and contains 8 exons, while that of human is 68 kb in length and displays two isoforms generated by alternative splicing of the 5(')-untranslated region and differential usage of translation start site . Based on sequence features and its subcellular location, we predict that pNO40 is a novel nucleolar protein with function related to ribosome maturation and/or biogenesis.

Biochem Biophys Res Commun, 2003 Aug 1, 307(3), 459 - 65
Centaurin-alpha(1) associates with and is phosphorylated by isoforms of protein kinase C; Zemlickova E et al.; Centaurin-alpha(1) is a member of the family of ADP-ribosylation factors (ARF) GTPase activating proteins (GAPs), although ARF GAP activity has not yet been demonstrated . The human homologue, centaurin-alpha(1) functionally complements the ARF GAP activity of Gcs1 in yeast . Although Gcs1 is involved in the formation of actin filaments in vivo, the function of centaurin remains elusive . We have identified a number of novel centaurin-alpha(1) binding partners; including CKIalpha and nucleolin . In this report, we have focused on the interaction of centaurin-alpha(1) with PKC . All groups of PKC associate directly through their cysteine rich domains . Centaurin-alpha(1) is also a substrate for all PKC classes and we have identified the two sites of phosphorylation . This is the first report of a kinase that phosphorylates centaurin-alpha(1).

Methods, 2003 Sep, 31(1), 90 - 5
In vivo protein-protein and protein-DNA crosslinking for genomewide binding microarray; Kurdistani SK et al.; Chromatin immunoprecipitation (ChrIP or ChIP) has commonly been used to map protein-DNA interaction sites at specific genomic loci through use of formaldehyde-induced crosslinking . However, formaldehyde alone has proved inadequate for crosslinking of certain proteins such as the yeast histone deacetylase Rpd3 . We report here a modified crosslinking procedure that includes a protein-protein crosslinking agent in addition to formaldehyde . Using this double crosslinking method, we have successfully mapped Rpd3 binding sites in vivo . We also describe the use of ChrIP in combination with DNA microarrays (ChrIP-array) to determine the pattern of Rpd3 binding genomewide . This approach couples the versatility of ChrIP with that of microarrays to identify binding patterns that would otherwise be hidden in a gene-by-gene survey.

Curr Opin Cell Biol, 2003 Aug, 15(4), 482 - 8
Staying in aerobic shape: how the structural integrity of mitochondria and mitochondrial DNA is maintained; Scott SV et al.; The structure and integrity of the mitochondrial compartment are features essential for it to function efficiently . The maintenance of mitochondrial structure in cells ranging from yeast to humans has been shown to require both ongoing fission and fusion . Recent characterization of many of the molecular components that direct mitochondrial fission and fusion events have led to a more complete understanding of how these processes take place . Further, mitochondrial fragmentation observed when cells undergo apoptosis requires mitochondrial fission, underlying the importance of mitochondrial dynamics in cellular homeostasis . Mitochondrial structure also impacts mitochondrial DNA inheritance . Recent studies suggest that faithful transmission of mitochondrial DNA to daughter cells might require a mitochondrial membrane tethering apparatus.

Int J Syst Evol Microbiol, 2003 Jul, 53(Pt 4), 1149 - 54
Caldisphaera lagunensis gen . nov., sp . nov., a novel thermoacidophilic crenarchaeote isolated from a hot spring at Mt Maquiling, Philippines; Itoh T et al.; Four novel, thermoacidophilic, crenarchaeotic cocci that grew anaerobically and heterotrophically were isolated from an acidic hot spring in the Philippines; two representative strains were characterized in detail . Most cells were regular cocci, 0.8-1.1 microm in width, which occurred singly or in pairs . They were non-motile and grew at 45-80 degrees C (optimum 70-75 degrees C) and pH 2.3-5.4 (optimum 3.5-4.0) . They utilized starch, glycogen, gelatin, beef extract, yeast extract and peptone as carbon and energy sources . Growth was stimulated by the presence of sulfur as an electron acceptor . The lipid fraction contained cyclic and acyclic tetraether core lipids . The DNA G + C content was 31 mol%; phylogenetic analysis based on 16S rDNA sequences showed that the novel cocci represent an independent lineage in the phylum Crenarchaeota, distantly related to Acidilobus aceticus and an allied strain, NC12 . Caldisphaera lagunensis gen . nov., sp . nov . is proposed to accommodate the four strains . The type strain is IC-154T (=JCM 11604T=MCC-UPLB 1331T=ANMR 0165T).

J Cell Physiol, 2003 Sep, 196(3), 541 - 56
Tumor suppressor pRB functions as a co-repressor of the CCAAT displacement protein (CDP/cut) to regulate cell cycle controlled histone H4 transcription; Gupta S et al.; The CCAAT displacement protein (CDP-cut/CUTL1/cux) performs a key proliferation-related function as the DNA binding subunit of the cell cycle controlled HiNF-D complex . HiNF-D interacts with all five classes (H1, H2A, H2B, H3, and H4) of the cell-cycle dependent histone genes, which are transcriptionally and coordinately activated at the G(1)/S phase transition independent of E2F . The tumor suppressor pRB/p105 is an intrinsic component of the HiNF-D complex . However, the molecular interactions that enable CDP and pRB to form a complex and thus convey cell growth regulatory information onto histone gene promoters must be further defined . Using transient transfections, we show that CDP represses the H4 gene promoter and that pRB functions with CDP as a co-repressor . Direct physical interaction between CDP and pRB was observed in glutathione-S-transferase (GST) pull-down assays . Furthermore, interactions between these proteins were established by yeast and mammalian two-hybrid experiments and co-immunoprecipitation assays . Confocal microscopy shows that subsets of each protein are co-localized in situ . Using a series of pRB mutants, we find that the CDP/pRB interaction, similar to the E2F/pRB interaction, utilizes the A/B large pocket (LP) of pRB . Thus, several converging lines of evidence indicate that complexes between CDP and pRB repress cell cycle regulated histone gene promoters .

J Am Diet Assoc, 2003 Aug, 103(8), 1015 - 9
Dietary intake and food sources of whole grains among US children and adolescents: data from the 1994-1996 Continuing Survey of Food Intakes by Individuals; Harnack L et al.; OBJECTIVE: This study characterizes whole grain consumption among a nationally representative sample of US children and adolescents . DESIGN: Data used in this study were collected as part of the 1994-1996 US Department of Agriculture Continuing Survey of Food Intakes by Individuals (CSFII) . SUBJECTS/SETTING: CSFII was designed to obtain a nationally representative sample of noninstitutionalized persons of all ages residing in the United States . Analyses reported in this article are limited to participants aged 2 to 18 years with two days of dietary recall data (n=4,802) . Foods reported in the survey were quantified in servings as defined by the Food Guide Pyramid using the US Department of Agriculture Pyramid Servings Database, which contains reference data for each food reported in CSFII in servings per 100 g for 30 Pyramid food groups, including whole grain and total grain . STATISTICAL ANALYSES: Means, frequencies, and logistic regression analyses were conducted as appropriate . RESULTS: Average whole grain intake ranged from 0.8 servings per day for preschool-aged children to 1.0 servings per day for adolescents . Ready-to-eat cereals, corn and other chips, and yeast breads were found to be the major food sources of whole grains accounting for 30.9%, 21.7%, and 18.1% of whole grain intake respectively among those aged two to 18 years . APPLICATIONS/CONCLUSIONS: Given the apparent low level of whole grain intake among most children and adolescents in the United States, interventions are needed to increase intake of whole-grain foods.

J Biol Chem . 2003 Jul 30; {Epub ahead of print}
The mouse APG10 homologue, an authentic E2-like enzyme for Apg12p-Apg5p conjugation system, facilitates MAP-LC3 processing; Nemoto T et al.; Autophagy is a process for the bulk degradation system of cytosolic compartments by lysosomes/vacuoles . Autophagosome formation involves dynamic membrane rearrangement for which two ubiquitin-like modifications, Apg12-Apg5 conjugation and MAP-LC3 processing, are essential . In yeast, Apg10p is an E2-like enzyme essential for Apg12p-Apg5p conjugation . However, a mammalian counterpart of yeast Apg10p has not been identified . In this paper, we report the isolation and characterization of a cDNA of the APG10 gene . The calculated molecular mass of mouse APG10 gene product is 24.3 kDa, and the region containing the active-site cysteine is conserved . Apg10p coprecipitates with Apg12p, its substrate, and Apg7p, an E1-like enzyme . A mutation of the active-site cysteine within Apg10 predominantly inhibits Apg12p-conjugation . The coexpression of Apg10p with Apg7p facilitates Apg12p-conjugation . Furthermore, the coexpression of Apg10p with Apg7p facilitates MAP-LC3 modification, while MAP-LC3 is not a substrate of Apg10p . Mammalian Apg10p is ubiquitously expressed in all tissues examined . These results indicate that mammalian Apg10p, an authentic E2-like enzyme essential for Apg12p, plays a pivotal role in the cooperative function of two modification systems.

Biochim Biophys Acta, 2003 Jul 28, 1628(2), 88 - 96
The human kallikrein protein 5 (hK5) is enzymatically active, glycosylated and forms complexes with two protease inhibitors in ovarian cancer fluids; Yousef GM et al.; The kallikrein family is a group of 15 serine protease genes clustered on chromosome 19q13.4 . Binding of kallikreins to protease inhibitors is an important mechanism for regulating their enzymatic activity and may have potential clinical applications . Human kallikrein gene 5 (KLK5) is a member of this family and encodes for a secreted serine protease (hK5) . This kallikrein was shown to be differentially expressed at the mRNA and protein levels in diverse malignancies . Our objective was to study the enzymatic activity and the interaction of recombinant hK5 protein with protease inhibitors . Recombinant hK5 protein was produced in yeast and mammalian expression systems and purified by chromatography . HPLC fractionation, followed by ELISA-type assays, immunoblotting and radiolabeling experiments were performed to detect the possible interactions between hK5 and proteinase inhibitors in serum . Enzymatic deglycosylation was performed to examine the glycosylation pattern of the protein . The enzymatic activity of hK5 was tested using trypsin and chymotrypsin-specific synthetic fluorogenic substrates . In serum and ascites fluid, in addition to the free ( approximately 40 kDa) form, hK5 forms complexes with alpha(1)-antitrypsin and alpha(2)-macroglobulin . These complexes were detected by hybrid ELISA-type assays using hK5-specific coating antibodies and inhibitor detection antibodies . The ability of hK5 to bind to these inhibitors was further verified in vitro . Spiking of serum samples with 125I-labeled hK5 results in the distribution of the protein in two higher molecular mass (bound) forms, in addition to the unbound form . The hK5 mature enzyme is active and shows trypsin, but not chymotrypsin-like, activity . The pro-form of hK5 is not active . Recombinant hK5 shows a higher than predicted molecular mass due to glycosylation . hK5 is partially complexed with alpha(1)-antitrypsin and alpha(2)-macroglobulin in serum and ascites fluid of ovarian cancer patients . The recombinant protein is glycosylated and its mature form shows trypsin-like activity.

Biochem Biophys Res Commun, 2003 Aug 15, 308(1), 170 - 6
CHD1 associates with NCoR and histone deacetylase as well as with RNA splicing proteins; Tai HH et al.; CHD1 is one of a family of nuclear proteins containing two chromodomains, a SWI/SNF-like helicase/ATPase domain and a DNA binding domain . We found that CHD1 co-immunoprecipitates with histone deacetylase (HDAC) activity and that CHD1 also associates with NCoR, a transcriptional corepressor, in yeast two-hybrid and in vitro pull-down assays . NCoR is known to associate with HDACs to effect its repressive activity, suggesting that the predicted chromatin remodeling activity of CHD1 plays a role in this repression . Yeast two-hybrid assays also showed that CHD1 interacts with splicing proteins mKIAA0164, Srp20, and SAF-B . Splicing assays show that CHD1 overexpression can affect alternative splicing . These results suggest that CHD1 may function in both chromatin mediated transcriptional repression and RNA splicing.

J Inherit Metab Dis, 2003, 26(2-3), 199 - 207
Mitochondrial disorders: clinical presentation and diagnostic dilemmas; Smeitink JA; The number of genes known to be involved in mitochondrial energy production and the elucidation of the function of their individual transcripts is still increasing . Although at this stage it is impossible to predict the number of human genes necessary for mitochondrial biogenesis and maintenance, the total number in humans will most probably exceed the number of mitochondrial genes found in, for example, the budding yeast, which is about 800 . Without doubt we have only seen the tip of the iceberg of the clinical spectrum of mitochondrial disorders . Recent findings such as mutations in structural complex II genes in certain tumours emphasize the need to think outside the classical clinical presentation . We propose the consideration of a mitochondrial disorder in every chronic, intermittent or progressive disorder with single system or multisystem involvement, even if lactic acid is normal, and discuss such dilemmas as whether we should 'scrape the barrel' in every patient that are raised by this statement . The characterization of mitochondrial and nuclear DNA mutations in patients with enzymatically established mitochondrial defects has taught us that several of the current clinical and diagnostic assumptions have to be altered or even eliminated . The most challenging future task will be the development of new diagnostic criteria covering the expanding clinical spectrum of mitochondrial disorders.

Biotechnol Bioeng, 2003 Sep 20, 83(6), 668 - 80
On-line monitoring of Phaffia rhodozyma fed-batch process with in situ dispersive Raman spectroscopy; Cannizzaro C et al.; Since the yeast Phaffia rhodozyma was first described some 35 years ago, there has been significant interest in the development of commercial processes to exploit its ability to produce carotenoids (approximately 80% astaxanthin) . However, the optimal conditions for carotenoid production are not well understood . A key limitation has been the lack of an appropriate sensor for on-line carotenoid quantification . In this study, an in situ Raman spectroscopy probe was used to monitor intracellular carotenoid production for three consecutive P . rhodozyma fed-batch experiments . Raman spectroscopy is particularly well suited to the study of carotenoids due to a resonance effect, which greatly enhances the intensity of the three fundamental carotenoid bands, nu(1) (1513 cm(-1), C(-) (-)C stretch), nu(2) (1154 cm(-1), C-C stretch), and nu(3) (1003 cm(-1), CH(3) rock) . For all three cultures, the peak height of these bands was linearly correlated with intracellular carotenoid content (1 to 45 mg/L) to a precision of better than 5%, and the correlation from one experiment was directly applicable to others .

J Biol Chem, 2003 Oct 10, 278(41), 40113 - 20 Epub 2003 Jul 29.
An evolutionarily conserved N-terminal acetyltransferase complex associated with neuronal development; Sugiura N et al.; We previously identified mNAT1 (murine N-terminal acetyltransferase 1) as an embryonic gene that is expressed in the developing brain and subsequently down-regulated, in part, by the onset of N-methyl-d-aspartate (NMDA) receptor function . By searching the data base we discovered a second closely related gene, mNAT2 . mNAT1 and mNAT2 are highly homologous to yeast NAT1, a gene known to regulate entry into the G0 phase of the cell cycle . However, in the absence of further characterization, including evidence that mammalian homologues of NAT1 encode functional acetyltransferases, the significance of this relationship has been unclear . Here we focus on mNAT1 . Biochemical analysis demonstrated that mNAT1 and its evolutionarily conserved co-subunit, mARD1, assemble to form a functional acetyltransferase . Transfection of mammalian cells with mNAT1 and mARD1 followed by immunofluorescent staining revealed that these proteins localize to the cytoplasm in both overlapping and separate compartments . In situ hybridization demonstrated that throughout brain development mNAT1 and mARD1 are highly expressed in areas of cell division and migration and are down-regulated as neurons differentiate . Finally, mNAT1 and mARD1 are expressed in proliferating mouse P19 embryonic carcinoma cells; treatment of these cells with retinoic acid initiates exit from the cell cycle, neuronal differentiation, and down-regulation of mNAT1 and mARD1 as the NMOA receptor 1 gene is induced . The results provide the first direct evidence that vertebrate homologues of NAT1 and ARD1 form an evolutionarily conserved N-terminal acetyltransferase and suggest that expression and down-regulation of this enzyme complex plays an important role in the generation and differentiation of neurons.

Trends Cell Biol, 2003 Aug, 13(8), 435 - 46
The formins: active scaffolds that remodel the cytoskeleton; Wallar BJ et al.; Evolutionarily conserved in eukaryotes, formin homology (FH) proteins, or formins, exert their effects on the actin and microtubule (MT) networks during meiosis, mitosis, the maintenance of cell polarity, vesicular trafficking, signaling to the nucleus and embryonic development . Once thought to be only molecular scaffolds that indirectly affected cellular functions through the binding of other proteins, recent in vitro studies have illustrated that they can function as actin nucleators in the formation of new filaments . The connection between formins and MTs is less well understood . In yeast, the MT effects appear to be dependent on the ability of formins to generate polarized actin cables whereas, in mammalian cells, formin signals that cause MT stabilization and polarization might be more direct . A subclass of formins, the Diaphanous-related formins (Drfs), can act as effectors for Rho small GTPases, yet it is not clear what GTPase binding contributes to formin function.

Mol Cell, 2003 Jul, 12(1), 87 - 100
The CCT chaperonin promotes activation of the anaphase-promoting complex through the generation of functional Cdc20; Camasses A et al.; The WD repeat protein Cdc20 is essential for progression through mitosis because it is required to activate ubiquitin ligation by the anaphase-promoting complex (APC/C) . Here we show in yeast that Cdc20 binds to the CCT chaperonin, which is known as a folding machine for actin and tubulin . The CCT is required for Cdc20's ability to bind and activate the APC/C . In vivo, CCT is essential for Cdc20-dependent cell cycle events such as sister chromatid separation and exit from mitosis . The chaperonin is also required for the function of the Cdc20-related protein Cdh1, which activates the APC/C during G1 . We propose that folding of the Cdc20 family of APC/C activators is an essential and evolutionary conserved function of the CCT chaperonin.

Plant J, 2003 Aug, 35(3), 373 - 85
The SPA1-like proteins SPA3 and SPA4 repress photomorphogenesis in the light; Laubinger S et al.; Suppressor of phyA-105 (SPA1) is a phytochrome A-specific signaling intermediate that acts as a light-dependent repressor of photomorphogenesis in Arabidopsis seedlings . SPA1 is part of a small gene family comprising three genes: SPA1-related 2 (SPA2), SPA1-related 3 (SPA3), and SPA1-related 4 (SPA4) . Here, we investigate the functions of SPA3 and SPA4, two very closely related genes coding for proteins with 74% identical amino acids . Seedlings with mutations in SPA3 or SPA4 exhibit enhanced photomorphogenesis in the light, but show no phenotype in darkness . While there are small differences between the effects of spa3 and spa4 mutations, it is apparent that SPA3 and SPA4 function to inhibit light responses in continuous far-red, red, and blue light . Phytochrome A is necessary for all aspects of the spa4 mutant phenotype, suggesting that SPA4, like SPA1, acts specifically in phytochrome A signaling . Enhanced photoresponsiveness of spa3 mutants is also fully dependent on phytochrome A in far-red and blue light, but not in red light . Hence, SPA3 function in red light may be dependent on other phytochromes in addition to phytochrome A . Using yeast two-hybrid and in vitro interaction assays, we further show that SPA3 as well as SPA4 can physically interact with the constitutive repressor of light signaling COP1 . Deletion analyses suggest that SPA3 and SPA4, like SPA1, bind to the coiled-coil domain of COP1 . Taken together, our results have identified two new loci coding for negative regulators that may be involved in fine tuning of light responses by interacting with COP1.

Biochem Soc Trans, 2003 Aug, 31(Pt 4), 828 - 32
Neuronal calcium sensor-1: a multifunctional regulator of secretion; Hilfiker S; Ca2+ ions play a crucial role not only as the trigger for neurotransmitter release, but also in other aspects of brain function, such as short-term and long-term modulation of synaptic efficacy, which may underlie certain forms of learning and memory . The actions of Ca2+ are mediated by Ca(2+)-binding proteins, including a group of proteins known as neuronal calcium sensor (NCS) proteins . The NCS family includes NCS-1, visinin-like proteins, recoverins, guanylate cyclase-activating proteins and potassium channel-interacting proteins . Some members of this family, such as recoverin and guanylate cyclase-activating protein, are only expressed in photoreceptor cells and have been implicated in the control of visual transduction pathways, while the functional roles of the other members are largely unknown . NCS-1 was originally identified in Drosophila in a screen for neuronal hyperexcitability mutants . NCS-1 is an N-terminally myristoylated protein that contains four EF-hand motifs, three of which are able to bind Ca2+ in the submicromolar range . Overexpression of NCS-1 has been shown to enhance evoked neurotransmitter release, paired-pulse facilitation and exocytosis in several neuronal and neuroendocrine cell types . Recent experiments suggest that NCS-1 interacts directly with phosphatidylinositol 4-hydroxykinase in yeast as well as mammalian cells, suggesting that it may enhance neuronal secretion by modulating cellular trafficking steps in a phosphoinositide-dependent manner . In contrast, an involvement of NCS-1 in the expression and regulation of voltage-gated Ca2+ channels and K+ channels has also been proposed, which may be attributed, at least in part, to the effects of NCS-1 on vesicular trafficking pathways . The present review describes current knowledge about the cellular functions and molecular mechanisms by which NCS-1 may regulate neurotransmitter release.

Avian Dis, 2003 Apr-Jun, 47(2), 254 - 60
Experimental infection of white-leghorn cockerels with Macrorhabdos ornithogaster (Megabacterium); Phalen DN et al.; Macrorhabdos ornithogaster is a newly described anamorphic ascomycetous yeast that has been reported to cause a chronic, debilitating disease in many species of birds, including poultry . Study of this organism is complicated by the limited ability to grow M . ornithogaster in vitro . In this study, we showed that the chicken can be used to amplify this organism and as a model to study its pathogenicity . An infection rate of 100% was achieved in day-old chicks orally inoculated with 10(5) M . ornithogaster derived from the budgerigar (Melopsittacus undulatus) . The organism was also determined to increase in number by greater than 10-fold 14 days after oral inoculation in these chicks . Chickens infected with M . ornithogaster demonstrated no sign of illness but had decreased feed conversion efficiency and consistent and characteristic histopathologic lesions in the proventriculus and isthmus of the stomach, suggesting that M . ornithogaster may represent a potential threat to the poultry industry.

What Is Functional Genomics?, What Is Genetic Engineering?, What Is Bioassay?, What Is Fermentation?, What Is Activated Sludge?, a, Microbes, c, Microorganisms, n, Microbiology, i, Bacteriology, o, Bacteria, s, Bacillus, o, Shigella, i, Mueller hinton broth, e, Biodegradation, o, Yeasts, e, Escherichia coli, e, Serratia, o, Gram negative, s, Yeasts, a, Listeriosis, e, Escherichia coli, o, Antibiotics, i, Bacillus, s, S. cerevisiae, i, Neisseria, a, Water treatment, o, Bactericidal, s, Fermentations, e, Halophilic bacterium, r, Escherichia coli, a, S. cerevisiae

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005