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Biomedical subjects

R Kobayashi

Publications and source records attributed to R Kobayashi.

At least 199 records · Page 11Linked to original sources
PubMed1996

Cdc53 targets phosphorylated G1 cyclins for degradation by the ubiquitin proteolytic pathway.

In budding yeast, cell division is initiated in late G1 phase once the Cdc28 cyclin-dependent kinase is activated by the G1 cyclins Cln1, Cln2, and Cln3. The extreme instability of the Cln proteins couples environmental signals, which regulate Cln synthesis, to cell division. We isolated Cdc53 as a Cln2-associated protein and show that Cdc53 is required for Cln2 instability and ubiquitination in vivo. The Cln2-Cdc53 interaction, Cln2 ubiquitination, and Cln2 instability all depend on phosphorylation of Cln2. Cdc53 also binds the E2 ubiquitin-conjugating enzyme, Cdc34. These findings suggest that Cdc53 is a component of a ubiquitin-protein ligase complex that targets phosphorylated G1 cyclins for degradation by the ubiquitin-proteasome pathway.

Amino Acid Sequence↗
PubMed1996

Cellular adherence elicits ligand-independent activation of the Met cell-surface receptor.

Cell adhesion has a fundamental role in the proliferation and motility of normal cells and the metastasis of tumor cells. To identify signaling pathways activated by the adherence of tumor cells, we analyzed the tyrosine phosphorylation of proteins in mouse melanoma cells before and after attachment to substrata. We discovered that cellular adherence activated the protein-tyrosine kinase of the cell surface receptor Met, whose ligand is hepatocyte growth factor and scatter factor. The activation was exceedingly prompt, affected the great majority of Met in the cells, persisted so long as the cells remained adherent, and was rapidly reversed as soon as the cells were detached from substrata. Activation of Met required that cells be adherent but not that they spread on the substratum, and it occurred in the absence of any apparent ligand for the receptor. Ligand-independent activation of Met occurred in several varieties of tumor cells but not in normal endothelial cells that express the receptor. The activation of Met described here may represent a means by which cells respond to mechanical as opposed to biochemical stimuli.

Animals↗
PubMed1996

The SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex is required for RNA polymerase II and III snRNA gene transcription and interacts with the TATA box binding protein.

The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.

Amino Acid Sequence↗
PubMed1996

Detection of latent infection by Epstein-Barr virus in peripheral blood cells of healthy individuals and in non-neoplastic tonsillar tissue from patients by reverse transcription-polymerase chain reaction.

A highly sensitive and specific reverse transcription-polymerase chain reaction (RT-PCR) assay was designed for detection of Epstein-Barr virus (EBV)-related sequences in nucleic acid extracted by the conventional phenol method. Using this EBV-infected malignant lymphoma cell line, Raji cells, a comparative study of this assay was carried out with EBER1 (EBV-encoded small RNA1) primer and conventional DNA-PCR with BamHI-W and EBER1 primers, respectively. The results revealed that this assay has sensitivity about 10(5)-fold higher than the conventional DNA-PCR method. The presence of EBER1 DNA and RNA was also investigated in 23 healthy individuals and 22 right and left tonsils of 11 healthy individuals. These results indicated that this assay is both sensitive and specific. Thus, EBV infection could be diagnosed easily determined and EBER1 was shown to be transcribed in peripheral blood cells and tonsils with quantitatively different grades. This assay can be used to diagnose EBV infection in clinical samples.

Burkitt Lymphoma↗
PubMed1996

Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene activation.

We report the cloning of a transcription-associated histone acetyltransferase type A(HAT A). This Tetrahymena enzyme is strikingly homologous to the yeast protein Gcn5, a putative transcriptional adaptor, and we demonstrate that recombinant Gcn5p possesses HAT activity. Both the ciliate enzyme and Gcn5p contain potential active site residues found in other acetyltransferases and a highly conserved bromodomain. The presence of this domain in nuclear A-type HATs, but not in cytoplasmic B-type HATs, suggests a mechanism whereby HAT A is directed to chromatin to facilitate transcriptional activation. These findings shed light on the biochemical function of the evolutionarily conserved Gcn5p-Ada complex, directly linking histone acetylation to gene activation, and indicate that histone acetylation is a targeted phenomenon.

Acetylation↗
PubMed1996

Biochemical characterization of the human cyclin-dependent protein kinase activating kinase. Identification of p35 as a novel regulatory subunit.

The activation of cyclin-dependent protein kinases (Cdks) is dependent upon site-specific phosphorylation and dephosphorylation reactions, as well as positive and negative regulatory subunits. The human Cdk-activating protein kinase (Cak1) is itself a Cdc2-related cyclin-dependent protein kinase that associates with cyclin H. The present study utilized specific anti-Cak1 antibodies and immunoaffinity chromatography to identify additional Cak1-associated proteins and potential target substrates. Immunoprecipitation of metabolically labeled human osteosarcoma cells revealed a number of Cak1-associated proteins, including p95, p37 (cyclin H), and a 35-kDa protein that was further characterized herein. Microsequence analysis obtained after limited proteolysis revealed peptide fragments that are similar, but not identical to, human and yeast cyclins, thus identifying p35 as a cyclin-like regulatory subunit. The greatest sequence similarity of human p35 is with Mcs2, a yeast cyclin that is essential for cell cycle progression. Immunoaffinity chromatography performed under nondenaturing conditions afforded the isolation of enzymatically active Cak1 from cell lysates, enabling studies of kinase autophosphorylation and comparative substrate utilization. Immunoaffinity-purified Cak1 phosphorylated monomeric Cdc2 and Cdk2, but not Cdk4; the phosphorylation of both Cdc2 and Cdk2 were increased in the presence of recombinant cyclin A. These studies indicate that the Cak1 catalytic subunit, like Cdc2 and Cdk2, associates with multiple regulatory partners and suggests that subunit composition may be an important determinant of this multifunctional enzyme.

Amino Acid Sequence↗
PubMed1996

Predominance of nocturnal sympathetic nervous activity in salt-sensitive normotensive subjects.

To assess the relation between salt sensitivity and autonomic nervous function by power spectral analysis of heart rate variability in normotensive subjects, low and high salt diets were given to 13 normotensive men (aged 25 to 39 years) for 4 days each. Autonomic function was assessed by power spectral analysis of R-R intervals based on an autoregressive algorithm from 24-h Holter electrocardiogram. Subjects whose mean blood pressure was increased more than 3 mm Hg by high salt diet were defined as salt sensitive (SS, n = 5), and the remainder as salt resistant (SR, n = 8). Using the low frequency (LF, 0.1 Hz) and high frequency (HF, 0.25 Hz) components, the LF to total power ratio (%LF) was used as a marker of sympathetic activity, and the HF to total power ratio (%HF) as a marker of parasympathetic activity. Compared to the daytime, SR revealed a decrease in %LF and an increase in %HF during the night on both diets. In SS, these circadian changes were observed only during low-salt diet. During the night, SS showed a higher %LF and a lower %HF than SR. Plasma catecholamines tended to be decreased by the high sodium diet in SR but not in SS subjects. These results suggest that the persistent nocturnal predominance of sympathetic nervous activity in a salt-sensitive men may contribute to the subsequent increase of blood pressure in these subjects.

Adult↗
PubMed1996

Insulin resistance and cardiovascular complications in patients with essential hypertension.

Hyperinsulinemia or insulin resistance is suggested to play a role in the pathogenesis of hypertension and its target organ diseases. We designed this study to evaluate the role of insulin resistance in cardiac function, cardiac hypertrophy, wall thickness of the common carotid artery, and endothelial function of the brachial artery in essential hypertensive patients without diabetes mellitus. Insulin resistance was evaluated by the constant glucose infusion rate (M value) during the euglycemic-hyperinsulinemic glucose clamp test. In correlation analysis for several indices of glucose metabolism, only M value correlated with left ventricular mass index (LVMI), ratio of peak velocity during atrial contraction to that during early left ventricular filling phase (E/A ratio) and intima-media complex (IMC). In stepwise regression analysis of various risk factors for cardiovascular diseases, only M value and age were dependent factors for LVMI, E/A ratio, and IMC. No indices of glucose metabolism or risk factors for cardiovascular diseases correlated with endothelium-dependent and -independent vasodilation. These results suggest that insulin resistance, but not glucose intolerance and hyperinsulinemia, partly accelerates cardiovascular complications such as left ventricular hypertrophy and wall thickening of the carotid artery in patients with essential hypertension.

Blood Flow Velocity↗
PubMed1996

Respiratory muscle strength after lung resection with special reference to age and procedures of thoracotomy.

Changes in respiratory muscle strength after lung resection were examined concerning age and procedures of thoracotomy. Maximum inspiratory (MIP) and expiratory (MEP) mouth pressure were measured before operation and 1, 2, 4, and 12 weeks after operation in 81 patients undergoing lung resection. In 48 patients undergoing pneumonectomy, lobectomy, or segmentectomy, patients older than 70 showed a significantly lower MIP and MEP before operation and throughout the postoperative period compared to younger ones (P < 0.01). Furthermore, the older patients showed a significantly lower percentage of postoperative MIP and MEP 4 weeks after operation than the younger ones (P < 0.01). In 31 patients undergoing lung wedge resection, patients undergoing limited thoracotomy (LT) and video-assisted thoracic surgery (VATS) showed significantly higher percentages of postoperative MIP and MEP than those undergoing posterolateral thoracotomy (PLT) 1 and 2 weeks after operation (P < 0.01 or 0.05). But there was no significant difference in the values between LT and VATS. We concluded that (1) elderly patients suffered respiratory muscle weakness before and after operation and their postoperative recovery of respiratory muscle strength was slower than in younger patients, and (2) VATS and LT resulted in more rapid recovery of respiratory muscle strength than PLT, but the difference between VATS and LT was not significant.

Adult↗
PubMed1996

The p55 subunit of Drosophila chromatin assembly factor 1 is homologous to a histone deacetylase-associated protein.

To gain a better understanding of DNA replication-coupled chromatin assembly, we have isolated the cDNA encoding the smallest (apparent molecular mass, 55 kDa; termed p55) subunit of Drosophila melanogaster chromatin assembly factor 1 (dCAF-1), a multisubunit protein that is required for the assembly of nucleosomes onto newly replicated DNA in vitro. The p55 polypeptide comprises seven WD repeat motifs and is homologous to the mammalian RbAp48 protein, which is associated with the HD1 histone deacetylase. dCAF-1 was immunopurified by using affinity-purified antibodies against p55; the resulting dCAF-1 preparation possessed the four putative subunits of dCAF-1 (p180, p105, p75, and p55) and was active for DNA replication-coupled chromatin assembly. Moreover, dCAF-1 activity was specifically depleted with antibodies against p55. Thus, p55 is an integral component of dCAF-1. p55 is localized to the nucleus and is present throughout Drosophila development. Consistent with the homology between p55 and the HD1-associated RbAp48 protein, histone deacetylase activity was observed to coimmunoprecipitate specifically with p55 from a Drosophila nuclear extract. Furthermore, a fraction of the p55 protein becomes associated with the newly assembled chromatin following DNA replication. These findings collectively suggest that p55 may function as a link between DNA replication-coupled chromatin assembly and histone modification.

Amino Acid Sequence↗
PubMed1996

RNA polymerase III transcription from the human U6 and adenovirus type 2 VAI promoters has different requirements for human BRF, a subunit of human TFIIIB.

Mammalian TFIIIB can be separated into two fractions required for transcription of the adenovirus type 2 VAI gene, which have been designated 0.38M-TFIIIB and 0.48M-TFIIIB. While 0.48M-TFIIIB has not been characterized, 0.38M-TFIIIB corresponds to a TBP-containing complex. We describe here the purification of this complex, which consists of TBP and a closely associated polypeptide of 88 kDa, and the isolation of a cDNA corresponding to the 88-kDa polypeptide. The predicted protein sequence reveals that the 88-kDa polypeptide corresponds to a human homolog of the Saccharomyces cerevisiae BRF protein, a subunit of yeast TFIIIB. Human BRF (hBRF) probably corresponds to TFIIIB90, a protein previously cloned by Wang and Roeder (Proc. Natl. Acad. Sci. USA 92:7026-7030, 1995), although its predicted amino acid sequence differs from that reported for TFIIIB90 over a stretch of 67 amino acids as a result of frameshifts. Immunodepletion of more than 90 to 95% of the hBRF present in a transcription extract severely debilitates transcription from the tRNA-type VAI promoter but does not affect transcription from the TATA box-containing human U6 promoter, suggesting that the 0.38M-TFIIIB complex, and perhaps hBRF as well, is not required for U6 transcription.

Adenoviruses, Human↗
PubMed1996

The SAP, a new family of proteins, associate and function positively with the SIT4 phosphatase.

SIT4 is the catalytic subunit of a type 2A-related protein phosphatase in Saccharomyces cerevisiae that is required for G1 cyclin transcription and for bud formation. SIT4 associates with several high-molecular-mass proteins in a cell cycle-dependent fashion. We purified two SIT4-associated proteins, SAP155 and SAP190, and cloned the corresponding genes. By sequence homology, we isolated two additional SAP genes, SAP185 and SAP4. Through such an association is not yet proven for SAP4, each of SAP155, SAP185, and SAP190 physically associates with SIT4 in separate complexes. The SAPs function positively with SIT4, and by several criteria, the loss of all four SAPs is equivalent to the loss of SIT4. The data suggest that the SAPs are not functional in the absence of SIT4 and likewise that SIT4 is not functional in the absence of the SAPs. The SAPs are hyperphoshorylated in cells lacking SIT4, raising the possibility that the SAPs are substrates of SIT4. By sequence similarity, the SAPs fall into two groups, the SAP4/SAP155 group and the SAP185/SAP190 group. Overexpression of a SAP from one group does not suppress the defects due to the loss of the other group. These findings and others indicate that the SAPs have distinct functions.

Amino Acid Sequence↗
PubMed1996

Drosophila NAP-1 is a core histone chaperone that functions in ATP-facilitated assembly of regularly spaced nucleosomal arrays.

We describe the cloning and analysis of Drosophila nucleosome assembly protein 1 (dNAP-1), a core histone-binding protein that functions with other chromatin assembly activities in a Drosophila chromatin assembly factor 1-containing fraction (dCAF-1 fraction) in the ATP-facilitated assembly of regularly spaced nucleosomal arrays from purified core histones and DNA. Purified, recombinant dNAP-1 acts cooperatively with a factor(s) in the dCAF-1 fraction in the efficient and DNA replication-independent assembly of chromatin. In the presence of histone H1, the repeat length of the chromatin is similar to that of native chromatin from Drosophila embryos. By coimmunoprecipitation analysis, dNAP-1 was found to be associated with histones H2A and H2B in a crude whole-embryo extract, which suggests that dNAP-1 is bound to the histones in vivo. Studies of the localization of dNAP-1 in the Drosophila embryo revealed that the factor is present in the nucleus during S phase and is predominantly cytoplasmic during G2 phase. These data suggest that NAP-1 acts as a core histone shuttle which delivers the histones from the cytoplasm to the chromatin assembly machinery in the nucleus. Thus, NAP-1 appears to be one component of a multifactor chromatin assembly machinery that mediates the ATP-facilitated assembly of regularly spaced nucleosomal arrays.

Adenosine Triphosphate↗
PubMed1996

Benign adult familial myoclonus epilepsy (BAFME): an autosomal dominant form not linked to the dentatorubral pallidoluysian atrophy (DRPLA) gene.

The genetic differences between two types of dominant inherited myoclonus epilepsy, dentatorubral pallidoluysian atrophy (DRPLA) and benign adult familial myoclonus epilepsy (BAFME), have been reported. A gene with a CAG repeat expansion responsible for DRPLA has been isolated. We have examined CAG repeat expansion in the DRPLA gene in five BAFME families, and the abnormal CAG expansion was not observed in the affected subjects. Linkage analysis using DNA polymorphisms in the DRPLA gene and the genes for gamma-aminobutyric acid (GABA) receptor subunits, GABAR beta 1, GABAR beta 3, and GABAR alpha 6, showed that these genes were not responsible for BAFME.

Atrophy↗
PubMed1996

Prenatal diagnosis of Duchenne muscular dystrophy using a single fetal nucleated erythrocyte in maternal blood.

We developed a method that allows prenatal diagnosis of Duchenne muscular dystrophy using a single nucleated erythrocyte (NRBC) isolated from maternal blood. Maternal blood was obtained at 8 to 20 weeks of gestation. NRBCs were separated with Percoll using a discontinuous density gradient method and then collected by micromanipulator under microscopic observation. The entire genome of a single cell was amplified by primer extension preamplification (PEP). Sex was determined from a small aliquot of the PEP reaction. After an NRBC was determined to be male and confirmed to be of fetal origin, dystrophin exons 4, 8, 12, 45, 48, 50, and 51 were determined from the same PEP reaction. This diagnostic method using maternal blood is safer than amniocentesis or cordocentesis and can be applied to other X-linked diseases.

Base Sequence↗
PubMed1996

N-tropomodulin: a novel isoform of tropomodulin identified as the major binding protein to brain tropomyosin.

We have identified and characterized two proteins in rat brain that bind to the neuron-specific tropomyosin isoform, TMBr3. The two proteins were identified by blot overlay assay, in which the proteins immobilized on the membrane were probed by epitope-tagged TMBr3, followed by detection with anti-epitope antibody. We have purified these proteins using a TMBr3 affinity column. Peptide sequencing as well as immunoblotting showed that one of the two proteins is identical to tropomodulin, a tropomyosin-binding protein originally identified in erythrocytes. The cDNA for the other protein was cloned from an adult rat brain cDNA library using degenerate oligonucleotides that we designed based on the peptide sequences. Sequence analysis of the cDNA clone revealed this protein to be a novel isoform of tropomodulin which is the product of a distinct gene, and is herein referred to as N-tropomodulin. Recombinant N-tropomodulin bound to TMBr3 as well as to other low molecular mass tropomyosins (TM5a or TM5), but not to high molecular mass tropomyosins (TM2 or TMBr1). Northern blotting and RNase protection assays as well as immunoblotting showed that N-tropomodulin is expressed predominantly in brain. Furthermore, RNase protection assays revealed no alternatively spliced regions within the coding sequence. Developmentally, N-tropomodulin was detected in rat brain as early as embryonic day 14 and reaches the adult level before birth. Immunofluorescence of primary frontal cortex cell cultures showed that N-tropomodulin is specifically expressed in neurons. The neuron-specific expression of N-tropomodulin strongly suggests specialized roles of this TM-binding protein in neurons.

Amino Acid Sequence↗