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Biomedical subjects

H Okayama

Publications and source records attributed to H Okayama.

At least 19 recordsLinked to original sources
PubMed1995

Fission yeast Rep2 is a putative transcriptional activator subunit for the cell cycle 'start' function of Res2-Cdc10.

In the yeast cell cycle 'start' requires sets of the Cdc10/ SWI family of transcriptional factors which activate the MCB cis elements contained in genes essential for S phase progression. Fission yeast possess two such overlapping systems, Res1-Cdc10 and Res2-Cdc10, both of which act to start the mitotic and meiotic cycles. We have recently isolated rep2+ as a multicopy suppressor of a temperature-sensitive cdc10 mutant which encodes a zinc finger protein. Here we show that the Rep2 zinc finger protein is an essential component of the active Res2-Cdc10 transcriptional regulator complex and likely to play a role in the control of cell cycle 'start'. Our data suggest that Rep2 is a transcriptional activator subunit which interacts with the MCB binding subunit complex formed by Res2 and Cdc10.

Amino Acid Sequence↗
PubMed1995

Requirement for tyrosine phosphorylation of Cdk4 in G1 arrest induced by ultraviolet irradiation.

Exposure to ultraviolet light arrests the function of mammalian fibroblasts in the G1 phase of the cell cycle, as well as the S and G2 phases. Although p21, an inhibitor of cyclin-dependent kinase (Cdk) that is induced by DNA damage may partly account for the arrest in G1 (ref. 1), the mechanism is little understood. Here we show that tyrosine phosphorylation of Cdk4 is required for this arrest. In rat fibroblast, Cdk4 is tyrosine-phosphorylated during G1 progression, and its dephosphorylation is required for S phase. When cells are ultraviolet-irradiated, their arrest in G1 is accompanied by an increase in phosphorylation level. Conversely, cells expressing unphosphorylatable Cdk4F17 fail to arrest in G1, and suffer significantly elevated chromosomal aberrations and cell death.

Animals↗
PubMed1995

A kinase from fission yeast responsible for blocking mitosis in S phase.

In virtually all eukaryotes, mitosis starts after the completion of DNA synthesis. This orderly process is ensured by the checkpoint mechanism that blocks the onset of mitosis while DNA is being synthesized or is damaged. In the fission yeast Schizosaccharomyces pombe, this mechanism involves some rad+ and hus+ genes. However, it is not known how the checkpoint system monitors these events. Recently a multicopy suppressor of a temperature-sensitive DNA polymerase-alpha mutant was isolated. This gene, named cds1+ (checking DNA synthesis), encodes a typical protein kinase. Here we report that this protein kinase is a key component of the DNA replication-monitoring S/G2 checkpoint system. Our data suggest that its primary role is to monitor DNA synthesis by interacting with DNA polymerase alpha and send a signal to block the onset of mitosis while DNA synthesis is in progress.

Amino Acid Sequence↗
PubMed1995

Localization of histamine N-methyltransferase messenger RNA in human nasal mucosa.

BACKGROUND: Histamine is metabolized mainly by histamine N-methyltransferase (HMT) to N tau-methylhistamine in human nasal mucosa. Human HMT cDNA has been cloned and expressed in COS cells. The purpose of this study was to determine the localization of HMT METHODS: The fragment (nucleotide residues 430-1055) of human HMT cDNA was subcloned in a Bluescript vector (Stratagene, La Jolla, Calif.), and HMT sense anti-sense RNA probes were made with T7 and T3 RNA polymerases. In situ hybridization with digoxigenin-labeled RNA probes was performed on surgical specimens of human nasal turbinates. RESULTS: HMT mRNA was localized in cells in the epithelium and submucosa, and densely in endothelial cells of vessels. No HMT mRNA was identified in the submucosal glands. The presence of HMT mRNA was confirmed by Northern blot analysis, and HMT activities were also detected in nasal mucosa. CONCLUSION: Our study indicates that endothelium expresses HMT mRNA, whereas cells in the epithelium and submucosa, which remain unidentified, are an additional source of HMT mRNA.

Blotting, Northern↗
PubMed1995

Liver fructose-1,6-bisphosphatase cDNA: trans-complementation of fission yeast and characterization of two human transcripts.

The SV40 early promoter is active both in mammalian cells and in the fission yeast Schizosaccharomyces pombe, and is used to drive full-length cDNA in polyvalent pcD-libraries. Two such liver libraries, of human and rat origin, were used to trans-complement a S. pombe mutant deficient in fructose-1,6-bisphosphatase (Fru-1,6-Pase) activity, a key gluconeogenic enzyme restricted to liver, kidney and intestine in mammals. A rat liver Fru-1,6-Pase cDNA was readily cloned and sequenced. Complementary PCR experiments revealed full-length Fru-1,6-Pase cDNA also present in the human liver library, however at a low abundance. Two human liver transcripts were thus characterized. Contrary to expectation, they were not differentially spliced products. They both encoded the same protein and were generated by a polyadenylation choice mechanism. The longest transcript comprised two polyadenylation signals and a consensus GT-rich element for the 3' processing of the upstream site. Rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) analysis of 3' ends from hepatic, renal and intestinal mRNA disclosed that both Fru-1,6-Pase transcripts are expressed in the three main gluconeogenic cell types and are subject to insulin differential modulation. On the other hand, overcoming liver cell heterogeneity problems, sequence analysis of 16 independent clones of 3' end-cDNA demonstrated that, in addition to a monocytic type corresponding to a previously described lambda gt11 clone, human liver does not contain a hepatic type Fru-1,6-Pase comprising a liver-specific carboxyl-terminal extension like its rat counterpart. This liver-specific extension is involved in enzyme up-regulation and appears to give a conclusive advantage to the rat hepatic enzyme over the human one when trans-complementing mutant yeast.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

The G1/S boundary-specific enhancer of the rat cdc2 promoter.

Multiple species of G1 cyclins and cyclin-dependent kinases are induced sequentially during G1 phase, and the expression of cyclin A and cdc2 genes is subsequently induced at the G1/S boundary. To analyze the mechanism of cdc2 promoter activation, the 5'-flanking region of the rat cdc2 gene was isolated and its structural features were characterized. The highly conserved sequence between human and rat cdc2 genes is present in the basal promoter region from positions -183 to -122, which contains the E box, SpI, and E2F motifs. The expression of 5' sequential deletion derivatives of the promoter fused to luciferase cDNA in rat 3Y1 cells revealed the presence of the enhancer element. The presumed enhancer region was further analyzed by the introduction of base substitutions and by the formation of DNA-protein complexes with cell extracts prepared at various times during the G1-to-S-phase progression. These analyses revealed that the enhancer sequence, AAGTTACAAATA, located from -276 to -265, confers strong inducibility on the basal promoter at the G1/S boundary. The base substitutions introduced into the motifs of transcription factors indicated that the E2F motif is essential for the enhancer-dependent activation of the cdc2 promoter at the G1/S boundary. Electrophoretic mobility shift assays and DNase I footprinting showed that a factor which interacts with the enhancer element is induced late in G1 phase.

Animals↗
PubMed1995

Relation between postpacing T wave changes and myocardial scintigraphic characteristics in patients with ventricular demand pacemaker.

The aim of this study was to elucidate the cause of T wave changes after ventricular pacing using thallium 201 single photon emission computed tomography (Tl-SPECT). Tl-SPECT was performed in 20 patients with sick sinus syndrome who had negative T wave after ventricular pacing and had had a ventricular demand pacemaker (VVI) implanted. Patients who have stenotic coronary arteries were excluded from the study. From the analysis of Tl-SPECT, extent score was calculated as the ischemic region. Data in patients with SSS were compared with those in 20 normal controls (NC). Thallium perfusion defects in patients with VVI were observed and distributed mainly in apex and inferior regions on the polar map. The extent score in patients with VVI was significantly higher than that in NC (56.4 +/- 21.6% vs 3.2 +/- 6.4%, P < 0.01). The washout rate of thallium 201 in the defect area was significantly lower in patients with VVI than in NC (27.4 +/- 10.2% vs 46.8 +/- 12.3%, P < 0.01). These results suggest that T wave changes after ventricular pacing are closely related to myocardial ischemia.

Aged↗
PubMed1994

A copy-number-controlled expression vector for the fission yeast Schizosaccharomyces pombe.

A novel expression vector for the fission yeast Schizosaccharomyces pombe carries the neomycin-resistance-encoding gene regulated by the SV40 early promoter, and its copy number is controlled by the level of Geneticin (G418). Foreign gene expression is driven by the human cytomegalovirus (hCMV) promoter which is transcriptionally active in S. pombe. Moreover, the vector expresses foreign genes at high levels, due to the 5'-untranslated region (5'-UTR) containing an A + T-rich sequence of about 50 nucleotides located between the TATA box of the hCMV promoter and the start codon. Recombinant human lipocortin I was produced at levels of up to 50% of the total soluble protein in the presence of 100-200 micrograms/ml of G418 in the media. Southern and Northern blotting showed that this high level of expression was due to an increase in copy number induced by G418, the high transcriptional activity of the hCMV promoter and the high translational efficiency of the 5'-UTR. We modified the vector into an 'ATG vector', named pTL2M, that maintains the 5'-UTR optimized for gene expression and into which any foreign gene, whose exact sequence is known, can be easily inserted.

Annexin A1↗
PubMed1994

Dnacin A1 and dnacin B1 are antitumor antibiotics that inhibit cdc25B phosphatase activity.

The p80cdc25 protein is a protein phosphatase directly involved in p34cdc2 protein kinase activation by dephosphorylation. The cdc25B gene is one of three human cdc25 homologs which can complement the temperature-sensitive cdc25 mutation of Schizosaccharomyces pombe, and is expressed a high levels in human cell lines, particularly in some cancer cells. A fusion protein of glutathione-S-transferase (GST) and the catalytic domain of cdc25B protein was constructed and found to retain phosphatase activity in the manner of a p80cdc25 phosphatase by using a chromogenic substrate, p-nitrophenylphosphate. Two benzoquinoid antitumor compounds, dnacin A1 and dnacin B1, inhibited phosphatase activity in a non-competitive manner.

Antibiotics, Antineoplastic↗
PubMed1994

A B-type cyclin negatively regulates conjugation via interacting with cell cycle 'start' genes in fission yeast.

In the fission yeast Schizosaccharomyces pombe, the cdc10+/SWI family members constitute the cell cycle 'start' genes. res1+ and res2+ are the newly identified members of this family and encode putative association partners of the Cdc10 protein. The Pat1 kinase plays a pivotal role in switching between vegetative growth and sexual development, and its inactivation in haploid cells induces unconditional growth arrest and subsequent meiosis. We have identified as an extragenic suppressor of a temperature sensitive pat1-114 mutant, a new B-type cyclin that negatively regulates conjugation by interacting with these 'start' genes. This cyclin, named Cyc17, is highly homologous with Cdc13, but has no detectable activity as a mitotic cyclin. Deletion of cyc17+ markedly enhances conjugation, despite the presence of nitrogen source, and accelerates growth arrest in G1 upon nitrogen starvation. Conversely, overexpression of the cyc17+ gene strongly inhibits conjugation. The cyc17+ gene is transcribed into 3.2 kb poly(A)+ and 3.0 kb poly(A)- RNAs. Only the poly(A)+ species is expressed during vegetative growth and periodically with a peak in the G1 and S phases of the cell cycle. On the other hand, the poly(A)- transcript is highly induced during conjugation. This induction is lost in res2- cells, whereas the poly(A)+ transcript is significantly reduced in res1- cells. However, the mating inhibition as well as the ability to rescue the pat1 mutation by overexpression of res1+ and res2+ are totally abolished in cyc17- cells. Thus, in S.pombe, a B-type cyclin, regulated by the newly identified cell cycle 'start' genes, plays a crucial role in the control of sexual development.

Amino Acid Sequence↗
PubMed1994

res2+, a new member of the cdc10+/SWI4 family, controls the 'start' of mitotic and meiotic cycles in fission yeast.

In the fission yeast Schizosaccharomyces pombe, the cdc10+ and res1+ genes play a crucial role in the start of mitotic and meiotic cycles. They encode structurally related transcriptional complex proteins and regulate some S phase-specific genes. Here we report the identification of a new member of this family named as res2+. res2+ has been isolated as a multicopy suppressor of a res1- null mutant and specifies a 73 kDa protein, which has two copies of the Swi/ankyrin motif and shares the highest sequence and structure similarity with the Res1 protein. res2+ is largely redundant in function with res1+ and is required for the initiation of mitotic and premeiotic DNA synthesis, but has an additional role in meiotic division. Unlike res1+, res2+ is highly induced during conjugation and strongly depends on cdc10+ for its activity. We conclude that the fission yeast contains two functionally overlapping parallel 'start' systems, Res1-Cdc10 and Res2-Cdc10, the former of which plays a major role in mitotic cycle whereas the latter in meiotic cycle.

Amino Acid Sequence↗
PubMed1994

A zinc finger protein controls the onset of premeiotic DNA synthesis of fission yeast in a Mei2-independent cascade.

In the fission yeast Schizosaccharomyces pombe, meiosis is initiated by the action of Mei2 in a complex cascade activated following conjugation. We have isolated a new gene named rep1+ that is required for the initiation of premeiotic DNA synthesis. rep1+ encodes a 53 kDa protein with one zinc finger motif that is essential for function, and effectively rescues a null mutant of the res1+ gene but only partially a temperature-sensitive mutant of the cdc10+ gene, both of which are required for the onset of mitotic, as well as premeiotic, S phase. Deletion of rep1+ has no apparent effects on the mitotic cell cycle or conjugation, but blocks the initiation of premeiotic DNA synthesis. However, this defect is partially suppressed when rapidly growing cells are induced to conjugate, indicating that the rep1+ function is at least partly substituted by those of the genes controlling the 'start' of the mitotic cell cycle. The rep1 null mutant fails to induce the res2+ gene, a newly identified res1+ homolog cooperating with Cdc10 and acting for the onset of mitotic and premeiotic DNA synthesis, as well as for meiotic division. The rep1+ gene itself is induced moderately during nitrogen starvation but highly during conjugation, and this induction is dependent on both ste11+ and mating pheromones but independent of mei2+. Thus, rep1+ controls the initiation of premeiotic DNA synthesis via induction and/or activation of Res2 and some other essential factors in a cascade independent of Mei2.

Amino Acid Sequence↗
PubMed1994

Cdc25A is a novel phosphatase functioning early in the cell cycle.

The cdc25+ tyrosine phosphatase is a key mitotic inducer of the fission yeast Schizosaccharomyces pombe, controlling the timing of the initiation of mitosis. Mammals contain at least three cdc25+ homologues called cdc25A, cdc25B and cdc25C. In this study we investigate the biological function of cdc25A. Although very potent in rescuing the S.pombe cdc25 mutant, cdc25A is less structurally related to the S.pombe enzyme. Northern and Western blotting detection reveals that unlike cdc25B, cdc25C and cdc2, cdc25A is predominantly expressed in late G1. Moreover, immunodepletion of cdc25A in rat cells by microinjection of a specific antibody effectively blocks their cell cycle progression from G1 into the S phase, as determined by laser scanning single cell cytometry. These results indicate that cdc25A is not a mitotic regulator but a novel phosphatase that plays a crucial role in the start of the cell cycle. In view of its strong ability to activate cdc2 kinase and its specific expression in late G1, cdc2-related kinases functioning early in the cell cycle may be targets for this phosphatase.

Amino Acid Sequence↗
PubMed1994

Id-related genes encoding helix-loop-helix proteins are required for G1 progression and are repressed in senescent human fibroblasts.

Three complete cDNA clones encoding Id-related helix-loop-helix (HLH) proteins lacking a basic region were isolated from a pcD2 cDNA expression library prepared from TIG-3 human diploid fibroblasts (HDF). Of these cDNAs (Id-1H, Id-1H', and Id-2H), two (Id-1H and Id-1H') appeared to be derived by alternative RNA splicing. Id-1H and Id-2H seem to be human homologues of mouse Id-1 and Id-2, respectively, and have potential to encode 154 and 135 amino acid proteins. The Id-1H and Id-2H mRNAs were barely detectable in quiescent early passage HDF; serum coordinately induced both mRNAs, with two peaks of expression, in early and late in G1. Antisense oligomers complementary to Id-1H and Id-2H mRNA prevented early passage HDF from entering the S phase of the cell cycle. The treatment of serum-stimulated early passage cells with the antisense Id-1H oligomer completely abolished Id-1H. In senescent cells, serum barely induced the Id-1H and Id-2H mRNAs, although the levels of c-myc expression induced were similar in early passage and senescent cells. The expression levels of these Id genes vary among immortal human cell lines. Both genes were overexpressed in VA4 SV40-transformed lung fibroblasts and EJ-1 bladder carcinoma cells, while these genes were expressed at a very low level in SVts8 cells derived from SV40 tsA-transformed TIG-3 cells. SVts8 cells may acquire some function redundant to Id proteins. HT1080 fibrosarcoma cells expressed the Id-1H gene but not the Id-2H gene, suggesting these Id genes may subserve redundant functions.

Alternative Splicing↗
PubMed1994

Highly sensitive detection of viral RNA genomes in blood specimens by an optimized reverse transcription-polymerase chain reaction.

A protocol was developed for a highly sensitive detection of viral RNA in blood specimens by reverse transcription coupled with a nested polymerase chain reaction. Using Japanese encephalitis virus (JEV) as a model, the optimized reverse transcription-polymerase chain reaction (ORTPCR) detects as few as 3-5 virions in 0.1 ml of whole blood specimens. The effectiveness of this assay system is confirmed by diagnosis of human hepatitis C viral (HCV) infection.

Animals↗
PubMed1994

Production of nonstructural proteins of hepatitis C virus requires a putative viral protease encoded by NS3.

Hepatitis C virus (HCV) is a positive strand RNA virus with certain similarity to flaviviruses and pestiviruses. To examine the processing and possible assembly of HCV proteins, we constructed a recombinant vaccinia virus that expresses a full-length genomic RNA, infected chimp liver cells with the virus, and analyzed HCV-related protein products by immunofluorescent antibody staining and Western blot detection with mouse monoclonal antibodies. The putative core, envelope, and NS1 and NS3 proteins that yielded from this recombinant were 22, 32, 53 to 58, and 65 kDa in size, respectively. The NS4 protein was unexpectedly small, with an estimated molecular weight of 7 kDa, and the NS5 protein was found to be further cleaved into 52-kDa NS5a and 58-kDa NS5b proteins, the latter of which contains a hallmark of RNA replicase. A point mutation in the putative protease domain of NS3 resulted in a failure in the production of NS3, NS4, NS5a, and NS5b, but coexpression of NS3 restored the proper processing of these proteins, demonstrating that NS3, the putative viral protease, is essential for the production of these nonstructural proteins. Thus, HCV strikingly resembles pestiviruses in the size and the processing mode of the nonstructural proteins, particularly NS4 and NS5.

Animals↗
PubMed1994

Magnesium regulates ion transport across canine tracheal epithelium.

We examined the effect of Mg2+ on potential difference (PD) and short circuit current (SCC) of the posterior epithelial membrane of canine trachea using an Ussing chamber. After the exchange to a Mg(2+)-free solution, PD and SCC rapidly increased, reaching maximal values within 3 min, followed by a gradual return towards the baseline over 60 min. In a Ca(2+)-free solution, Mg2+ removal did not alter PD and SCC values. Increased Mg2+ in the solution produced significant gradual decreases in PD and SCC. The decreases in PD and SCC were reversed by the addition of excessive Ca2+ to the solution. Mg2+ removal did not alter significantly isoproterenol-induced increases in PD and SCC values, while increased Mg2+ significantly reduced the increases. These findings indicate that extracellular Mg2+ is an important determinant in ion transport across the airway epithelium, probably through antagonistic actions of Mg2+ and Ca2+.

Amiloride↗