Biomedical subjects

H Okayama

Publications and source records attributed to H Okayama.

At least 19 recordsLinked to original sources

PubMed2000

Fission yeast Fizzy-related protein srw1p is a G(1)-specific promoter of mitotic cyclin B degradation.

Downregulation of cyclin-dependent kinase (Cdk)-mitotic cyclin complexes is important during cell cycle progression and in G(1) arrested cells undergoing differentiation. srw1p, a member of the Fizzy-related protein family in fission yeast, is required for the degradation of cdc13p mitotic cyclin B during G(1) arrest. Here we show that srw1p is not required for the degradation of cdc13p during mitotic exit demonstrating that there are two systems operative at different stages of the cell cycle for cdc13p degradation, and that srw1p is phosphorylated by Cdk-cdc13p only becoming dephosphorylated during G(1) arrest. We propose that this phosphorylation targets srw1p for proteolysis and inhibits its activity to promote cdc13p turnover.

CDC2 Protein Kinase↗

PubMed2000

Loop-mediated isothermal amplification of DNA.

We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10(9) copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.

Base Sequence↗

PubMed2000

Role of TGF-beta in EGF-induced transformation of NRK cells is sustaining high-level EGF-signaling.

We have been isolating and analyzing NRK cell mutants, which fail to transform by epidermal growth factor (EGF) and transforming growth factor (TGF)-beta. One such mutant, R14, can respond to the growth inhibitory signal of TGF-beta to the same extent as parental NRK but fail to respond to the growth stimulatory signal of EGF. This mutant has a defect in EGF receptor (EGFR) expression. When R14 mutant expressed a high level of EGFR, however, EGF not only induced proliferation in this mutant but also induced transformation without the aid of TGF-beta. These findings suggest that the major role of TGF-beta in this transformation system should be to counteract the ligand-dependent down-regulation of EGFR, thereby sustaining high-level EGF-signaling.

Animals↗

PubMed2000

The polyubiquitin gene is essential for meiosis in fission yeast.

We isolated a novel sporulation-deficient mutant of Schizosaccharomyces pombe. The mutant did not have a mitotic growth defect but aborted meiosis at the first or the second division with condensed chromosomes that failed to separate, abnormal spindle(s), and disintegrated spindle pole bodies (SPBs). During the first division, the centromeres were pulled to near the spindle poles but condensed divalent chromosomes remained at the center. The failure to proceed to anaphase was also observed during a time-lapse recording of a SPB protein tagged with green fluorescent protein. The polyubiquitin gene ubi4(+), which encoded eight ubiquitins fused in tandem, complemented this mutant. The mutation, an A to G substitution, was identified within the ubi4(+) gene at the ATG initiation codon. Disruption of the ubi4(+) gene produced the same phenotypes. The ubi4(+) mRNA was strongly induced for meiosis. However, ubiquitin increases only slightly, suggesting that the role of the polyubiquitin gene is to supply ubiquitin that is consumed by unidentified mechanisms. Before the ubi4 mutant cells entered meiosis, ubiquitin was greatly decreased indicating that shortage of ubiquitin caused abortion of meiosis. This work provides insights for the role of polyubiquitin gene and importance of ubiquitination in SPB integrity at the meiotic divisions.

Amino Acid Sequence↗

PubMed2000

Genetic studies with the fission yeast Schizosaccharomyces pombe suggest involvement of wee1, ppa2, and rad24 in induction of cell cycle arrest by human immunodeficiency virus type 1 Vpr.

Accessory protein Vpr of human immunodeficiency virus type 1 (HIV-1) arrests cell cycling at G(2)/M phase in human and simian cells. Recently, it has been shown that Vpr also causes cell cycle arrest in the fission yeast Schizosaccharomyces pombe, which shares the cell cycle regulatory mechanisms with higher eukaryotes including humans. In this study, in order to identify host cellular factors involved in Vpr-induced cell cycle arrest, the ability of Vpr to cause elongated cellular morphology (cdc phenotype) typical of G(2)/M cell cycle arrest in wild-type and various mutant strains of S. pombe was examined. Our results indicated that Vpr caused the cdc phenotype in wild-type S. pombe as well as in strains carrying mutations, such as the cdc2-3w, Deltacdc25, rad1-1, Deltachk1, Deltamik1, and Deltappa1 strains. However, other mutants, such as the cdc2-1w, Deltawee1, Deltappa2, and Deltarad24 strains, failed to show a distinct cdc phenotype in response to Vpr expression. Results of these genetic studies suggested that Wee1, Ppa2, and Rad24 might be required for induction of cell cycle arrest by HIV-1 Vpr. Cell proliferation was inhibited by Vpr expression in all of the strains examined including the ones that did not show the cdc phenotype. The results supported the previously suggested possibility that Vpr affects the cell cycle and cell proliferation through different pathways.

Cell Cycle↗

PubMed2000

Fission yeast Eso1p is required for establishing sister chromatid cohesion during S phase.

Sister chromatid cohesion is essential for cell viability. We have isolated a novel temperature-sensitive lethal mutant named eso1-H17 that displays spindle assembly checkpoint-dependent mitotic delay and abnormal chromosome segregation. At the permissive temperature, the eso1-H17 mutant shows mild sensitivity to UV irradiation and DNA-damaging chemicals. At the nonpermissive temperature, the mutant is arrested in M phase with a viability loss due to a failure to establish sister chromatid cohesion during S phase. The lethal M-phase arrest phenotype, however, is suppressed by inactivation of a spindle checkpoint. The eso1(+) gene is not essential for the onset and progression of DNA replication but has remarkable genetic interactions with those genes regulating the G(1)-S transition and DNA replication. The N-terminal two-thirds of Eso1p is highly homologous to DNA polymerase eta of budding yeast and humans, and the C-terminal one-third is homologous to budding yeast Eco1p (also called Ctf7p), which is required for the establishment of sister chromatid cohesion. Deletion analysis and determination of the mutation site reveal that the function of the Eco1p/Ctf7p-homologous domain is necessary and sufficient for sister chromatid cohesion. On the other hand, deletion of the DNA polymerase eta domain in Eso1p increases sensitivity to UV irradiation. These results indicate that Eso1p plays a dual role during DNA replication. The C-terminal region acts to establish sister chromatid cohesion, and the N-terminal region presumably catalyzes translesion DNA synthesis when template DNA contains lesions that block regular DNA replication.

Acetyltransferases↗

PubMed2000

Infection of left atrial thrombus associated with mitral stenosis: A case report.

We describe the first reported case of an infected left atrial thrombus. The case of the 65-year-old male patient in this report was associated with mitral stenosis and involved Escherichia coli, and was treated successfully with surgical resection of the infected thrombus. This case suggests that such infection should be considered as a possible complication of intracardiac thrombus when bacteremia is present.

Aged↗

PubMed1999

Oncogenic stimulation recruits cyclin-dependent kinase in the cell cycle start in rat fibroblast.

The rat fibroblast NRK cells are transformed reversibly by a combination of growth factors. When stimulated with serum, NRK cells rely on cyclin-dependent kinase 4 (Cdk4) for their S phase entry. However, when stimulated with serum containing oncogenic growth factors, they come to rely on either Cdk4 or Cdk6, and their S phase entry cannot be blocked unless both Cdk4 and Cdk6 are immunodepleted. Such change of dependence does not occur in the NRK cell mutants defective in an oncogenic signal pathway and, therefore, deficient in anchorage-independent cell cycle start ability, correlating Cdk6 dependence with this remarkable, cancer-associated phenotype. However, both Cdk4 and Cdk6 are activated upon serum stimulation, and neither the amounts of Cdk6, Cdk4, cyclin D1, and cyclin-dependent kinase inhibitors nor the activities or subcellular localization of Cdk6 and Cdk4 are significantly influenced by oncogenic stimulation. Thus, oncogenic stimulation invokes Cdk6 to participate in a critical step of the cell cycle start in a rat fibroblast, but by a mechanism seemingly unrelated to the regulation of the kinase. Given that many hematopoietic cells employ predominantly Cdk6 for the cell cycle start and perform anchorage-independent growth by nature, our results raise the possibility that the oncogenic stimulation-induced anchorage-independent cell cycle start of NRK is elicited by a mechanism similar to the one used for hematopoietic cell proliferation.

Animals↗

PubMed1999

Role of human Cds1 (Chk2) kinase in DNA damage checkpoint and its regulation by p53.

In response to DNA damage, mammalian cells adopt checkpoint regulation, by phosphorylation and stabilization of p53, to delay cell cycle progression. However, most cancer cells that lack functional p53 retain an unknown checkpoint mechanism(s) by which cells are arrested at the G(2)/M phase. Here we demonstrate that a human homolog of Cds1/Rad53 kinase (hCds1) is rapidly phosphorylated and activated in response to DNA damage not only in normal cells but in cancer cells lacking functional p53. A survey of various cancer cell lines revealed that the expression level of hCds1 mRNA is inversely related to the presence of functional p53. In addition, transfection of normal human fibroblasts with SV40 T antigen or human papilloma viruses E6 or E7 causes a marked induction of hCds1 mRNA, and the introduction of functional p53 into SV40 T antigen- and E6-, but not E7-, transfected cells decreases the hCds1 level, suggesting that p53 negatively regulates the expression of hCds1. In cells without functional ataxia telangiectasia mutated (ATM) protein, phosphorylation and activation of hCds1 were observed in response to DNA damage induced by UV but not by ionizing irradiation. These results suggest that hCds1 is activated through an ATM-dependent as well as -independent pathway and that it may complement the function of p53 in DNA damage checkpoints in mammalian cells.

Ataxia Telangiectasia Mutated Proteins↗

PubMed1999

Cell cycle start from quiescence controlled by tyrosine phosphorylation of Cdk4.

In mammals Cdk4 (or Cdk6 in some cell types) is required for starting the cell cycle. Recently we showed that Cdk4 is regulated by tyrosine phosphorylation and dephosphorylation, and that this regulation is required for a DNA damage-induced G1 arrest. We report here that a generic anti-phosphotyrosine antibody can detect tyrosine-phosphorylated Cdk4 and that as revealed by immunoblot detection and kinase assay, this regulation is employed for DNA damage-responsive checkpoint control during cell cycle start from quiescence. In rat fibroblasts traversing G1 or arrested in G1 by deprivation of anchorage, Cdk4 does not undergo tyrosine phosphorylation. Tyrosine phosphorylation occurs only during cell's arrest in quiescence and dephosphorylation during their cell cycle start. Ultraviolet irradiation blocks dephosphorylation and concomitant activation of Cdk4, thereby preventing the start of cell cycling. Thus, unlike tyrosine phosphorylation of Cdc2, which controls phase transition in the regular cell cycle, tyrosine phosphorylation of Cdk4 is employed for controlling cell cycle start from quiescence in a rat fibroblast.

3T3 Cells↗

PubMed1999

A double-strand break repair component is essential for S phase completion in fission yeast cell cycling.

Fission yeast rad22(+), a homologue of budding yeast RAD52, encodes a double-strand break repair component, which is dispensable for proliferation. We, however, have recently obtained a cell division cycle mutant with a temperature-sensitive allele of rad22(+), designated rad22-H6, which resulted from a point mutation in the conserved coding sequence leading to one amino acid alteration. We have subsequently isolated rad22(+) and its novel homologue rti1(+) as multicopy suppressors of this mutant. rti1(+) suppresses all the defects of cells lacking rad22(+). Mating type switch-inactive heterothallic cells lacking either rad22(+) or rti1(+) are viable, but those lacking both genes are inviable and arrest proliferation with a cell division cycle phenotype. At the nonpermissive temperature, a synchronous culture of rad22-H6 cells performs DNA synthesis without delay and arrests with chromosomes seemingly intact and replication completed and with a high level of tyrosine-phosphorylated Cdc2. However, rad22-H6 cells show a typical S phase arrest phenotype if combined with the rad1-1 checkpoint mutation. rad22(+) genetically interacts with rad11(+), which encodes the large subunit of replication protein A. Deletion of rad22(+)/rti1(+) or the presence of rad22-H6 mutation decreases the restriction temperature of rad11-A1 cells by 4-6 degrees C and leads to cell cycle arrest with chromosomes incompletely replicated. Thus, in fission yeast a double-strand break repair component is required for a certain step of chromosome replication unlinked to repair, partly via interacting with replication protein A.

Amino Acid Sequence↗

PubMed1999

Fission yeast cdc24 is a replication factor C- and proliferating cell nuclear antigen-interacting factor essential for S-phase completion.

At the nonpermissive temperature the fission yeast cdc24-M38 mutant arrests in the cell cycle with incomplete DNA replication as indicated by pulsed-field gel electrophoresis. The cdc24(+) gene encodes a 501-amino-acid protein with no significant homology to any known proteins. The temperature-sensitive cdc24 mutant is effectively rescued by pcn1(+), rfc1(+) (a fission yeast homologue of RFC1), and hhp1(+), which encode the proliferating cell nuclear antigen (PCNA), the large subunit of replication factor C (RFC), and a casein kinase I involved in DNA damage repair, respectively. The Cdc24 protein binds PCNA and RFC1 in vivo, and the domains essential for Cdc24 function and for RFC1 and PCNA binding colocalize in the N-terminal two-thirds of the molecule. In addition, cdc24(+) genetically interacts with the gene encoding the catalytic subunit of DNA polymerase epsilon, which is stimulated by PCNA and RFC, and with those encoding the fission yeast counterparts of Mcm2, Mcm4, and Mcm10. These results indicate that Cdc24 is an RFC- and PCNA-interacting factor required for DNA replication and might serve as a target for regulation.

Amino Acid Sequence↗

PubMed1999

Isolation of a mammalian homologue of a fission yeast differentiation regulator.

In the fission yeast Schizosaccharomyces pombe the nrd1(+) gene encoding an RNA binding protein negatively regulates the onset of differentiation. Its biological role is to block differentiation by repressing a subset of the Ste11-regulated genes essential for conjugation and meiosis until the cells reach a critical level of nutrient starvation. By using the phenotypic suppression of the S. pombe temperature-sensitive pat1 mutant that commits lethal haploid meiosis at the restrictive temperature, we have cloned ROD1, a functional homologue of nrd1(+), from rat and human cDNA libraries. Like nrd1(+), ROD1 encodes a protein with four repeats of typical RNA binding domains, though its amino acid homology to Nrd1 is limited. When expressed in the fission yeast, ROD1 behaves in a way that is functionally similar to nrd1(+), being able to repress Ste11-regulated genes and to inhibit conjugation upon overexpression. ROD1 is predominantly expressed in hematopoietic cells or organs of adult and embryonic rat. Like nrd1(+) for fission yeast differentiation, overexpressed ROD1 effectively blocks both 12-O-tetradecanoyl phorbol-13-acetate-induced megakaryocytic and sodium butyrate-induced erythroid differentiation of the K562 human leukemia cells without affecting their proliferative ability. These results suggest a role for ROD1 in differentiation control in mammalian cells. We discuss the possibility that a differentiation control system found in the fission yeast might well be conserved in more complex organisms, including mammals.

Amino Acid Sequence↗

PubMed1999

Angioplasty-bypass surgery combination therapy: case study of an elderly patient with unstable angina and colon cancer.

An 83-year-old man was admitted with refractory unstable angina and severe anemia. Colonofiberscopy revealed hemorrhagic colon cancer in the transverse colon. Coronary angiography showed total occlusion of the right coronary artery (RCA), diffuse, calcified 90% stenosis of the middle portion of the left anterior descending artery (LAD); and fair collaterals from the LAD to the RCA. Coronary revascularization was considered prior to colectomy, but because of the patient's advanced age and hemorrhagic cancer, conventional coronary aorta bypass grafting (CABG) using extracorporeal circulation, as well as coronary stenting requiring antiplatelet therapy, were regarded as inadvisable. Percutaneous transluminal coronary angioplasty (PTCA) for the LAD carried the risk of suboptimal coronary stenting. Thus, the patient was first treated with PTCA for the occluded RCA, followed 7 days later by a left internal thoracic artery graft to the LAD on the beating heart without extracorporeal circulation. The patient was stable thereafter. This approach to coronary revascularization may be suitable for patients for whom anticoagulation or antiplatelet therapy are contraindicated, or when complete revascularization would be difficult with CABG or PTCA alone.

Aged↗

PubMed1999

The role of alcohol dehydrogenase 2 and aldehyde dehydrogenase 2 genotypes in alcohol-induced vasospastic angina.

Alcohol ingestion often provokes attacks in patients with vasospastic angina. Type 2 aldehyde dehydrogenase (ALDH2) deficiency, which is based on a single point mutation (Glu487Lys) of the ALDH2 gene, is common in the Japanese population, but rare among the Caucasian population. We investigated how the genotype of ALDH2 affects the characteristics of alcohol-induced vasospastic angina. Ninety-one patients with vasospastic angina who had ingested alcohol daily or occasionally were studied. Patients had been diagnosed as vasospastic angina by a provocation test with an intracoronary injection of ergonovine or acetylcholine during coronary angiography. The Glu487Lys mutation was detected by allele specific PCR. We interviewed the patients to obtain information concerning the relationship between alcohol ingestion and anginal attacks. Alcohol ingestion induced attacks in 16 of 66 patients without the Glu487Lys mutation, 8 of 22 in heterozygotes, and 1 of 3 in mutant homozygotes. The intervals between alcohol ingestion and the onset of anginal attacks were shorter in homozygotes (0.17 hours) and heterozygotes (1.5+/-0.6 hours) for ALDH2*2 than in normal homozygotes for ALDH2*1 (5.4+/-0.6 hours). The amount of ethanol which induced attacks was significantly greater in normal homozygotes than in homozygotes (11 ml) and heterozygotes (42.5+/-7.1 ml) for ALDH2*2 (96.1+/-13.4 ml in normal patients). The frequency of anginal attacks induced by alcohol ingestion did not differ between ALDH deficient and normal homozygotes. In ALDH deficient patients, however, anginal attacks were induced by a smaller amount of alcohol immediately after its ingestion. Thus, the ALDH2 genotype modifies the characteristics of the anginal attacks as a co-factor for the induction of vasospastic angina after alcohol ingestion.

Alcohol Dehydrogenase↗

PubMed1998

Arg506Gln mutation of the coagulation factor V gene not detected in Japanese pulmonary thromboembolism.

The incidence of pulmonary thromboembolism (PTE) is lower in Japanese than in Caucasians. The basis for the different incidence has not been clarified. A poor anticoagulant response to activated protein C based on a single point mutation of the factor V gene (Arg506Gln) was found to be a pathogenetic factor for venous thrombosis and PTE in North America and Europe. We investigated whether the Arg506Gln mutation of factor V is responsible for the occurrence of PTE among Japanese. We analyzed genomic DNA prepared from fresh peripheral blood of 25 patients with PTE of unknown etiology (12 of acute type and 13 of chronic type) and that of 110 controls without respiratory or circulatory disorders. To detect the Arg506Gln mutation, 267 bp DNA fragments of the factor V gene including the Arg506Gln region were amplified by PCR, digested by MnlI and electrophoresed. After digestion of PCR products with MnlI, DNA fragments of 163 bp length, but not DNA fragments of 200 bp length, were identified in all samples, indicating the absence of the Arg506Gln mutation in the patients with PTE and control subjects. These results suggest that the Arg506Gln mutation is absent or very rare and not an important pathogenetic factor for PTE in Japanese.

Aged↗

PubMed1998

Dexamethasone suppresses gene expression and production of IL-13 by human mast cell line and lung mast cells.

BACKGROUND: IL-13 has been shown to induce IgE production in B cells by promoting class switching to IgE. Mast cells are known to play an important role in the pathogenesis of allergic diseases. We evaluated the ability of human mast cells to produce IL-13 using human mast cell line HMC-1 and freshly isolated lung mast cells and then examined the effect of dexamethasone on the gene expression and production of IL-13 by these cells. METHODS: HMC-1 cells and lung mast cells were cultured with 10 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 micromol/L ionomycin and with 5 microg/ml phytohemagglutinin (PHA) and 10 ng/ml PMA, respectively, in the presence of dexamethasone. The gene expression of IL-13 at 3 hours (HMC-1 cells) or 12 hours (human lung mast cells) after stimulation was assessed semiquantitatively by sequential reverse transcription-polymerase chain reaction and Southern blot analysis. IL-13 production at 12 hours after stimulation was assayed by ELISA. RESULTS: The gene expression of IL-13 by HMC-1 cells and human lung mast cells, which was detected at a low level in an unstimulated condition, was increased by PMA/ionomycin and suppressed by dexamethasone. The supernatant of HMC-1 cells and human lung mast cells showed a low level of IL-13, which was increased by the stimulation and suppressed by dexamethasone. CONCLUSION: These findings indicate that HMC-1 cells and human lung mast cells produce IL-13 and that dexamethasone suppresses the production of IL-13 by these cells through an inhibitory action on the gene expression.

Cell Line↗

PubMed1998

Increased contraction of myocytes isolated from the young spontaneously hypertensive rat: relationship between systolic and diastolic function.

This study was designed to assess heart performance in young (10-week-old) spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats, in terms of whole heart function in vivo and mechanics of isolated ventricular myocytes in vitro. The data suggest that left ventricular pressure (LVP) generation is greater, and the maximal velocities of LVP generation and decline are faster in SHR than in WKY. Two-dimensional morphologic measurements show that SHR myocytes are hypertrophied and that augmented contractile function is also present in isolated cells as determined by the extent of shortening and velocity of shortening. Relaxation is also faster at the myocyte level as determined by velocity of relengthening. However, the slope of the relationship between myocyte peak shortening and velocity of relaxation was similar in both groups. These results suggest that hyperdynamic myocyte relengthening may reflect changes in elastic recoil from increased shortening rather than intrinsic changes in cellular mechanisms, which are independent of shortening.

Animals↗