Biomedical subjects

H Okayama

Publications and source records attributed to H Okayama.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1998

Constitutive association of EGF receptor with the CrkII-23 mutant that inhibits transformation of NRK cells by EGF and TGF-beta.

Crk belongs to the adapter proteins that participate in many signalling pathways from cell surface receptors. We have characterised the CrkII-23 mutant that inhibits the transformation of NRK cells induced by epidermal growth factor (EGF) and transforming growth factor (TGF)-beta. To study the biochemical difference, cDNAs of the wild-type CrkII and the CrkII-23 mutant were introduced stably into NIH 3T3 cells expressing EGF receptor (EGFR). Both CrkII and CrkII-23 were phosphorylated on tyrosine upon EGF simulation with similar time course and dose dependency. Whereas the wild-type CrkII bound to EGFR only after EGF stimulation, CrkII-23 bound to EGFR from before stimulation. Mutation in the Src homology (SH) 2 or amino-terminal SH3 domain did not abolish the binding of CrkII-23 to EGFR in the quiescent cells, suggesting that the binding is mediated by a novel mechanism. These CrkII-23-derived mutants, however, did not suppress transformation of NRK cells by EGF and TGF-beta. Hence, both the SH2 and amino-terminal SH3 domains are required to inhibit transformation of NRK cells. These results suggest that persistent signalling from CrkII-23 bound to EGFR suppresses transformation by EGF and TGF-beta in NRK23 cells.

3T3 Cells↗

PubMed1998

Functional domains of rep2, a transcriptional activator subunit for Res2-Cdc10, controlling the cell cycle "start".

In the fission yeast Schizosaccharomyces pombe, passage from G1 to S-phase requires the execution of the transcriptional factor complex that consists of the Cdc10 and Res1/2 molecules. This complex activates the MluI cell cycle box cis-element contained in genes essential for S-phase onset and progression. The rep2(+) gene, isolated as a multicopy suppressor of a temperature-sensitive cdc10 mutant, has been postulated to encode a putative transcriptional activator subunit for the Res2-Cdc10 complex. To identify the rep2(+) function and molecularly define its domain organization, we reconstituted the Res2-Cdc10 complex-dependent transcriptional activation in Saccharomyces cerevisiae. Reconstitution experiments, deletion analyses using one and two hybrid systems, and in vivo Res2 coimmunoprecipitation assays show that the Res2-Cdc10 complex itself can recognize but cannot activate MluI cell cycle box without Rep2, and that consistent with its postulated function, Rep2 contains 45-amino acid Res2 binding and 22-amino acid transcriptional activation domains in the middle and C terminus of the molecule, respectively. The functional essentiality of these domains is also demonstrated by their requirement for rescue of the cold-sensitive rep2 deletion mutant of fission yeast.

Binding Sites↗

PubMed1998

Novel factor highly conserved among eukaryotes controls sexual development in fission yeast.

In the fission yeast Schizosaccharomyces pombe, the onset of sexual development is controlled mainly by two external signals, nutrient starvation and mating pheromone availability. We have isolated a novel gene named rcd1+ as a key factor required for nitrogen starvation-induced sexual development. rcd1+ encodes a 283-amino-acid protein with no particular motifs. However, genes highly homologous to rcd1+ (encoding amino acids with >70% identity) are present at least in budding yeasts, plants, nematodes, and humans. Cells with rcd1+ deleted are sterile if sexual development is induced by nitrogen starvation but fertile if it is induced by glucose starvation. This results largely from a defect in nitrogen starvation-invoked induction of ste11+, a key transcriptional factor gene required for the onset of sexual development. The striking conservation of the gene throughout eukaryotes may suggest the presence of an evolutionarily conserved differentiation controlling system.

Amino Acid Sequence↗

PubMed1998

An RNA binding protein negatively controlling differentiation in fission yeast.

The fission yeast Schizosaccharomyces pombe starts sexual development when starved for nutrients and simultaneously activated by mating pheromones. We have identified a new gene regulating the onset of this process. This gene, called nrd1(+), encodes a typical RNA binding protein that preferentially binds poly(U). Deletion of nrd1(+) causes cells to initiate sexual development without nutrient starvation. We have found that the biological role of nrd1(+) is to block the onset of sexual development by repressing the Ste11-regulated genes essential for conjugation and meiosis until cells reach a critical level of starvation.

Amino Acid Sequence↗

PubMed1998

Methacholine bronchial hyperresponsiveness in chronic sinusitis.

The coexistence of chronic sinusitis (CS) may deteriorate the clinical condition of lower airway diseases such as bronchial asthma (BA) or chronic bronchitis (CB). However, the bronchial hyperresponsiveness (BH) in CS without any apparent lower airway disease is not fully understood nor are the effects of treatment. We examined lower airway hyperresponsiveness to methacholine (MCh) in 42 subjects with CS but without allergic rhinitis (AR) who had normal lung functions without any pulmonary symptoms, comparing it with that of 50 subjects with stable BA, 50 subjects with simple CB and 40 subjects with AR, and further examined the effect of endoscopic sinus surgery in 7 CS subjects with BH. The BH to MCh was measured in terms of the minimum dose (Dmin), defined as the cumulative dose at the point where respiratory conductance began to decrease. A Dmin <50 units was defined as BH. Seventy-one percent of CS subjects showed BH without relation to the severity or duration of CS, or atopic status. BH in CS subjects, which was less than that in BA subjects, was similar to that in simple CB or AR in both its prevalence and degree. After the surgical treatment of CS, BH significantly decreased (p < 0.01) with improvements in both nasal symptoms and sinus lesions. These findings suggest that CS itself induces BH to a degree similar to simple CB and AR without any relationship to the clinical background, and that adequate treatment of CS reduces BH.

Adolescent↗

PubMed1998

Surfactant protein A2 gene expression by human airway submucosal gland cells.

To determine whether human airway submucosal glands produce and secrete surfactant proteins, we examined their protein and gene expression in submucosal glands from trachea and bronchi obtained from operated and autopsied lungs within 4 h of death. Using a monoclonal antibody (PE-10) against surfactant protein A (SP-A), a positive immunoperoxidase stain was observed over serous cells of submucosal glands in histologic sections of airway walls. Measurement of SP-A in culture medium samples using single-step enzyme-linked immunosorbent assay showed a significant secretion of SP-A by isolated submucosal glands (1.2 +/- 0.08 ng/ml/h, SEM, n = 40). In gene expression experiments by reverse transciption-polymerase chain reaction, the SP-A complementary DNA (cDNA) segment was amplified from isolated submucosal glands, indicating the presence of SP-A messenger RNA (mRNA) in airway submucosal glands. Bronchial superficial epithelial cells failed to show the presence of SP-A mRNA. No cDNA segment of SP-B, SP-C, or SP-D cDNA was amplified from isolated submucosal glands or superficial epithelial cells, whereas all were amplified from alveolar tissue. Furthermore, in contrast to the control alveolar tissue, which expressed both SP-A1 and SP-A2 genes, SP-A2 gene transcript alone was detected in isolated submucosal glands by Southern analysis that included the digestion of the amplified SP-A cDNA fragment with the restriction enzyme Apa I. These findings indicate that human airway submucosal gland cells can transcribe the SP-A2 gene and produce SP-A protein in a manner different from peripheral airways and alveoli, playing a role in the airway defense mechanism.

Adult↗

PubMed1998

Paroxysmal atrial fibrillation as a cause of potentially lethal ventricular arrhythmia with myocardial ischemia in hypertrophic cardiomyopathy--a case report.

The mechanism(s) of myocardial ischemia in hypertrophic cardiomyopathy remain unclear. In this report, the authors present a 75-year-old Japanese woman with nonobstructive hypertrophic cardiomyopathy in whom paroxysmal atrial fibrillation caused severe myocardial ischemia and induced sustained ventricular tachycardia. Her coronary angiogram showed normal findings, and no ischemic changes were provoked by either physical exercise testing or dobutamine stress echocardiography under sinus rhythm. In view of these findings, the rapid ventricular response in the absence of atrial contraction may aggravate or induce myocardial ischemia and predispose patients with hypertrophic cardiomyopathy to develop lethal ventricular arrhythmia.

Aged↗