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Biomedical subjects

H Okayama

Publications and source records attributed to H Okayama.

At least 91 records · Page 5Linked to original sources
PubMed1995

Contractile properties of left ventricular myocytes isolated from spontaneously hypertensive rats: effect of angiotensin II.

OBJECTIVE: This study was undertaken to clarify whether the myocardial dysfunction observed in the hypertensive heart is an intrinsic property of the myocyte or not. MATERIALS AND METHODS: We investigated left ventricular function and contractile function of myocytes from 30-week-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). We also evaluated the effect of angiotensin II on contractile function of myocytes from both rats. RESULTS: The time constant of isovolumic pressure fall was significantly greater in SHR (13.2 +/- 0.6 ms) than in WKY (10.3 +/- 0.5 ms). The extent of shortening in isolated myocytes was significantly higher in SHR (11.3 +/- 0.4%) than in WKY (9.8 +/- 0.4%, P<0.01). Both the normalized maximal velocity of shortening and the normalized maximal velocity of relengthening were significantly greater in SHR (2.12 +/- 0.08 and 2.10 +/- 0.08 s-1; both P<0.01) than in WKY (1.76 +/- 0.06 and 1.75 +/- 0.07 s-1). Angiotensin II caused significant decreases in the extent of shortening, the normalized maximal velocity of shortening and the normalized maximal velocity of relengthening in isolated myocytes from SHR, but these parameters were unchanged in WKY. CONCLUSIONS: These results suggest that left ventricular diastolic dysfunction in SHR is not due to an intrinsic abnormality of the cardiac myocytes, and that angiotensin II suppresses the function of myocytes from hypertrophied hearts.

Animals↗
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↗
PubMed1994

Glucocorticoids suppressed production and gene expression of interleukin-5 by peripheral blood mononuclear cells in atopic patients and normal subjects.

BACKGROUND: Interleukin-5 (IL-5) is known to play a major role in regulating eosinophil function in atopic diseases, including bronchial asthma. Glucocorticoids are most effective agents for treating these diseases. However, their mechanism remains unclear. We examined the effects of glucocorticoids on the production and gene expression of IL-5 in atopic patients and normal subjects. METHODS: Human peripheral blood mononuclear cells (PBMCs) in five atopic and four normal subjects were cultured with phytohemagglutinin and phorbol 12-myristate 13-acetate (PMA) in the presence of dexamethasone. IL-5 secreted by PBMCs was assayed by ELISA. Gene expression of IL-5 by PBMCs was assessed semiquantitatively by sequential reverse transcription-polymerase chain reaction, and Southern blot analysis. RESULTS: Phytohemagglutinin/PMA-stimulated PBMCs from all atopic patients and three normal subjects secreted detectable amounts of IL-5, which were suppressed by dexamethasone in a dose-dependent manner, with 85.8% suppression at 10(-6) mol/L. Gene expression of IL-5 was detected by reverse-transcription polymerase chain reaction in PBMCs from all subjects, even when not stimulated; was increased by stimulation; and was suppressed by dexamethasone. The concentration of dexamethasone resulting in 50% inhibition in IL-5 gene expression did not differ between atopic patients and normal subjects. CONCLUSION: These findings indicate that dexamethasone suppressed IL-5 production in atopic human PBMCs through an inhibitory action on the gene expression. These results suggest that the suppression of IL-5 production through the suppression of IL-5 gene expression is one of the most important mechanisms by which glucocorticoids inhibit eosinophil functions in the treatment of atopic diseases, including bronchial asthma.

Base Sequence↗
PubMed1994

High-level expression of human lipocortin I in the fission yeast Schizosaccharomyces pombe using a novel expression vector.

We have developed a novel expression system that allows the fission yeast, Schizosaccharomyces pombe, to be used for the efficient overproduction of heterologous proteins. As an example of the utility of this system, human lipocortin I was expressed to 50 percent of soluble protein, and 150 mg of highly purified material was obtained from 10 grams of wet cell paste. Expression of lipocortin I was driven by the human cytomegalovirus (hCMV) promoter in a vector that also contains a neomycin resistance gene (neo) under the control of the SV40 early promoter, permitting selection for increasing copy-number with increasing concentrations of the antibiotic G418. The purified protein was equivalent to its native counterpart with respect to antigenicity and biochemical properties such as phospholipase A2 inhibition, actin binding and N-terminal acetylation. We have also used this system to produce comparable amounts of other proteins including rat arginase, rat NDP-kinase and human interleukin-6.

Animals↗
PubMed1994

Contractile dysfunction in the diabetic-rat heart is an intrinsic abnormality of the cardiac myocyte.

1. In order to clarify whether the myocardial dysfunction observed in diabetic-rat hearts is an intrinsic property of the myocytes or not, we investigated cardiac function and myocyte contractile function in diabetic rats 5 weeks after the injection of streptozotocin. 2. Maximal and minimal dP/dt and time constant of isovolumic pressure fall were measured using a micromanometer in diabetic and age-matched control rats. 3. Isolated myocytes were enzymically obtained from each rat heart and were stimulated at 1 Hz (37 degrees C) in a buffer containing 1.5 mmol/l Ca2+. The images of myocyte contractions were recorded by a video system. Normalized maximal velocity of shortening (maximal velocity of cell shortening/resting cell length; s-1), normalized maximal velocity of relengthening (maximal velocity of cell relengthening/resting cell length; s-1) and extent of shortening [(twitch amplitude/resting cell length) x 100; %] were analysed by a digitalized computer as contractile functions of the myocyte. 4. The maximal and minimal dP/dt in diabetic rats (7876, 5341 mmHg/s) were significantly lower than those in control rats (9349, 7876 mmHg/s). The time constant of isovolumic pressure fall in diabetic rats (12.7 ms) was significantly greater than that in control rats (8.6 ms). Moreover, the normalized maximal velocity of shortening, normalized maximal velocity of relengthening and extent of shortening in myocytes from diabetic rats (1.40 s-1, 1.38 s-1, 9.5%) were significantly lower than those in myocytes from control rats (1.64 s-1, 1.60 s-1, 11.8%). 5. These results suggest that contractile impairment in this diabetic-rat heart model is mainly due to an intrinsic abnormality of the cardiac myocytes.

Animals↗
PubMed1994

[Intra-ventricular pressure gradient and systolic anterior movement of the mitral valve associated with administration of dopamine in a 91-year-old patient].

A 91-year-old woman had decreased systemic blood pressure accompanied by dyspnea and general fatigue after resection of a face tumor (Merkel cell carcinoma). Dopamine was administered to elevate systemic blood pressure, but it did not sufficiently restore the hemodynamic status. Left ventricular apical aneurysm was observed on echocardiogram. Furthermore, SAM (systolic anterior movement of the mitral valve) was observed on M-mode echocardiogram and a pressure gradient of 34.6 mmHg in the midventricular position was confirmed by continuous wave Doppler using the Bernoulli equation. After volume overload and discontinuation of dopamine, SAM completely disappeared and midventricular pressure gradient markedly decreased. Her general condition improved. Because of the narrowed outflow tract, hypercontractile state induced by beta adrenergic stimulation seemed to be one of the important factors for the genesis of SAM and pressure gradient.

Aged↗
PubMed1993

Raf-1 is not a major upstream regulator of MAP kinases in rat fibroblasts.

RCR cells are NRK clones in which Raf-1 production is blocked by the expression of an antisense RNA, and consequently they are refractory to transformation by various oncogenes. In RCR cells, MAP kinases (ERK1 and ERK2) were activated to an extent and in a time course similar to those of the original NRK cells, irrespective of whether the stimulus was oncogenic or non-oncogenic. Moreover, there was no significant elevation of ERK activities in oncogene-transformed NRK cells. These results indicate that Raf-1 kinase is not the major upstream activator of ERK's in NRK cells and that neither ERK1 nor ERK2 are likely to mediate oncogenic signals from Raf-1 kinase.

Animals↗