Biomedical subjects

S Kawashima

Publications and source records attributed to S Kawashima.

At least 217 records · Page 12Linked to original sources

PubMed1995

Unaltered size of right ventricular infarct in dogs with right ventricular hypertrophy induced by pressure overload.

We investigated whether right ventricular infarct size is increased in the hypertrophied right ventricle. In dogs, we produced chronic pressure overload-induced right ventricular hypertrophy (RVH) and acute pulmonary stenosis (APS) with a similar right ventricular systolic pressure to RVH. Then, in an open-chest model, right coronary artery occlusion for 4 h followed by 1 h of reperfusion was performed in animals with RVH or APS or in controls. Regional myocardial blood flow was measured with nonradioactive colored microspheres. Compared with the control group, regional myocardial oxygen consumption in the right ventricle was increased in the APS but not in the RVH group. The risk area and collateral blood flow were comparable in the three groups. However, the infarct size relative to risk area was increased in the APS group (42.3 +/- 4.9%), but that in the RVH group (21.1 +/- 4.4%) was not different from controls (26.1 +/- 4.2%). Thus right ventricular infarction in the heart with RVH was not increased compared with the normal heart.

Acute Disease↗

PubMed1995

C1Q complexes with beta-2-microglobulin and amyloid P-component in sera of patients with dialysis-associated amyloidosis.

We studied complexes composed of C1q and beta 2-microglobulin (C1q.beta 2M) or serum amyloid P-component (C1q.SAP) in the sera of 38 patients receiving hemodialysis (HD) and 20 healthy control subjects using an enzyme-linked immunoassay. We also compared complex levels in HD patients with and without symptoms of HD-associated amyloidosis (HA). Serum C1q-beta 2M levels were significantly higher in HD patients without HA than in healthy control subjects and significantly higher in HD patients with HA than in HD patients without HA. Serum C1q.SAP levels were significantly lower in patients with HA than in those without HA. These results suggest that C1q.beta 2M may contribute to HA.

Amyloidosis↗

PubMed1995

Inhibition of endothelial nitric oxide synthase activity by protein kinase C.

Nitric oxide (NO) is an important molecular messenger accounting for endothelium-derived relaxing factor. Recently, NO synthase (NOS) from cultured endothelial cells has been purified and molecularly cloned. To evaluate the effect of phosphorylation by protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA) on endothelial constitutive NOS catalytic activity, we incubated purified endothelial NOS with PKC or PKA. Endothelial NOS was stoichiometrically phosphorylated by PKC and PKA. In intact bovine aortic endothelial cells (BAECs), NOS was phosphorylated by stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA). NOS activity measured by the conversion of [3H]arginine to [3H]citrulline in homogenates of BAECs treated with TPA or phorbol 12,13-dibutyrate was reduced by 30%, whereas dibutylyl cyclic AMP did not affect NOS activity. Moreover, we measured NO release from cultured BAECs by a chemiluminescence method to examine the effect of PKC and PKA on endothelial NOS activity. In cultured BAECs, ATP gamma S and A23187 induced NO release in time- and dose-dependent manners. Phorbol esters such as TPA and phorbol 12,13-dibutyrate dose dependently inhibited NO release stimulated by A23187 as well as ATP gamma S. Reduction of NO release by TPA was almost completely prevented by pretreatment with staurosporine, an inhibitor of PKC. NO release by A23187 was increased in PKC-downregulated BAECs. In contrast, dibutylyl cyclic AMP or 8-bromo cyclic GMP had no effect on NO release from BAECs induced by A23187 or ATP gamma S. These results indicate that phosphorylation of NOS by PKC is associated with a reduction of its catalytic activity in vascular endothelial cells.

Amino Acid Oxidoreductases↗

PubMed1995

Low concentration of oxidized low-density lipoprotein and lysophosphatidylcholine upregulate constitutive nitric oxide synthase mRNA expression in bovine aortic endothelial cells.

Endothelium-dependent relaxation is markedly reduced in atherosclerotic arteries. Recently, the endothelium-dependent relaxing factor has been identified as nitric oxide (NO). We used RNase protection assay and immunoblotting to elucidate the effect of atherogenic lipoprotein on the expression of constitutive NO synthase (cNOS) mRNA and protein levels in bovine aortic endothelial cells. Twenty-four-hour exposure to a low concentration of oxidized low-density lipoprotein (10 micrograms protein/mL) upregulated cNOS mRNA levels (2.4 +/- 0.4-fold, P < .01). However, native low-density lipoprotein and high-density lipoprotein did not have any effect on cNOS mRNA levels. Furthermore, 5 micrograms/mL of lysophosphatidylcholine (LPC) also upregulated cNOS mRNA levels (2.6 +/- 0.5-fold, P < .01) at 8 hours. This action of LPC was abolished with cycloheximide but not with staurosporine. We concluded that atherogenic lipoproteins upregulate cNOS mRNA and protein levels in bovine aortic endothelial cells. This observation supports the hypothesis that an impairment of endothelium-dependent vasodilatation in atherosclerotic vessels may not be due to a decrease in cNOS expression. Moreover, the LPC action on cNOS mRNA levels requires new protein synthesis.

Amino Acid Oxidoreductases↗

PubMed1995

Reperfusion of rat heart after brief ischemia induces proteolysis of calspectin (nonerythroid spectrin or fodrin) by calpain.

Rat myocardium expresses the 240- and 235-kD polypeptides antigenically related to alpha- and beta-subunits of brain calspectin (nonerythroid spectrin or fodrin), respectively. In the subcellular fractions of the myocardium, alpha-calspectin was found in the 600g, 10,000g, and 100,000g pellets, whereas beta-calspectin was localized to the 10,000g pellet. On the basis of the Na+,K(+)-ATPase activity and the contents of a gap junction protein, the sarcolemma was distributed to the 10,000g and 100,000g pellets, and the intercalated disks were enriched in the 10,000g pellet. Both alpha- and beta-calspectin were proteolyzed by calpain in vitro. The two subunits were also proteolyzed in vivo, when the rat hearts underwent 10 to 60 minutes of global ischemia followed by 30 minutes of reperfusion. The reperfusion following the ischemia induced the proteolysis of alpha-calspectin in the 10,000g and 100,000g pellets, producing the 150-kD fragment. A synthetic calpain inhibitor, calpain inhibitor-1, suppressed the degradation of calspectin in vivo, which indicates that calpain is responsible for the reperfusion-induced proteolysis of calspectin. The inhibitor also improved myocardial stunning. Immunohistochemical study revealed that the proteolysis of alpha-calspectin occurs at the intercalated disks and the sarcolemma after postischemic reperfusion, in accord with the biochemical data. These results suggest that degradation of calspectin partly accounts for the contractile failure of the myocardium after postischemic reperfusion by disrupting the membrane skeleton and the intercalated disks.

Animals↗

PubMed1995

Three distinct phases of fodrin proteolysis induced in postischemic hippocampus. Involvement of calpain and unidentified protease.

BACKGROUND AND PURPOSE: Fodrin, a neuronal cytoskeleton protein, is proteolyzed by calpain after ischemic insult. We examined proteolysis of fodrin induced by global forebrain ischemia in gerbil hippocampus in spatial terms by using the antibody specific to the calpain-proteolyzed form of fodrin. METHODS: In gerbils, a 10-minute forebrain ischemia was produced by occlusion of both carotid arteries. After recirculation, the hippocampus was processed for immunohistochemical and immunoblot study with the antibody against the calpain-proteolyzed form of fodrin. Additionally, short-term ischemia was studied to find the threshold of fodrin proteolysis. RESULTS: Three phases of fodrin proteolysis distinct in chronology and distribution arose: (1) an early predegeneration phase in the molecular layer and stratum oriens of the CA1 and CA3 sectors within the first 15 minutes, which lasted up to 4 hours; (2) a late predegeneration phase in the whole CA1 sector, except for the pyramidal cells, between 12 hours and 2 days; and (3) a postdegeneration phase in the cytoplasm of the CA1 neurons, which arose in 3 to 7 days. A 4-minute (not a 3-minute) forebrain ischemia induced the late predegeneration phase of fodrin proteolysis and delayed neuronal death in CA1. Immunoblotting showed that the primary product of calpain action was further proteolyzed by an unidentified protease. CONCLUSIONS: Calpain induced proteolysis of fodrin in ischemic hippocampus, and the late predegeneration phase of the proteolysis was closely associated with the delayed neuronal death in the CA1 sector. Calpain and another protease may play a role in the development of neuronal death after transient forebrain ischemia.

Animals↗

PubMed1995

Modification of pancreatic digestive function by pituitary grafting in mice.

The effect of prolactin on the digestive potency of the acinar pancreas was examined in pituitary-grafted hyperprolactinemic mice, because our previous experiment showed that a marked proliferation of pancreatic acinar cells was induced by pituitary grafting in mice. To know whether the digestive function is modified, the tissue contents of pancreatic digestive enzymes, such as chymotrypsin, lipase alpha-amylase and ribonuclease, were measured in the hyperprolactinemic mice. Pituitary grafting significantly increased the contents of chymotrypsin and lipase in the pancreas on day 12 after the operation without affecting intake of food, when compared to those in the sham-operated controls. On day 30, however, the differences between pituitary-grafted and control mice were no more discernible. Thus, the digestive enzyme activities are easily modified soon after the increase of circulating prolactin level. This effect of prolactin on the function of the pancreas may be responsible for "homeorhetic" control of nutrients during lactation. In another set of experiments in adrenalectomized-castrated or castrated mice, pituitary grafting induced an increase in the weight of the pancreas. In addition, adrenalectomy in combination with castration did not alter the pancreatic contents of chymotrypsin and lipase but decreased the amylase content. These results taken together seem to indicate that the effect of prolactin on the exocrine pancreas is not mediated by gonadal and adrenal steroid hormones.

Adrenalectomy↗

PubMed1995

The difference in autofluorescence features of lipofuscin between brain and adrenal.

Lipofuscin is the autofluorescent material, which accumulates with aging in the cells of various tissues. However, its autofluorescence characteristics are different among tissues. In the present study, the autofluorescence features of lipofuscin in the brain and adrenal were compared. In 18-21-month-old rats, the brain lipofuscin was granular and its autofluorescence was bright whitish-yellow to bright orange. On the contrary, the adrenal lipofuscin was not demarkated as granules, and its autofluorescence was subdued orange. The emission maximum of the bright whitish-yellow brain lipofuscin was 540 nm to 570 nm and that of the adrenal lipofuscin was 640 nm to 660 nm, when excited at 330 nm to 380 nm. When the spectra were drawn after correcting the wavelength-dependent bias of microspectrofluorometer, the autofluorescence spectra were consistent with microscopically observable tint. To conclude, the present results showed that the autofluorescence features of the bright whitish-yellow brain lipofuscin and the adrenal lipofuscin were quite different.

Adrenal Glands↗

PubMed1995

Expression of a dopamine transporter in PC12h cells: an immunohistochemical study with an antipeptide antibody.

Two kinds of oligopeptides, based on the amino acid sequences of No. 217-232 and 374-387 of a rat dopamine transporter, were designed and chemically synthesized. Five clones of the monoclonal antibodies against these peptides were produced with the in vitro immunization method. Two of them have recognized a protein of M(r) approximately 85,000 in a synaptosomal fraction of the rat striatum. It is probable that the protein M(r) approximately 85,000 corresponds to a dopamine transporter in the nerve terminal of the dopamine neurons in the striatum. The expression of dopamine transporter was immunohistochemically examined with these antibodies in PC12h cells. The immunoreactivity was detected on the surface membrane of the cells.

Animals↗

PubMed1995

[An attempt of applying the image processing for the automatic estimation of sampled airborne pollen].

Development of techniques to rapidly and easily estimate an airborne pollen quantity is necessitated in order to make out a appropriate prescription for an allergy patient from medical clinical viewpoints, and in order to research a movement of allergen from medical basic viewpoint. The measurement of airborne pollen quantity required a large labour and time, because the amount of pollen grains is visually measured by naked eye. This paper, as a first step to estimate an airborne pollen quantity full-automatically, discuss techniques to measure the quantity of sampled airborne cedar pollen automatically and rapidly using the image processing techniques. As a result, the following facts are cleared. Automatic measurement is possible to some degree without any special image processing. It is important to eliminate a noise on image as blurs on basefilm for high accuracy measurement. The precision has improved fairly with level correction for image. Sharpening filter is the most appropriate process to improve the accuracy of automatic measurement of sampled airborne pollen. This filtering process has a merit that the operativeness is easy.

Air Pollutants↗

PubMed1994

The calpain-calpastatin system is regulated differently during human neuroblastoma cell differentiation to Schwannian and neuronal cells.

Changes in expression of calpains and calpastatin during differentiation in GOTO cells were examined using antibodies specific to calpains and calpastatin. Neuronal differentiation induced by dibutyryl cyclic AMP elicited a remarkable decrease in m-calpain. No marked changes in the levels of calpains were found in bromodeoxyuridine-induced Schwannian differentiation. Calpastatin was detected as a single band of 110k in undifferentiated and in neuronally differentiated cells by Western blot analysis. However, the appearance of a 120k species was detected in Schwannian differentiation associated with morphological change. The data show that marked changes in m-calpain and calpastatin occur in a differentiation-specific manner.

Bromodeoxyuridine↗

PubMed1994

Distinct kinetics of subunit autolysis in mammalian m-calpain activation.

Subunit autolysis of mammalian m-calpain upon activation was examined in kinetic terms using a set of antibodies recognizing different portions of the protease. Activation of m-calpain by calcium resulted in no apparent autolysis in the large catalytic subunit, whereas the small regulatory subunit underwent immediate autolysis followed by substrate proteolysis. This profile of subunit autolysis is distinct from that of the other ubiquitous isozyme, mu-calpain, in which autolysis of the large subunit and then of the small subunit precedes substrate proteolysis under the normal conditions. The activation state of m-calpain thus is not reflected by the large subunit autolysis. The mode and role of autolysis may vary among calpain isozymes.

Amino Acid Sequence↗

PubMed1994

Spatial resolution of the primary beta-amyloidogenic process induced in postischemic hippocampus.

Proteolytic modifications of amyloid precursor protein (APP) play key roles in the development of Alzheimer's disease. However, each specific in vivo process has not yet been fully resolved in spatial terms because the orthodox approach employing electrophoretic analysis requires homogenization of samples and thus provides limited information on the localization of the process. To acquire such spatial information for the primary process involved in beta-amyloidogenesis, we have designed and developed a novel antibody exclusively specific to APP fragments possessing the exact amino terminus of the major beta-amyloid (A beta) peptide. Use of this antibody revealed that cleavage of APP at the amino terminal position of the A beta sequence is a normal steady-state process in gerbil hippocampus. Furthermore, nonfatal transient (10 min) forebrain ischemia followed by reperfusion enhanced the initial beta-amyloidogenic reaction mainly in pyramidal cells of CA1 sector and of dentate gyrus prior to and along with delayed neuronal degeneration. The APP fragments accumulated in cell bodies and dendrites of the neurons. These results suggest that beta-amyloidogenesis may involve a process that is also activated in postischemic brain and that ischemia-like conditions may contribute to pathogenic A beta accumulation.

Alzheimer Disease↗

PubMed1994

Purification and characterization of an endogenous inhibitor specific to the Z-Leu-Leu-Leu-MCA degrading activity in proteasome and its identification as heat-shock protein 90.

We previously identified a benzyloxycarbonyl(Z)-Leu-Leu-Leu-4-methylcoumaryl-7-amide (ZLLL-MCA) degrading activity in proteasome as a candidate for the regulator of neurite outgrowth. As its counterpart, we purified a protein from bovine brain that specifically inhibits the ZLLL-MCA degrading activity in proteasome. This protein is heat stable and has no effect on the other catalytic activities in proteasome, or on the activities of trypsin, chymotrypsin, or m- and mu-calpains either. The molar ratio of inhibitor-to-proteasome that inhibits 50% of the ZLLL-MCA degrading activity of proteasome is 1:1. The inhibitory mechanism of the inhibitor against proteasome is non-competitive. Finally, the inhibitor was identified as heat-shock protein 90 (HSP90) by partial amino acid sequencing and immunodetection. The results suggest that HSP90 initiates neurite outgrowth through the inhibition of the ZLLL-MCA degrading activity in proteasome.

Amino Acid Sequence↗

PubMed1994

Stimulation of protein-tyrosine phosphorylation in gerbil hippocampus after global forebrain ischemia.

Tyrosine phosphorylation in the gerbil hippocampus after a transient ischemia was analyzed by immunoblotting and immunohistochemistry. In control hippocampus, the phosphotyrosine was detected in many proteins of 165 to 10 kDa and the immunostain showed a distinct distribution. The ischemic insult induced various alterations of the phosphotyrosine immunoreactivities in both ischemia-resistant and -vulnerable neurons which were associated with alterations in the expression of 165 to 19 kDa-immunoreactive bands. These results suggest that tyrosine phosphorylation is involved in the ischemic hippocampus to play a role in the development of early and delayed neuronal deaths in CA4 and CA1 neurons, respectively.

Animals↗

PubMed1994

Overproduction of a Ca(2+)-independent protein kinase C isozyme, nPKC epsilon, increases the secretion of prolactin from thyrotropin-releasing hormone-stimulated rat pituitary GH4C1 cells.

Rat pituitary GH4C1 cells express protein kinase C (PKC) transcripts for cPKC alpha, cPKC beta II, nPKC delta, nPKC epsilon, nPKC eta, and aPKC zeta, but not for cPKC gamma or nPKC theta. Of the transcripts produced, the nPKC epsilon isoform is the most abundant. Transfection of GH4C1 cells with an expression plasmid containing nPKC epsilon cDNA leads to the transient overexpression of cellular nPKC epsilon and confers enhanced phorbol ester binding activity. Transient expression of an inactive point mutant (nPKC epsilon K-->R) of nPKC epsilon, where Lys436 at the putative ATP-binding site is replaced with Arg, also confers elevated binding activity. However, only overproduction of the wild type in transfected cells increases the basal levels and stimulates the secretion of prolactin (PRL) by 12-O-tetradecanoylphorbol-13-acetate or thyrotropin-releasing hormone (TRH). In stable clones overexpressing nPKC epsilon, immunocytofluorescence and immunoblot experiments indicated that TRH causes the rapid translocation and down-regulation of an appreciable fraction of nPKC epsilon. Both the basal and TRH-stimulated levels of PRL secretion are clearly correlated with the expression level of nPKC epsilon but not with the TRH receptor densities in these clones. The dose dependence of TRH-stimulated secretion were similar in all cells overexpressing cPKC alpha, cPKC beta II, nPKC epsilon, and nPKC delta, but the enhancement of PRL secretion was specific for the overproduction of nPKC epsilon, no effect was found when other isozymes were overproduced. These findings clearly demonstrate that the expression level of nPKC epsilon in GH4C1 cells is rate-limiting for basal and TRH-stimulated PRL secretion, and they provide the first direct evidence that nPKC epsilon plays a key role in hormonal secretory processes.

Animals↗

PubMed1994

Widespread tissue distribution of multiple mitochondrial DNA deletions in familial mitochondrial myopathy.

We used Southern blot analysis and the polymerase chain reaction to analyze the tissue distribution of multiple mitochondrial DNA (mtDNA) deletions in a 45-year-old man with familial mitochondrial myopathy-Southern blots showed two major types of abnormal mtDNA with approximately 4- and 8-kilobase deletions in the skeletal and extraocular muscles. The amount of muscle mtDNA with deletions correlated approximately with the severity of muscle involvement. The polymerase chain reaction showed multiple mtDNA deletions even in clinically asymptomatic tissues, the pattern of which differed with the type of tissue. Nucleotide sequences of several mtDNAs with deletions showed that the deletions were flanked by direct repeats consisting of 3 to 12 nucleotides. Leukocytes from the patient's affected sister and his mother exhibited the same mtDNA deletion pattern. Most of the same deletions were present in leukocytes obtained from the patient's father.

Adolescent↗

PubMed1994

Characterization of cDNA-encoding N-terminal region of the quail lutropin receptor.

For understanding the evolutionary relationships between gonadotropins [GTHs: lutropin (LH) and follitropin (FSH)] and their receptors, we attempted to characterize the extracellular domain of the receptors, which is thought to be a key region of hormone binding, in nonmammalian species, and to compare the information to that of the known mammalian data. For this purpose, we designed two sets of sense and antisense oligonucleotides as polymerase chain reaction (PCR) primers, referring to the known mammalian GTH receptors, such as LH receptors of human, pig, and rat, and FSH receptors of human and rat. All possible combinations of the primers showed the successful amplification of cDNA of LH receptor without contamination of FSH receptor cDNA from rat testicular RNA samples. With these primers, reverse transcription (RT)-PCR was applied to the gonads of nonmammalian species (quail, snake, tortoise, newt, and bullfrog). Only the quail, however, showed the specific amplification when only one set of primers was used. Thus, the PCR product of the quail was used as a probe for Northern blot and in situ hybridization. By Northern blot analysis, a single size of mRNA (3 kb) was identified from quail testicular poly A+ RNA. The distribution of mRNA visualized by in situ hybridization was limited only on Leydig cells of quail testis. These results suggest that a part of the quail LH receptor cDNA was amplified by RT-PCR. The nucleotide and predicted peptide sequences of this amplified cDNA were compared with those of mammalian receptors. The size of characterized cDNA sequence was 519 bp, which is completely identical with those of mammalian LH receptors. The homology of both cDNA and predicted peptide was about 70% of those of mammalian LH receptors (intramammalian, about 80%). In spite of the relatively low homology, the positions of cystein residues and potential N-linked glycosylation sites in the peptide were completely conserved in all species compared (human, pig, rat, and quail). The conserved portions indicate their importance for the molecular conformation and specific ligand binding activity of LH receptors.

Animals↗