Biomedical subjects

S Kawashima

Publications and source records attributed to S Kawashima.

At least 145 records · Page 8Linked to original sources

PubMed1997

The calpain proteolytic system in neonatal hypoxic-ischemia.

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (HI, unilateral occlusion of the common carotid artery +7.70% O2 for 100 min) and allowed to recover for up to 14 days. Calpain caseinolytic activity was found to increase in both hemispheres for at least 20 hr. Hypoxic exposure per se increased the activity of calpains, more pronounced in a membrane-associated fraction, probably through interaction with cellular components, whereas HI introduced a loss of activity, most likely through consumption and loss of proteases. Consecutive tissue sections were stained with antibodies against calpastatin, alpha-fodrin, the 150-kDa breakdown product of alpha-fodrin (FBDP, marker of calpain proteolysis) or microtubule-associated protein 2 (MAP-2, marker of dendrosomatic neuronal injury). Areas with brain injury displayed a distinct loss of MAP-2, which clearly delineated the infarct. FBDP accumulated in injured and borderline regions ipsilaterally, and a less conspicuous, transient increase in FBDP also occurred in the contralateral hemisphere, especially in the white matter. The cytosolic fraction (CF) and the membrane and microsomal fraction (MMF) of cortical tissue were subjected to Western blotting and stained with antibodies against calpain, calpastatin and the 150-kDa breakdown product of alpha-fodrin (FBDP). Calpain immunoreactivity decreased bilaterally in the CF during the insult (62-68% of controls) and remained significantly lower during early recovery, whereas the MMF showed no significant changes. This translocation of calpains coincided with the appearance of FBDP in the ipsilateral, HI hemisphere, displaying a significantly higher level of FBDP from immediately after the insult until at least 1 day of recovery (204-292% of controls). No significant changes in FBDP were found in the contralateral, undamaged hemisphere, despite translocation of calpains in both hemispheres, a prerequisite for calpain activation. This discrepancy may be related to changes in the endogenous inhibitor, calpastatin. Calpastatin protein was found to decrease during and shortly after HI in the ipsilateral, but not the contralateral, hemisphere. The inhibitory activity of calpastatin also tended to decrease after HI, indicating that a reduction of calpastatin may be necessary for extensive calpain activation to occur. The mRNA of m-calpain increased in the HI hemisphere 48 hr after the insult (167%, p < 0.001), a time point when the protein was also increased. In summary, our findings indicate that calpains are activated during HI and in the early phase of reperfusion after HI, preceding neuronal death.

Animals↗

PubMed1997

Molecular interactions between dynamin and G-protein betagamma-subunits in neuroendocrine cells.

Dynamin and G-proteins both are guanosine triphosphate (GTP) binding proteins, with dynamin active in cellular membrane trafficking and G-proteins in intracellular signal transduction. Here we demonstrate that dynamin physically and functionally interacts with G-protein betagamma-subunits in neuroendocrine GH4C1 cells, on stimulation with thyrotropin-releasing hormone and somatostatin. The interaction appears to be of high affinity and inhibitory on dynamin GTPase activity, mediated by the pleckstrin homology domain and regulated both by the G-protein alpha-subunit and by guanosine nucleotides. Thus, dynamin may target particular sites for receptor-mediated endocytosis by sharing betagamma-subunits with the alpha subunit of G-proteins in neuroendocrine cells.

Animals↗

PubMed1997

Carnosine-induced inward currents in rat olfactory bulb neurons in cultured slices.

Olfactory neurons are rich in carnosine, which is regarded as a possible neurotransmitter between olfactory neurons and the olfactory bulb neurons. In the present study, we used the organotypic slice culture technique to culture olfactory bulb neurons and explore carnosine's role in the olfactory system. Inward current responses to carnosine upon increases in membrane conductance were recorded under whole-cell voltage-clamp conditions. The inward currents were desensitized during continuous application of carnosine. At carnosine concentrations above 50 microM, the current responses increased with increases in carnosine concentrations. The present results suggest that carnosine serves as an excitable neuroeffector between olfactory neurons and olfactory bulb neurons.

Animals↗

PubMed1997

Identification of mu-, m-calpains and calpastatin and capture of mu-calpain activation in endothelial cells.

The presence of the calpain-calpastatin system in human umbilical vein endothelial cells (HUVEC) was investigated by means of ion exchange chromatography, Western blot analysis, and Northern blot analysis. On DEAE anion exchange chromatography, calpain and calpastatin activities were eluted at approximately 0.30 M and 0.15-0.25 M NaCl, respectively. For half-maximal activity, the protease required 800 microM Ca2+, comparable to the Ca2+ requirement of m-calpain. By Western blot analysis, the large subunit of mu-calpain (80 kDa) was found to be eluted with calpastatin (110 kDa). Both the large subunit of m-calpain (80 kDa) and calpastatin were detected in the respective active fractions. By Northern blot analysis, mRNAs for large subunits of mu- and m-calpains were detected in single bands, each corresponding to approximately 3.5 Kb. Calpastatin mRNA was observed in two bands corresponding to approximately 3.8 and 2.6 Kb. Furthermore, the activation of mu-calpain in HUVEC by a calcium ionophore was examined, using an antibody specifically recognizing an autolytic intermediate form of mu-calpain large subunit (78 kDa). Both talin and filamin of HUVEC were proteolyzed in a calcium-dependent manner, and the reactions were inhibited by calpeptin, a cell-permeable calpain specific inhibitor. Proteolysis of the cytoskeleton was preceded by the appearance of the autolytic intermediate form of mu-calpain, while the fully autolyzed postautolysis form of mu-calpain (76 kDa) remained below detectable levels at all time points examined. These results indicate that the calpain-calpastatin system is present in human endothelial cells and that mu-calpain may be involved in endothelial cell function mediated by Ca2+ via the limited proteolysis of various proteins.

Antibody Specificity↗

PubMed1997

Cytoplasmic processing of human profilaggrin by active mu-calpain.

The differentiation of keratinocytes involves numerous steps including the formation of the cornified envelope and the aggregation of keratin filaments by filaggrin monomer molecules. In this study, we investigated whether mu-calpain is involved in the processing of profilaggrin to filaggrin monomers by using both an active mu-calpain specific antibody and a 27-mer synthetic calpastatin peptide, a cell-permeable calpain-specific inhibitor. Upon incubation of cultured keratinocytes with Ca2+ for 96 hours, active mu-calpain with a molecular mass of 76 kDa appeared preceded by an increase in mu-calpain mRNA. In synchrony with the appearance of active mu-calpain, the processing of profilaggrin occurred. Furthermore, the Ca2+-induced activation of mu-calpain and the processing of profilaggrin were affected by the addition of the synthetic calpastatin inhibitor. These results indicate that the activation of mu-calpain plays a major role in the profilaggrin processing.

Amino Acid Sequence↗

PubMed1997

Vascular endothelial growth factor increases endothelin-converting enzyme expression in vascular endothelial cells.

Endothelin-converting enzyme-1 (ECE-1) is a key enzyme in endothelin processing. Although it has been revealed that ECE-1 expression increases in vascular wall after balloon injury in vivo experimental models, the regulation of ECE-1 expression in vitro remains undefined. In this study, we demonstrated that the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF) increased ECE-1 expression in cultured bovine aortic endothelial cells (BAEC). Northern blot analysis revealed that VEGF increased ECE-1 mRNA expression in 12-24 hours by 2-fold in BAEC, and this effect was dose-dependent. VEGF also increased ECE-1 protein expression detected by immunoblotting in 36 hours in BAEC. Therefore, VEGF increased ECE-1 expression in BAEC, which suggests that ECE-1 induction by VEGF may be involved in endothelin-system upregulation under pathological conditions such as neointimal formation and atherosclerosis.

Animals↗

PubMed1997

Activity-dependent survival of rat cerebellar granule neurons is not associated with sustained elevation of intracellular Ca2+.

Ca2+ plays a pivotal role for the activity-dependent survival of neurons. In primary culture of cerebellar granule neurons, we found that there is no significant difference in intracellular Ca2+ level in the survival-promoting condition (cultures in the presence of 25 mM KCl) and that in the apoptosis-inducing condition (cultures in the presence of 5 mM KCl). This was not due to the inactivation of voltage-dependent L-type Ca2+ channels in the survival-promoting condition, but due to the enhanced rate of the influx and the efflux of Ca2+ in the survival-promoting condition compared to that in the apoptosis-inducing condition. These results suggest that the activity-dependent survival of the granule neurons is not associated with sustained rise of intracellular Ca2+ but associated with the enhanced turnover rate of Ca2+.

Animals↗

PubMed1997

Overexpression of a neuropeptide nociceptin/orphanin FQ precursor gene, N23K/N27K, induces neurite outgrowth in mouse NS20Y cells.

We recently discovered N23K and its splicing variant N27K as transcripts upregulated in mouse NS20Y cells after differentiation induced by dibutylyl cyclic AMP (dbcAMP) treatment. N23K and N27K encode precursor proteins for an opioid neuropeptide, nociceptin/orphanin FQ, but the transient expression of N23K and N27K suggests that it may be involved in neuronal differentiation. In the present study, we report that NS20Y cells transfected with N23K and/or N27K but not with vector alone formed neurites, with the expressed protein distributed in the perinuclear region and distal parts of the neurites. The granular staining of the N23K and N27K proteins was also colocalized with secretogranin I, indicating incorporation into large dense core vesicles. This cellular targeting of the N23K and/or N27K protein is similar to that of dbcAMP-induced processes in nontransfected NS20Y cells. In addition, the neurites of transfectants that expressed both N23K and N27K were longer than those of the transfectants that expressed N23K or N27K alone. Our results demonstrate that N23K and N27K participate in the regulation of neurite outgrowth.

Amino Acid Sequence↗

PubMed1997

Activity-dependent expression of parathyroid hormone-related protein (PTHrP) in rat cerebellar granule neurons. Requirement of PTHrP for the activity-dependent survival of granule neurons.

To identify genes whose expression is neuronal activity-dependent, we used an mRNA differential display technique and discovered that parathyroid hormone-related protein (PTHrP) is expressed in an activity-dependent manner in primary cultures of rat cerebellar granule neurons. PTHrP mRNA was expressed as early as 1 h by the addition of KCl to a final concentration of 25 mM to the culture medium. This expression was induced by Ca2+ influx through voltage-dependent L-type Ca2+ channels and regulated at the transcriptional step. PTHrP mRNA was persistently expressed before and after the time of commitment of granule neurons to apoptosis when they are cultured in the presence of 25 mM KCl or both 150 microM N-methyl-D-aspartic acid and 15 mM KCl, both of which promote the survival of these neurons. PTHrP was rapidly secreted into the culture medium in a depolarization-dependent manner. Parathyroid hormone/PTHrP receptor mRNA was also expressed in the primary cultures, and its expression was up-regulated by KCl and/or N-methyl-D-aspartic acid. The addition of anti-PTHrP antiserum to the culture medium resulted in a reduction of the activity-dependent survival of the granule neurons. These results suggest that PTHrP is involved in an autocrine loop and required for the survival of granule neurons.

Animals↗

PubMed1997

Up- and down-regulation of calpain inhibitor polypeptide, calpastatin, in postischemic hippocampus.

Based on our previous observation that transient forebrain ischemia induces calpain-catalyzed proteolysis in gerbil hippocampus in a region-specific manner, we examined the effect of ischemia on the quantity and localization of the endogenous calpain-specific inhibitor protein, calpastatin, in the tissue. Brief (5 min) forebrain ischemia followed by reperfusion induced an overall increase of calpastatin immunoreactivity in hippocampus, particularly in pyramidal cells, in 4 h as analyzed by Western blotting and immunohistochemistry. The amount of calpastatin, however, decreased to the preischemic level and lower in 24 h to 7 days due to proteolysis except in CA2 showing continuously elevated calpastatin immunoreactivity. Because calpastatin is not only a potent inhibitor but also a preferred substrate for calpain and because CA2 neurons are less vulnerable to ischemic stress than the adjacent CA1 neurons, these observations imply involvement of calpastatin in calpain regulation as a bait substrate and, possibly, in neuroprotection under ischemic conditions. Calpastatin may participate in the stress responses together with the previously known ischemia-induced stress proteins such as heat shock proteins.

Animals↗

PubMed1997

Reduction of plasma glutamyl aminopeptidase activity in sporadic Alzheimer's disease.

Previously, we hypothesized that aminopeptidase-catalyzed proteolysis may limit the rate of beta-amyloid catabolism in brain and that reduction of a certain aminopeptidase activity may lead to deposition of peptidic metabolites represented by beta-amyloid and thus to Alzheimer's disease (AD). To explore this possibility in clinical situations and to seek a possible biochemical marker for the disease, we quantitated four classes of aminopeptidase activities in cerebrospinal fluids and heparinized plasma from sporadic AD patients and agematched controls collected in two independent medical institutions. We found that only plasma glutamyl aminopeptidase activity was significantly and consistently lower in AD patients. Although the mechanism leading to such a biochemical change in plasma remains to be elucidated, the results provide support for the aminopeptidase hypothesis and indicate that the enzyme activity may potentially be used as a diagnostic/ predictive marker for AD.

Aged↗

PubMed1997

Disparity between serotonin- and acetylcholine-provoked coronary artery spasm.

BACKGROUND AND HYPOTHESIS: Intrinsic vasoactive substances, such as serotonin and acetylcholine, are known to provoke coronary artery spasm in patients with vasospastic angina. It remains unclear, however, whether these different agents, which activate different receptors, produce spasms at the same sites in these patients. The present study was designed to clarify the disparity of receptor agonist-induced coronary artery spasms in the same patients. METHODS: We conducted sequential provocative tests of coronary artery spasm by acetylcholine, serotonin, and ergonovine in 20 patients with rest angina examined with quantitative coronary angiography. RESULTS: Coronary artery spasms were provoked in all patients at 27 spastic sites. In 13 patients, ergonovine provoked spasms and in 10 of 13 patients who were diagnosed with variant angina, both acetylcholine and serotonin provoked spasms at the same sites where ergonovine also did. In 4 of 13 patients, spasms were provoked by serotonin but not by acetylcholine. In the remaining seven patients, whose spasms were induced by ergonovine, spasms were produced by acetylcholine but not by serotonin. On coronary angiography, the spastic sites for both acetylcholine and serotonin, and those for serotonin alone, were located in the proximal segments of coronary arteries, whereas the spastic sites for acetylcholine alone were located in the distal segments. CONCLUSIONS: This study documented the disparity between serotonin- and acetylcholine-induced spasms. Provocative tests using agents that activate different receptors may produce coronary artery spasms at the same and/or different sites, and this disparity may reflect the clinical heterogeneity of vasospastic ischemic syndrome.

Acetylcholine↗

PubMed1997

Spontaneous subcapsular hepatic hemorrhage associated with pregnancy: report of a case.

We report herein a case of spontaneous subcapsular hepatic hemorrhage which occurred in a 33-year-old woman 1 day after she had been delivered of her second child by cesarean section following an uneventful pregnancy. She complained of right upper quadrant pain on the 1st postoperative day, and computed tomography (CT) showed subcapsular low-density masses in both liver lobes, while extravasation was demonstrated by CO2 intraarterial digital subtraction angiography (IADSA). The hemorrhage was successfully controlled by transcatheter arterial embolization (TAE). However, on the 3rd day after TAE, an exploratory laparotomy was performed to establish an exact diagnosis to explain the persistent abdominal pain and abnormal liver function tests. Subcapsular hematomas in both lobes were confirmed and no visible laceration was present. The patient recovered gradually by spontaneous absorption of the hematomas and was discharged on the 22nd postoperative day. Spontaneous hepatic hemorrhage associated with pregnancy is a very rare complication, and establishing a correct diagnosis and initiating appropriate therapy are essential for this life-threatening disease.

Adult↗

PubMed1997

A major glycoprotein of Xenopus egg vitelline envelope, gp41, is a frog homolog of mammalian ZP3.

A predominant glycoprotein in the vitelline envelope (VE) of the anuran Xenopus laevis is gp41, known to be proteolytically converted from gp43 of the coelomic egg envelope concomitant with the acquisition of egg fertilizability. To characterize the protein core of gp41, purified gp41 from VE was digested with lysyl endopeptidase, and peptides isolated from the digests were sequenced for amino acids to design degenerate primers for polymerase chain reaction. By reverse transcription-polymerase chain reaction with a poly(A)+ RNA from the ovary of an ovulated female Xenopus, a specifically amplified band was obtained and sequenced. The upstream and downstream sequences of the sequenced region were completed by 5'- and 3'-rapid amplification of cDNA ends, respectively. The cDNA, referred to as gp43 cDNA, comprises 1423 base pairs and contains one open reading frame with a sequence for 460 amino acids. The predicted amino acid sequence of gp43 cDNA has a close similarity with that of mammalian ZP3. Northern blot and in situ hybridization studies indicated that gp43 mRNA is expressed in oocytes, particularly in the previtellogenic oocytes. A comparison of the N-terminal sequences of gp41 and gp43 strongly suggested that gp41 is generated at least by processing of the N-terminal portion of gp43 with oviductin.

Amino Acid Sequence↗

PubMed1997

Downregulation of calpastatin in rat heart after brief ischemia and reperfusion.

The activities of calpain and its endogenous inhibitor, calpastatin, were measured in the soluble fraction of perfused rat heart after ischemia for 5-20 min and reperfusion for up to 30 min. The method for m-calpain measurement was modified: washing of the DEAE-cellulose column with 0.18 M NaCl instead of 0.15 M NaCl increased the m-calpain activity 12.5-fold. Ischemia for 20 min followed by reperfusion for 30 min did not affect the m-calpain activity but decreased the calpastatin activity. m-Calpain was enriched in the nucleus-myofibril fraction but was not further translocated on ischemia-reperfusion. Mu-calpain was below the limit of detection on immunoblotting or casein zymography, but its mRNA was substantially expressed, as detected on Northern blotting. Casein zymography also revealed a novel Ca2+-dependent protease without the typical characteristics of mu- or m-calpain. The immunoblotting of myocardial fractions showed that calpastatin was proteolyzed on ischemia-reperfusion. The calpastatin proteolysis was suppressed by a calpain inhibitor, Ac-Leu-Leu-norleucinal. Calpastatin may sequester calpain from its substrates in the normal myocardium, but may be proteolyzed by calpain in the presence of an unidentified activator in the early phase of calpain activation during ischemia-reperfusion, resulting in the proteolysis of calpastatin and then other calpain substrates.

Animals↗

PubMed1997

Identification of endogenous substrates for Drosophila calpain from a salt-extracted fraction of Drosophila ovaries.

Drosophila calpain (Dm-calpain) produced in Escherichia coli has a distinct Ca2+-dependent activity. By using a recombinant Dm-calpain, we searched for its substrates occurring in Drosophila ovaries, where Dm-calpain is expressed. Among a number of major proteins, several proteins in a salt-extracted fraction were selectively degraded by Dm-calpain in a Ca2+-dependent manner. The major substrates were identified by microsequencing the lysylendopeptidase-digested proteins. Three ribosomal proteins, the L5, L7, and L8 subunits of the 60S ribosome, were found to be potential Dm-calpain substrates. In addition, the alpha subunit of elongation factor-1 (EF-1alpha), a multi-functional protein involved in both protein synthesis and cytoskeletal regulation, was shown to be cleaved by Dm-calpain into several distinct fragments when expressed as a GST-fusion protein. Endogenous EF-1alpha in ovary extracts was also shown by western blot analysis to be similarly degraded. These observations suggest that Dm-calpain may regulate protein synthesis and cytoskeletal structure through its degradative or processing activity.

Amino Acid Sequence↗

PubMed1997

Nitric oxide inhibits neutrophil adhesion to cytokine-activated cardiac myocytes.

Neutrophils play important roles in myocardial injury in which an inflammatory reaction ensues. We investigated the role of nitric oxide (NO) in neutrophil adhesion to cardiac myocytes. Isolated adult rat myocytes were coincubated with human neutrophils, and the number of neutrophils adherent to myocytes was microscopically counted. The adhesion increased up to 2.4-fold when both myocytes and neutrophils were activated by interleukin-1 beta (IL-1 beta) and platelet-activating factor (PAF), respectively. The NO donors, S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP), inhibited the adhesion by 50% when administered to neutrophils before activation. However, when activated neutrophils were added to myocytes that had been incubated with NO donors during IL-1 beta stimulation, the inhibition of adhesion was not observed. Pretreatment of neutrophils with SNAP or 8-bromoguanosine 3',5'-cyclic monophosphate did not reduce PAF-induced CD11b/CD18 expression determined by flow cytometry, nor did simultaneous treatment of myocytes with IL-1 beta and SNAP decrease IL-1 beta-induced intercellular adhesion molecule-1 expression determined by immunofluorescence staining and enzyme-linked immunosorbent assay. Thus NO inhibits neutrophil-myocyte adhesion, mainly acting on neutrophils without quantitatively affecting the upregulation of CD11b/CD18.

Animals↗

PubMed1997

Lesions of gonadotropin-releasing hormone-immunoreactive terminal nerve cells: effects on the reproductive behavior of male dwarf gouramis.

Functions of the terminal nerve (TN) are largely unknown. To examine whether gonadotropin-releasing hormone (GnRH)-immunoreactive TN cells (TN-GnRH cells) are involved in the control of reproductive behavior, effects of lesions of TN-GnRH cells were studied in male dwarf gouramis, Colisa lalia. After bilateral electrolytic lesion, a characteristic impairment was observed in one of the repertoires of male reproductive behavior, nest-building. The occurrence of mating trials in which males showed no nest-building was increased. However, the incidence of nest-building behavior during postoperative trials was not affected by the lesion. No impairment was observed in other reproductive repertoires. These results suggest that (1) TN-GnRH cells are involved in the control of the threshold for nest-building behavior initiation and (2) TN-GnRH cells are not a prerequisite for other aspects of reproductive behavior in the male gouramis.

Animals↗