Search PubMed⌕ Search

Biomedical subjects

S Kawashima

Publications and source records attributed to S Kawashima.

At least 199 records · Page 11Linked to original sources
PubMed1995

Myristoylation of endothelial cell nitric oxide synthase is important for extracellular release of nitric oxide.

Endothelial cell nitric oxide synthase (NOS) is known to have a N-myristoylation consensus sequence. Such a consensus sequence is not evident in the macrophage, smooth muscle and neuronal NOS. A functional role for this N-terminal myristoylation is not clear yet. In the present study, we examined the effect of N-terminal myristoylation on the NOS activity determined by the conversion of L-[3H]arginine to L-[3H]citrulline and extracellular NO release determined by nitrite production in the conditioned medium from the COS-7 cells transfected with wild-type bovine aortic endothelial cell (BAEC) NOS cDNA or non-myristoylated BAEC-NOS mutant cDNA. NOS activity of wild type BAEC-NOS in COS-7 cells was localized in the particulate fraction and that of mutant NOS was in the cytosolic fraction. In contrast, nitrite production from COS-7 cells transfected with wild type BAEC-NOS cDNA was greater than that of mutant cDNA in a time dependent and a concentration dependent manner. These results suggest that membrane localization of NOS with myristoylation facilitates extracellular transport of NO and leads to enhanced NO signaling on the vascular smooth muscle cells and the intravascular blood cells including neutrophils, macrophages and platelets.

Animals↗
PubMed1995

The role of the calpain-calpastatin system in thyrotropin-releasing hormone-induced selective down-regulation of a protein kinase C isozyme, nPKC epsilon, in rat pituitary GH4C1 cells.

We have examined the mechanism for the selective down-regulation of protein kinase C epsilon (nPKC epsilon) in rat pituitary GH4C1 cells responding to thyrotropin-releasing hormone (TRH) stimulation. Among various low molecular weight protease inhibitors examined, only a cysteine protease inhibitor (calpain inhibitor I, N-acetyl-Leu-Leu-norleucinal) blocked the down-regulation of nPKC epsilon. Furthermore, the introduction of a synthetic calpastatin peptide, an exclusively specific inhibitor of calpain, into the cells also reduced the down-regulation, suggesting the involvement of calpain among all the intracellular cysteine proteases in this process. In accordance, we observed TRH-induced translocation of m-calpain from the cytosol to the membrane and the concomitant up-regulation of calpastatin isoforms; presumably, the former represents activation of the protease initiating the kinase degradation, while the latter constitutes a negative feedback system protecting the cells from activated calpain. These results suggest that in GH4C1 cells, TRH mobilizes both protease (m-calpain) and inhibitor (calpastatin) as a strictly regulating system for the nPKC epsilon pathway mediating TRH signals.

Amino Acid Sequence↗
PubMed1995

The number of 5-fluoro-2'-deoxyuridine-5'-monophosphate binding sites and reduced folate pool in human colorectal carcinoma tissues: changes after tegafur and uracil treatment.

Thymidylate synthase (TS) is a target enzyme of 5-fluorouracil and is inhibited by 5-fluoro-dUMP (FdUMP) to form an inactive ternary complex. We investigated the changes in the number of FdUMP binding sites in human colorectal carcinoma tissues after treatment with 5-fluorouracil derivatives and also examined the mechanisms underlying these changes. The number of FdUMP binding sites was significantly increased in patients who received tegafur and uracil preoperatively [UFT (+) group, n = 14] compared with those who did not [UFT (-) group, n = 36; P < 0.0001]. No amplification of the TS gene was observed in the carcinoma tissues in either group. The level of TS mRNA in the carcinoma tissues showed no significant difference between UFT (-) and UFT (+) groups. There was a significant correlation between the level of TS mRNA and TS in the UFT (-) group, as shown by simple linear regression analysis (P < 0.05). In the UFT (+) group, 9 of 12 cases showed TSf corresponding to their level of TS mRNA. It seemed that the augmentation of TStot is the result of accumulation of ternary complex. Thus, TS inhibition by FdUMP will be insufficient in the absence of methylenetetrahydrofolate in the cytosol, because translation of TS from TS mRNA has been shown to continue in the presence of FdUMP.

Base Sequence↗
PubMed1995

Molecular cloning of cytidine monophospho-N-acetylneuraminic acid hydroxylase. Regulation of species- and tissue-specific expression of N-glycolylneuraminic acid.

Cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylase, which is the key enzyme for the synthesis of N-glycolylneuraminic acid (NeuGc), has been purified from the cytosolic fraction of mouse liver, as described in our previous paper. The amino acid sequences of the purified CMP-NeuAc hydroxylase, and peptides obtained by lysylendopeptidase digestion, were used to synthesize specific oligonucleotide primers. A mouse cDNA clone of the enzyme was obtained by a combination of the polymerase chain reaction and rapid amplification of cDNA ends. The sequence of the clone contained an open reading frame coding for a protein of 577 amino acids with a predicted molecular mass of 66 kDa. The deduced sequence included the amino acid sequences obtained for the purified enzyme and peptides, and a complete match was obtained for 159 residues. The enzyme has neither a signal peptide sequence nor a membrane spanning domain, which is consistent with localization of the enzyme in the cytosol. Transfection of a cDNA construct to COS-1 cells increased the enzyme activity and the amount of NeuGc. Comparison of the sequence with GenBank data indicated that no similar sequence has been reported so far. Northern blot analysis of various mouse tissues with the enzyme cDNA as a probe indicated that expression of NeuGc is related to the level of CMP-NeuAc hydroxylase mRNA. On Southern blot analysis with the same probe, cross-hybridizing bands were detected in the human and fish genomes.

Amino Acid Sequence↗
PubMed1995

Fodrin degradation and subcellular distribution of calpains after neonatal rat cerebral hypoxic-ischemia.

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (unilateral occlusion of the common carotid artery + 7.70% O2 for 100 min). Ipsi-and contralateral parietal cerebral cortex was assayed with Western blotting for fodrin breakdown product (FBDP). Calpain immunoreactivity was assayed in the cytosolic fraction (CF) and the membrane and microsomal fraction (MMF). 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 relative redistribution of calpains coincided with the appearance of FBDP in the left, ipsilateral 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 could be detected in the right, contralateral hemisphere, indicating that although redistribution of calpains occurred, hypoxia per se did not suffice to initiate fodrin degradation in this model of neonatal hypoxic-ischemia.

Animals↗
PubMed1995

Characterization of gonadotropin-releasing hormone (GnRH)-immunoreactive protein in the rat pineal gland.

The aim of the present study was to characterize GnRH-like substance(s) in the rat pineal gland using a monoclonal antibody, LRH13, as a probe. The epitope of LRH13 is between 2nd and 5th amino acid residues of the mammalian GnRH, and its immunological characters were previously defined by us. LRH13 could show strong immunological signal on the rat pineal gland. Immunoblot after SDS-PAGE of the pineal gland preparations showed a LRH13 immunoreactive band with apparent mol wt 52 kilo-Dalton (kD), which is much bigger than that of hypothalamic GnRH precursor (10 kD). The 52 kD protein, however, was detected from insoluble fraction of the pineal homogenate and liberated from the fraction by Triton X-100 (2%) treatment. On the other hand, NaCl (140 mM and 500 mM) or EDTA (10 mM) treatment failed to liberate. Two-dimensional gel electrophoresis showed that the 52 kD protein is a mixture of two proteins with different isoelectric points (pI approximately 6.8 and 7.0). Both proteins showed identical patterns of peptide mapping by V8 protease digestion, and they might be originated from the same peptide. These results suggest that the rat pineal GnRH-immunoreactive substance has a unique property as a membrane associate protein.

Animals↗
PubMed1995

Immunocytochemical localization of sGnRH and cGnRH-II in the brain of goldfish, Carassius auratus.

The immunocytochemical distribution of salmon gonadotropin-releasing hormone (sGnRH) and chicken GnRH-II (cGnRH-II) neurons in the brain of goldfish was examined using respective antisera. Salmon GnRH-immunoreactive (ir) cell bodies were localized in the area between the olfactory nerve and the olfactory bulb (the terminal nerve ganglion), the ventral telencephalon, the preoptic area, and the hypothalamus. Chicken GnRH-II-ir cell bodies were observed in the same areas as were those of sGnRH, although the number of cell bodies were fewer than those of sGnRH. In addition, chicken GnRH-II-ir cell bodies were also observed in the midbrain tegmentum where no sGnRH-ir cell bodies were found. Both sGnRH-ir and cGnRH-II-ir fibers were distributed not only in the hypothalamus and the pituitary gland but also in various brain areas from the olfactory bulb to the spinal cord. The wide distribution of GnRH-ir fibers suggests that in the goldfish, sGnRH and cGnRH-II not only regulate gonadotropin release from the pituitary gland but also function as neuromodulators in various brain regions.

Animals↗
PubMed1995

Identification and characterization of membrane-bound calpains in clathrin-coated vesicles from bovine brain.

Calpains are intracellular cysteine proteases activated in a Ca(2+)-dependent manner. Previously, we found that the differentiation of K562 cells induced by 4 beta-phorbol 12-myristate 13-acetate treatment is accompanied by an increase in m-calpain levels and, at the same time, m-calpain becomes localized on the inside of plasma membranes, coated pits, and coated vesicles [Nakamura, M., Mori, M., Morishita, Y., Mori, S. & Kawashima, S. (1992) Exp. Cell Res. 200, 513-522]. We also reported that mu-calpain plays an essential role in morphological changes and membrane fusion of erythrocytes through the degradation of spectrin, a lining protein [Hayashi, M., Saito, Y. & Kawashima, S. (1992) Biochim. Biophys. Res. Commun. 182, 939-946]. Thus, calpains are implicated in endocytosis and/or exocytosis, processes stimulated by Ca2+ and involving intracellular membrane fusion. In this study, we report the biochemical characterization of calpains as components of purified coated vesicles from bovine brain. It was found by Western-blot analysis and chemical cross-linking of proteins that calpains are bound to the membranes of coated vesicles, and not to the coats. The binding of m-calpain to vesicles is Ca(2+)-dependent, while that of mu-calpain is less dependent on the presence of Ca2+. We also identified substrate proteins for calpains in coated vesicles. Upon activation of endogenous calpains, component proteins of coated vesicles such as the clathrin light chain, tubulins, and adaptins, but not the clathrin heavy chain, are highly sensitive to calpain digestion. In the case of exogenously added calpains, low concentrations degraded the same protein components. The degradation pattern differs slightly between added mu-calpain and m-calpain. These results strongly suggest that calpains are involved in the formation of coated vesicles and/or vesicle fusion to endosomes.

Animals↗
PubMed1995

Multiple gonadotropin-releasing hormone (GnRH)-immunoreactive systems in the brain of the dwarf gourami, Colisa lalia: immunohistochemistry and radioimmunoassay.

The present study characterizes gonadotropin-releasing hormone (GnRH) neuronal groups that are located in several different brain regions by investigating GnRH molecular species and projection patterns in an anabantid fish, Colisa lalia. First, we examined the molecular species of GnRHs in extracts of the brain and the pituitary by reverse-phase high-performance liquid chromatography followed by radioimmunoassays. We found salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and an unfamiliar GnRH-like substance. Next, to examine the distribution of each GnRH molecule in different GnRH neuronal groups, we performed immunohistochemistry using four kinds of antisera and an antibody. Furthermore, we performed brain lesioning experiments of terminal nerve (TN) cells, the most conspicuous GnRH-immunoreactive cells in Colisa lalia. Comparisons of immunoreactive structures between TN-lesioned fish and untreated fish elucidated the projection area of each neuronal group. Three major neuronal groups were observed. TN-GnRH cells, which are located in the transitional area between the olfactory bulb and the telencephalon, showed strong sGnRH and weaker cGnRH-II immunoreactivity. TN-GnRH cells projected to wide areas of the central nervous system from the olfactory bulb to the spinal cord. The second group, located in the preoptic area, showed only sGnRH immunoreactivity and projected only to the pituitary. The third one, located in the midbrain tegmentum, exhibited strong cGnRH-II and weaker sGnRH immunoreactivity. This cell group projected mainly to brain regions posterior to the hypothalamus and the spinal cord. These different projection patterns suggest functional differentiation of each GnRH neuronal group.

Animals↗
PubMed1995

Identification, characterization, and intracellular distribution of cofilin in Dictyostelium discoideum.

We identified and purified an actin monomer-binding protein of apparent molecular weight of 15,000 from Dictyostelium discoideum. The 15-kDa protein depolymerized actin filaments in a pH-dependent manner. The protein also had an activity to decrease apparent viscosity of actin solutions in a dose-dependent manner. This activity was inhibited by phosphatidyl inositides. Molecular cloning of genes encoding this protein revealed that the protein is 42% identical in its primary sequence to yeast cofilin. We concluded that the 15-kDa protein is cofilin of this organism. D. discoideum cells contain two cofilin genes (DCOF1 and DCOF2) whose nucleotide sequences were entirely identical in their exsons while the promoter and intron regions were different. Promoter assay experiments revealed that DCOF1 is expressed both in vegetative and differentiating cells and that DCOF2 is not expressed under any conditions examined. Gene disruption experiments suggested that DCOF1 might be essential for the proliferation of D. discoideum cells whereas the disruption of DCOF2 was proven not to alter any phenotypes. Indirect immunofluorescence microscopic observations showed that cofilin is distributed diffusely throughout cytoplasm in vegetative cells. In flattened cells under starvation stress, cofilin localized at dramatically reorganizing actin-cytoskeletons in ruffling membranes of the leading edge, but not at rigid actin meshwork in focal adhesion plaques. These results suggest that cofilin may be involved in dynamic reorganization of membranous actin cytoskeletons.

Actin Depolymerizing Factors↗
PubMed1995

In situ detection of gonadotropin-releasing hormone (GnRH) receptor mRNA expression in the rat ovarian follicles.

To clarify the target cells of GnRH in the ovary, in vivo expression of ovarian GnRH receptor mRNA was examined histologically by in situ hybridization in immature rats treated with PMSG only or in combination with hCG. Strong hybridization signals were observed in the granulosa cells of atretic follicles. However, no significant signals were found in the granulosa cells of healthy small, preantral, or early antral follicles. Healthy Graafian and preovulatory follicles also showed intense signals in their mural granulosa cells, but no signals were detected in the cumulus oophorus cells. Corpora lutea showed only weak signals, but luteinizing follicles probably after atresia exhibited signals of moderate intensity in their luteinized and remaining granulosa cells. No signals were detected in the theca cells and oocytes in all the follicles. Interstitial cells sometimes exhibited hybridization signals of moderate intensity, when the cells were eosinophilic. Pretreatment with different combinations of gonadotropins yielded different ovarian histology, but this had no influence on the localization of hybridization signals. These results, showing that the authentic GnRH receptor mRNA was expressed in a certain cell population in the rat ovary, suggest that the receptor is involved in the control of various ovarian functions including follicular development, atresia, ovulation, and luteinization after ovulation and follicular atresia.

Animals↗
PubMed1995

The possible self-down-regulation of calpain triggered by cell membranes.

In order to confirm whether the binding sites for mu-calpain on the inner surface of erythrocyte membranes are substrate proteins themselves, we examined the binding properties of mu-calpain to mu-calpain-pretreated inside-out membranes. When native mu-calpain was incubated with mu-calpain-pretreated membranes, however, newly added calpain was degraded rapidly in a time- and Ca(2+)-dependent manner. Although the degradation of mu-calpain was not inhibited by various proteinase inhibitors, it was strongly inhibited by digestible substrates for calpain that possess the ability to inhibit the binding of mu-calpain to erythrocyte membranes. On the other hand, when mu-calpain inactivated by N-ethylmaleimide was incubated with mu-calpain-pretreated membranes, no degradation was observed. These results indicate that the degradation of mu-calpain occurs on the surface of mu-calpain-modified membranes and that it depends on the autoproteolytic activity of mu-calpain itself. It seems likely that the autoproteolytic activity of mu-calpain is accelerated markedly by some component(s) exposed on the surface of membranes during the pretreatment with mu-calpain. The possibility is thus proposed that cell membranes possess the ability to down-regulate calpain to protect cell membranes from overdegradation by excessively bound calpain. The active factor(s) in the membranes that can accelerate the autoproteolytic degradation of mu-calpain could be almost completely removed from mu-calpain-modified membranes by treatment with Triton X-100.

Animals↗
PubMed1995

Simultaneous repair of an abdominal aortic aneurysm and resection of bronchogenic carcinoma: report of a case.

A 69-year-old man successfully underwent simultaneous repair of an abdominal aortic aneurysm and resection of bronchogenic carcinoma during the same operation. Pulmonary lobectomy was performed following complete closure of the abdomen after aneurysmectomy to prevent any contamination. If the patients are carefully selected, an operation for abdominal aortic aneurysm and bronchogenic carcinoma can be safely performed with the advantage of treating both lesions simultaneously and thus sparing the patients from having to undergo the physical and psychosomatic pain, as well as the risk, of a second operation.

Aged↗
PubMed1995

Cerebellar ataxia in patients with Leber's hereditary optic neuropathy.

We report the cases of a mother and son with Leber's hereditary optic neuropathy (LHON), where a point mutation of mitochondria DNA from guanine to adenine on nucleotide position 11778 was verified. Both also had cerebellar ataxia and dysarthria and in both cases cerebellar atrophies were detected by computed tomography or magnetic resonance imaging. It was not possible to elucidate the relationship between LHON and the cerebellar atrophy, but it should be kept in mind that various neurological complications may occur in LHON.

Adult↗
PubMed1995

Dominant and differential deposition of distinct beta-amyloid peptide species, A beta N3(pE), in senile plaques.

We analyzed an amino-terminal modification of beta-amyloid (A beta) peptide in brain, using anti-A beta antibodies that distinguish distinct molecular species. Examination of cortical sections from 28 aged individuals with a wide range in senile plaque density revealed that a molecular species distinct from the standard A beta is deposited in the brain in a dominant and differential manner. This modified A beta peptide (A beta N3(pE)) starts at the 3rd aminoterminal residue of the standard A beta, glutamate, converted to pyroglutamate through intramolecular dehydration. Because plaques composed of A beta N3(pE) are present in equivalent or greater densities than those composed of standard A beta bearing the first amino-terminal residue (A beta N1) and because deposition of the former species appears to precede deposition of the latter, as confirmed with specimens from Down's syndrome patients, the processes involved in A beta N3(pE) production and retention may play an early and critical role in senile plaque formation.

Adult↗
PubMed1995

Effects of monocyte-macrophage colony-stimulating factor (M-CSF) on in vitro erythropoiesis of marrow progenitor cells from patients with renal anemia.

We examined the influence of monocyte-macrophage colony-stimulating factor (M-CSF) on erythropoiesis both in vitro and in vivo in 98 patients with chronic renal failure who were undergoing hemodialysis. Serum levels of M-CSF and the clinical response to therapy with human recombinant erythropoietin (Epo) were analyzed. The following results were obtained: 1) The serum level of M-CSF was 6.90 +/- 2.41 ng/ml in the patient population (n = 98), but only 2.0 +/- 0.3 ng/ml in 10 healthy donors. 2) 41 of the 98 anemic patients were treated with various doses of Epo for 3 months, and the average increase in the blood hemoglobin level during this period was 26.1 +/- 12.5 mg/dl/unit of Epo/kg patient's b.w./week. Lower levels of M-CSF before treatment significantly predicted a better response to subsequent Epo therapy (r = -0.496, p < 0.001). 3) When cultured with a maximally stimulatory amount of Epo (10 IU/ml), the number of marrow early erythroid progenitor cells (burst-forming unit for erythroid, BFU-E) in patients was identical to that in normal donors, while the number of late progenitors (colony-forming unit for erythroid, CFU-E) was relatively lower in patients. 4) The addition of recombinant M-CSF to the culture resulted in suppression of erythroid progenitor cell growth in the patient population, but induced enhancement in normal donors. The inhibitory effect of M-CSF on the patients' cells was not eliminated by the addition of antibodies against interleukin-1 alpha/beta, tumor necrosis factor-alpha, or interferon-alpha/beta/gamma. Supernatants from marrow mononuclear cells cultured in the presence of M-CSF carried this inhibitory effect on marrow CD34+ cells obtained from patients. Together, these results suggest that M-CSF aggravates a previously existing decreased sensitivity of erythroid progenitor cells to Epo in some patients with renal anemia.

Adolescent↗