Biomedical subjects

S Nakashima

Publications and source records attributed to S Nakashima.

At least 19 recordsLinked to original sources

PubMed1998

Increased activity of oleate-dependent type phospholipase D during actinomycin D-induced apoptosis in Jurkat T cells.

Apoptosis is an active form of cell death that can be induced by a wide variety of agents and conditions. In response to actinomycin D, hydrogen peroxide (H2O2), or TNF-alpha, Jurkat T cells underwent typical apoptosis. Phospholipase D (PLD) activity in intact cells determined by phosphatidylbutanol generation was up-regulated by these agents. The PLD activation was in a time-dependent manner during apoptosis. It was also shown that the PLD activity measured by using exogenous substrate in the lysate from apoptotic cells was higher than that in the lysate from control untreated cells. The PLD activity in lysate from control untreated cells was stimulated by unsaturated fatty acids (UFA), but not by guanosine 5'-O-(3-thiotriphosphate). However, the PLD activity in the apoptotic cell lysate was no longer enhanced by the addition of oleate, suggesting that the increased PLD activity during apoptosis was attributed to the PLD of UFA-dependent type, but not the small G protein-dependent one. In fact, the release of free UFA was increased during apoptosis. The caspase inhibitors, z-DEVD and z-VAD, effectively suppressed PLD activation and apoptosis, but UFA release was unaffected. These results suggest the possibility that UFA-dependent type PLD may be implicated in apoptotic process in Jurkat T cells. This is the first demonstration that the PLD of UFA-dependent type would be involved in cellular responses.

Apoptosis↗

PubMed1998

Cloning and disruption of caPLB1, a phospholipase B gene involved in the pathogenicity of Candida albicans.

The Candida albicans PLB1 gene was cloned using a polymerase chain reaction-based approach relying on degenerate oligonucleotide primers designed according to the amino acid sequences of two peptide fragments obtained from a purified candidal enzyme displaying phospholipase activity (Mirbod, F., Banno, Y., Ghannoum, M. A., Ibrahim, A. S., Nakashima, S., Yasuo, K., Cole, G. T., and Nozawa, Y. (1995) Biochim. Biophys. Acta 1257, 181-188). Sequence analysis of a 6.7-kilobase pair EcoRI-ClaI genomic clone revealed a single open reading frame of 1818 base pairs that predicts for a pre-protein of 605 residues. Comparison of the putative candidal phospholipase with those of other proteins in data base revealed significant homology to known fungal phospholipase Bs from Saccharomyces cerevisiae (45%), Penicillium notatum (42%), Torulaspora delbrueckii (48%), and Schizosaccharomyces pombe (38%). Thus, we have cloned the gene encoding a C. albicans phospholipase B homolog. This gene, designated caPLB1, was mapped to chromosome 6. Disruption experiments revealed that the caplb1 null mutant is viable and displays no obvious phenotype. However, the virulence of strains deleted for caPLB1, as assessed in a murine model for hematogenously disseminated candidiasis, was significantly attenuated compared with the isogenic wild-type parental strain. Although deletion of caPLB1 did not produce any detectable effects on candidal adherence to human endothelial or epithelial cells, the ability of the caplb1 null mutant to penetrate host cells was dramatically reduced. Thus, phospholipase B may well contribute to the pathogenicity of C. albicans by abetting the fungus in damaging and traversing host cell membranes, processes which likely increase the rapidity of disseminated infection.

Amino Acid Sequence↗

PubMed1998

Changes in the expression of protein kinase C (PKC), phospholipases C (PLC) and D (PLD) isoforms in spleen, brain and kidney of the aged rat: RT-PCR and Western blot analysis.

The age-dependent changes of expression of protein kinase C (PKC), phospholipase C (PLC) and phospholipase D (PLD) isozymes were analyzed in spleen, brain and kidney of young-adult (12-16 week-old) and aged (82-88 week-old) rats. The activities of spleen cPKC and nPKC were significantly decreased by nearly 35 and 30% in aged rats compared to those of young adults, respectively (P < 0.05). The level of PKC beta1 was significantly decreased in aged rats as assessed by RT-PCR and Western blot analyses. In aged rat brain where the activity of cPKC was significantly decreased by nearly 25% (P < 0.05), PKC alpha and beta1 isozymes were significantly down-regulated. In kidney, the level of PKC beta2 was decreased. In spleen the both mRNA and protein levels of PLC beta2 and gamma2 were significantly down-regulated in aged rat (P < 0.05). PLC beta1 was also significantly lower in aged rat brain (P < 0.05) as assessed by RT-PCR and Western blotting. Moreover, PLC beta1 was significantly down-regulated in both mRNA and protein levels in aged rat kidney (P < 0.05). In contrast, the tissues examined, the expressions of PLD isozymes (PLD1a, 1b and 2) were rather stable in the course of aging. These results indicate that mRNAs of PLD isozymes were rather stable but that particular PKC and PLC isozymes were down-regulated in different tissues during aging, suggesting age-dependent decline of specific PKC and PLC isozymes in organs which may, at least in part, be implicated in tissue dysfunction with aging.

Aging↗

PubMed1998

Molecular cloning and cell-cycle-dependent expression of a novel NIMA (never-in-mitosis in Aspergillus nidulans)-related protein kinase (TpNrk) in Tetrahymena cells.

With the intention of investigating the signal-transduction pathway that mediates the cold-stress response in Tetrahymena, we isolated a gene that encodes a novel protein kinase of 561 amino acids, termed Tetrahymena pyriformis NIMA (never-in-mitosis in Aspergillus nidulans)-related protein kinase (TpNrk), by differential display from Tetrahymena cells exposed to temperature shift-down. TpNrk possesses an N-terminal protein kinase domain that is highly homologous with other NIMA-related protein kinases (Neks) involved in the control of the cell cycle. The TpNrk protein is 42% identical in its catalytic domain with human Nek2, 41% identical with mouse Nek1 and 37% with A. nidulans NIMA. In addition, TpNrk and these NIMA-related kinases have long, basic C-terminal extensions and are therefore similar in overall structure. In order to further explore the function of the TpNrk gene and the association of the cold stress with the cell cycle of Tetrahymena, changes of TpNrk mRNA were determined during the course of the synchronous cell division induced by the intermittent heat treatment. The level of TpNrk transcription increased immediately after the end of the heat treatment, with a peak at 30 min, and declined thereafter reaching the minimum level when nearly 80% of the cells synchronously entered cell division (75 min after the end of heat treatment). The accumulation of TpNrk mRNA starting from 0 min to 30 min after the end of the heat treatment was assumed to be a prerequisite for the start of synchronous cell division. These results suggest that TpNrk may have a role in the cell cycle of Tetrahymena, and that mRNA expression, at least, is under tight cell-cycle control.

Amino Acid Sequence↗

PubMed1998

Molecular cloning of a gene encoding phospholipase D from the pathogenic and dimorphic fungus, Candida albicans.

A phospholipase D gene (CaPLD) has been cloned from the Candida albicans genomic DNA library. The CaPLD is a member of a highly conserved gene family of PLD and has the highest homology to Saccharomyces cerevisiae PLD (SPO14) with an overall homology of 42%. Phylogenetic analysis indicated that fungus PLDs including CaPLD composed one of the three clusters of PLD genes.

Amino Acid Sequence↗

PubMed1998

Ceramide formation leads to caspase-3 activation during hypoxic PC12 cell death. Inhibitory effects of Bcl-2 on ceramide formation and caspase-3 activation.

PC12 cells undergo apoptosis as well as necrosis following exposure to hypoxia. Following a 6-h hypoxic treatment, a time-dependent increase in intracellular ceramide level was observed with a concurrent decrease in sphingomyelin. It was also shown that the hypoxia-induced ceramide accumulation resulted from activation of neutral magnesium-dependent sphingomyelinase. Comparative kinetic analyses of the neutral sphingomyelinase in the cells under normoxia and hypoxia showed that hypoxia increased Vmax but did not affect Km of the enzyme. In PC12 cells overexpressing Bcl-2 which show strong resistance to hypoxia, sphingomyelin hydrolysis was decreased and activation of neutral sphingomyelinase was reduced. Addition of exogenous C2-ceramide induced cell death and activated caspase-3 as markedly as the hypoxia treatment. On the other hand, in PC12 cells overexpressing Bcl-2, significant decreases in cell death and inhibition of caspase-3 activation were observed after exogenous addition of C2-ceramide. The inhibitors of caspase-3 prevented cell death by either hypoxia or C2-ceramide. These results suggest that ceramide generated by activation of neutral magnesium-dependent sphingomyelinase mediates hypoxic cell death and that Bcl-2 has inhibitory effects on ceramide formation and caspase activation.

Animals↗

PubMed1998

Molecular cloning of a cDNA encoding a serine protease homologous to complement C1s precursor from rat C6 glial cells and its expression during glial differentiation.

A cDNA of rat C6 cells was cloned, which was considered to be involved in glial cell differentiation induced by dibutyryl cyclic AMP and theophylline. The cDNA fragment of the gene, termed r-gsp, was originally isolated by mRNA fingerprinting using arbitrarily primed polymerase chain reaction, and was homologous to complement C1s precursors of hamster and human. It encodes a protein of 694 amino acids containing a potential signal peptide, an epidermal growth factor-like domain surrounded by two complement C1r/C1s-related repeats, and a putative trypsin-type serine protease domain. Since the hamster and human C1s, and a protein encoded by r-gsp shared high similarity in primary structure, the r-gsp gene could encode a C1s counterpart of the rat. Messenger RNA expression of this gene was markedly increased during cyclic AMP-induced glial cell differentiation. Its expression profile was well correlated with those of glial fibrillary acidic protein (GFAP) and S100B, which are known as glial differentiation markers. It was, moreover, observed that the r-gsp expression in brain increased considerably after birth, like those of S100B and GFAP. The results presented here suggest that the rat C1s gene would be also implicated in glial differentiation besides the complement cascade.

Amino Acid Sequence↗

PubMed1998

Increased mRNA expression of phospholipase D (PLD) isozymes during granulocytic differentiation of HL60 cells.

In response to dibutyryl cyclic AMP (dbcAMP) and all-trans retinoic acid (ATRA), HL60 cells differentiate into granulocyte-like cells. Membrane-associated phospholipase D (PLD) activity in response to guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or phorbol myristate acetate (PMA) was upregulated by these treatments. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses revealed that both hPLD1a and hPLD1b mRNAs were expressed in HL60 cells and that their expression levels increased during differentiation. hPLD2 mRNA levels rose dramatically during differentiation. These results suggest that the PLD genes undergo changes in transcriptional regulation during granulocytic differentiation of HL60 cells.

Bucladesine↗

PubMed1998

Involvement of tyrosine phosphorylation in inhibition of fMLP-induced PLD activation by N-acetyl-L-cysteine in differentiated HL60 cells.

N-acetyl-L-cysteine (NAC), which is known as a multipotential agent; an antioxidant, a thiol reagent, or a tyrosine kinase inhibitor, inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced phospholipase D (PLD) activation in HL60 cells in a concentration-dependent manner (IC50 = 2 mM). Its inhibitory mechanism was examined in this study to gain insight into the regulation of PLD activity. NAC had no direct effect on membrane PLD activity in an in vitro assay system. fMLP-induced formation of inositol phosphates via phospholipase C (PLC) was not affected by the drug, suggesting that the receptor-G protein coupling was not inhibited. H2O2, which is known to induce PLD activation in several types of cells, failed to activate PLD in HL60 cells. Pretreatment of 3-amino-1,2,4-triazole (ATZ), a catalase inhibitor, did not enhance fMLP-induced PLD activation. NAC inhibited fMLP-induced tyrosine phosphorylation of several protein bands (42, 44, 64, and 138 kDa) in a concentration-dependent manner. The temporal and concentration-dependent inhibitory profiles for tyrosine phosphorylation of 64- and 138-kDa proteins were well correlated with PLD activation. However, thiol reagents, 1 mM 2,3-dimercapto-l-propanol (2,3-DMP), 1 mM dithiothreitol (DTT), and 2 mM cysteine also did not suppress protein tyrosine phosphorylation or PBut formation by fMLP. Wortmannin, a selective phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, inhibited these two tyrosine phosphorylation bands. These results suggest that NAC inhibits fMLP-induced PLD activation through blockage of protein tyrosine phosphorylation, which is located at the downstream of PI-3 kinase.

Acetylcysteine↗

PubMed1998

Meningeal melanocytoma in the left frontal region.

We report a rare case of meningeal melanocytoma in the left frontal region. A 45-year-old man complained of a headache. Magnetic resonance (MR) scanning showed characteristic patterns: a slightly high signal intensity mass in the left frontal region on the T1-weighted image and a low signal intensity on the T2-weighted image. The patient underwent gross total removal of the tumor. The postoperative course was uneventful. After two years, there was a small local recurrence. The histological finding of the tumor showed meningeal melanocytoma. To our knowledge, this is the second published report of a meningeal melanocytoma in the supratentorial region.

Brain Neoplasms↗

PubMed1998

Possible involvement of mitogen-activated protein kinase in phospholipase D activation induced by H2O2, but not by carbachol, in rat pheochromocytoma PC12 cells.

We have previously reported that hydrogen peroxide (H2O2) induced a considerable increase of phospholipase D (PLD) activity and phosphorylation of mitogen-activated protein (MAP) kinase in PC12 cells. H2O2-induced PLD activation and MAP kinase phosphorylation were dose-dependently inhibited by a specific MAP kinase kinase inhibitor, PD 098059. In contrast, carbachol-mediated PLD activation was not inhibited by the PD 098059 pretreatment whereas MAP kinase phosphorylation was prevented. These findings indicated that MAP kinase is implicated in the PLD activation induced by H2O2, but not by carbachol. In the present study, H2O2 also caused a marked release of oleic acid (OA) from membrane phospholipids in PC12 cells. As we have previously shown that OA stimulates PLD activity in PC12 cells, the mechanism of H2O2-induced fatty acid liberation and its relation to PLD activation were investigated. Pretreatment of the cells with methylarachidonyl fluorophosphonate (MAFP), a phospholipase A2 (PLA2) inhibitor, almost completely prevented the release of [3H]OA by H2O2 treatment. From the preferential release of OA and sensitivity to other PLA2 inhibitors, the involvement of a Ca2+-independent cytosolic PLA2-type enzyme was suggested. In contrast to OA release, MAFP did not inhibit PLD activation by H2O2. The inhibitory profile of the OA release by PD 098059 did not show any correlation with that of MAP kinase. These results lead us to suggest that H2O2-induced PLD activation may be mediated by MAP kinase and also that H2O2-mediated OA release, which would be catalyzed by a Ca2+-independent cytosolic PLA2-like enzyme, is not linked to the PLD activation in PC12 cells.

Animals↗

PubMed1998

Detection of lymph node metastasis using desmoglein 1 expression in superficial esophageal cancer in relation to the endoscopic mucosal resection.

Endoscopic mucosal resection (EMR) has recently been standardized for mucosal cancer of the esophagus. It may be hypothesized that EMR may be considered to be curative for superficial esophageal cancer (SEC), if the possibility of lymph node metastasis can be excluded beforehand. Ninety patients with p-T1 tumours who underwent esophagectomy were studied. Their primary lesions were stained with anti-Desmoglein 1 antigens using the ABC method. The p-T1 tumors were subdivided into three categories: carcinoma limited to the lamina propria mucosae (19 patients, SEC1), carcinoma invading the lamina muscularis mucosae or with invasion just into the submucosa (27 patients, SEC2), and carcinoma definitely invading the submucosa (44 patients, SEC3). Lymph node metastasis was not observed in the SEC1 patients but was observed in 19% of the SEC2 patients and 41% of the SEC3 patients. None of the SEC1 or SEC2 patients had lymph node metastasis when preserved Desmoglein 1 expression was obtained. The EMR appears to be appropriate therapy for SEC1. Our findings indicate that, for SEC2, preserved expression of Desmoglein 1 may be a helpful aid to exclude the possibility of lymph node metastases. Transthoracic esophagectomy with lymphadenectomy should be selected in the SEC3 patients.

Aged↗

PubMed1998

The expression pattern of the gene for NPK1 protein kinase related to mitogen-activated protein kinase kinase kinase (MAPKKK) in a tobacco plant: correlation with cell proliferation.

Mitogen-activated protein kinase (MAPK) cascades consist of members of three families of protein kinases: the MAPK family, the MAPK kinase family, and the MAPK kinase kinase (MAPKKK) family. Some of these cascades have been shown to play central roles in the transmission of signals that control various cellular processes including cell proliferation. Protein kinase NPK1 is a structural and functional tobacco homologue of MAPKKK, but its physiological function is yet unknown. In the present study, we have investigated sites of expression of the NPK1 gene in a tobacco plant and developmental and physiological controls of this expression. After germination, expression of NPK1 was first detected in tips of a radicle and cotyledons, then in shoot and root apical meristems, surrounding tissues of the apical meristems, primordia of lateral roots, and young developing organs. No expression was, however, observed in mature organs. Incubation of discs from mature leaves of tobacco with both auxin and cytokinin induced NPK1 expression before the division of cells. It was also induced at early stages of the development of primordia of lateral roots and adventitious roots. Thus, NPK1 expression appears to be tightly correlated with cell division or division competence. Even when an inhibitor of DNA synthesis was added during the germination or the induction of lateral roots by auxin, NPK1 expression was detected. These results showed that the NPK1 expression precedes DNA replication. We propose that NPK1 participates in a process involving the division of plant cells.

Base Sequence↗

PubMed1998

Successful polymerase chain reaction-based diagnosis of fungal meningitis in a patient with chronic granulomatous disease.

Meningitis is not a common complication of chronic granulomatous disease (CGD). Here, we present details of a 3-year-old boy with X-linked CGD, who suffered from fungal meningitis. While 19 samplings using conventional cerebrospinal fluid (CSF) cultures failed to detect any organisms, fungal DNA was identified in the CSF by a new polymerase chain reaction (PCR)-based method. The patient recovered without any sequelae after treatment with a combination of antifungal agents, interferon-gamma and granulocyte infusions. This case report demonstrates that fungal meningitis must be included in the differential diagnosis of infections in CGD patients and that the PCR-based detection of fungal DNA is a powerful tool for diagnosis.

Child, Preschool↗

PubMed1998

Ureterocolic fistula secondary to colonic diverticulitis.

We describe the case of a 45-year-old woman with a ureterocolic fistula caused by colonic diverticulitis. She had a 10-year history of intermittent left flank pain that had not been treated. The fistulous tract between the left ureter and sigmoid colon was confirmed by retrograde urography and a barium enema. A nephroureterectomy was successfully performed.

Barium Sulfate↗

PubMed1998

Studies on blood compatibility of terpolymers composed of methyl methacrylate, methoxypolyethyleneglycol methacrylate, and dimethylsiloxane methacrylate.

Terpolymers composed of methyl methacrylate (MMA), polydimethylsiloxane methacrylate (PDMSMA), and methoxypolyethyleneglycol methacrylate (MPEGMA), having different compositions were synthesized. Platelets were not adsorbed onto terpolymer surfaces composed of 50 wt% MMA, 25 wt% PDMSMA and 25 wt% MPEGMA, while on terpolymers with the other compositions, platelet adsorption and fibrin clot were observed. It was shown that PDMS segment was predominant on these terpolymer surfaces via XPS. Receding contact angles of terpolymers, on which no platelet was observed, showed intermediate values between PDMS- and MPEG-rich surfaces. It was suggested that these terpolymers had blood compatibility.

Adult↗

PubMed1998

[Molecular cloning of Candida albicans phospholipase D].

Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine, a major substrate, to phosphatidic acid and choline, and its activity is regulated by a variety of hormones, growth factors, and other extracellular signals in mammalian cells. Thus, it is now recognized as a signal transducing enzyme such as phosphatidylinositol-specific phospholipase C, adenylate cyclase, or protein tyrosine kinases. Furthermore, recent findings that regulation by members of the ADP-ribosylation factor (ARF) and Rho families of monomeric GTP-binding protein suggest roles of PLD in intracellular vesicle traffi-cking, morphological changes, and mitogenic signaling process. In Saccharomyces cerevisiae, PLD gene has been cloned and revealed to be essential for meiosis. In contrast, little is known about PLD in Candida albicans. As a first step to understand possible physiological roles of PLD in C. albicans, we cloned a PLD gene from a C. albicans genomic DNA library. Deduced amino acid sequence analysis showed the structural similarity to mammalian, yeast, and plant PLDs. It was also suggested employing RT-PCR (reverse transcriptase polymerase chain reaction) that an isozyme of C. albicans PLD was present.

Candida albicans↗

PubMed1998

Hyperintense basal ganglia on T1-weighted MR images in a patient with central nervous system lupus and chorea.

We describe a patient with central nervous system lupus and choreatic movements in whom both basal ganglia showed high signal intensity on T1-weighted MR images, while the signal on T2-weighted images remained low. Within 8 months after onset, the choreatic movements had disappeared, with a corresponding decrease in the hyperintense T1 signal. The emergence of the choreatic movement disorder in this patient might have been related to the T1 hyperintensity of the basal ganglia, which, in turn, might have resulted from a vascular insult associated with central nervous system lupus.

Adult↗