Biomedical subjects

M Nishijima

Publications and source records attributed to M Nishijima.

At least 19 recordsLinked to original sources

PubMed1998

Genetic evidence that phosphatidylserine synthase II catalyzes the conversion of phosphatidylethanolamine to phosphatidylserine in Chinese hamster ovary cells.

Phosphatidylserine (PS) in mammalian cells is synthesized through the exchange of free L-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine (PE). The serine base exchange in Chinese hamster ovary (CHO) cells is catalyzed by at least two enzymes, PS synthase (PSS) I and II. A PSS I-lacking mutant of CHO-K1 cells, PSA-3, which exhibits approximately 2-fold lower serine base exchange activity than CHO-K1, is defective in the conversion of phosphatidylcholine to PS but has the ability to convert PE to PS. The PSA-3 mutant requires exogenous PS or PE for cell growth. In the present study, from PSA-3 mutant cells, we isolated a mutant, named PSB-2, with a further decrease in the serine base exchange activity. The activity in the homogenate of PSB-2 mutant cells was approximately 10% that of PSA-3 mutant cells and approximately 5% that of CHO-K1 cells. The PSB-2 mutant exhibited an approximately 80% reduction in the PSS II mRNA level relative to that in PSA-3 mutant and CHO-K1 cells. These results showed that the PSB-2 mutant is defective in PSS II. Like the PSA-3 mutant, the PSB-2 mutant grew well in medium supplemented with PS. However, in the medium supplemented with PE, the PSB-2 mutant was incapable of growth, in contrast to the PSA-3 mutant. In the medium with exogenous PE, the PSB-2 mutant was defective in PS biosynthesis, whereas the PSA-3 mutant synthesized a normal amount of PS. A metabolic labeling experiment with exogenous [32P]PE revealed that the PSB-2 mutant was defective in the conversion of exogenous PE to PS. This defect and the growth and PS biosynthetic defects of the PSB-2 mutant cultivated with exogenous PE were complemented by the PSS II cDNA. In addition, the cDNA of the other PS synthase, PSS I, was shown not to complement the defect in the conversion of exogenous PE to PS of the PSB-2 mutant, implying that PSS I negligibly contributes to the conversion of PE to PS in CHO-K1 cells. These results indicated that PSS II is critical for the growth and PS biosynthesis of PSA-3 mutant cells cultivated with exogenous PE and suggested that most of the PS formation from PE in CHO-K1 cells is catalyzed by PSS II.

Animals↗

PubMed1998

Control of phosphatidylserine biosynthesis through phosphatidylserine-mediated inhibition of phosphatidylserine synthase I in Chinese hamster ovary cells.

Phosphatidylserine (PtdSer) synthesis in Chinese hamster ovary (CHO) cells occurs through the exchange of L-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine. The synthesis is depressed on the addition of PtdSer to the culture medium. A CHO cell mutant named mutant 29, whose PtdSer biosynthesis is highly resistant to this depression by exogenous PtdSer, has been isolated from CHO-K1 cells. In the present study, the PtdSer-resistant PtdSer biosynthesis in the mutant was traced to a point mutation in the PtdSer synthase I gene, pssA, resulting in the replacement of Arg-95 of the synthase by lysine. Introduction of the mutant pssA cDNA, but not the wild-type pssA cDNA, into CHO-K1 cells induced the PtdSer-resistant PtdSer biosynthesis. In a cell-free system, the serine base-exchange activity of the wild-type pssA-transfected cells was inhibited by PtdSer, but that of the mutant pssA-transfected cells was resistant to the inhibition. Like the mutant 29 cells, the mutant pssA-transfected cells grown without exogenous PtdSer exhibited an approximately 2-fold increase in the cellular PtdSer level compared with that in CHO-K1 cells, although the wild-type pssA-transfected cells did not exhibit such a significant increase. These results indicated that the inhibition of PtdSer synthase I by PtdSer is essential for the maintenance of a normal PtdSer level in CHO-K1 cells and that Arg-95 of the synthase is a crucial residue for the inhibition.

Animals↗

PubMed1998

Parasitism by a protozoan in the hemolymph of the giant clam, Tridacna crocea.

A parasitism by a protozoan was found in the giant clam, Tridacna crocea. The parasites were spindle-shaped, 8.6 +/- 0.5 micro m in length and 2.5 +/- 0.3 micro m in width. Structural features of the apical complex of the parasite and a molecular phylogenetic analysis of its 18S rRNA gene sequence indicate that the protozoan belongs to the Apicomplexa. No flagellum was observed in the parasitic protozoan. It infected the eosinophilic granular hemocyte, one of the three types of hemocytes in the clam hemolymph, but it is not known whether it influenced the growth of the clam.

Animals↗

PubMed1998

Reduction of caveolin-1 expression in tumorigenic human cell hybrids.

Studies on human cell hybrids of a cervical carcinoma cell line, HeLa, and normal fibroblasts have indicated that the tumorigenicity of these cells is under the control of a putative tumor suppressor on chromosome 11, although the nature of this suppressor remains unknown. We examined the expression of caveolin-1, a protein component of caveolae of the plasma membrane in these cell hybrids. The non-tumorigenic cell hybrid, CGL1, and normal fibroblast WI38 cells expressed 21-24 kDa caveolin-1, whereas in tumorigenic hybrid CGL4 as well as in the parental HeLa cells, the level of caveolin-1 was markedly reduced. Caveolin-1 expression was also reduced in gamma-ray-induced tumorigenic clones (GIMs) isolated from CGL1 cells, whereas non-tumorigenic irradiated cells expressed the same level of caveolin-1 as CGL1 cells. In accordance with these changes, the cellular level of caveolin-1 mRNA was reduced in the tumorigenic CGL4 cells and GIMs without any detectable changes in the caveolin-1 gene. However, the in vivo tumor growth of CGL4 cells was not altered when caveolin-1 was stably overexpressed through the transfection of a human caveolin-1 cDNA. These results suggest that reduction of caveolin-1 expression is necessary but not sufficient for emergence of the tumorigenic phenotypes of HeLa cell hybrids. Possible roles of the putative tumor suppressor in the control of gene expression are also discussed.

Blotting, Northern↗

PubMed1998

Infrequent mutations in the PTEN/MMAC1 gene among primary breast cancers.

Recently PTEN/MMAC1, a candidate tumor suppressor gene, was isolated from chromosome 10q23-24 and somatic mutations of this gene were detected in several malignancies including brain, prostate, and breast tumors. To investigate further the potential role of this gene in mammary carcinogenesis, we examined 69 primary breast cancers for mutations in PTEN/MMAC1 by means of polymerase chain reaction single-strand conformation polymorphism and sequencing analysis. We detected only one somatic missense mutation, a change from T to C at codon 59 (TCA to CCA) resulting in substitution of Pro for Ser in the predicted protein. This site is located outside of phosphatase or phosphate-acceptor motifs, but this codon encodes a residue that is conserved in homologous proteins, tensin and auxilin and is likely to be crucial for normal function of PTEN/MMAC1. Among the 69 tumors examined, three low-frequency polymorphisms were found as well, one in the non-coding region of exon 1 and one each in introns 2 and 7. Our results suggested that mutation of the PTEN/MMAC1 gene is not a major factor in the development of most primary breast cancers.

Adult↗

PubMed1998

Direct packing of the isolated sinus in patients with dural arteriovenous fistulas of the transverse-sigmoid sinus.

OBJECT: The goal of this study was to evaluate the efficacy of direct packing of the isolated sinus (occluded both distally and proximally) in patients with dural arteriovenous fistulas (AVFs) of the transverse-sigmoid sinus. METHODS: Eight patients were included in this study. There were seven men and one woman, ranging in age from 47 to 75 years (mean 60.4 years). Five patients presented with intracranial hemorrhage or venous infarction, one with convulsions, and two with pulsatile tinnitus. Prominent retrograde cortical venous drainage due to sinus isolation was angiographically demonstrated in all patients. All patients were treated by a small craniotomy and direct sinus packing with microcoils; the procedure was performed with the aid of digital subtraction angiography. Five patients were pretreated with transarterial embolization to reduce arterial inflow before the procedure, and intrasinus pressure and sinus blood gases were monitored throughout the operation. Postsurgery, the dural AVF was completely obliterated in all patients. The sinus pressure was 29 to 58% of systemic blood pressure, and sinus blood gas levels were purely arterial before packing. There was no morbidity related to direct sinus packing; however, one patient died as a result of acute myocardial infarction. Over a follow-up period ranging from 1 to 5 years, a faint asymptomatic dural AVF recurred in one patient on the cortex adjacent to the occluded sinus but regressed spontaneously within 1 year. CONCLUSIONS: Direct sinus packing was found to be highly effective for the treatment of dural AVFs that empty into the isolated sinus. Measurement of changes in sinus pressure and sinus blood gas levels was useful for monitoring the progress of direct sinus packing.

Aged↗

PubMed1997

A mammalian homolog of the yeast LCB1 encodes a component of serine palmitoyltransferase, the enzyme catalyzing the first step in sphingolipid synthesis.

Serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzes the initial step dedicated to sphingolipid biosynthesis and is thought to be a key enzyme for regulating cellular sphingolipid content. For SPT activity, the yeast Saccharomyces cerevisiae requires two genes, LCB1 and LCB2. We isolated mammalian LCB1 cDNA homologs from mouse and Chinese hamster ovary (CHO) cells and an LCB2 cDNA homolog from CHO cells. The mammalian LCB1 proteins are predicted to have about 35% amino acid identity to the yeast Lcb1 protein, whereas the CHO LCB2 protein is predicted to have about 40% amino acid identity to the yeast Lcb2 protein. Northern blot analysis of mRNA isolated from various mouse tissues revealed that the tissue distribution of both LCB1 and LCB2 messengers followed a similar pattern. Transfection of an SPT-defective CHO mutant strain with a CHO LCB1-expressing plasmid restored both SPT activity and de novo sphingolipid synthesis to the wild type levels, whereas transfection of the mutant strain with a CHO LCB2-expressing plasmid did not exhibit any recovery effects, indicating that the SPT defect in the mutant cells is specifically complemented by the CHO LCB1 homolog. Furthermore, when the SPT-defective mutant cells were transfected with a plasmid encoding a His6-tagged CHO LCB1 protein, SPT activity bound to a Ni2+-immobilized resin. These results indicate that the CHO LCB1 homolog encodes a component of SPT.

Acyltransferases↗

PubMed1997

Inhibitory effect on curcumin on mammalian phospholipase D activity.

Curcumin, the major yellow pigment of turmeric (Curcuma longa), has strong anti-carcinogenic and anti-inflammatory activities. We examined the effects of curcumin on enzyme activities of the following phospholipases in a cell-free system: G protein-mediated phospholipase D (PLD), phosphatidylinositol-specific phospholipase C, and phospholipase A2 from mouse macrophage-like cell line J774.1 cells, sphingomyelinase from bovine brain, and phosphatidylcholine-phospholipase C from Bacillus cereus. Curcumin inhibited several types of phospholipases, most effectively PLD among those tested. It also inhibited 12-O-tetradecanoylphorbol-13-acetate-induced PLD activation in intact J774.1 cells in a dose-dependent manner. These results suggest that the anti-inflammatory and anti-carcinogenic action of curcumin is partly due to the inhibition of PLD.

Animals↗

PubMed1997

Phosphatidylserine synthase I and II of mammalian cells.

Phosphatidylserine (PtdSer) in mammalian cells is synthesized through an exchange of free L-serine for the base moiety of pre-existing phospholipids. Studies on PtdSer biosynthesis in Chinese hamster ovary (CHO) cells have suggested that the serine base-exchange is catalyzed by at least two different enzymes; one, named PtdSer synthase I (PSS I), uses phosphatidylcholine (PtdCho) and possibly phosphatidylethanolamine (PtdEtn) as phosphatidyl donors for the serine base-exchange, and the other, named PtdSer synthase II (PSS II), uses PtdEtn but not PtdCho as a phosphatidyl donor. Recently, cDNAs of the PSS I and II have been isolated from CHO-K1 cells. This review will briefly describe the current understanding of PtdSer synthases of mammalian cells, mainly CHO cells.

Amino Acid Sequence↗

PubMed1997

Structure and alternative promoters of the mouse glutamic acid decarboxylase 67 gene.

gamma-Aminobutyric acid is synthesized by glutamic acid decarboxylase (GAD), which has two forms, GAD65 and GAD67. Genomic clones coding mouse GAD67 (mGAD67) have been isolated. The restriction map of the overlapping clones covers a region of more than 45 kb of genomic DNA. The mGAD67 gene contains 16 translated exons in addition to an exon which is preferentially expressed in foetal brain. The rapid amplification of 5'-cDNA ends showed that mGAD67 gene transcripts have two different 5'-untranslated regions. Analysis of the genomic clones encompassing the 5'-exons revealed that the two transcripts arose from a single gene by alternative splicing using two different donor sites and a common acceptor. The exons were found 1.5 and 0.6 kb upstream of exon 1. The corresponding promoter regions of these exons have a number of putative regulatory elements, including Sp1- and Krox-24-binding sites. Analysis of mGAD67 transcripts demonstrated that each of the 5'-untranslated exons was expressed in mouse brain. In contrast, exon 0A, but not exon 0B, was expressed in mouse testis and pancreas. These results suggest that these transcripts may be regulated under the control of independent promoters.

Alternative Splicing↗

PubMed1997

Cloning of a Chinese hamster ovary (CHO) cDNA encoding phosphatidylserine synthase (PSS) II, overexpression of which suppresses the phosphatidylserine biosynthetic defect of a PSS I-lacking mutant of CHO-K1 cells.

Phosphatidylserine (PtdSer) in mammalian cells is synthesized through the exchange of free L-serine for the polar head group (base) of preexisting phospholipid. We previously showed the presence of two different enzymes catalyzing the serine base exchange in Chinese hamster ovary (CHO) cells and isolated the cDNA of one of the enzymes, PtdSer synthase (PSS) I, which also catalyzes the exchange of the base moiety of phospholipid(s) for ethanolamine and choline. In this study, we cloned a CHO cDNA, designated as pssB, which encodes a protein exhibiting 32% amino acid sequence identity with CHO PSS I. Introduction of the pssB cDNA into CHO-K1 cells resulted in striking increases in both the serine and ethanolamine base exchange activities. In contrast to the PSS I cDNA, the pssB cDNA was incapable of increasing the choline base exchange activity. The expression of the pssB gene in Sf9 insect cells also results in striking increases in both serine and ethanolamine base exchange activities. The pssB cDNA was found to transform a PtdSer-auxotrophic PSS I-lacking mutant of CHO-K1 cells to PtdSer prototrophy. The PtdSer content of the resultant transformant grown without exogenous PtdSer for 2 days was 4-fold that of the mutant and similar to that of CHO-K1 cells, indicating that the pssB cDNA complemented the PtdSer biosynthetic defect of the PSS I-lacking mutant. These results suggested that the pssB cDNA encoded the second PtdSer synthase PSS II, which catalyzed the serine and ethanolamine base exchange, but not the choline base exchange.

Amino Acid Sequence↗

PubMed1997

Involvement of diacylglycerol production in activation of nuclear factor kappaB by a CD14-mediated lipopolysaccharide stimulus.

Exposure of Chinese hamster CHO-K1 transfectant cells expressing mouse CD14 (CHO/CD14 cells) to lipopolysaccharide (LPS) induced rapid elevation of the cellular diacylglycerol (DAG) and choline/phosphocholine levels and nuclear translocation of nuclear factor kappaB (NFkappaB). When cells were incubated with short-chain DAG analogues or bacterial phospholipase C, NFkappaB activation occurred even without the LPS stimulus. Treatment of CHO/CD14 cells with tricyclo[5.2.1.0(2.6)]decyl-(9[8])xanthogenate (D609), an inhibitor of phosphatidylcholine-specific phospholipase C and phospholipase D, almost completely inhibited not only the LPS-dependent production of DAG and choline/phosphocholine but also the LPS-dependent NFkappaB activation. In contrast, treatment of cells with 1-(6-{[3-methoxyoestra-1,3, 5(10)-trien-17beta-yl]-1H-pyrrole-2,5-dione (U73122), an inhibitor of phosphatidylinositol-specific phospholipase C in vitro, did not affect the LPS-dependent activation of NFkappaB. Production of DAG and activation of NFkappaB after the LPS stimulus were observed in mouse macrophage-like J774.1 cells, and this response to LPS by J774. 1 cells was also inhibited by D609. These results suggest that the production of DAG from phosphatidylcholine was upstream of NFkappaB activation in response to a CD14-mediated LPS stimulus.

Animals↗

PubMed1997

Involvement of heparan sulfate proteoglycans in the binding step for phagocytosis of latex beads by Chinese hamster ovary cells.

Chinese hamster ovary (CHO) K1 cells, typical nonprofessional phagocytes, exhibited intense phagocytosis of latex beads when incubated under serum-free conditions. Under the serum-free conditions, the recognition mechanism of latex beads by cells was investigated. Exogenous heparin and heparan sulfate but not chondroitin sulfate effectively inhibited the binding of latex beads to cells. The binding of latex beads to cells was also inhibited by treatment of cells with heparitinase more effectively than by treatment of cells with chondroitinase. Furthermore, CHO mutant cells defective in biosyntheses of both heparan sulfate and chondroitin sulfate proteoglycans almost completely lacked binding activity of latex beads. Another mutant, which is deficient in heparan sulfate proteoglycans but rather overproduces chondroitin sulfate proteoglycans, also showed lower binding activity, compared with wild-type cells. Coculture of these proteoglycan-less mutants and the wild-type cells did not restore the binding activity of the mutant cells, suggesting that membrane-bound rather than secretory proteoglycans were responsible for the binding of latex beads. These results indicated that heparan sulfate proteoglycans at the cell surface were involved in the binding step for phagocytosis of latex beads by CHO cells.

Animals↗

PubMed1997

Morphological and Functional Characterization of Hemocytes in the Giant Clam Tridacna crocea

In order to understand the cellular defense system in the giant clam Tridacna crocea, which harbors symbiotic microalga zooxanthella, hemocytes of the giant clam were characterized using light and electron microscopy. Three types of hemocytes, which we named eosinophilic granular hemocytes, agranular cells, and morula-like cells, were recognized. The eosinophilic granular hemocyte contained granules approximately 0.6 &mgr;m in diameter. The agranular cell adhered to glass surfaces and contained electron-lucent granules and few electron-dense granules. The morula-like cell was packed with large (approximately 3 &mgr;m in diameter), electron-dense granules. The eosinophilic granular hemocyte was acid phosphatase positive and showed phagocytic ability against latex particles. The agranular cell and the morula-like cell lacked phagocytic ability. When the hemolymph was exposed to seawater, the hemocytes coagulated to make large clots. The agranular cells were mainly found in the core space of the clots. The morula-like cells released the large granules in the aggregation process.

Journal Article↗

PubMed1997

Ester-type cephalotaxus alkaloids from Cephalotaxus harringtonia var. drupacea.

Three alkaloids, neoharringtonine, homoneoharringtonine and 3'S-hydroxyneoharringtonine, were isolated from the leaves and stems of Cephalotaxus harringtonia var. drupacea. Their structures were established by spectroscopic methods, including two-dimensional NMR and CD spectra, and their antileukaemic activity was evaluated using P-388 leukaemia cells.

Alkaloids↗

PubMed1997

Should C-section be applied to fetal arrhythmia?

OBJECTIVE: To investigate the efficacy of fetal arterial oxygen saturation (SpO2) monitoring in cases of fetal arrhythmia. STUDY DESIGN: Continuous fetal SpO2 was obtained by infrared pulse oximetry (Nellcor Puritan Bennett Inc.). The relationship between SpO2 and FHR pattern was studied in 47 cases. Then three cases of congenital complete A-V block and two cases of supraventricular tachyarrhythmia were prospectively managed by SpO2 monitoring during labor. RESULTS: Whenever the FHR was reassuring, the range of SpO2 was stable between 40 and 80%. Although a variation in SpO2 values were noted during decelerative patterns, a more acidotic tendency was found in cases where SpO2 was below 40% prior to delivery. SpO2 values of all five cases of fetal arrhythmia were stable between 40 and 80% and safe vaginal delivery proved to be possible. CONCLUSIONS: Fetal SpO2 monitoring seemed to be a useful method to evaluate fetal oxygenation during labor in cases of fetal arrhythmia.

Adult↗

PubMed1997

Role of CD14 molecules in internalization of Actinobacillus actinomycetemcomitans by macrophages and subsequent induction of apoptosis.

We report the evidence for apoptosis in J774.1 cells by the periodontopathic bacterium Actinobacillus actinomycetemcomitans, suggesting that the ability of A. actinomycetemcomitans to promote apoptosis might be important in the initiation and development of periodontitis. In this study, we examined the role of macrophage CD14, anchored by a glycerophosphatidylinositol tail, in the induction of apoptosis by A. actinomycetemcomitans infection by using the parent J774.1 cells and CD14-defective mutant (LR-9) cells. A small number of A. actinomycetemcomitans Y4 cells inside the LR-9 cells compared with the number in J774.1 cells was detected by confocal scanning microscopy. We found that LR-9 cells showed a weak cytotoxic effect after being infected with A. actinomycetemcomitans Y4. Apoptotic cell death of LR-9 cells infected with A. actinomycetemcomitans Y4, compared with that of the parent J774.1 cells was almost undetectable, as shown by the proportion of fragmented DNA in agarose gel electrophoresis and by the terminal deoxynucleotidyl transferase-mediated dUTP end-labeling method. Flow cytometric cell cycle analysis of J774.1 cells infected with A. actinomycetemcomitans Y4 revealed the increased percentage of apoptotic cells with hypodiploid DNA. However, LR-9 cells infected with A. actinomycetemcomitans Y4 showed no increase in population of apoptotic nuclei compared with the noninfected cells. These findings suggest that the CD14 molecules may contribute to the phagocytosis of A. actinomycetemcomitans by J774.1 cells and regulate, at least in part, apoptotic cell death of macrophages infected with A. actinomycetemcomitans.

Actinobacillus Infections↗

PubMed1997

Brain tumors: detection with C-11 choline PET.

PURPOSE: To evaluate the effectiveness of positron emission tomography (PET) with carbon-11 choline in brain tumor imaging. MATERIALS AND METHODS: A rat glioma cell line (C6) was incubated with C-14 choline; the time course of uptake and metabolism was determined in vitro. C-11 choline was injected intravenously in tumor-bearing rats; the time course of distribution in organs was determined. C-11 choline also was injected intravenously in 20 patients (aged 6-86 years) with brain tumors and two volunteers (aged 38 and 58 years); distribution of the tracer in the brain was determined. Regional cerebral blood flow was measured by using oxygen-15 water on the same day. RESULTS: C-14 choline was metabolized to phosphoryl choline in glioma cells. The uptake of C-11 choline by glioblastoma cells was three to four times higher than that in the rat brain. All brain tumors took up more C-11 choline than did normal brain; thus, brain tumors that were not treated, as well as those that were treated with surgery or radiation therapy, were depicted. The tumor-normal brain uptake ratio of C-11 choline in brain tumor did not correlate with the O-15 water regional blood flow in the corresponding area. CONCLUSION: C-11 choline PET can depict brain tumors effectively. This method was clinically useful in patients who had undergone surgery.

Adult↗