PubMed Health⌕ Search

Biomedical subjects

T Kamitani

Publications and source records attributed to T Kamitani.

At least 91 records · Page 5Linked to original sources
PubMed1992

Inhibition of glycosylphosphatidylinositol anchor formation by mannosamine.

Many eucaryotic cell surface proteins are anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI), of which the core region is highly conserved from protozoa to mammalian cells. Previous studies (Lisanti, M. P., Field, M. C., Caras, I. W., Menon, A. K., and Rodiguez-Boulan, E. (1991) EMBO J. 10, 1969-1977) showed that mannosamine blocked the expression of a recombinant GPI-anchored protein in Madin-Darby canine kidney cells and converted this protein to an unpolarized secretory product. In the present study, we examined the effect of mannosamine on the formation of the glycan portion of the GPI anchor precursors. This amino sugar inhibited the incorporation of mannose into the glycan portion, and the inhibition was dose-dependent. Mannosamine was shown to be incorporated into the glycan as mannosamine, probably mostly in the second mannose position and thereby to block the further addition of mannose and other anchor components. The products formed in the presence of this drug were characterized by gel filtration and high resolution TLC both before and after deamination with nitrous acid and dephosphorylation by HF. Galactosamine and trehalosamine were inactive in this system, whereas glucosamine also inhibited mannose incorporation into GPI intermediates.

Animals↗
PubMed1992

Identification of a missing link in glycosylphosphatidylinositol anchor biosynthesis in mammalian cells.

A large number of mammalian proteins are anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Biosynthetic intermediates of the GPI anchor have been identified in mammalian cells. The early GPI precursors are sensitive to phosphatidylinositol (PI)-specific phospholipase C (PLC). However, all of the later GPI precursors, which contain 1 or more mannose residues, are PI-PLC-resistant, suggesting that there is another unidentified precursor. Here, we report the identification of this missing link. This GPI precursor can only be labeled with glucosamine and inositol, and is resistant to PI-PLC but sensitive to GPI-phospholipase D. It accumulates in large quantity only in mutants which are defective in the addition of the first mannose residue to the elongating GPI core. Thus, fatty acylation of glucosaminylphosphatidylinositol, to render it PI-PLC-resistant, is an obligatory step in the biosynthesis of mammalian GPI anchor precursors.

Animals↗
PubMed1992

Regulation of Na,K-ATPase gene expression by thyroid hormone in rat cardiocytes.

Synthesis and activity of the enzymatic equivalent of the sodium pump, Na,K-ATPase, are regulated by thyroid hormone in responsive tissues. The purpose of this study was to determine whether triiodothyronine (T3) regulates the level of the messenger RNA (mRNA) coding for Na,K-ATPase alpha- and beta-subunits in the heart. The expression of Na,K-ATPase mRNAs in in vitro myocardial cells was directly assayed by Northern and slot blot hybridization using Na,K-ATPase alpha- and beta-isoform-specific cDNA probes. Exposure of cultured neonatal rat cardiocytes to 10(-8) M T3 resulted in 1) threefold to fourfold increase in alpha 1- and beta 1-mRNA accumulation, with a maximum elevation at 48 hours, 2) sevenfold increase in alpha 2-mRNA accumulation with a peak elevation at 72 hours, and 3) transient threefold increase in alpha 3-mRNA within the first 24 hours followed by a deinduction thereafter. The increase in alpha 1-mRNA accumulation by T3 occurred over the physiological T3 concentration range with an EC50 of 5 x 10(-10) M. This was associated with a twofold increase in alpha 1-subunit protein accumulation and an increase in Na,K-ATPase transport activity. The half-life of alpha 1-mRNA analyzed by actinomycin D chase was less than 3 hours and was not affected by T3. Transfection experiments with the luciferase reporter gene revealed that thyroid hormone response sequences are located within the 5'-flanking regions of each alpha-isoform gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Differential expression of glycosylphosphatidylinositol-anchored proteins in a murine T cell hybridoma mutant producing limiting amounts of the glycolipid core. Implications for paroxysmal nocturnal hemoglobinuria.

A T cell hybridoma mutant, which expressed a markedly reduced level of glycosylphosphatidylinositol (GPI)-anchored proteins on the cell surface, was characterized. The surface expression level of Thy-1 was approximately 17% of the wild-type level, whereas the surface expression of Ly-6A was approximately 2.4% of the wild-type level. We show here that these cells synthesized limiting amounts of the GPI core and that the underlying defect in these cells was an inability to synthesize dolichyl phosphate mannose (Dol-P-Man) at the normal level. The defect in Ly-6A expression could be partially corrected by tunicamycin, which blocked the biosynthesis of N-linked oligosaccharide precursors and shunted Dol-P-Man to the GPI pathway. Full restoration of Thy-1 and Ly-6A expression, however, required the stable transfection of a yeast Dol-P-Man synthase gene into the mutants. These results revealed that when the GPI core is limiting, there is a differential transfer of the available GPI core to proteins that contain GPI-anchor attachment sequences. Our findings also have implications for the elucidation of the defects in paroxysmal nocturnal hemoglobinuria.

Animals↗
PubMed1992

Studies on cerebral protective agents. I. Novel 4-arylpyrimidine derivatives with anti-anoxic and anti-lipid peroxidation activities.

Novel 4-arylpyrimidine derivatives were synthesized by the oxidation of 4-aryl-1,4-dihydropyrimidines, and their effects on anti-anoxic (AA) activity in mice and anti-lipid peroxidation (ALP) activity in rat brain mitochondria were investigated. Among these compounds, ethyl 6-methyl-2-phenyl-4-(4-pyridyl)-5-pyrimidinecarboxylate (4b) has AA activity (10 mg/kg, i.p.) and ethyl 6-methyl-4-(3-nitrophenyl)-2-phenyl-5-pyrimidinecarboxylate (4f) has ALP activity (73% inhibition at 10(-5) g/ml). The latter compound (100 mg/kg, i.p.) was also effective on arachidonate-induced cerebral edema in rats with comparable potency to that of vitamin E.

Animals↗
PubMed1991

Functional analysis of T-cell mutants defective in the biosynthesis of glycosylphosphatidylinositol anchor. Relative importance of glycosylphosphatidylinositol anchor versus N-linked glycosylation in T-cell activation.

The glycosylphosphatidylinositol (GPI) anchor, potentially capable of generating a number of second messengers, such as diacylglycerol, phosphatidic acid, and inositol phosphate glycan, has been postulated to be involved in signal transduction in various cell types, including T-cells. We have identified a panel of T-cell hybridoma mutants that are defective at various steps of GPI anchor biosynthesis. Since they were derived from a functional T-T hybridoma, we were able to determine the precise role of the GPI anchor in T-cell activation. Two mutants were chosen for this analysis. The first mutant is defective at the first step of GPI anchor biosynthesis, i.e. in the transfer of N-acetylglucosamine to a phosphatidylinositol acceptor. Thus, it cannot form any GPI precursors or GPI-like compounds. Interestingly, this mutant can be activated by antigen, superantigen, and concanavalin A in a manner comparable to the wild-type hybridoma. These data strongly suggest that the GPI anchor, its precursor, or its potential cleavage product, inositol phosphate glycan, is not required for the early phase of T-cell activation. The second mutant is able to synthesize the first two GPI precursors, but is not able to add mannose residues to them due to a deficiency in dolichol-phosphate-mannose (Dol-P-Man) biosynthesis. Unexpectedly, all of the Dol-P-Man mutants are defective in activation by antigen, suprantigen, and concanavalin A despite normal T-cell receptor expression. Here, we show that the activation defect was due to a pleiotropic glycosylation abnormality because Dol-P-Man is required for both GPI anchor and N-linked oligosaccharide biosynthesis. When the yeast Dol-P-Man synthase gene was stably transfected into the mutants, full expression of surface GPI-anchored proteins was restored. However, N-linked glycosylation was either partially or completely corrected in different transfectants. Reconstitution of activation defects correlates well with the status of N-linked glycosylation, but not with the expression of GPI-anchored proteins. These results thus reveal an unexpected role of N-linked glycosylation in T-cell activation.

Antigens, CD↗
PubMed1991

Interleukin-2-activated murine cell lines with macrophage- and B-lymphoblast-lytic activity.

Interleukin-2 (IL-2)-activated murine killer cell lines with macrophage- and B-lymphoblastic-lytic activity were established, and their target specificity, surface markers, recognition-related structures, and requirements for optimal cell growth were characterized. Sustained growth of IL-2-activated lymphocytes was supported by the combination of IL-2 and IL-4-enriched T cell conditioned medium (CM), but was not supported by IL-2 alone or the combination of IL-2 and IL-3-containing CM in the presence of macrophages (M phi). The established line required continuous contact with M phi to maintain anti-M phi cytolytic activity. Flow cytometric analysis showed that the original line isolated by the first cloning was Thyl+, CD4-, and weakly CD8+, FcR+. The majority of these cells were CD3+ and TCR-V beta 8+. From this line, the CD3+, TCR-V beta 8+ and CD3-, TCR-V beta 8- clones were isolated by subcloning. The former clone showed Thyl+, CD3+, CD4-, CD8-, TCR-V beta 8+, FcR(+)-phenotype, and the latter clone showed Thyl+, CD3-, CD4-, CD8-, TCR-V beta 8-, FcR- phenotype. The original line and subclones showed a similar target specificity and killed resident or thioglycollate (TG)-induced peritoneal M phi and B-lymphoblasts, but did not kill T-lymphoblasts. Allogeneic M phi, M phi-like cell line P388D1, and B cell hybridoma were sensitive, whereas fresh lymphocytes, T cell lymphoma BW5147, natural killer (NK)-sensitive YAC-1, and NK-resistant P815 tumor cells were resistant to lysis by these cytotoxic lines. The addition of anti-H-2 heteroserum, anti-MHC class 1, anti-MHC class II, anti-CD3, or anti-TCR-V beta 8 monoclonal antibody (mAb) to assay cultures did not inhibit the anti-M phi cytolysis by these killer cells. In addition, the CD3- TCR-V beta 8- clone killed M phi and B lymphoblasts better than the CD3+, TCR-V beta 8+ clone. These results suggest that cytotoxic lines established in this study do not use the T cell receptor (TCR) molecules to recognize target cells and the MHC molecules are not involved in recognition. Anti-LFA-1 mAb partially inhibited anti-M phi-lysis, suggesting that the cell contact between targets and effectors is important in cytolysis. Our present data suggest that the culture condition containing IL-2, IL-4, and M phi may support the continuous growth of non-MHC-restricted killer cells with relative target specificity against M phi and B-lymphoblasts.

Animals↗
PubMed1991

An antibody reacting with splenic red pulp macrophages in the sera of patients with rheumatic diseases.

An antibody was detected in the sera of patients with certain rheumatic diseases that reacted with the cytoplasm of the splenic red pulp (SRP) cells of adult mice. This antibody was detected in the sera of all patients with mixed connective tissue disease (MCTD), 53% of patients with systemic lupus erythematosus (SLE), 42% with Sjögren's syndrome (SS), and 10% with rheumatoid arthritis (RA). However, this antibody was found neither in the sera from patients with other types of rheumatic diseases nor in healthy volunteers. The screening of this antibody may be useful in diagnoses of MCTD, SLE, and SS. In the present study, we also performed the characterization of the cells reacting with this antibody. The cells proved to be acid phosphatase positive phagocytes in the SRP, that is, red pulp macrophages. Moreover, a histochemical analysis of the reacting antigen in these cells has demonstrated that its antigenic activity is NaIO4 and RNase sensitive, suggesting that the antigen may be associated with RNA.

Aging↗
PubMed1991

Characterization of inhibitory effects of an anti-murine CD2 monoclonal antibody on the proliferation of T cell clones.

Though the functional roles of human CD2 are well characterized, murine studies have been lacking until very recent years. Our previous work showed that a mAb against the T11(1)-like domain of CD2 clearly inhibited T cell proliferation induced by mitogenic and allo-antigenic stimuli, but the degree of inhibition was much smaller than had been demonstrated in the human system. In the present study, we observed functional aspects of murine CD2 on CD4+ T cell clones. It was shown that all T cell clones tested were CD2-positive, and that proliferation induced by APC + Ag was inhibited by anti-CD2 mAb. The maximum inhibition was also partial (40-60% inhibition) but CD2-mediated inhibition was observed even at concentrations as low as 0.2 microgram/ml. In contrast to the APC + Ag stimulation, the proliferation induced by lymphokines or immobilized anti-CD3 mAb was not inhibited at all. Taken together, these findings indicate that: (1) CD2 is also involved in the interaction between HTL and APC in murine system; but (2) perturbation of murine CD2 does not influence TCD/CD3- and lymphokine-mediated signal transduction.

Animals↗
PubMed1991

Interleukin 6 stimulates growth of vascular smooth muscle cells in a PDGF-dependent manner.

We have investigated the effect of interleukin 6 (IL-6) on the growth of vascular smooth muscle cells (VSMC) isolated from rat aortas. Murine recombinant IL-6 significantly increased the number of VSMC and stimulated tritiated thymidine incorporation into VSMC in a dose-dependent manner. The IL-6-induced thymidine incorporation into VSMC was totally inhibited by the Ca2+ channel blocker verapamil; however, IL-6 showed no effects on the intracellular Ca2+ level ([Ca2+]i) in VSMC. Antibody against platelet-derived growth factor (PDGF) also totally inhibited the IL-6-induced thymidine uptake. PDGF caused a significant increase in the [Ca2+]i, which was totally inhibited by verapamil. IL-6 mRNA was not detected in unstimulated "quiescent" VSMC, but its expression was stimulated by exposure of VSMC to 10% fetal bovine serum. Immunohistochemical study using anti-PDGF antibody showed that IL-6 stimulated PDGF production in VSMC. These results support the premise that IL-6 is released by VSMC in an autocrine manner and promotes the growth of VSMC via induction of endogenous PDGF production.

Animals↗
PubMed1991

Augmented Na,K-ATPase gene expression in spontaneously hypertensive rat hearts.

Abnormalities in cardiovascular Na,K-ATPase ion-transport function and regulation may play an important role in the pathogenesis of hypertension. However, it is not known whether these abnormalities are secondary to the effects of hypertension, such as increased pressure, or reflect an intrinsic abnormality in Na,K-ATPase gene expression and regulation. A genetic model of hypertension was used to address this issue. Na,K-ATPase alpha subunit gene expression in hearts was compared between spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Pre-hypertensive, 4-week old SHR hearts exhibited an approximately 4 fold elevation in alpha 1 and 8 fold elevation in alpha 2 mRNA levels compared with age-matched WKY hearts. These SHR mRNA levels remained almost equivalent throughout the development of hypertension at 8 and 16 weeks of age. WKY alpha 1 and alpha 2 mRNA levels exhibited a progressive increase during the same time period. The neonatal alpha 3 mRNA isoform was detected only in pre-hypertensive (4-week) SHR hearts. We conclude that cardiac Na,K-ATPase alpha subunit gene expression is significantly altered in SHR even before the onset of hypertension. These findings suggest that an abnormality in cardiac Na,K-ATPase gene expression constitutes an early, if not primary, event in spontaneous hypertension.

Animals↗
PubMed1990

Ventricular cells in culture from adult spontaneously hypertensive rats exhibit decreased growth.

We have investigated the growth characteristics of adult rat ventricular cells (ARVC) in culture from 8-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Protein synthesis in ARVC estimated from tritiated leucine uptake was augmented in both strains after exposure of cells to either norepinephrine or fetal bovine serum (FBS), but the response was significantly smaller in SHR than in WKY. A relative increase in the protein content of ARVC incubated with norepinephrine or FBS was also smaller in SHR than in WKY. These results demonstrate that SHR cardiac myocytes are not genetically growth-accelerated and show a decreased growth response to growth stimuli such as norepinephrine and serum during the adult period.

Aging↗
PubMed1990

Presence of autoantibody against nasal mucosal cells in patients with Wegener's granulomatosis.

We examined whether or not patients with Wegener's granulomatosis have autoantibodies against the respiratory tract and kidney, by using the direct avidin-biotin-glucose oxidase complex method. On immunostaining, the IgG fractions obtained from the sera of 2 patients with Wegener's granulomatosis and of 7 controls were coupled with biotin, and each couple was used as the primary antibody. The serum IgG sample from 1 patient with Wegener's granulomatosis definitely reacted with epithelial cells of the nasal mucosa, with the strongest reaction in basal layer. The sample from another patients with Wegener's granulomatosis showed a similar but weaker reaction. Control sera showed negative reactions in 6 subjects (3 with rheumatoid arthritis, 1 with allergic granulomatous angiitis and 2 normal volunteers) and a very weak reaction in 1 patient with poly-arteritis nodosa. These observations suggest the presence of an autoantibody reacting with some cell components of the nasal epithelium in patients with Wegener's granulomatosis.

Adult↗