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Biomedical subjects

Y Akamatsu

Publications and source records attributed to Y Akamatsu.

At least 109 records · Page 6Linked to original sources
PubMed1985

A temperature-sensitive Chinese hamster ovary cell mutant pleiotropically defective in protein export.

We have developed a new selection procedure for mammalian cell mutants defective in protein export by the use of diphtheria toxin, and devised a new screening method for defective protein secretion using nitrocellulose membranes. By the combination of these procedures, we have isolated a temperature-sensitive mutant clone of Chinese hamster ovary cells which shows a pleiotropic defect in protein export. This mutant, designated DS28-6, is temperature-sensitive for growth. Secretion of a series of proteins is markedly inhibited at the non-permissive temperature. These proteins seem to be normally synthesized and accumulated within the cell at the non-permissive temperature and secreted upon shift down to the permissive temperature. When this mutant is infected with vesicular stomatitis virus, oligosaccharide processing of G-protein is arrested at an endoglycosidase-H-sensitive stage at the non-permissive temperature. The lesion of this mutant appears to be in the endoplasmic reticulum or the cis Golgi or both.

Animals↗
PubMed1985

A novel type of Chinese hamster ovary cell mutant defective in oligosaccharide-lipid synthesis.

A novel type of Chinese hamster ovary cell mutant has been isolated with a constitutive defect in the synthesis of lipid-linked oligosaccharides. This mutant, designated AS15-1, incorporates 30-fold less glucosamine into an oligosaccharide-lipid fraction than the wild type. A gel filtration analysis has shown that a small amount of oligosaccharide-lipid corresponding to Man5GlcNAc2-lipid is formed in the mutant. This mutant shows temperature sensitivity for both growth and adhesion to substratum, and constitutively secretes several unusual proteins in large amounts.

Animals↗
PubMed1985

Alteration in the characters of CDP-choline synthetase and phospholipid-choline exchange enzyme upon choline starvation in Chinese hamster ovary cells.

When CHO-K1 cells are cultivated under choline-deficient conditions, the specific activity of CDP-choline synthetase increases and conversely phospholipid-choline exchange enzyme activity decreases, whereas the other three known enzyme activities related to synthesis of phosphatidylcholine remain unchanged. The changes of the former two enzyme activities take place immediately after removal of choline from the medium. The altered activities readily revert to the control levels upon resupplementation of choline to the starved cell culture. The changes upon choline starvation are sensitive to cycloheximide, while the restoration processes are insensitive to the drug. The activity of CDP-choline synthetase in unstarved control cells is found in both the soluble and membrane fractions. The Km value of the enzyme in the soluble fraction for choline phosphate differs from that in the membrane fraction. Asolectin alters the Km value of the former to a value close to that of the latter and raises its Vmax value, whereas it hardly affects the Km and Vmax values of the latter. In choline-starved cells, the activity is exclusively found in the membrane fraction. The change in the subcellular distribution of the activity upon choline starvation is sensitive to cycloheximide. The altered subcellular distribution reverts to the initial status upon resupplementation of choline even in the presence of cycloheximide. The activity of the phospholipid-choline exchange enzyme is exclusively found in the membrane fraction for both starved and control cells. The properties of the enzyme are altered upon choline starvation with respect to the Vmax value for choline and the Km and Vmax values for Ca2+. These altered kinetic parameters are changed by egg yolk phosphatidylcholine so as to be indistinguishable from those in unstarved control cells. We discuss the mechanism of the alterations in the characters of both enzymes in response to choline starvation.

Animals↗
PubMed1985

Reciprocal effects of phenothiazines and naphthalene sulfonamides on the external ATP-dependent permeability change in Chinese hamster ovary cells.

External ATP causes a great increase in passive permeability to phosphorylated metabolites in several transformed cells, but not in untransformed cells. We have previously demonstrated that the external ATP-dependent permeability change was induced in Chinese hamster ovary cells, CHO-K1, only in the presence of a mitochondrial inhibitor (rotenone, KCN) or a cytoskeleton-attacking agent, vinblastine (Kitagawa, T. and Akamatsu, Y. Biochim. Biophys. Acta 649, 76-82 (1981); 734, 25-32 (1983]. A similar ATP-dependent permeability change was also induced in CHO cells when the cells were treated with 10-30 microM trifluoperazine. This permeability change, like the previously mentioned ones, was found to be reversible and the treated cells remained viable. The permeability change induced by ATP and trifluoperazine was independent of changes in cellular ATP concentration and this property was the same as that of the permeability change with external ATP and vinblastine. Since trifluoperazine is known to interact with calmodulin and to inhibit calmodulin-dependent cellular functions, these results may indicate that calmodulin associated with the cytoskeleton plays an important role in control of the permeability change, although nonspecific perturbation by the drug of the membranes cannot be ruled out. Chlorpromazine and a naphthalene sulfonamide, W-7, also induced an ATP-dependent permeability change. However, these drugs, like mitochondrial inhibitors, reduced the cellular ATP concentration to induce the permeability change. Thus, a clear difference in the action of these drugs in intact cells was also shown in this study. Possible mechanisms for the ATP-dependent permeability change in mammalian cells are discussed.

Adenosine Triphosphate↗
PubMed1985

Macrophage activation by monosaccharide precursors of Escherichia coli lipid A.

Certain Escherichia coli mutants defective in phosphatidylglycerol biosynthesis accumulate two novel glycolipids, designated X and Y. Lipid X is a diacylglucosamine 1-phosphate bearing beta-hydroxymyristoyl groups at positions 2 and 3, and lipid Y has the same structure as X, except for the additional presence of a palmitoyl moiety on the N-linked beta-hydroxymyristate. We have examined the activities of X, Y, and several related compounds as activators of macrophages. Both X and Y induce morphological changes (spreading), prostaglandin E2 synthesis, and killing of tumor cells by mouse peritoneal macrophages in vitro, properties with which lipopolysaccharide and lipid A are also endowed. Both glycolipids have similar effects on the macrophage-like mouse cell line J774.1. Selective removal from lipid X of either the ester-linked beta-hydroxymyristate at position 3 or the phosphate at position 1 abolishes activity. Our results show that the monosaccharides X and Y retain some of the properties of intact lipopolysaccharide and lipid A with respect to macrophage activation. Because the structures of X and Y are defined, our findings should facilitate the elucidation of the molecular mechanism of macrophage activation by lipid A.

Animals↗
PubMed1985

Isolation of a somatic-cell mutant defective in phosphatidylserine biosynthesis.

Mutant clones of Chinese hamster ovary (CHO) cells defective in the base-exchange reaction of phospholipids with choline were isolated by using an in situ enzymatic assay for the reaction in cell colonies immobilized on polyester cloth. The specific activities of the choline-exchange reaction in extracts of one of the mutants (designated 64) grown at 33 degrees C and 40 degrees C were 13% and 6% of those in parental (CHO-K1) cells, respectively. The choline-exchange activity in the mutant was more thermolabile in cell extracts than that in the parent, suggesting that a mutation in the structural gene for the choline-exchange enzyme might have been induced in this mutant. In culture medium supplemented with lipoprotein-deficient serum, mutant 64 grew almost normally at 33 degrees C but divided only twice at 40 degrees C and then stopped growing. Labeling of intact cells with [32P]Pi showed that mutant 64 was also strikingly defective in the biosynthesis of phosphatidylserine at 40 degrees C but was normal at 33 degrees C. Most temperature-resistant revertants of mutant 64 exhibited nearly normal ability to synthesize phosphatidylserine at 40 degrees C and also showed choline-exchange activity similar to that in parental cells. The addition of phosphatidylserine to medium supplemented with newborn calf serum, in which mutant 64 grew more slowly than parental cells at 40 degrees C, restored the growth rate of the mutant to the parental level. Our findings suggest that the choline-exchange enzyme functions as the major route for the formation of phosphatidylserine and that the temperature-sensitive growth of mutant 64 is due to a defect in phosphatidylserine biosynthesis at 40 degrees C.

Animals↗
PubMed1984

pH-dependent Ca2+ interaction with phospholipids related to phosphatidylethanolamine N-methylation.

The interactions of PE and its N-methylated derivatives (PME, PDE AND PC) WITH Ca2+ were examined. PE and the intermediate phospholipids of PE N-methylation (PME and PDE) interacted with Ca2+ in a pH-dependent and reversible manner. When these phospholipids were present in the heptane phase, Ca2+ in the aqueous phase was translocated into the heptane phase at alkaline pH but not at acidic pH. PDE was also effective for the translocation even at around neutral pH, while PC hardly translocated Ca2+ at pH 6.0-9.2. The amounts of Ca2+ interacting with these phospholipids were in the following order: PDE is greater than PME is greater than PE is much greater than PC. P1, phosphatidic acid and PS interacted with Ca2+ in the whole pH range examined. The Ca2+ interactions with P1 and phosphatidic acid were independent of pH, while PS interacted with more Ca2+ at alkaline pH. These phospholipids interacted with Ca2+ most strongly among the cations studied. Liposomes containing PDE also bound the highest amounts Ca2+ among PE and its N-methylated derivatives. Furthermore, mammalian cultured cell membranes, which contain increased amounts of PDE by in vivo modification with N,N'-dimethylethanolamine, bound more Ca2+ than those prepared from choline-treated control cells.

Animals↗
PubMed1984

Regulation of phosphatidylcholine metabolism in mammalian cells. Isolation and characterization of a Chinese hamster ovary cell pleiotropic mutant defective in both choline kinase and choline-exchange reaction activities.

By means of an in situ autoradiographic assay for the base-exchange reaction of phospholipids with L-serine in Chinese hamster ovary cell colonies immobilized on filter paper ( Esko , J.D. and Raetz , C.R.H. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 1190-1193), a mutant (designated 89.1) was isolated in which the specific activity of the serine-exchange enzyme was about 2-fold less than in the parent. Unexpectedly, it was demonstrated that in extracts of the mutant the specific activities of both ATP:choline phosphotransferase (choline kinase) (EC 2.7.1.32) and the enzyme that catalyzes the base-exchange of phospholipids with choline (choline-exchange enzyme) were strikingly reduced (3- to 4-fold and 10- to 15-fold, respectively), while the specific activities of other enzymes of phosphatidylcholine synthesis were normal. Several lines of evidence presented here suggested that the partial defect of serine-exchange activity in this mutant was due to a decrease of acceptor phospholipid(s) for the reaction. The growth rates and phospholipid compositions of the mutant and parent were quite similar. However, mutant 89.1 exhibited a significant defect in its ability in vivo to synthesize phosphatidylcholine. The fact that the mutant was also defective in phosphorylcholine biosynthesis in vivo, together with the finding of an enzymatic lesion of the mutant in choline kinse in vitro as described above, clearly demonstrated that with respect to the reduced phosphatidylcholine biosynthesis the primary defect was at the level of choline kinase. In addition to the decreased synthetic rate of phosphatidylcholine, the turnover rate of phosphatidylcholine was also reduced approximately 2-fold in this mutant. These decreased rates of both synthesis and degradation of phosphatidylcholine probably account for the identical phosphatidylcholine contents between the mutant and parent. As a conclusion, it may be given that strain 89.1 is a pleiotropic mutant which possesses several alterations in phosphatidylcholine metabolism, and such mammalian mutants have not been isolated previously.

Animals↗
PubMed1984

Protein kinase activity on the cell surface of a macrophage-like cell line, J774.1 cells.

Protein kinase activity was demonstrated on the cell surface of a murine macrophage-like cell line, J774.1 cells, and was characterized in detail. When intact cells were incubated with [gamma-32P]ATP, a transfer of [32P]phosphate into acid-insoluble materials of the cells occurred. This reaction was Mg2+-dependent but cAMP-independent, and Mg2+ could be substituted for by Mn2+. The reaction products were found to be proteins, as revealed by SDS-polyacrylamide gel electrophoresis and autoradiography, with phosphomonester linkages to serine and threonine residues, but not to tyrosine. The results of experiments with chemical and enzymatic treatments as well as Con A-Sepharose column chromatography ruled out the possibility that an acyl-phosphate linkage or phosphomannosylglycopeptide was present in the reaction products. The protein kinase(s) and the reaction products were located on the cell surface of the cells, as shown by the fact that the products were removed by mild trypsinization of cells carefully controlled so that the cells remained in an intact state. Phosphorylation of exogenous proteins (phosvitin and casein) by intact cells further supported the location of the enzyme. The phosphorylated proteins of the cells were found to be metabolically stable and remained on the cell surface even at 120 min after the phosphorylation reaction. Possible roles of ecto-protein kinase activity in macrophage functions and macrophage-activation are also discussed.

Animals↗
PubMed1983

Modulation of passive permeability by external ATP and cytoskeleton-attacking agents in cultured mammalian cells.

External ATP causes a passive permeability change in several transformed cells, but not in untransformed cells. We previously demonstrated that in CHO-K1 cells, a transformed clone of Chinese hamster ovary cells, the external ATP-dependent permeability change was induced when the intracellular ATP concentration was reduced by a mitochondrial inhibitor (Kitagawa, T. and Akamatsu, Y. (1981) Biochim. Biophys. Acta 649, 76-82). A permeability change with similar characteristics was also observed when the CHO cells were treated with external ATP and a cytoskeleton-attacking agent such as vinblastine or cytochalasin B. Just like mitochondrial inhibitors, vinblastine could increase the sensitivity of transformed 3T3 cells to external ATP but showed no effect on passive permeability of normal 3T3 cells. However, in contrast with the effect of the mitochondrial inhibitors, the cytoskeleton drugs caused the permeability change with little reduction of intracellular ATP concentration, suggesting different actions of these two kinds of drug on the permeability change. The present results suggest an important role of cytoskeletal structures in controlling the external ATP-dependent permeability change in transformed cells. Possible effects of intracellular ATP on cytoskeletal structures are also discussed.

Adenosine Triphosphate↗
PubMed1982

Phospholipid modification retards intracellular transport and secretion of immunoglobulin G1 by mouse MOPC-31c plasmacytoma cells.

The intracellular transport and secretion of immunoglobulin G1(IgG1) by mouse MOPC-31C plasmacytoma cells were analyzed from the viewpoint of the roles of phospholipids. The membrane phospholipids were modified by culturing cells in a medium supplemented with choline analogues, N,N'-dimethylethanolamine or N-monomethylethanolamine, and accordingly the membranes were enriched in phosphatidyl-N,N'-dimethylethanolamine or phosphatidyl-N-monomethylethanolamine (Maeda, M., Tanaka, Y. and Akamatsu, Y. (1980) Biochem. Biophys. Res. Commun. 96, 876-881). The modified cells were pulse-labeled with L-[35S]methionine and the secretion of labeled IgG1 was chased. Half of the IgG1 was exported to the extracellular medium 1-1.5 h and 2-3 h after synthesis by choline- and dimethylethanolamine-supplemented cells, respectively. However, most of the newly synthesized IgG1 was not secreted by monomethylethanolamine-supplemented cells, even after 5 h; it remained within the cells. The sensitivity of intracellular IgG1 to endoglycosidase H was examined for probing the movement of IgG1 from the rough endoplasmic reticulum to the Golgi complex. Half of the newly synthesized IgG1 acquired resistance to endoglycosidase H after 30-45 min, 1-1.5 h and 2-3 h in choline-, dimethylethanolamine- and monomethylethanolamine-supplemented cells, respectively. Thus, the transport of IgG1 was markedly retarded by the modification with choline analogues, dimethylethanolamine or monomethylethanolamine, at least in the following two processes, from the rough endoplasmic reticulum to the Golgi complex and from the Golgi to the outside of cells. Modification with monomethylethanolamine was more effective than that with dimethylethanolamine in slowing down the transport of IgG1 and appeared to cause accumulation of IgG1 within the cells. A morphological study was also carried out for the three kinds of cell. The roles of phospholipids in the processes of membrane flow are discussed.

Animals↗
PubMed1981

Control of passive permeability of Chinese hamster ovary cells by external and intracellular ATP.

External ATP causes passive permeability change in several transformed cells, but not in untransformed cells. We studied the effect of external ATP on the passive permeability of CHO-K1 cells, a transformed clone of Chinese hamster ovary cells. Treatment of the cells with external ATP alone did not produce a permeability change, and this was observed only when a mitochondrial inhibitor, such as rotenone or oligomycin, was present together with ATP. These inhibitors reduced the concentration of intracellular ATP and a permeability change by external ATP was observed when intracellular ATP was decreased more than 70%. This requirement for permeability change of CHO-K1 cells was quite unique, since passive permeability change of other transformed cells so far tested was induced by ATP alone. Treatment of CHO-K1 cells with cyclic AMP analogues increased their sensitivity to external ATP about 2-fold. The roles of external and intracellular ATP in controlling passive permeability are discussed.

Adenosine Triphosphate↗
PubMed1981

Presence of the phospholipid methylation pathway in mammalian cultured cells.

Stepwise N-methylation of phosphatidylethanolamine to phosphatidylcholine was examined by measuring incorporation of the radioactive methyl group of S-adenosylmethionine into phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N'-dimethylethanolamine and phosphatidylcholine by membranes of five mammalian cultured cell lines (BHK-21 cells, Chang liver cells, PC-12 h cells, MOPC-31C cells and LM cells). The three successive methylation steps were found in all the cells studied, though the total incorporation and the distribution profile of the radioactivity among the products differed with different cells. Furthermore, increase in the amounts of phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N'-dimethylethanolamine in the amine and N,N'-dimethylethanolamine, respectively, resulted in increased methylation of phospholipids. This remarkable enhancement of methylation seems to be a characteristic effect of such modification of membrane phospholipids in mammalian cultured cells.

Animals↗