Yuzuru Akamatsu

CD14 Is a pattern recognition receptor

Septic shock caused by a diverse group of bacterial pathogens is a serious human disease. Recognition of bacterial envelope constituents is one mechanism used by mammalian cells to initiate responses leading to bacterial killing or, unfortunately, responses that also cause fatal septic shock. Here we show that CD14 plays a key role in initiating cell activation by a group of bacterial envelope components from Gram-negative and Gram-positive microorganisms, as well as mycobacteria. We propose that CD14 is a receptor used by mammalian cells to recognize and signal responses to a diverse array of bacterial constituents. This finding defines the molecular basis for innate microbial immunity; implicit in these findings are new possibilities for therapeutics.

Regulation of glucose transport activity and expression of glucose transporter mRNA by serum, growth factors and phorbol ester in quiescent mouse fibroblasts

We have investigated the effects of growth factors such as serum, platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) on glucose transport activity in quiescent mouse Swiss 3T3 cells. DNA synthesis was synchronously induced by either calf serum, or platelet-poor plasma in combination with PDGF or FGF. Early stimulation of glucose transport in the quiescent cells was also caused by serum, or by either PDGF or FGF. The time courses for the stimulation of transport were identical for serum, PDGF and FGF, and the stimulated uptake in each case was associated with a 5-6-fold increase in Vmax. There were no detectable changes in apparent Km. Expression of glucose transporter mRNA was also enhanced by these growth factors. By contrast, EGF, insulin and platelet-poor plasma had little effect on glucose transport and transporter-gene expression, although uridine uptake was enhanced by all of these growth factors. These results suggest that cell cycle-dependent stimulation of glucose transport and expression of the transporter mRNA are regulated by a specific class of growth factors such as PDGF and FGF. The tumor promoter phorbol 12-myristate 13-acetate (PMA) also stimulated glucose transport and expression of transporter mRNA in quiescent 3T3 cells. These stimulations were absent in PMA-pretreated cells. However, serum, PDGF and FGF were able to stimulate glucose transport as well as expression of the transporter mRNA in PMA-pretreated cells, suggesting that there are at least two independent pathways for regulating glucose transport and glucose transporter mRNA level in quiescent fibroblasts.

Solubilization and properties of a phosphatidylethanolamine-dependent methyltransferase system for phosphatidylcholine synthesis from mouse liver microsomes.

Summary A method was developed for solubilization of a phosphatidylethanolamine-dependent methyltransferase system for phosphatidylcholine synthesis in mouse liver microsomes. A preparation with high specific activity was obtained in good yield by treating 0.3% deoxycholate-treated microsomes with 0.2% Triton X-100. With this preparation, methyl incorporation into phospholipids was activated by phosphatidylethanolamine and its methylated intermediates. The specific activity of the preparation with phosphatidylethanolamine was 4 times that of intact microsomes. The reaction products with the solubilized preparation were phosphatidylcholine and methylated intermediates of phosphatidylethanolamine.

Immunochemical identification of the pssA gene product as phosphatidylserine synthase I of Chinese hamster ovary cells

We have previously shown that a Chinese hamster ovary (CHO) cell mutant defective in phosphatidylserine synthase I recovers the enzyme activity on transfection with a pssA cDNA clone isolated from the parental CHO‐K1. The resultant transfectant, CDT‐1, exhibited about 20‐fold higher specific activity of the enzyme in the membrane fraction than CHO‐K1 cells. Polyclonal antibodies against two peptides of the predicted pssA product cross‐reacted with a membrane protein having an apparent molecular mass of 42 kDa, which was overproduced in CDT‐1 cells. By immunoprecipitation with the antibody, phosphatidylserine synthase I activity as well as the 42‐kDa protein was eliminated from solubilized membrane proteins of CDT‐1 cells. Both the enzyme activity and the 42‐kDa protein of CHO‐K1 cells were enriched in the mitochondria‐associated membrane fraction and the microsome fraction, but neither was enriched in the mitochondria fraction or the cytosol fraction. These results suggest that the pssA gene encodes phosphatidylserine synthase I.

External ATP-induced passive permeability change and cell lysis of cultured transformer cells: action in serum-containing growth media

External ATP causes a marked increase in the passive permeability to phosphorylated metabolites in several types of transformed cells in alkaline medium containing low concentrations of Ca2+, but not in untransformed cells. Such increased membrane permeability with external ATP was also observed in B16 melanoma cells at pH 7.4-7.5 in both Tris-buffered saline and a growth medium containing 10% calf serum and divalent ions at normal concentrations, although a higher concentration of ATP was required. The permeability change in the growth medium was significantly enhanced by calmodulin-interacting drugs, such as trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) and chlorpromazine (CPZ). As expected, prolonged exposure of the cells to ATP in the serum-containing medium led to cell lysis. This ATP-dependent cell lysis was observed only in several transformed cell lines, and not in untransformed mouse fibroblasts. These results indicate that the effect of ATP on the membrane permeability in transformed cells is elicited under the physiological conditions and this would be useful in some limited way for cancer chemotherapy management.

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.

Abortive infection with Sindbis virus of a Chinese hamster ovary cell mutant defective in phosphatidylserine and phosphatidylethanolamine biosynthesis

The effects of phosphatidylserine starvation on the infection with Sindbis virus (an enveloped RNA virus) have been investigated in a Chinese hamster ovary (CHO) cell mutant (strain PSA-3) which requires exogenously added phosphatidylserine for cell growth because it lacks the ability to synthesize this phospholipid. When PSA-3 cells were grown in the absence of phosphatidylserine, the cellular contents of phosphatidylserine and also phosphatidylethanolamine produced through decarboxylation of phosphatidylserine decreased. Sindbis virus production in the mutant cells decreased immediately upon phosphatidylserine deprivation as did the contents of phosphatidylserine and phosphatidylethanolamine, whereas the cell growth, viability, and syntheses of protein, DNA and RNA remained normal for approx. 40 h phosphatidylserine starvation. Although PSA-3 cells grown without phosphatidylserine for 24 h were able to bind and internalize Sindbis virus almost normally, viral RNA synthesis was greatly reduced in the cells, suggesting that nucleocapsids of internalized Sindbis virus are not normally released into the cytoplasm. Unlike mammalian cell mutants defective in endosomal acidification, PSA-3 cells grown without phosphatidylserine were not resistant to diphtheria toxin. Furthermore, the yield of virions and viral RNA synthesis in PSA-3 cells were not completely restored on brief exposure of the cells to low pH medium following virus adsorption, which is known to induce artificial fusion of the viral envelope with the plasma membrane of normal host cells and then injection of viral nucleocapsids into the cytoplasm. Our data demonstrate the requirement of membrane phospholipids, such as phosphatidylserine and/or phosphatidylethanolamine, in CHO cells for Sindbis virus infection, and we discuss their possible roles.

Functional reconstitution of sphingomyelin synthase in Chinese hamster ovary cell membranes

Sphingomyelin synthase (phosphatidylcholine:ceramide phosphocholinetransferase) activity in the membranes of Chinese hamster ovary cells was found to be detectable with a fluorescent ceramide analog, containing a short acyl chain, as a substrate. We developed a method for the functional reconstitution of sphingomyelin synthase in detergent-treated membranes. Treatment of membranes with 1.5% octyl glucoside in the absence of exogenous phosphatidylcholine resulted in almost complete loss of sphingomyelin synthase activity, even after removal of the detergent by dialysis. In contrast, membranes treated with the detergent in the presence of exogenous phosphatidylcholine showed partial activity and, after dialysis of this mixture, enzyme activity was restored to almost the same level as the activity in dialyzed intact membranes. The effects of various lipids on enzyme activity in this reconstitution system suggested that L-alpha-phosphatidylcholine was the environmental lipid essential for the functional reconstitution of the enzyme. Furthermore, diacylglycerol was suggested to serve as an inhibitory regulator of sphingomyelin synthesis.

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.

Control of membrane permeability by external ATP in mammalian cells: Isolation of an ATP-resistant variant from Chinese hamster ovary cells

External ATP causes a great increase in the passive permeability of the plasma membrane for phosphorylated metabolites and other small molecules in cultured mammalian cells. We previously demonstrated that in CHO-K1 cells an ATP-dependent permeability change was induced in the presence of a mitochondrial inhibitor (KCN or rotenone), a cytoskeleton-attacking agent (vinblastine) and a calmodulin antagonist (trifluoperazine). These permeability changes were reversible but long exposure, for 30-60 min, to ATP together with a mitochondrial inhibitor significantly reduced the cell viability of the treated cells. Since this cell lysis was shown to be due to the ATP-dependent permeability change, we could isolate several clones resistant to the action of the external ATP from CHO-K1 cells after repeated treatment with ATP and rotenone. In 9.1 cells, one of the isolated clones, little or no ATP-dependent permeability change was observed in the presence of either a mitochondrial inhibitor, vinblastine or trifluoperazine. This CHO variant could be specifically resistant as to the change in membrane permeability induced by external ATP, since the permeabilities for the 2-deoxyglucose and drugs used in the present studies were similar to those in the case of the parent cells. These results suggest that a specific defect or alteration in the plasma membrane is involved in the ATP-dependent permeability change. It is also reported that Mg2+-dependent ATPase activity was found on the cell surface of both CHO-K1 and 9.1 cells, and this activity was shown to be not involved in the permeability change controlled by external ATP.