PubMed Health⌕ Search

Biomedical subjects

S Nojima

Publications and source records attributed to S Nojima.

At least 73 records · Page 4Linked to original sources
PubMed1988

The primary structure of rat platelet phospholipase A2.

In our previous report (Hayakawa, M., Kudo, I., Tomita, M., & Inoue, K. (1988) J. Biochem. 103, 263-266), we have shown that phospholipases A2 purified from rat platelet membrane fractions and an extracellular medium of thrombin-stimulated rat platelets were essentially identical to each other. Both purified enzymes were digested with proteases, and the resulting peptides were subjected to primary sequence determination. The sequence analysis of the HPLC-separated peptides and the alignment of the sequences showed a tentative primary structure of rat platelet phospholipase A2, which was composed of 125 amino acid residues. It showed 47% homology with snake venom Agkistrodon halys blomhoffii phospholipase A2.

Amino Acid Sequence↗
PubMed1987

Activation of mouse peritoneal macrophages by synthetic glyceroglycolipid liposomes.

Liposomes composed of chemically synthesized glyceroglycolipids, such as 1,2-dipalmityl-[beta-cellobiosyl-(1'----3)]- glycerol (Cel-DAG), 1,2-dipalmityl-[beta-lactosyl-(1'----3)]-glycerol, or 1,2-dipalmityl-[beta-maltosyl-(1'----3)]-glycerol, were found to enhance protective immunity against transplantable tumor cells (sarcoma 180) in ICR mice. Peritoneal exudate cells prepared from mice treated in vivo with Cel-DAG showed cytostatic activity in vitro against the mouse leukemia cell line, EL-4. Adherent cells separated from this preparation showed similar activity. Peritoneal cells from polypeptone-injected mice acquired appreciable cytostatic activity when incubated in vitro in the presence of glyceroglycolipid liposomes. The adherent cell fraction alone showed rather weak cytostatic activity when pretreated with the glyceroglycolipids, and full activity was restored by supplementing with the nonadherent cell fraction. The ability of glycolipids to induce tumoricidal effects was affected by cholesterol content: with increasing cholesterol content, the activities decreased. Cholesterol-free glycolipid liposomes were taken more efficiently by macrophages than cholesterol-containing liposomes. Cholesterol modifies the surface property of glyceroglycolipid liposomes. Activation of macrophages is responsible for enhancement of protective immunity against tumor cells by injection of these glycolipids in vivo.

Animals↗
PubMed1987

Antitumor activity of synthetic alkylphospholipids with or without PAF activity.

1-O-Octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OMe) has been reported to possess definite antitumor activity in vivo. Twenty-two alkyl lysophospholipid analogs were chemically synthesized, and their antitumor activity against mouse experimental tumors (Sarcoma 180, MM46, P388) was examined. Among them, 1-O-octadecyl-2-O-acetoacetyl-rac-glycero-3-phosphocholine was found to show antitumor activity similar to ET-18-OMe with less acute toxicity. Intravenous injection of the ET-18-OMe with sn-3 configuration retarded the subcutaneous growth of Sarcoma 180 cells effectively, while the growth inhibition by the sn-1 isomer was much less effective. This stereospecificity was similar to that observed in their activities as platelet-activating factor (PAF) agonists. The acetoacetyl compound, another PAF agonist, showed similar stereospecific antitumor action in vivo. These findings suggest that some alkyl lysophospholipids may activate host cells to a cytostatic stage against tumor cells in vivo through binding to a PAF receptor. Our preliminary results indicated that the responsible cells under these conditions might be primarily immature macrophages present in the bone marrow. No appreciable or even adverse stereospecificity was observed in the different sets of experiments where the activity of ET-18-OMe against MM46 tumor cells in vivo or the direct cytotoxicity against human promyelocytic leukemia HL-60 cells in vitro was examined. Under some conditions, the antitumor activity of ET-18-OMe in vivo may be revealed through direct cytotoxicity and/or modulation of the host defense system by "nonspecific" mechanisms. Some alkylphospholipids without PAF activity may also show antitumor activity through similar "nonspecific" mechanisms.

Animals↗
PubMed1987

Purification and characterization of phospholipase A2 released from rat platelets.

It was found that phospholipase A2 and lysophospholipase, both of which were released from thrombin-stimulated rat platelets, had high affinity to insolubilized heparin. Phospholipase A2 released from rat platelets was purified by the sequential use of column chromatography on heparin-Sepharose and TSK gel G2000SW (high-performance liquid chromatography, HPLC). The enzyme was near homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC, and its Mr was estimated to be 13,500. The purified enzyme was labile and lost its activity within 1 h when incubated at 37 degrees C. Phospholipids or detergent in the solution protected the enzyme against inactivation. Phospholipase activity was inhibited by p-bromophenacylbromide, but not by diisopropylfluorophosphate or iodoacetamide. Lysophospholipase, which was also released from rat platelets, was separated from phospholipase A2 by chromatography on heparin-Sepharose.

Animals↗
PubMed1987

Selective release of phospholipase A2 and lysophosphatidylserine-specific lysophospholipase from rat platelets.

Rat platelets released phospholipase A2 and lysophospholipase upon activation with thrombin or ADP. The release of phospholipases was energy-dependent and was not in parallel with that of a known lysosomal marker enzyme, N-acetyl-beta-D-glucosaminidase. The phospholipases are derived from other granules (dense granules or alpha-granules) rather than lysosomal granules of the cells. All of the activities of both phospholipases in the cell free fraction obtained from the activated platelet reaction mixture was recovered in the supernatant after centrifugation at 105,000 X g. The degree of hydrolysis of phospholipids by the phospholipase A2 followed the order: phosphatidylethanolamine (PE) greater than phosphatidylserine (PS) greater than phosphatidylcholine (PC). Phospholipase A2 shows a broad pH optimum (greater than pH 7.0) and absolutely requires Ca2+. Lysophospholipase was specific to lysophosphatidylserine (lysoPS), and neither lysophosphatidylethanolamine (lysoPE) nor lysophosphatidylcholine (lysoPC) was hydrolyzed appreciably. Both 1-acyl- and 2-acyl-lysophosphatidylserine were equally hydrolyzed. Lysophospholipase activity shows similar pH optimum to phospholipase A2. The lysophospholipase activity was lost easily at 60 degrees C. The activity was reduced by the presence of EDTA, though low but distinct activity was observed even in the presence of EDTA. Addition of Ca2+ to the mixtures restores the full activity.

Animals↗
PubMed1987

Characteristics of phospholipid transacylase of Escherichia coli.

We have previously demonstrated the activity(ies) of phospholipid transacylase in Escherichia coli extract (Homma & Nojima (1982) J. Biochem. 91, 1093-1101), which catalyzed a new type of reaction of acyl transfer from diacylphospholipids to lysophospholipids. In this communication we report the specificities and characteristics of this enzyme activity. The activity catalyzed a reversible transfer of an acyl group between diacylphospholipids and lysophospholipids. The acyl group in the 1-position of the glycerol backbone was selectively transferred, and palmitic acid was the only fatty acid species transferred. Presumably, neutral lipids do not serve as substrates. The transacylase was firmly associated with the envelope fraction of E. coli. Neither potassium chloride nor urea was effective in solubilization of the activity and only about half of the activity was solubilized with Triton X-100. This observation was consistent with the equal distribution of the activity between the outer membrane and the inner membrane of E. coli. Functional aspects of this phospholipid transacylase are also discussed.

Acyltransferases↗
PubMed1987

Metabolism of extracellular phospholipids in Tetrahymena pyriformis.

We studied the metabolism of phospholipids exogenously added to cultures of the protozoan, Tetrahymena pyriformis. Tetrahymena cells were found to metabolize the extracellular phospholipids and the fatty acyl chains of the latter were accumulated predominantly as a form of triacylglycerol in the cells. This metabolism was considered to be initiated via endocytosis of phospholipid vesicles, as judged from the following facts: Cytochalasin B, an inhibitor of endocytosis, suppressed the metabolism almost completely. Phospholipid vesicles were incorporated into a phagosome-like structure in Tetrahymena cells, as observed under an electron microscope. When phospholipids doubly labeled with 14C and 3H at the glycerol moiety and fatty acyl chain, respectively, were incubated with Tetrahymena cells, the glycerol moiety and fatty acyl chain at the sn-2-position of the exogenous phospholipids were incorporated into the cellular triacylglycerol fraction in a 1 to 1 ratio. Monoacylglycerol acyltransferase activity was detected in the microsomal fraction of Tetrahymena cells. From these results, together with those of our previous study on lysosomal phospholipid hydrolysis in Tetrahymena (J. Biochem. 99, 125-133 (1986)), it is suggested that the extracellular phospholipids which were taken up by the cells via endocytosis were hydrolyzed through the action of lysosomal phospholipases A1 and C, and also that one of the products, sn-2-monoacylglycerol, served as an acyl acceptor for the synthesis of triacylglycerol via the microsomal "monoacylglycerol pathway."

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1987

Amino acid composition and NH2-terminal amino acid sequence of rat platelet secretory phospholipase A2.

The amino acid composition and partial NH2-terminal amino acid sequence of the phospholipase A2 secreted by stimulated rat platelets were determined. The most predominant amino acid in the phospholipase A2 was cysteine followed by lysine, suggesting that it is a basic one. This finding is consistent with its high affinity to a cation exchange column. The NH2-terminal 24 amino acids were found to be as follows: X-Leu-Leu-Glu-Phe-Gly-Gln-Met-Ile-Leu-Phe-Lys-Thr-Gly-Lys-Arg-Ala-Asp- Val-Ser-Tyr-Gly-Phe-Tyr-Gly- The enzymes contains 5Phe, 8Met, 9Ile, 24Tyr, and 25Gly residues, all of which are conserved in the sequenced pancreatic phospholipase A2. This is the first report of the tentative characterization of a eukaryotic phospholipase A2, the cellular source of which is known, i.e., it does not originate from a venom or the pancreas.

Amino Acid Sequence↗
PubMed1987

Antibody to platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF).

Specific antibodies to platelet activating factor (PAF) were prepared by immunizing rabbits with a hapten-bovine serum albumin (BSA) conjugate. As the hapten we used the synthetic PAF derivative which is resistant against enzymatic inactivation by plasma or tissues and which can bind to BSA through covalent bonding. Antibody activity was determined by an enzyme-linked immunosorbent assay (ELISA). Anti-PAF IgG reacted strongly with PAF. By means of the ELISA inhibition assay, we found that the antibody did not cross-react with phosphocholine, glycerophosphocholine, dilaurylglycerophosphocholine or PAF analogues which have ethanolamine-type polar head groups instead of choline group.

Animals↗
PubMed1987

Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase from Streptomyces cacaoi.

A 19 kb SphI DNA fragment containing the gene for the extracellular active-site serine beta-lactamase of Streptomyces cacaoi KCC-SO352 was cloned in Streptomyces lividans TK24 using the high-copy-number plasmid pIJ702 as vector. A 30-fold higher yield of beta-lactamase was obtained from S. lividans strain ML1, carrying the recombinant plasmid pDML51, than from S. cacaoi grown under optimal production conditions. In all respects (molecular mass, isoelectric point, kinetics of inhibition by beta-iodopenicillanate) the overproduced S. lividans ML1 beta-lactamase was identical to the original S. cacaoi enzyme. A considerable reduction of beta-lactamase production was caused by elimination of a 12.8 kb portion of the 19 kb DNA fragment by cleavage at an internal SphI site located more than 3 kb upstream of the beta-lactamase structural gene. The beta-lactamase gene was located within a 1.8 NcoI-BclI fragment but when this fragment was cloned in S. lividans pIJ702, the resulting strain produced hardly any more beta-lactamase than the original S. cacaoi.

Cloning, Molecular↗
PubMed1986

Effect of serine phospholipid structure on the enhancement of concanavalin A-induced degranulation in rat mast cells.

The correlations among the potentiating activity of various PS analogs on concanavalin A (Con A)-induced rat mast cell degranulation, the hemolytic activity and the incorporation into the mast cell membrane were studied. The following results were obtained. Lysophosphatidylserine (LysoPS) caused rat mast cell activation (degranulation) in the presence of Con A. The order of the activity was as follows: 1-stearoyl lysoPS = 1-palmitoyl lysoPS greater than 1-myristoyl lysoPS greater than 1-lauroyl lysoPS. The relative hemolytic activity of these compounds was similar to that observed in the mast cell activation. Dilauroyl PS, which shows similar hemolytic activity to 1-myristoyl lysoPS, did not activate mast cells appreciably. The relative activity of these phospholipids in the binding to mast cells was 1-stearoyl lysoPS greater than dilauroyl PS greater than 1-lauroyl lysoPS. Hemolytic activity, as well as activity on mast cells, of lysoPS analogs was well correlated to mast cell membrane incorporation, whereas such a correlation was not found with PS analogs. Dilauroyl PS could be accumulated in the mast cell membrane and showed hemolytic activity, but did not activate histamine secretion.

Animals↗
PubMed1986

Metabolism of lysophosphatidylserine, a potentiator of histamine release in rat mast cells.

Lysophosphatidylserine (lysoPS) strongly enhances degranulation of rat mast cells induced by concanavalin A (Con A). In the present paper, the metabolism of exogenous lysoPS in intact mast cells was investigated. Incubation of mast cells with 1-stearoyl-sn-glycero-3-phospho-[3-3H]serine resulted in the rapid binding of lysoPS to mast cells and the time-dependent formation of a considerable amount of [3H]phosphatidylserine. No other radiolabeled lipid metabolites were detected. These results suggest that phosphatidylserine (PS) is synthesized through acylation of lysoPS incorporated into mast cells. Most of the lysoPS associated with mast cells was removed by washing with bovine serum albumin, whereas PS newly formed from lysoPS was not. The cells washed with albumin showed no appreciable histamine release upon subsequent addition of Con A. A different set of experiments was performed using lysoPS analogs which were modified at the hydroxyl group at position 2 of glycerol to avoid acylation. 1-Stearoyl-2-O-methyl-glycero-3-phosphoserine showed almost the same potentiating activity as 1-stearoyl-lysoPS, although the former does not have the free hydroxyl moiety at position 2 of the glycerol residue. The enhancing activity of another lysoPS analog, 1-stearyl-propanediol-3-phosphoserine, which lacks the hydroxyl group altogether, was quite similar to that of 1-stearyl-lysoPS. From these results we conclude that the acylation of lysoPS bears no relation to its potentiating activity and that lysoPS acts toward mast cells as lysoPS itself without any conversion to PS. The effect of replacement of an ester bond at position 1 of glycerol in lysoPS with an ether bond, and the phospholipid composition of rat mast cells are also discussed.

Animals↗
PubMed1986

Properties of acid phospholipases in lysosome and extracellular medium of Tetrahymena pyriformis.

Phospholipase activity in the lysosomes of the protozoan Tetrahymena pyriformis strain NT-1 was studied using phospholipids radioactively labeled in the fatty acid moieties. Lysosomal homogenates showed high phospholipase activity with an acidic pH optimum. Unlike the phospholipases in rat liver lysosomes, almost all activity was recovered from the membranous fraction of the lysosomes. The activity was partially solubilized by treatment of the membranes with a detergent or trypsin. Using specifically labeled phospholipids revealed that phospholipase. A1 and C are predominant in Tetrahymena lysosomes, no appreciable phospholipase A2 or lysophospholipase activity was detected in the fraction. There are two catabolic pathways of the hydrolysis of phospholipid: Hydrolysis is initiated by deacylation at the 1-position by phospholipase A1 and the 2-acyllysophospholipid thus formed is successively attacked by (lyso)phospholipase C; hydrolysis is initiated by cleavage of phosphodiester by phospholipase C and the diacylglycerol thus formed is attacked by lipase. Both pathways give the same end products, free fatty acid and 2-monoacylglycerol. The former pathway might be predominant in Tetrahymena lysosomes under physiological conditions since the pathway is independent of detergent. Phospholipases A1 and C activities were partially released into the medium. At least two different phospholipases C are present in the medium as judged by chromatographic behavior and their substrate specificities.

Animals↗
PubMed1985

The activity of membranes reconstituted from HVJ envelope proteins and lipids to induce hemolysis and fusion between liposomes and erythrocytes.

A simple method for preparation of lipid-free envelope proteins (HN protein and F protein) of HVJ (Sendai virus) was developed. Reconstituted 'envelopes' were then prepared from envelope proteins and various lipids by the detergent dialysis method, and the activity to induce hemolysis and fusion between liposome and erythrocyte was studied. Lipid-free envelope protein aggregates could induce hemolysis and liposome-erythrocyte fusion. The activity was however greatly augmented by incorporation of envelope proteins into membrane of viral total lipids. Hemolytic and fusogenic activity was somewhat augmented by incorporation of envelope proteins into dipalmitoylphosphatidylcholine/cholesterol (1:1, molar ratio) and dimyristoylphosphatidylcholine/cholesterol (1:1), though the augmentation was lower than that observed with viral total lipids. When 'envelopes' were reconstituted with the proteins and viral total lipids supplemented with phosphatidylethanolamine, two kinds of 'envelopes' were prepared; one was permeable to Dextran (Mr 75000) and hemolytic, and the other was impermeable to Dextran and nonhemolytic. The latter acquired hemolytic activity after subjection to freezing and thawing, and its barrier function was lost concomitantly. The study suggests that envelope proteins (HN protein and F protein) could function without lipids but their activity was greatly influenced by not only the composition of additional lipids but also mode of arrangement of components on the reconstituted membranes.

Cell Fusion↗
PubMed1985

Peroxidation of liposomes in the presence of human erythrocytes and induction of membrane damage of erythrocytes by peroxidized liposomes.

Hemolysis (Kobayashi, T., Takahashi, K., Yamada, A., Nojima, S. and Inoue, K. (1983) J. Biochem. 93, 675-680) and shedding of acetylcholinesterase-enriched membrane vesicles (diameter 150-200 nm) were observed when human erythrocytes were incubated with liposomes of phosphatidylcholine which contained polyunsaturated fatty acyl chains. These events occurring on erythrocyte membrane were inhibited by radical scavengers or incorporation of alpha-tocopherol into liposomes, suggesting that lipid peroxidation is involved in the process leading to membrane vesiculation and hemolysis. The idea was supported by findings that generation of chemiluminescence, formation of thiobarbituric acid reactive substance, accumulation of conjugated diene compounds in liposomes and decrease of polyunsaturated fatty acids in liposomes occurred concomitantly during incubation. Hemolysis was also suppressed by the addition of extra liposomes, insensitive to peroxidation, or of serum albumin even after the completion of peroxidation of liposomes. These results suggest that peroxidized lipids, responsible for vesiculation and hemolysis, may be formed first in liposomes and then gradually transferred to erythrocyte membranes. The accumulation of these lipids peroxides may eventually cause membrane vesiculation followed by hemolysis.

Egg Yolk↗