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Biomedical subjects

N Okamura

Publications and source records attributed to N Okamura.

At least 181 records · Page 10Linked to original sources
PubMed1987

Production and characterization of monoclonal antibodies to human tumor necrosis factor.

Five stable hybridoma cell lines secreting antibodies to recombinant human tumor necrosis factor (TNF) were established. All monoclonal antibodies belong to the IgG1 subclass. One monoclonal antibody (MAB) (designated as 3B10) neutralizes the L929 cytotoxic activity of natural and recombinant human TNF. Other MABs bind to human TNF but do not neutralize cytotoxic activity. A sandwich enzyme immunoassay specific to human TNF molecule has been developed using 3B10. This assay measures only biologically active human TNF molecules and is as sensitive as a bioassay which measures the cytotoxic activity on L929 cells. These results show that 3B10 is quite useful in studying the biological functions of TNF.

Animals↗
PubMed1987

Effects of calcitonin gene-related peptide (CGRP) and isoproterenol on the contractility and adenylate cyclase activity in the rat heart.

The occurrence of calcitonin gene-related peptide (CGRP) has been predicted by the study of the calcitonin gene of rats [1]. Recently CGRP was isolated from porcine spinal cord [3], and thus, CGRP is assumed to be a neuropeptide. It has been shown that CGRP exhibits potent positive chronotropic and inotropic effects on the atrial muscles of rats and guinea-pigs [5, 9, 11] . We have previously demonstrated the presence of nonadrenergic noncholinergic (NANC) nerves in the atria of rats and guinea-pigs [10] and have suggested that CGRP is a neurotransmitter of the NANC nerves in the guinea-pig atria [9, 11]. It is supposed that the positive inotropic response mediated by the beta-adrenoceptor results from a stimulation of the adenylate cyclase (AC) activity and subsequent elevation of cyclic AMP (cAMP) content. In the present study, the effects of calcitonin gene-related peptide (CGRP) and isoproterenol (ISO) on the myocardial contractility and adenylate cyclase (AC) activity were compared in cardiac muscles of the rat. In atrial muscles, CGRP exerted a positive inotropic effect and stimulated the AC activity at the similar dose range. On the contrary, the concentrations of ISO needed to stimulate the AC activity were nearly 10-fold higher than those needed for eliciting positive inotropic responses. In ventricular muscles, CGRP produced neither a positive inotropic responses nor a stimulation of AC activity, while ISO induced both responses. Inasmuch as CGRP-like immunoreactive (CGRP-I) nerves were found to be present in the atria but rarely in the ventricles, CGRP was assumed to be physiologically important in the atria rather than in the ventricles.

Adenylyl Cyclases↗
PubMed1987

Significance of phosphorylation/dephosphorylation of 46K protein(s) in regulation of superoxide anion production in intact guinea pig polymorphonuclear leukocytes.

Superoxide anion (O2-) production stimulated by concanavalin A (Con A) in guinea pig polymorphonuclear leukocytes (PMNL) was suppressed by addition of methyl-alpha-mannoside, a Con A inhibitor, and resumed upon readdition of Con A. The reversible change in the O2- production was assumed to reflect the change in NADPH oxidase activity measured for the 30,000 X g particulate fraction. The stimulation by Con A of the phosphorylation of 46K protein(s), as observed previously with several membrane-perturbing agents in parallel with an activation of NADPH oxidase in intact guinea pig PMNL (Okamura, N., et al. (1984) Arch. Biochem. Biophys. 228, 270-277), was also suppressed by methyl-alpha-mannoside and resumed upon readdition of Con A. Similar parallelism between the phosphorylation and NADPH oxidase activity was also observed in the case of stimulation by N-formyl-methionyl-leucyl-phenylalanine (FMLP) and phorbol 12-myristate 13-acetate (PMA), though both processes were reversible after the stimulation by FMLP but not reversible after that by PMA. Thus, such a parallelism observed in both intact PMNL and 30,000 X g particulate fraction indicates possible involvement of the protein phosphorylation in the regulation of the production of active oxygen metabolites in PMNL.

Animals↗
PubMed1987

Antimicrobial resistance of Campylobacter jejuni and Campylobacter coli with special reference to plasmid profiles of Japanese clinical isolates.

A total of 111 clinical isolates of Campylobacter jejuni and 10 clinical isolates of Campylobacter coli were characterized by their susceptibility to nine antimicrobial agents and by their plasmid profiles on agarose gel electrophoresis. All of the C. jejuni isolates were susceptible to chloramphenicol, ciprofloxacin, erythromycin, kanamycin, and nalidixic acid, but 55% were tetracycline resistant. In the 10 C. coli isolates, a high prevalence of multiple-antibiotic resistance was noted. Plasmids were found in 82% of the tetracycline-resistant and 15% of the tetracycline-susceptible C. jejuni isolates. Tetracycline resistance in six randomly selected C. jejuni isolates, which contained 50- or 135-kilobase (kb) plasmids, was transferred by conjugation to a Campylobacter fetus subsp. fetus recipient with recovery of a 50- or a 45-kb plasmid from transconjugants. From one multiple-antibiotic-resistant C. coli isolate, resistance to tetracycline, kanamycin, and chloramphenicol was transferred concomitantly with a 58-kb plasmid, pNR9589. Nonconjugative 98-kb plasmids, pNR9131 and pNR9581, from C. coli isolates with resistance to tetracycline, kanamycin, and erythromycin were shown by cloning experiments to code for at least kanamycin resistance. Restriction digests revealed that 50-kb plasmids from tetracycline-resistant C. jejuni isolates were identical, although plasmids from multiple-antibiotic-resistant C. coli isolates shared partial DNA homology to each other. Cloning of the kanamycin and chloramphenicol resistance genes of pNR9589 into Escherichia coli showed that the two genes are closely linked or clustered. Double-digestion analysis of the fragments encoding the kanamycin resistance of pNR9131, pNR9581, and pNR9589 showed that these three plasmids contain a common fragment related to kanamycin resistance.

Anti-Bacterial Agents↗
PubMed1987

Inhibition of superoxide anion production in guinea pig polymorphonuclear leukocytes by a seleno-organic compound, ebselen.

Production of superoxide anion (O2-) induced by tetradecanoyl phorbol acetate (TPA) in intact guinea pig polymorphonuclear leukocytes (PMNL) was markedly inhibited by a seleno-organic compound, 2-phenyl-1,2-benzisoselenazol-3(2H)-one (Ebselen), with glutathione peroxidase-like activity. The compound almost completely inhibited O2- production by a particulate fraction prepared from TPA-treated PMNL at a concentration as low as 250 nM.

Animals↗
PubMed1986

Involvement of protein kinase C in the phosphorylation of 46 kDa proteins which are phosphorylated in parallel with activation of NADPH oxidase in intact guinea-pig polymorphonuclear leukocytes.

Two proteins (Mr 46,000, pI 6.4 and 7.0), the phosphorylation of which was increased by any of the membrane-perturbing agents in parallel with activation of NADPH oxidase in intact guinea-pig polymorphonuclear leukocytes in our previous study (Okamura, N., Ohashi, S., Nagahisa, N. and Ishibashi, S. (1984) Arch. Biochem. Biophys. 228, 270-277), were also phosphorylated in a cell-free system prepared from the leukocytes. The in vitro phosphorylation of these two proteins was stimulated by the addition of phosphatidylserine in the presence of higher concentrations of Ca2+ (300-500 microM). The phosphorylation was further increased when protein kinase C partially purified from guinea-pig brain was added to the system. At a low concentration of Ca2+ (about 10 microM), stimulation of the phosphorylation was not attained by phosphatidylserine alone but required the addition of diacylglycerol or phorbol myristate acetate. On the other hand, the increase in the phosphorylation was inhibited by H-7, an inhibitor for protein kinase C. These results indicate that protein kinase C is involved in the phosphorylation of the two proteins, which may be related to the superoxide anion production stimulated by various membrane-perturbing agents.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1986

Determination of gangliosides as 2,4-dinitrophenylhydrazides by high-performance liquid chromatography.

A specific, sensitive and easily performed method for the determination of gangliosides in tissue was developed. After removal of water-soluble compounds, total lipids were extracted from tissue and then treated with 2,4-dinitrophenylhydrazine hydrochloride and dicyclohexylcarbodi-imide in dimethylformamide at 0 degrees C to form ganglioside hydrazides. After removal of excess reagents by column chromatography on silicic acid, the ganglioside 2,4-dinitrophenylhydrazides were eluted from the column and analysed by h.p.l.c. with the use of a silica-gel normal-phase column eluted with an isocratic chloroform/methanol/water/acetic acid system. The addition of CaCl2 improved the separation of GM3 ganglioside containing N-acetylneuraminic acid from that containing N-glycollylneuraminic acid. 2,4-Dinitrophenylhydrazide peaks were measured by the absorbance at 342 nm. Quantification of GM3, GM2, GM1, GD1a, GD1b, GT1b and LM1 gangliosides was linear in a range 0.02-1.6 nmol. GM4, GD3, GT1a and GQ1b gangliosides also yielded distinct peaks, although the range of linearity was not examined. This method was applied to the analysis of the total lipids of rat brain and hepatocytes.

Animals↗
PubMed1986

Metabolism of cerebrosides and sulfatides in the nervous system of Xenopus tadpole during metamorphosis.

Xenopus laevis tadpoles undergoing metamorphosis were used to study the turnover of cerebrosides and sulfatides in the nervous system of the frog. Tadpoles at the beginning of metamorphosis were treated by intraperitoneal injection with [U-14C]glucose and radioactivity incorporated into galactosphingolipids of brain and tail was measured after various times. The specific activity of brain cerebrosides increased rapidly for the first 24 hr after injection, reached a plateau after 48 hr, and then declined 40% by 7 days. The specific activity of sulfatides changed somewhat more slowly. Hydroxy fatty acid-containing galactosphingolipids had nearly twice the specific activity compared with their nonhydroxy counterparts in brain. Despite the complete regression of tail nerve cord, metabolism of glycosphingolipids in this tissue also indicated active synthesis as well as degradation during this period. The specific activities of these lipids were similar and all reached a peak 24 hr after injection. Examination of the components of these galactosphingolipids disclosed that only a small fraction (7-25%) of the radioactivity was in the galactose moiety in both brain and tail. The ratios of the radioactivity in fatty acid to that in the sphingoid base were much higher for hydroxycerebroside and hydroxysulfatide than for the nonhydroxy isomers.

Animals↗
PubMed1986

Accumulated strain mechanism for length determination of thick filaments in skeletal muscle. I. Experimental bases.

The kinetics of dissociation of myosin from both ends of thick filaments in glycerinated skeletal muscle fibres and myofibrils was studied in the presence of MgATP by use of an optical diffraction method and phase-contrast microscopy. The dissociation velocity, v (identical to -dL/dt where L is the length of thick filaments at time t), increased with increasing KCl concentration (0.225 to 0.5 M), or increasing pH (6.5 to 8.0) but hardly changed with temperature (5 and 25 degrees C), micromolar concentrations of Ca2+ or sarcomere length (2.4 and 2.75 micron). Over a wide range of filament length, the dissociation velocity could be expressed by v0exp(alpha L), where v0 and alpha are positive constants depending upon the dissociation condition. When the effects of crossbridge formation are minimized it was thus shown that the structural stability of thick filaments in a muscle fibre and a myofibril gradually decreases from the central part to the tips of the filaments. On the basis of these results we propose that the length of thick filaments is largely regulated by an accumulated strain mechanism in which the free energy of association of myosin molecules increases with filament length.

Animals↗
PubMed1986

Lowered levels of bicarbonate in seminal plasma cause the poor sperm motility in human infertile patients.

Both the adenylate cyclase activity and the motility of human sperm were stimulated by bicarbonate with the same concentration dependency. The correlation between bicarbonate levels in semen and the motility of sperm from the patients with male infertility was investigated. Bicarbonate in semen was found to originate mainly from the seminal vesicles, and a significant positive correlation was observed between bicarbonate levels and volume of semen. The motility of infertile sperm was also found to correlate positively to the seminal levels of bicarbonate. These results suggest that the lowered levels of bicarbonate in semen are at least in part responsible for the poor sperm motility in infertile patients, as a result of the failure in the activation of sperm adenylate cyclase.

Acid Phosphatase↗
PubMed1986

Facile release of NADPH oxidase from polymorphonuclear leukocyte membrane by mild pressure treatment.

NADPH oxidase, a complex enzyme system in the cell membrane responsible for the bactericidal function of polymorphonuclear leukocytes through the production of superoxide anion, was facilely released by mild treatment with a press. At the pressure where almost all NADPH oxidase activity was released, releases of the activities of lactate dehydrogenase, 5'-nucleotidase, lysozyme, and N-acetyl-beta-glucosaminidase, and of the amount of total protein were negligible. This method can be useful for the elucidation of NADPH oxidase.

Cell Membrane↗
PubMed1986

Isolation and characterization of multilayered sheath membrane rich in glucocerebroside from shrimp ventral nerve.

A membrane fraction rich in glucocerebroside was isolated from homogenates of ventral nerves of pink shrimp (Penaeus duorarum) by sucrose gradient centrifugation. The membrane fraction was observed at 0.15 M sucrose and was rich in lipids (lipid/protein ratio approximately 15:1). Electron microscopy showed that the fraction was derived from myelin-like multilayered glial membrane ensheathing axons, which has morphological similarities to myelin. Most of the lipids in shrimp nerve, including glucocerebroside, sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and ethanolamine-plasmalogen, as well as cholesterol, appeared to be concentrated in this fraction. The fatty acids of these phospholipids were exclusively saturated or monounsaturated with C14-C26 chain lengths. The aldehyde moiety of plasmalogens contained only saturated C14-C18 carbon chains. Like glucocerebrosides, the sphingoid base of sphingomyelin consisted mainly of C14-C16 sphingenines and sphinganines, but they also contained significant amounts of C19 and C20 sphinganines. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins in this fraction showed several bands in the 23,000-85,000 Mr range. Radioimmunoassay, however, did not show cross-reactivity with antibodies to myelin basic protein. The functional role of this membrane in relation to mammalian myelin is discussed.

Animals↗
PubMed1985

Diverse involvements of Ni protein in superoxide anion production in polymorphonuclear leukocytes depending on the type of membrane stimulants.

Effects of islet-activating protein (IAP) were examined to assess the involvement of the guanine nucleotide-binding regulatory protein responsible for inhibition of adenylate cyclase system (Ni protein) in the superoxide anion (O-2) production in polymorphonuclear leukocytes (PMNL) stimulated with various agents. N-Formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated O-2 production was inhibited by the pretreatment with IAP. O-2 production induced by each of phorbol myristate acetate, concanavalin A, and A23187, however, was rather resistant to the pretreatment with IAP. This observation indicates that the Ni protein does not involve in the common pathway for the O-2 production. in PMNL, and the involvement is rather specific for the FMLP-induced production. O-2 production in PMNL stimulated with various membrane perturbing agents was also diverse in the requirement of extracellular Ca2+.

Adenylate Cyclase Toxin↗
PubMed1985

Sodium bicarbonate in seminal plasma stimulates the motility of mammalian spermatozoa through direct activation of adenylate cyclase.

Recently, a low molecular weight factor, which specifically stimulates sperm adenylate cyclase, was found in porcine seminal plasma (Okamura, N., and Sugita, Y. (1983) J. Biol. Chem. 258, 13056-13062). The purified factor was analyzed by 1H NMR, 13C NMR, infrared spectroscopy, and elementary analysis and identified as sodium bicarbonate. The effects of sodium bicarbonate both on adenylate cyclase activity in porcine spermatozoa and on sperm motility have been studied. Sperm adenylate cyclase was found to be specifically activated by bicarbonate over the physiological concentration range. In contrast, the adenylate cyclase activity in other tissues was not affected. The same concentration range of bicarbonate which resulted in activation of adenylate cyclase also stimulated sperm motility. The motility and enzyme activity of spermatozoa in all species so far tested (human, bovine, rat, mouse, and dog) were found to be similarly sensitive to bicarbonate concentration. These results show that the bicarbonate-sensitive adenylate cyclase system regulates sperm motility and suggest that this system is common to all mammals.

Adenylyl Cyclases↗
PubMed1985

Phylogenetic dichotomy of nerve glycosphingolipids.

Galactocerebrosides and sulfatides are major characteristic components of vertebrate myelin. In contrast, glucocerebroside is the major glycosphingolipid of shrimp nerve. In this study, the concentrations of these glycosphingolipids in the nervous systems of animals from several evolutionary branches were determined by use of high-performance liquid chromatography. In nerves of protostome animals only glucose-containing glycosphingolipids were detected, whereas glycosphingolipids from deuterostomes contained predominantly galactose. Neither the glycolipids containing alpha-hydroxy fatty acids nor sulfate esters of the glycolipids, both of which always accompany galactocerebrosides in deuterostome myelin, were present in protostome nerves. This correlation suggests an evolutionary trend from gluco- to galactocerebrosides, which corresponds with changes in the nervous system from loosely structured membrane-enwrapped axons to multilamellar highly structured myelin.

Animals↗
PubMed1985

Lipid composition of the nervous system of earthworms (Lumbricus terrestris).

As part of a systematic study of the evolution of the nervous system, the lipid composition of the ventral nerves of earthworms was examined. The nerve axons are wrapped in copious layers of loosely bound membrane, superficially resembling the myelin sheath of vertebrates. However, neither galactocerebroside nor sulfatide, both of which are considered to be markers for myelin, was present, and only traces of glucocerebroside, which is abundant in shrimp nerve, were detected. The lipids were rich in cholesterol (15.3 mumol/g of fresh tissue) and phospholipids (21.7 mumol/g of fresh tissue). The phospholipids were composed of phosphatidylethanolamine, -choline, -serine, and -inositol in the ratio of 41:44:8:7. Most of the ethanolamine-containing phospholipids were in the form of plasmalogens. The fatty acid moieties of these phospholipids were predominantly 18:1, 18:0, and 20:1, whereas the aldehyde moieties of plasmalogen were mostly 18:0. Sphingomyelin, which is considered a ubiquitous component of animal membranes, was not detected. How the unique structure of the membranes of earthworm nerves may be related to the function of the nervous system in this organism is discussed.

Animals↗