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Biomedical subjects

M Kaneda

Publications and source records attributed to M Kaneda.

At least 145 records · Page 8Linked to original sources
PubMed1987

The respiratory burst oxidase of neutrophils. Separation of an FAD enzyme and its characterization.

Pig blood neutrophils were briefly activated by various fatty acids and then fractionated into membrane vesicles with different NADPH oxidase activities. Treatment of these membranes with a detergent, octyl glucoside, resulted in a high yield of solubilized oxidase, which was subjected to isoelectric focusing on gels (pI 4.0-8.0). 1) A distinct band staining with NADPH-nitroblue tetrazolium focused at pI 5.0. The enzyme (pI 5.0) showed high specificity for NADPH and similar characteristics to the oxidase involved in the respiratory burst. 2) The enzyme was extracted from gel slices and analyzed. When measured promptly after its extraction, its NADPH oxidase activity was high, but there was apparent superoxide dismutase-insensitive cytochrome c reduction, probably due to direct electron transfer to the heme protein. However, it could produce superoxide anion (O2-) under some micelle conditions. 3) Therefore, the formation of the enzyme-substrate complex of yeast cytochrome c peroxidase was employed for the detection of H2O2. A fresh extract of stimulated cells catalyzed equimolar NADPH oxidation and H2O2 production of 306 and 300 nmol min-1 (mg protein)-1, respectively. The Km value of the enzyme for NADPH was 30 +/- 13 (S.D.) microM. The recovery of the extract (pI 5.0) was 19% of the total activity. 4) The enzyme extract contained 1.1-1.9 nmol of FAD/mg of protein, giving a turnover number of 300-600 min-1 in terms of O2- generation/FAD. No heme protein was found in the enzyme. The enzyme was mainly of 67-kDa molecular mass.

Animals↗
PubMed1987

Carbazomycins C, D, E and F, minor components of the carbazomycin complex.

Carbazomycins C (III), D (IV), E (V) and F (VI), the minor components of the carbazomycin complex, were isolated from the cultured broth of Streptoverticillium ehimense together with carbazomycins A (II) and B (I). Among them, III and IV were shown to be new substances and their structures were elucidated as 4-hydroxy-3,6-dimethoxy-1,2-dimethylcarbazole and 3,4,6-trimethoxy-12-dimethylcarbazole, respectively, by spectroscopic and chemical means. The other components, V and VI, were found to contain an aldehyde function and were identified as carbazomycinal and 6-methoxycarbazomycinal, respectively. The antimicrobial activity of III and IV are also reported.

Anti-Bacterial Agents↗
PubMed1987

[An immunohistochemical study using a double staining method for regional lymph nodes in gastric cancer].

We have performed a double immunofluorescent staining of two monoclonal antibodies, obtained from gastric cancer patients, that were directed against lymphocyte surface antigens on the regional lymph nodes. Through this method we were able to identify the lymphoid cell subset in detail. As a result of our double staining, most OKT8+ lymphocytes were also found to be Leu 15- lymphocytes and these OKT8+ Leu 15- lymphocytes were regarded as being cytotoxic effector cells or precursors. A few OKT8+ Leu 15+ lymphocytes (suppressor T lymphocytes) were obtained. More Leu 3a+ Leu 8- lymphocytes (helper T lymphocytes) were observed than Leu 3a+ Leu 8- lymphocytes (suppressor inducer T lymphocytes). To investigate whether these T lymphocytes were activated, we used an OKT 9 monoclonal antibody directed against the transferrin receptor and an OKIa1 monoclonal antibody directed against the HLA-DR framework. Such double stained lymphocytes (activated T lymphocytes) were few.

Antibodies, Monoclonal↗
PubMed1987

[Effect of splenectomy on cancers induced with ENNG (N-ethyl-N'-nitro-N-nitroso-guanidine) in the alimental canal].

The effect of splenectomy on cancers in the alimental canal induced with ENNG was investigated. Wistar rats were given 100 micrograms/ml ENNG in tap water ad libitum for four months. Splenectomy was carried out one month on group A (n = 19), two months on group B (n = 18), three months on group C (n = 19) and four months on group D (n = 16) after administration of ENNG. In the control group splenectomy was not performed and ENNG was only given. For the results, the size of tumors in group A and B rats were grossly reduced compared with group C and D rats. Histologically, epithelial tumors seemed to be differentiated in group A and B rats compared with those of group C and D rats. In the early stage when it was difficult to determine the tumors macroscopically and histologically, splenectomy seemed to inhibit the growth of alimental tumors induced with ENNG and splenectomy itself seemed to decrease the growth of alimental tumors induced with ENNG.

Animals↗
PubMed1986

Effect of saturated and unsaturated fatty acids on the oxidative metabolism of human neutrophils. The role of calcium ion in the extracellular medium.

The ability of fatty acids to stimulate the generation of superoxide anion (O2-) by human neutrophils was investigated with respect to their Krafft points. Saturated (myristic acid) and unsaturated (elaidic and oleic acid) induced a marked O2- generation and release from human neutrophils at pH 7.4 in the absence of Ca2+, while 0.3 mM Ca2+ inhibited both myristic acid and elaidic acid-induced O2- release. [14C]Myristic acid association with neutrophils was reduced by addition of Ca2+, whereas oleic acid association was not affected. When the pH of the reaction mixture was lowered to 6.4, 0.6 mM Ca2+ did not inhibit the O2- generation by human neutrophils. These results indicate that the inhibitory effect of Ca2+ on the fatty acid-induced O2- generation might be due to the ionic interaction between the carboxyl group of the fatty acid and Ca2+. Furthermore, 11-methyltridecanoic acid, a branched isomer of myristic acid, which showed the low Krafft point even in the presence of Ca2+, stimulated O2- generation by human neutrophils not only in the absence but also in the presence of 0.6 mM Ca2+. The effect of Ca2+ on the fatty acid-induced O2- generation by neutrophils was discussed with reference to its possible relationship to the Krafft point.

Calcium↗
PubMed1986

Electron spin resonance studies on a flavoprotein in neutrophil plasma membranes. Redox potentials of the flavin and its participation in NADPH oxidase.

Plasma membrane fractions of stimulated and resting cells were isolated from pig blood neutrophils. The midpoint redox potential (Em) of the membrane-bound flavin was determined potentiometrically by analysis of the flavin free-radical signal by electron spin resonance (ESR) spectroscopy. In both stimulated and resting cells, a peak position of the titration curve gave an Em value of -280 mV at pH 7.0 (Em7). The flavin free radical showed an ESR spectrum at g = 2.004 with a peak to peak width of 19 G, which indicates that the redox intermediate is a neutral semiquinone. Redox titrations were anaerobically examined at 25 degrees C with NADPH in place of dithionite. Addition of NADPH to plasma membranes of stimulated cells resulted in a rapid change in potential, accompanied by the formation of the ESR signal of flavin free radical. Computer simulation of the titration points gave an ambient midpoint potential of -280 mV (Em7). In contrast, those of resting cells showed a very slow change in potential and no g = 2.00 signal formation. Power saturation behavior of the ESR signal showed a marked difference between those of stimulated and resting cells. ESR characteristics of the flavin are discussed in relation to the membrane-bound NADPH oxidase.

Animals↗
PubMed1986

Hypertonic glycerol induces a respiratory burst in leukocytes.

Exposure to hypertonic glycerol induced cyanide-insensitive oxygen consumption and formation of superoxide anion (O-2) in leukocytes such as porcine blood polymorphonuclear leukocytes, guinea pig peritoneal leukocytes and guinea pig alveolar macrophages. Generation of O-2 occurred after a short lag time, remained maximal for a certain time and then stopped. Its termination was not due to cell damage, since cells exposed to glycerol did not release cytosolic enzymes such as lactate dehydrogenase and exhibited a subsequent respiratory burst upon addition of other stimulators such as myristic acid and phorbol myristate acetate. The period of O-2 generation increased linearly as a function of the glycerol concentration; cells exposed to 20% (v/v) glycerol produced O-2 for 10 min. The maximal velocity of O-2 generation also increased with the concentration of glycerol, reaching a plateau at 10% glycerol. Membrane vesicles isolated from the cells exposed to 20% glycerol showed high activity of NADPH-dependent O-2 generation as compared to those of unexposed cells. Activation of leukocytes by glycerol was not accompanied by degranulation, unlike stimulation by phagocytosis. A marked change in shape of the cell membrane of glycerol-treated cells was observed by light and scanning electron microscopies.

Animals↗
PubMed1986

Isolation and characterization of proteinases from the sarcocarp of snake-gourd fruit.

Seven proteinases were isolated from the fruit of snake-gourd, Trichosanthes cucumeroides Maxim. Their isozymes are all serine proteinases, and homologous in their respective molecular weights, amino acid compositions, and enzymatic properties. Their molecular weight was estimated to be about 50,000. Using casein as a substrate, the maximum activity was found in the alkaline pH region. The optimum temperature using casein was 70 degrees C at pH 7.3. The enzymes were strongly inhibited by diisopropyl fluorophosphate and not inhibited by inhibitors of sulfhydryl or metalloproteases. The reduced and S-carboxymethylated insulin B-chain was used as a substrate in an investigation of the specificity. The enzyme was found to have a wide specificity for this substrate but preferentially hydrolyzed the peptide bonds involving the carboxyl groups of charged amino acid such as S-cm-cysteine, glutamic acid, histidine, arginine, and lysine. Experimental evidence indicated that the snake-gourd proteinases are similar in their properties to cucumisin, which is isolated from the sarcocarp of melon fruit.

Amino Acids↗
PubMed1986

Is cytochrome b558 translocated into the plasma membrane from granules during the activation of neutrophils?

Two laboratories (Borregaard et al. (1984) J. Biol. Chem. 259, 47; Ohno et al. (1985) J. Biol. Chem. 260, 2409) have reported that a b-type cytochrome (b558) was translocated into plasma membranes from specific granules in activated neutrophils. In an attempt to examine the cytochrome b translocation, porcine neutrophils were activated by treatment with surface-active agents such as myristate (MA) and phorbol myristate acetate (PMA), and then the postnuclear supernatants of both activated and unactivated cells were fractionated by Percoll density gradient centrifugation with a Zonal rotor. In activated neutrophils, high O2- generating activity was found in the plasma membrane fraction, which showed a peak of Na, K-ATPase activity as a marker enzyme. Cytochrome b558 was recovered 74 to 78% in the plasma membrane fraction and 14 to 16% in granules in either activated or unactivated cells. No change in specific content of cytochrome b558 was observed in plasma membranes before and after activation of cells. Furthermore, in both activated and unactivated cells, vitamin B12-binding protein, a specific granule marker, was mainly found in the bottom fractions and scarcely at all in plasma membranes. These results suggest that no translocation of cytochrome b558 occurs during activation of neutrophils.

Animals↗