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Biomedical subjects

J Nath

Publications and source records attributed to J Nath.

At least 109 records · Page 6Linked to original sources
PubMed1982

Effects of taxol on human neutrophils.

Taxol, a plant alkaloid, promotes and stabilizes microtubule assembly in cells and cellfree systems. In the present study, the effects of taxol on various functional, morphologic, and biochemical phenomena in human peripheral blood PMN (Hypaque-Ficoll) were examined. Taxol (10(-7) M) inhibited PMN chemotaxis stimulated by N-formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe) or endotoxin-activated serum by more than 60%. The inhibition was not readily reversed by washing, and taxol itself was not a chemoattractant, nor is it a secretagogue. Spontaneous nondirected migration, cell spreading on a glass surface, and orientation of cell organelles in response to a chemoattractant gradient were also inhibited by taxol. Taxol (10(-5) M) decreased killing of Staphylococcus aureus, but did not alter phagocytosis of heat-killed Candida or hexose monophosphate shunt activity in resting or stimulated PMN. Ultrastructural studies showed that PMN incubated in f-met-leu-phe, taxol, or both had increased (p less than 0.001) numbers of centrosome-associated microtubules, and the microtubules of cells incubated in taxol with or without f-met-leu-phe were organized into bundles. Taxol (10(-5) M) markedly inhibited post-translational tyrosinolation of alpha-chains of tubulin in both resting and f-met-leu-phe-stimulated PMN. The data indicate that taxol inhibits PMN locomotion and bacterial killing, supporting a role for microtubules in these processes. The ultrastructural and biochemical data also support the view that taxol mediates its effects on PMN by its effect on microtubules.

Alkaloids↗
PubMed1981

Cytological evidence for DNA chain elongation after UV irradiation in the S phase.

Human cells irradiated with UV light synthesize lower molecular weight DNA than unirradiated cells. This reduction in molecular weight is greater in xeroderma pigmentosum (XP) cells than in normal cells. The molecular weight of DNA is further reduced by the addition of caffeine to XP cells. By several hours after irradiation, DNA fragments are barely detectable. Cells from excision-proficient and excision-deficient XP patients were studied autoradiographically to produce cytological evidence of DNA chain elongation. Replicate cultures with and without caffeine were synchronized and irradiated with UV light during the S phase. Caffeine was removed in G2, and the cells were labeled with 3H-thymidine. Results showed significantly increased labeling during G2 of excision-deficient XP cells. Labeling was dependent on the time of irradiation and presence of caffeine. The XP variant cells had no increase in labeling for any irradiation time.

Adult↗
PubMed1981

Stimulation of tubulin tyrosinolation in rabbit leukocytes evoked by the chemoattractant formyl-methionyl-leucyl-phenylalanine.

Cellular tubulin is subject to a posttranslational modification involving the reversible addition to tyrosine through peptide linkage to the C-terminal glutamate of the alpha-chain. The synthetic peptide chemoattractant, N-formyl-methionyl-leucyl-phenylalanine, causes a specific, dose-dependent stimulation of tubulin tyrosinolation in rabbit leukocytes. This stimulation is prevented by carbobenzoxy-phenylalanyl-methionine, benzoyl-tyrosine ethylester, and nordihydroguaiaretic acid, which are all inhibitors of chemotaxis presumed to act via membrane-associated events. The combination of 3-deazaadenosine and homocysteine thiolactone, which inhibits phospholipid methylation, and quinacrine, an inhibitor of phospholipase A2, also abolishes the response to the peptide. Colchicine, however, which causes a marked disassembly of cellular microtubules in these cells and also inhibits chemotaxis, does not have any inhibitory effect on the basal or peptide-stimulated rate of tubulin tyrosinolation. In contrast, taxol, a microtubule-stabilizing agent, has an inhibitory effect on both the basal and peptide-stimulated tyrosine incorporation. Taxol also inhibits chemotaxis in rabbit leukocytes. The results strongly suggest the role of closely linked membrane-cytoskeleton interactions in leukocyte chemotaxis, in which tyrosinolation of tubulin may be functionally involved.

Alkaloids↗
PubMed1980

Acid phosphatase changes associated with development of male sterile and fertile maize (Zea mays L.).

Free and bound acid phosphatase were investigated biochemically and electrophoretically in male sterile and fertile plants. Scutella at the 72-hr seedling stage and anthers with enclosed reproductive tissue at the premeiosis, meiosis, and mature developmental stages were tested. Biochemical data show that while the total amount of free acid phosphatase behaved similarly in fertile and sterile plants, specific activities decreased in fertile plants and remained unchanged or increased in sterile plants. Total amounts of bound acid phosphatase increased significantly in fertile plants while specific activities decreased. In sterile anthers both specific activity and amount of bound acid phosphatase decreased significantly (except cms-S). Electrophoretic results indicated that the basic form of the enzyme was very similar in each location.

Acid Phosphatase↗
PubMed1980

An apparent paradox in the occurrence, and the in vivo turnover, of C-terminal tyrosine in membrane-bound tubulin of brain.

Tubulin tyrosine ligase catalyzes the reversible addition of tyrosine to the C-terminus of tubulin alpha chains. By using ligase and carboxypeptidase A in conjunction, we have previously shown that brain cytoplasmic tubulin exists in three forms: 15-40% already has C-terminal tyrosine, another 10-30% can accept additional tyrosine, and about one-half is an uncharacterized species which is not a ligase substrate. A membrane-bound fraction of brain tubulin, purified by vinblastine precipitation from a detergent extract, has been found to differ by the complete absence of preexisting tyrosine. The membrane fraction from which tubulin was extracted also contained masked forms of both ligase and a distinct detyrosylating enzyme, which can be released by detergent extraction. The turnover of alpha-chain C-terminal tyrosine in vivo was studied by incubating brain mince with labeled tyrosine, or injecting it intracerebrally, under conditions where protein synthesis was inhibited. Tyrosine appeared to turn over to about the same extent in membrane-bound, as in soluble, tubulin. This apparently paradoxical result was not due to ATPase in the membrane fraction, which might have allowed ligase-catalyzed exchange between free and fixed tyrosine. Authentic [14C]tyrosylated tubulin added to the brain membrane fraction was not detyrosylated or subject to endoprotease digestion during subsequent procedures to isolate tubulin. The unexpected finding that tubulin tyrosylated at the C-terminal in vivo appears to be in the "non-substrate" fraction points toward a possible resolution of the paradox.

Animals↗
PubMed1979

Tubulin tyrosylation in vivo and changes accompanying differentiation of cultured neuroblastoma-glioma hybrid cells.

Changes in a posttranslational modification of tubulin, which accompany differentiation, have been studied in neuroblastoma-glioma hybrid cultured cells. The modification consists of the reversible enzymatic addition of a tyrosine to the COOH terminus of the alpha chain. Cytoplasmic tubulin purified from undifferentiated cells resembled that from adult mammalian brain in that half was in a form which can not accept tyrosine; of the remainder, which is a substrate for tubulin-tyrosine ligase, a higher proportion had COOH-terminal tyrosine. In the tubulin from differentiated cells, in which there had been extensive assembly of axonal microtubules from a preformed pool of subunits, the nonsubstrate tubulin was almost entirely replaced by the species with COOH-terminal tyrosine. In living cells, in the absence of protein synthesis, there was fixation of labeled tyrosine into cytoplasmic alpha chains which was extensive enough to be consistent with turnover, during the course of an hour, of the pre-existing COOH-terminal tyrosine. The alpha chain in the particulate fraction of the cells was comparably labeled, along with some unidentified low molecular weight components.

Animals↗
PubMed1979

Purification of bovine adrenocortical and brain tubulin. A comparative study.

Microtubules from the cow adrenal cortex and brain were purified by three cycles of the temperature-dependent polymerization-depolymerization procedure. Whereas tubulin comprised approximately 8--10% of soluble brain protein, it comprised only 0.5-1.0% of the soluble adrenocortical protein. The partially purified tubulin from both sources gave similar results in the following studies: (1) [3H]colchicine binding examined by Scatchard analysis revealed an apparent Ka of 1 . 10(6) M-1 and a colchicine/tubulin molar binding ratio of 0.4-0.6; (2) tyrosylation studies using a specific tubulin-tyrosine ligase (which adds a tyrosine residue to the C-terminal glutamate or glutamine of the alpha-chain) in conjunction with carboxypeptidase A (which recovers the tyrosine) and (3) amino acid analysis. Examination of protein bands, in addition to the tubulin doublet of 55 000 molecular weight, on sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a difference between the two tubulin preparations. The adrenocortical preparation had protein bands corresponding to apparent molecular weight of 36 000, 60 000, and 68 000. In contrast the brain preparation had only proteins of molecular weights greater than 200 000 (these bands were absent in all adrenal preparations). It would thus appear that if proteins which copurify with tubulin through repeated cycles of polymerization-depolymerization play a role in either microtubule formation or function there is a distinct difference between neural and non-neural tissue.

Adrenal Cortex↗
PubMed1977

Possible mitochondrial involvement in mechanism of cytoplasmic male sterility in maize (Zea mays L.).

The mechanism of cytoplasmic male sterility was investigated in maize by isolating mitochondria from seedlings and various anther stages and analyzing cytochrome oxidase and succinic dehydrogenase biochemically and electrophoretically. Sterile anthers exhibited a lack of biochemical activity and fewer isozymatic bands for cytochrome oxidase. No apparent differences were detected biochemically or electrophoretically between fertile and sterile anthers for succinate dehydrogenase.

Electron Transport Complex IV↗
PubMed1976

Cyclic nucleotides, thioldisulfide status of proteins, and cellular control processes.

It is shown that cyclic nucleotides can have a variety of effects on cell division, cell shape, cell adhesion, and cell movement, depending on the cells selected and the conditions under which they are used. For example, while CHO cells elongate under the influence of exogenous dibutyryl CAMP, Y-1 adrenal tumor cells round up and polyoma-transformed 3T3 cells show no change in shape. The totality of experience with cyclic nucleotides suggests that where they have been used by cells as control elements involving the four processes listed above, they are superimposed on basic cellular processes that progress in their absence--that is, they must be acting indirectly. In attempting to understand the inhibitory action of methyl xanthines on egg development, we were forced to abandon the idea that they acted through cyclic nucleotides. We found that methyl xanthines inhibited the activation of glutathione reductase and that glutathione oxidizing agents act as mitotic inhibitors. Further, we found that tubulin polymerizability, NAD-kinase activity, and a mitotic apparatus associated Ca+2-ATP-ase were all inhibited by oxidation of some of their sulfhydryls and were activated by reduction of the resulting disulfides. These results are discussed in terms of reported cycles and activations of glutathione reductase (GR) in cells and reports that mixed disulfides of glutathione and proteins can act as substrates for GR. Using the fact that a CAMP-dependent protein kinase has been reported to be activated by glutathione, we have suggested potential sites where sulfhydryl control processes and cyclic nucleotide control processes and cyclic nucleotide control processes may interact in certain restricted cases.

Adenosine Triphosphatases↗
PubMed1976

Effects of caffeine and other methylxanthines on the development and metabolism of sea urchin eggs. Involvement of NADP and glutathione.

Methylxanthines (MX) inhibit cell division in sea urchin and clam eggs. This inhibitory effect is not mediated via cAMP. MX also inhibit respiration in marine eggs, at concentrations which inhibit cleavage. Studies showed that no changes occurred in ATP and ADP levels in the presence of inhibitory concentrations of MX, indicating an extra-mitochondrial site of action for the drug. Subsequent studies revealed decreased levels of NADP+ and NADPH, when eggs were incubated with inhibitory concentrations of MX, but no change in levels of NAD+ and NADH. MX did not affect the pentose phosphate shunt pathway and did not have any effect on the enzyme NAD+ -kinase. Further studies showed a marked inhibitory effect on the glutathione reductase activity of MX-treated eggs. Reduced glutathione (GSH) could reverse the cleavage inhibitory effect of MX. Moreover, diamide, a thiol-oxidizing agent specific for GSH in living cells, caused inhibition of cell division in sea urchin eggs. Diamide added to eggs containing mitotic apparatus (MA) could prevent cleavage by causing a dissolution of the formed MA. Both MX and diamide inhibit a Ca2+-activated ATPase in whole eggs. The enzyme can be reactivated by sulfhydryl reducing agents added in the assay mixture. In addition, diamide causes an inhibition of microtubule polymerization, reversible with dithioerythritol. All experimental evidence so far suggests that inhibition of mitosis in sea urchin eggs by MX is mediated by perturbations of the in vivo thiol-disulfide status of target systems, with a primary effect on glutathione levels.

Adenosine Triphosphate↗