Biomedical subjects
Publications and source records attributed to M Oka.
The structures of new antitumor antibiotics, glidobactins A (Ia), B (Ib) and C (Ic) were elucidated by a combination of chemical and enzymatic degradations and spectral analyses. They have in common a cyclized tripeptide nucleus composed of L-threonine, 4(S)-amino-2(E)-pentenoic acid and erythro-4-hydroxy-L-lysine, and differ from each other in the unsaturated fatty acid moiety attached to the peptide.
Glidobactin deacylating activity was found in a bacterial strain of Pseudomonas sp. Glidobactamine, a key intermediate for acyl analogues of glidobactin, was isolated from the enzymatic degradation products of glidobactins after treatment using a column of fibrous active gel on which the cells of the Pseudomonas strain were immobilized. The chemical structure of glidobactamine was confirmed as the intact peptide moiety of glidobactins by chemical reformation of glidobactin A from glidobactamine and 2,4-dodecadienoic acid which is the constitutive fatty acid of glidobactin A.
Polyangium brachysporum sp. nov. strain ATCC 53080 produces a novel type of antifungal and antitumor antibiotic complex, glidobactins A, B and C. Enhanced production of minor components, glidobactins B and C, was achieved by medium modification. The addition of soybean oil or corn oil, which are rich in unsaturated C18 fatty acids, to the fermentation medium led to an increased production of components B and C. Productivity of component C was selectively enhanced by the addition of oleic acid-rich oils, olive oil and Tween 80 (polyoxyethylene sorbitan mono-oleate). Furthermore, precursing palmitoleate, linoleate and oleate permitted the direct biosynthesis of components A, B and C, respectively. The fermentation with 3% addition of an appropriate oil at initial time provided an optimal production of component B or C.
A variety of glidobactin analogs modified at the fatty acid, L-threonine and nucleus moieties of the molecule were synthesized and their structure-activity relationships examined. The antitumor and antifungal activity was greatly influenced by modification of the fatty acid glidobactin, with the dodecanoyl and tetradecanoyl analogs exhibiting better antitumor activity than the parent antibiotics. Replacement of the L-threonine with other amino acids greatly reduced the activity and reduction of the double bond of the nucleus completely eliminated the biological activity of glidobactin.
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The effect of intravenous administration of dopamine (DA) or L-3,4-dihydroxyphenylalanine (L-DOPA), its immediate precursor, on the level of DA sulfate in dog plasma was examined, to clarify the source and physiological significance of DA sulfate which is present at high level in the plasma. After DA administration, the plasma level of free DA increased markedly, but the level of DA sulfate did not change. However, after administration of L-DOPA, the levels of both free DA and DA sulfate increased greatly. After a single injection of L-DOPA, increase in the level of free DA was transient, but that of DA sulfate persisted for a long time. These results suggest that some of the DA sulfate in dog plasma is formed from circulating L-DOPA, not from circulating DA, and that formation of DA conjugate may play a role in regulating the plasma level of free DA.
Flavin adenine dinucleotide synthetase (ATP:FMN adenylyltransferase, EC 2.7.7.2) was purified about 10,000-fold from the high-speed supernatant of rat liver by a sequence of ammonium sulfate fractionation and column chromatographies on DEAE-Sephadex (A-50), chromatofocusing, FMN-agarose affinity, and Sephadex G-200. The specific activity of the purified enzyme was 133 units (nanomoles of FAD formed per min at 37 degrees C)/mg of protein. This preparation was free from contaminating FAD pyrophosphatase. The apparent molecular weight was estimated to be 97,000 by gel filtration on Sephadex G-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an apparent subunit molecular weight of 53,000. Hence, the enzyme is a dimer of approximately 100,000. The enzyme was found most active at pH 7.1, requires Mg2+, and is essentially irreversible in the direction of FAD formation. Kinetic analysis gave Km values of 9.6 microM for FMN and 53 microM for ATP.
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In isolated bovine adrenal medullary cells, vasoactive intestinal polypeptide (VIP) stimulated 14C-catecholamine synthesis from 14C-tyrosine, but not from 14C-DOPA. This stimulatory effect of VIP on 14C-catecholamine synthesis was not dependent upon extracellular Ca2+. VIP did not affect the intracellular cyclic AMP (cAMP) level. The stimulatory effect of VIP on 14C-catecholamine synthesis was additive with that of carbamylcholine, which was dependent upon extracellular Ca2+, but not with that of phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA), an activator of protein kinase C. Moreover, 1-(isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, inhibited not only TPA-stimulated, but also VIP-stimulated 14C-catecholamine synthesis from 14C-tyrosine. These results suggested that VIP stimulated catecholamine synthesis by activation of tyrosine hydroxylase and that protein kinase C was involved in this stimulatory mechanism.
The interaction of soluble tyrosine hydroxylase with isolated chromaffin granule membranes was studied. The incubation of the granule membranes with the soluble fraction in the mixture that may approximate the intracellular environment of the resting cell resulted in a marked increase in the enzyme activity recovered in the membrane fraction, and enzyme activity precipitated with the granule membranes was increased according to incubation time and dependent on amounts of both the granule membranes and soluble fraction. The association of the soluble enzyme with the granule membranes was reversible and specific, whereas a decrease in activity of the soluble enzyme was observed when the soluble fraction was incubated with the granule membranes in the mixture in which association of the soluble enzyme with the granule membranes might occur. These results seem to indicate that activity of the soluble enzyme is presumably modulated by the granule through association of the soluble enzyme with the surface of the granule, thus suggesting that interaction between tyrosine hydroxylase and the chromaffin granule may play a possible role in regulation of catecholamine biosynthesis as a consequence of modulating activity of the rate-limiting enzyme within the cell.
The effects of a newly synthesized alpha 2-adrenoceptor agonist (an imidazole derivative, DJ-7141) on catecholamine secretion from isolated bovine adrenal medullary cells were examined. DJ-7141 did not affect basal catecholamine secretion, but inhibited catecholamine secretion induced by stimulation of the nicotinic ACh receptor. This inhibitory effect of DJ-7141 was less than that of clonidine, another alpha 2-agonist. DJ-7141 also inhibited [45Ca]2+ uptake by the cells induced by nicotinic stimulation. DJ-7141 did not affect catecholamine secretion induced by high K+ concentration. Its inhibitory effect on nicotine-induced catecholamine secretion was not restored by increase in either the nicotine or Ca2+ concentration of the medium, suggesting that it interfered with the coupling between nicotinic ACh receptor stimulation and Ca2+-channel activation. The inhibitory effect of DJ-7141 seemed to be independent of its effect on alpha 2-adrenoceptors, because its effect was not antagonized by the alpha 2-adrenoceptor antagonists yohimbine and DG-5128, which both had no effect on either basal or nicotine-induced catecholamine secretion.