Hemagglutinating activity in extracts of mycelia from submerged mushroom cultures.

Extracts from mycelia of seven different mushrooms agglutinated erythrocytes of several species. More than one agglutinating factor was identified in the extracts of three different mycelia. Agglutination was partially inhibited nonspecifically by high concentrations of glucose, galactose, mannose, fucose, and rhamnose.     

Immobilization of yeast cells containing beta-galactosidase

Cultural conditions optimum for beta-galactosidase production by Saccharomyces anamensis are pH 4.5, temperature 26 +/- 2 degrees C, and 30 h of incubation period. Addition of lactose at 24 h fermentation greatly increase the level of enzyme. Optimum pHl, temperature, pH stability, and thermostability of yeast beta-galactosidase are negligibly affected by immobilization. The K(m) values of enzyme in the native and immobilized cells are 102mM and 148mM, respectively. Glucose noncompetitively inhibits the enzyme activity. Addition of substances such as dithioerythritol, glutathione, and bovine serum albumin to the native cell during assay procedure and immobilized cell prior to immobilization have stimulatory effects on enzyme activity. Metal ions like Ca(2+), Mg(2+) enhance the beta-galactosidase activity for both intact and bound cells. Immobilized cells retain 68.6% of the beta-galactosidase activity of intact cells and there is no significant loss of activity on storage at 4 degrees C for 28 days.     

Preparation and X-ray characterization of four new crystal forms of jacalin, a lectin from Artocarpus integrifolia

Four new crystal forms of the anti-T lectin from jackfruit (Artocarpus integrifolia) have been prepared and characterized. Three of them, two monoclinic (P21, a = 59.4 A, b = 83.3 A, c = 63.5 A, beta = 107.7 degrees; C2, a = 106.1 A, b = 53.9 A, c = 128.0 A, beta = 95.0 A) and one orthorhombic (C222(1), a = 98.1 A, b = 67.3 A, c = 95.1 A) were grown with 2-methylpentan-2,4-diol (MPD) as the precipitant while the fourth, an hexagonal form (P6(1)22, a = b = 129.6 A, c = 157.9 A), was obtained in the presence of methyl-alpha-D-galactopyranoside with polyethylene glycol 4000 as the precipitant. The reported relative molecular mass (Mr) of the lectin was found to be inconsistent with the solvent content of the crystals estimated using measured densities. The Mr was redetermined using size-exclusion chromatography in the presence of methyl-alpha-D-galactopyranoside and Ferguson-plot analysis of mobilities in polyacrylamide gel electrophoresis. The redetermined Mr (66,000) is consistent with the measured crystal densities. The orthorhombic and the hexagonal forms, which have one half molecule and one molecule, respectively, in the asymmetric unit, are suitable for high-resolution X-ray analysis.     

Stereoselective enzymatic hydrolysis of (exo,exo)-7-oxabicyclo[2.2.1]heptane-2,3-dimethanol diacetate ester in a biphasic system

Summary A key chiral intermediate lactol(3)[3aS (3a,4,7,7a)]-hexahydro-4,7-epoxy-isobenzofuran-1 (3H)-one was prepared for the total synthesis of a new thromboxane antagonist. The stereoselective hydrolysis of (exo,exo)-7-oxabicyclo[2.2.1]heptane-2,3-dimethanol, diacetate ester (1) to the corresponding chiral monoacetate ester (2) was carried out with lipases, among which Amano P-30 lipase from Pseudomonas sp. was most effective since it gave the desired enantiomer of monoacetate ester. A yield of 75 mol% and optical purity of >99% was obtained when the reaction was conducted in a biphasic system with 10% toluene at 5 g/l of the substrate. Lipase P-30 was immobilized on Accurel polypropylene (PP) and the immobilized enzyme was reused (five cycles) without loss of enzyme activity, productivity or optical purity. The reaction process was scaled-up to 80 1 (400 g substrate) and monoacetate (2) was isolated in 80 mol% yield with 99.3% optical purity as determined by chiral HPLC and nuclear magnetic resonance (NMR) analysis. A gas chromatography of 99.5% and specific rotation, []_D of -7.6° was obtained. The chiral monoacetate ester (2) was oxidized to its corresponding aldehyde and subsequently hydrolyzed to give lactol (3).     

Excavations in drug chirality: 1. Cyclothiazide

There is a great deal of current interest in the role and importance of chirality in the development of new drugs, but little attention is being paid to the stereochemistry of older drugs. Indeed, many older chiral drugs were introduced without adequate information on their stereochemical identity or composition. We have examined one such drug, the antihypertensive diuretic agent cyclothiazide. Standard sources of drug information and the research literature do not provide data on the stereochemical composition of clinically used cyclothiazide, although scattered reports indicate that the drug may consist of "several stereoisomers." Inspection of the chemical structure of the drug, 6-chloro-3,4-dihydro-3-(5-norbornen-2-yl)-2H-1,2,4-benzothiadiazin e-7- sulfonamide 1,1-dioxide, shows that it can exist as eight stereoisomers that may form four racemates. Using synthesis, fast-atom-bombardment mass spectrometry, gas-liquid chromatography, chiral and nonchiral high-performance liquid chromatography, and nuclear magnetic resonance spectroscopy, we determined that pharmaceutical cyclothiazide is in fact a mixture of the eight stereoisomers in the form of the four racemates. The two racemates with endo configuration at the norbornene moiety predominate over the exo racemates, and small but significant differences in isomer distribution between different batches of the drug were observed. We urge that in studies of older drugs the stereochemical details be considered.     

Purification and characterization of protease from a newly isolated Rhizopus oryzae

A newly isolated Rhizopus oryzae was found to exhibit some unusual phenomenon of secreting alkaline protease which was purified and characterized. The molecular weight was determined to be 28,600 dalton in gel electrophoresis. The enzyme is stable in the pH range from 3 to 11 and most active at pH 8. The temperature optimum of this thermostable biocatalyst is at 60 °C. The enzyme is sensitive to metal chelators, most of the metal ions (excepting a few monovalent cations) and inhibitor like PMSF. This indicates that the protease of isolated Rhizopus oryzae falls under alkaline serine group.     

Effect of retinoic acid on adenosine diphosphate and collagen-induced alterations in enzymes of GSH-linked antioxidant defence system of human blood platelets in vitro

Collagen stimulation of blood platelets resulted in significant increases in malondialdehyde (MDA) formation and activity of glucose-6-phosphate dehydrogenase (G6PDH) and a decrease in catalase and glutathione peroxidase (GPx). Retinoic acid (RA) pretreatment did not show any appreciable changes except for a decrease in G6PDH activity as compared with collagen alone. RA pretreatment of human blood platelets resulted in an increase in the activities of catalase and GPx, two important radical scavenging enzymes, with significant decrease in MDA formation when compared with ADP alone. It is suggested that RA has a significant effect on the antioxidant defence system in ADP stimulated platelets but not in the collagen stimulated platelets.     

Differential effect of retinoic acid on ADP and collagen induced platelet aggregation

Retinoic acid (RA) was found to inhibit ADP induced but not collagen induced aggregation of human platelets and the differential action is related to intraplatelet Ca2+ reflux. RA was active at concentrations as low as 10(-7) M and required 20 min prior incubation with platelet suspension in order to inhibit aggregation by ADP. All the steps in ADP induced but not collagen induced platelet activation, viz. hydrolysis of phosphatidyl inositol, phosphorylation of 20, 47 and 250 kDa proteins as well as increased association of actin with Triton X-100 insoluble cytoskeletal matrix were inhibited by RA. RA when used as an agent for differentiation induction of cell progenitor is likely to affect the platelet aggregation and thereby the haemostatic process.     

Mechanism of reductive activation of cobalamin-dependent methionine synthase: an electron paramagnetic resonance spectroelectrochemical study

The mechanism of reductive methylation of cobalamin-dependent methionine synthase (5-methyltetrahydrofolate:homocysteine methyltransferase, EC 2.1.1.13) has been investigated by electron paramagnetic resonance (EPR) spectroelectrochemistry. The enzyme as isolated is inactive, and its UV/visible absorbance and EPR spectra are characteristic of cob(II)alamin. There is an absolute requirement for catalytic amounts of AdoMet and a reducing system for the formation and maintenance of active enzyme during in vitro turnover. The midpoint potentials of the enzyme-bound cob(II)alamin/cob(I)alamin and cob(III)alamin/cob(II)alamin couples have been determined to be -526 +/- 5 and +273 +/- 4 mV (versus the standard hydrogen electrode), respectively. The presence of either CH3-H4folate or AdoMet shifts the equilibrium distribution of cobalamin species observed during reduction by converting cob(I)alamin to methylcobalamin. The magnitude of these shifts is however vastly different, with AdoMet lowering the concentration of cob(II)alamin at equilibrium by a factor of at least 3 X 10(7), while CH3-H4folate lowers it by a factor of 19. These studies of coupled reduction/methylation reactions elucidate the absolute requirement for AdoMet in the in vitro assay system, in which the ambient potential is approximately -350 mV versus the standard hydrogen electrode. At this potential, the equilibrium distribution of cobalamin in the presence of CH3-H4folate would be greatly in favor of the cob(II)alamin species, whereas in the presence of AdoMet the equilibrium favors methylated enzyme. In these studies, a base-on form of cob(II)alamin in which the dimethylbenzimidazole substituent of the corrin ring is the lower axial ligand for the cobalt has been observed for the first time on methionine synthase.(ABSTRACT TRUNCATED AT 250 WORDS)