Two glyceraldehyde-3-phosphate dehydrogenase isozymes from the koningic acid (heptelidic acid) producer Trichoderma koningii

The sesquiterpene lactone koningic acid (heptelidic acid) irreversibly inactivated glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde 3-phosphate: NAD+ oxidoreductase (phosphorylating)] (EC 1.2.1.12) (GAPDH) and thus inhibits glycolysis. The koningic-acid-producing strain of Trichoderma koningii M3947 was shown to contain the koningic-acid-resistant GAPDH isozyme (GAPDH I) under conditions of koningic acid production. In peptone-rich medium, however, no koningic acid production was observed, and the koningic-acid-sensitive GAPDH isozyme (GAPDH II), in addition to the resistant enzyme, was produced. Both enzymes were tetramer with a molecular mass of 152 kDa (4 x 38 kDa) and lost enzyme activity when two of the four cysteine residues reacted with koningic acid. The apparent Km values of GAPDH I and II for glyceraldehyde 3-phosphate were 0.54 mM and 0.33 mM, respectively. The former isozyme was inhibited 50% by 1 mM koningic acid but not affected at 0.1 mM, while the latter isozyme was inhibited 50% at 0.01 mM. The immunochemical properties and partial amino acid sequences suggested that the two isozymes have different molecular structures. These results suggest that GAPDH I is responsible for the glycolysis in T. koningii when koningic acid is produced.     

Comparison of the intestinal bacteria in specific pathogen free mice from different breeders

Specific pathogen free BALB/c mice from 3 commercial laboratory animal breeders in Japan were compared on the composition of caecal flora revealed by selective and nonselective cultivation as well as direct microscopical observation on smears, and relative caecal weight. Large differences were detected in viable counts of total bacteria and almost all bacterial groups, while direct microscopical counts which consisted mainly of fusiform bacteria were almost equal, resulting in diverse recovery rates among 3 breeders. Eubacterium and spiral shaped organisms were not detected from all breeders. There also existed variations within breeders, especially those in the number of Enterobacteriaceae. Relative caecal weight also differed among breeders, suggesting the participation of variety of caecal bacteria in determining this parameter. As these variations in bacteriological status of animals can influence experimental results, standardization of intestinal microbial flora is required.     

The late-replicating X chromosome in digynous mouse triploid embryos

Using BrdU-labeling and acridine orange staining, the behavior of X-chromosome replication was studied in 28 XXX and 19 XXY digynous mouse triploids. In some of these the paternal and maternal X chromosome could by cytologically distinguished. Such embryos were obtained by mating chromosomally normal females with males carrying Cattanach's X chromosome which contains an autosomal insertion that substantially increases the length of this chromosome. In the XXX triploids there were two distinct cell lines, one with two late-replicating X chromosomes, and the other with only one late-replicating X. The XXY triploids were also composed of two cell populations, one with a single late-replicating X and the other with no late replicating X chromosome. Assuming that the late-replicating X is genetically inactive, in both XXX and XXY triploids, cells from the embryonic region tended to have only one active X chromosome, whereas those from the extra-embryonic membranes tended to have two active X chromosomes. The single active X chromosome was either paternal or maternal in origin, but two active X chromosomes were overwhelmingly maternal in origin, suggesting paternal X-inactivation in extra-embryonic tissues.     

Effects of angiotensin I-converting enzyme inhibitor, SQ 14225, in nomal men.

Effects of an orally active angiotensin I-converting enzyme inhibitor, SQ 14225, on the actions of angiotensin I (AI) infused intravenously for 120 to 390 min were studied in 5 normal men. When 20 ng/kg/min of AI infusion was started immediately after a single oral administration of 100 mg of SQ 14225, a significant rise in blood pressure (BP) was observed for the first 15 min, but BP began to fall from 17 min and returned to the pretreatment level at 45 min. This BP level continued at least to 120 min and in one subject to 180 min. In this subject BP began to rise again from 185 min and reached the level of 15 min at 390 min. Plasma AI level increased gradually from 45 min. At 15 min plasma renin activity (PRA) decreased and plasma aldosterone (PA) increased, but then PRA began to increase and PA began to decrease. At 120 min the values of PRA and PA were similar to the pretreatment values. In one subject plasma AI and PRA began to decrease and PA began to increase after 120 or 180 min. On the other hand, in the 5 men sole AI infusion caused a continued BP rise, PRA decrease and PA increase, and sole SQ 14225 administration caused increases in plasma AI and PRA and a decrease in PA but no BP change. From these results it was concluded that complete blockade and partial inhibition of AI conversion by 100 mg of oral SQ 14225 lasted for about 2.5 and 6.5 hr, respectively and that BP rise, PRA suppression and aldosterone stimulation after AI infusion were entirely due to the actions of angiotensin II converted from AI.     

Inhibition of in vitro cholesterol synthesis by fatty acids.

Inhibitory effect of 44 species of fatty acids on cholesterol synthesis has been examined with a rat liver enzyme system. In the case of saturated fatty acids, the inhibitory activity increased with chain length to a maximum at 11 to 14 carbons, after which activity decreased rapidly. The inhibition increased with the degree of unsaturation of fatty acids. Introduction of a hydroxy group at the alpha-position of fatty acids abolished the inhibition, while the inhibition was enhanced by the presence of a hydroxy group located in an intermediate position of the chain. Branched chain fatty acids having a methyl group at the terminal showed much higher activity than the corresponding saturated straight chain fatty acids with the same number of carbons. With respect to the mechanism for inhibition, tridecanoate was found to inhibit acetoacetyl-CoA thiolase specifically without affecting the other reaction steps in the cholesterol synthetic pathway. The highly unsaturated fatty acids, arachidonate and linoleate, were specific inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA synthase. On the other hand, ricinoleate (hydroxy acid) and phytanate (branched-chain acid) diminished the conversion of mevalonate to sterols by inhibiting a step or steps between squalene and lanosterol.     

Quantitative determination of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane

Quantitative determination of the sulfated glycoproteins present in tissue and secretion fluid was performed. After digestion of the specimen with pronase in order to convert glycoproteins to glycopeptides, the sulfated glycopeptides were separated from a mixture of acidic glycans (glycosaminoglycans, sialoglycopeptides and sulfated glycopeptides) by two-dimensional electrophoresis on cellulose acetate membrane [(1986) J. Biochem. Biophys. Methods 12, 239-246]. After staining with alcian blue, the spot of sulfated glycopeptide on the cellulose acetate membrane was cut out, and then only the dye bound to the sulfated glycopeptide was extracted with a 5% cetylpyridinium chloride solution at 100 degrees C for 15 min. The extract was then measured by absorbance at 615 nm using an authentic sulfated glycopeptide as a standard. This method facilitated the determination of sulfated glycopeptides, which were separated from other acidic glycans, within the range 0-25 micrograms.     

Use of ryanodine for functional removal of the calcium store in smooth muscle cells of the guinea-pig

Calcium store of the skinned fibers of the guinea-pig portal vein, pulmonary artery and taenia caeci consisted of two classes: one with both Ca-induced Ca release (CICR) and inositol 1,4,5-trisphosphate (IP3)-induced Ca release (IICR) mechanisms (S alpha) and the other only with IICR mechanisms (S beta). Ryanodine, applied during the CICR was activated, locked the CICR channels open, but the drug had practically no effect on the IICR mechanism. Thus, after the ryanodine treatment the Ca store with the CICR (S alpha) lost its capacity to hold Ca. Changes in the agonist-evoked contraction of intact muscle due to the ryanodine treatment suggest that agonists release Ca from S alpha which produces the initial phase of contractures.     

The rbcX gene product promotes the production and assembly of ribulose-1,5-bisphosphate carboxylase/oxygenase of Synechococcus sp. PCC7002 in Escherichia coli

The operon encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the cyanobacterium Synechococcus sp. PCC7002 contains three rbc genes, rbcL, rbcX and rbcS, in this order. Introduction of translational frameshift into the rbcX gene resulted in a significant decrease in the production of large (RbcL) and small (RbcS) subunits of the Rubisco protein in Synechococcus sp. PCC7002 and in Escherichia coli. To investigate the function of the rbcX gene product (RbcX), we constructed the expression plasmid for the rbcX gene and examined the effects of RbcX on the recombinant Rubisco production in Escherichia coli. The coexpression experiments revealed that RbcX had marked effects on the production of large and small subunits of Rubisco without any significant influence on the mRNA level of rbc genes and/or the post-translational assembly of the Rubisco protein. The present rbcX coexpression system provides a novel and useful method for investigating the Rubisco maturation pathway.