Influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase in rat striatal membranes

The influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase activity were investigated in rat striatal membranes. GTP promoted and inhibited the activity at 1 and 100 microM, respectively. The inhibitory effects of GTP were abolished by pretreatment of the membranes with pertussis toxin. GppNHp (guanyl-5'-y1-beta,gamma-imidodiphosphate) exerted only stimulatory effects and pertussis toxin did not affect the effects of GppNHp. GDP at 10 and 100 microM caused significant inhibition which was completely suppressed by pertussis toxin. It is suggested that guanine nucleotide regulates the affinity of as in stimulatory GTP-binding regulatory protein to either beta gamma or catalytic units of adenylate cyclase in a flip-flop manner. Inhibitory GTP-binding regulatory protein seems to play a regulatory role in inhibiting alpha s activity supplying the beta gamma heterodimer.     

Isolation of U1-snRNP(s) from mouse teratocarcinoma cells using immunochemical and biochemical procedures: high proportion of U1a-snRNP to U1b-snRNP

An attempt was made to immunochemically and biochemically purify and characterize the U1-snRNP(s) of mouse embryonal carcinoma cells. The results obtained by RNA analysis of U1-snRNP(s) purified immunochemically from embryoid bodies, F9 cells and PYS-2 cells indicated that the U1-snRNP(s) in these cells consisted of U1a-snRNP and U1b-snRNP. The proportion of U1a-snRNP to U1b-snRNP was also found to be high in the embryoid bodies and F9 cells. The U1a-snRNP predominance in U1-snRNP population was also detected in PYS-2 cells. The immunochemically purified U1-snRNP population from liver nuclei of 129 syngeneic male mouse (129/sv), a host mouse for transplantable tetratocarcinoma OTT6050, and ICR male mouse, contained approximately equal levels of the two U1-snRNP species (U1a- and U1b-snRNP). Partially purified U1-snRNP from embryoid bodies was also obtained by elution from a DEAE-Sepharose column at around 0.18 M NH4Cl or by fractionation by 5-20% linear sucrose gradient centrifugation. The electrophoretic RNA profiles of the partially purified U1-snRNP of embryoid bodies were similar to those obtained immunochemically.     

Aldose reductase inhibitors: flavonoids, alkaloids, acetophenones, benzophenones, and spirohydantoins of chroman

The inhibitory activity of various compounds, including 12 flavonoids, 10 alkaloids, 15 benzophenones, 5 acetophenones, and 7 spirohydantoins of chroman, was tested on rabbit lens aldose reductase, an enzyme involved in complications of diabetes. Almost all compounds tested were found to inhibit the enzyme at low concentrations (10(-5) M). The most potent inhibitor was 2R,4S-6-chloro-2-methylspiro(chroman-4,4'-imidazo-lidine+ ++)-2',5'-dione with an I50 value of 4.7 x 10(-8) M; other spirohydantoins showed similar potency. Polyhydroxybenzophenones were also potent inhibitors with an I50 value of about 10(-7) M. The possible structure-inhibitory activity relationships of the compounds tested are discussed.     

Isolation of porcine follicular fluid inhibin of 32K daltons

Purification of ovarian inhibin from porcine follicular fluid was performed by using an bioassay based upon the suppression of spontaneous FSH release from cultured cells of rat anterior pituitary. The presence in the follicular fluid of four molecular forms of inhibin activity corresponding to Mr 100K, 80K, 55K and 32K was revealed by SDS-gel electrophoresis under non-reducing conditions. The smallest inhibin amongst them, named 32K inhibin, eliciting about 70% of the total activity in the follicular fluid, was separated by gel filtration in the presence of 8 M urea. By subsequent ion-exchange chromatography, followed by RP-HPLC, 32K inhibin was purified to homogeneity with a 8,000 fold purification factor in a yield of 12%. The purified 32K inhibin was found to comprise two polypeptide subunits (Mr 20K and 13K), linked by disulfide bridges and to specifically suppress the secretion of FSH, but not of LH from the pituitary cells.     

Direct-acting mutagenicity of N4-aminocytidine derivatives bearing alkyl groups at the hydrazino nitrogens.

To investigate the mechanism of N4-aminocytidine-induced mutagenesis, N'-alkyl-N4-aminocytidines and N4-alkyl-N4-aminocytidines were prepared and their mutagenicity on bacteria were assayed. N'-Methyl-N4-aminocytidine, N'-(2-hydroxyethyl)-N4-aminocytidine and N',N'-dimethyl-N4-aminocytidine showed direct-acting mutagenicity on S. typhimurium TA100 and E. coli WP2 uvrA, tester strains that are sensitive to base-pair substitutions. In contrast, N4-methyl-N4-aminocytidine, N4-(2-hydroxyethyl)-N4-aminocytidine and N4,N'-dimethyl-N4-aminocytidine were not mutagenic on these bacteria. Since N'-methyl-N4-aminocytidine does not form hydrazones, the possibility that N4-aminocytidine causes mutation due to its reactivity with carbonyl compounds has been excluded. Furthermore, the fact that only those alkyl N4-aminocytidines having a hydrogen on the nitrogen at position 4 are mutagenic is consistent with the previously proposed mechanism in which the tautomerization between the amino and the imino forms of N4-aminocytosine allowing an ambiguous base pairing is the cause of the mutagenesis.     

Effect of Streptococcus faecalis BIO-4R on intestinal flora of weanling piglets and calves.

The effect of oral administration of Streptococcus faecalis BIO-4R, an antibiotic-resistant lactic acid bacterium, on the intestinal flora of weanling piglets and cows reared on antibiotic-containing diet was investigated. Fourteen days after administration of the bacteria, the intestinal flora of the piglets was examined. Animals of the administered group had stabilized lactic flora such as bifidobacteria, streptococci, and lactobacilli, whereas most animals of control group had reduced lactic flora. On the other hand, abundant yeasts were detected from the cecum, colon, and feces of the control animals, but the levels were significantly lower in the animals given strain BIO-4R. The density of Salmonella in the intestine appeared to be reduced after the administration of strain BIO-4R. The number of BIO-4R cells was shown to be 10 times lower in the duodenum and jejunum than in the ileum, suggesting that strain BIO-4R might have grown transiently in the ileum. The similar trend toward stabilization of the lactic flora was also observed in cows after administration of BIO-4R. In addition, an antagonistic effect of the strain against yeasts and Salmonella was suggested. These findings indicate that the oral administration of strain BIO-4R is one of the useful methods whereby the potentially deleterious effect of antibiotics on the intestinal flora of farm animals may be minimized.     

A temperature-sensitive mutant of E. coli exhibiting slow processing of exported proteins

A temperature-sensitive E. coli mutant with a mutation in the spc ribosomal protein operon was found to have a conditional defect in the processing of precursor proteins destined for the periplasmic space or the outer membrane. At high temperatures, significant amounts of precursor proteins having unprocessed signal sequences are detected in the mutant cell by pulse-labeling. The precursors are processed at very slow rates during a subsequent chase. Genetic analysis indicates that the mutation impairs the function of a gene, termed secY, located at the promoter-distal part of the spc operon. The secY gene is distinct from those genes previously known to specify ribosomal proteins, yet it is within the spc operon. It is suggested that the product of the secY gene is a component of the cellular apparatus that is essential for protein secretion across the cytoplasmic membrane. The gene secY is probably identical with prlA, previously identified as a suppressor of signal sequence mutations.