The stage-dependent inhibitory effect of porcine follicular cells on the development of preantral follicles.

The objective of this study was to examine the effects of follicular cells on the in vitro development of porcine preantral follicles. In Experiment 1, one preantral follicle alone (Trt 1) was cocultured with a follicle of the same size with oocytes (Trt 2) or without oocytes (Trt 3). Preantral follicles cultured alone in vitro for 12 days had greater follicle diameters (1017 +/- 96 microm versus 706 +/- 69 or 793 +/- 72 microm, P < 0.05), growth rates (201 +/- 0.3 versus 103 +/- 0.2 or 128 +/- 0.2, P < 0.05) and oocyte survival rates (73% versus 48, or 25%, P < 0.05) than other groups. The inhibitory effects of follicle cells on the growth of preantral follicles and oocyte survival rates were not enhanced by the addition of oocytectomized preantral follicles (Experiment 2). Follicles were cocultured with different sources of follicular cells in other experiments. Coculture with cumulus cells enhanced oocyte survival compared to the control (without coculture) and mural follicular cell groups (Experiment 3). The growth and survival rates of oocytes collected from the group of follicles cocultured with cumulus cells from large antral follicles (>3 mm) were greater (P < 0.05) than those from small antral follicles (<3 mm), or than the control group (without cumulus cells, experiment 4). No significant differences in the follicular diameters (674 +/- 30 microm versus 638 +/- 33 and 655 +/- 28 microm) and growth rate (105% versus 94 and 105%) were observed among the preantral follicles of the different treatments (P > 0.05). Taken together, coculture with the cells from large antral follicles (>3 mm) exerted a significant positive effect on oocyte survival. The growth and oocyte survival of preantral follicle cocultured with the same size of follicles (with or without oocyte) were inhibited. Growth and survival rates of preantral follicles and oocytes are improved by coculturing them with the cumulus cells derived from larger antral follicles.

Genetic toxicity of N-methylcarbamate insecticides and their N-nitroso derivatives.

N-Methylcarbamate esters are an important group of insecticides. They have lower acute toxicity to vertebrates than organophosphates, although their genotoxicity has not been adequately studied. Here we investigate the cytotoxicity and genotoxicity of N-methylcarbamate insecticides and their N-nitroso derivatives in Chinese hamster V79 cells, using the hprt locus as a marker, and also assess inhibition of gap junctional intercellular communication. N-Methylcarbamate insecticides were chemically N-nitrosated to obtain the N-nitroso derivatives. N-Nitrosation greatly increased the cytotoxicity and mutagenicity of N-methylcarbamates at the hprt locus in Chinese hamster V79 cells. The mutagenic potential of N-nitroso-N-methylcarbamates was much higher than those of many other known mutagenic nitroso compounds, as well as some non-nitroso mutagenic alkylating agents. Parental N-methylcarbamates themselves were not mutagenic, however, they inhibited gap junctional intercellular communication half as effectively as the well-studied tumor promoter 12-O-tetradecanoylphorbol-13-acetate. The findings show that N-methylcarbamate insecticides and their N-nitroso derivatives have the potential to act through mediation of epigenetic and genotoxic mechanisms respectively in the multiple stages of chemical carcinogenesis.

Litebamine N-homologues: preparation and anti-acetylcholinesterase activity.

Litebamine N-homologues were easily prepared from laurolitsine, generally via three reaction steps (N-alkylation, solvolysis with 1 M NH4OAc under reflux, and the Mannich reaction) in more than 80% overall yield. Among the prepared compounds, N-propyl-, N-isobutyl-, and N-isopropylnorlitebamines exhibited moderate antiacetylcholinesterase activity (IC50 ca. 7.0 microM), while the corresponding N-metho salt of N-propylnorlitebamine showed potent activity (IC50 2.70 microM).

Purification and characterization of a glycerol oxidase from Penicillium sp. TS-622.

A novel extracellular glycerol oxidase was purified 39-fold from wheat bran culture of a soil-isolated Penicillium strain TS-622 with an overall yield of 3%. The addition of Triton X-100 into the extraction buffer improved the extraction yield by 90 times, indicating that the enzyme is bound to the cell surface. The molecular weight of this enzyme was 400,000 as determined by size-exclusion high-performance liquid chromatography. The optimum pH was from 6 to 7 and the optimum temperature was 45 degrees C. This enzyme showed high specificity toward dihydroxyacetone and glycerol. It was inhibited by KCN, NaN3, and hydroxylamine.

Purification and partial characterization of an alkaline lipase from Pseudomonas pseudoalcaligenes F-111.

An extracellular alkaline lipase of alkalophilic Pseudomonas pseudoalcaligenes F-111 was purified to homogeneity. The apparent molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 32,000, and the isoelectric point was 7.3. With p-nitrophenyl esters as its substrates, the enzyme shows preference for C12 acyl and C14 acyl groups. It was stable in the pH range of 6 to 10, which coincides with the optimum pH range.

NMR assignments of territrems A, B, and C and the structure of MB2, the major metabolite of territrem B by rat liver microsomal fraction.

The 1H- and 13C-nmr assignments of territrems A, B, and C [1-3] were made by using nOe and 2D nmr techniques. Following the same methods, the structure of MB2 [4], the major product of territrem B incubated with rat liver microsomal fraction, was determined as a hydroxylation product at the pro S methyl group of C-4 of territrem B.

Biotransformation of territrems by S9 fraction from rat liver.

Male Wistar rats were pretreated with phenobarbital, 3-methyl-cholanthrene, or polychlorinated biphenyl. The S9 fraction was isolated from their livers. An amount of 40-microliters territrem (TRA, B, or C) (1 mg/ml methanol) was added to 5-ml reaction mixtures containing S9 (8 mg protein), NADP sodium salt (20 mumol), glucose-6-phosphate monosodium salt (25 mumol), MgCl2 (40 mumol), KCl (165 mumol), and sodium phosphate buffer, 0.1 M, pH 7.4, after preincubation for 5 min. Further incubation was carried out for 30 min by shaking (100 ocillations/min). The reaction was stopped by adding 2 ml acetone. The acetone was then removed by evaporation in a hood at room temperature. The residue was lyophilized and extracted with 2 ml methanol 3 times. When TRB was a substrate, at least four blue fluorescent products, designated as MB1, MB2, MB3, and MB4, were found in the methanol extract by TLC under view of long-wave UV light. MB2 was the major product. When TRA or TRC was a substrate, two products, MA1 (the major product) and MA2 from TRA, and one product, MC from TRC, were, respectively, detected in the methanol extract. The formation of the products was dependent on the presence of S9, NADP, glucose-6-phosphate, and territrem. The reaction was enhanced by pretreatment of rats with phenobarbital. It was demonstrated that MB2 and MA1 are hydroxylated products of the methyl group at the C4 position of TRB and TRA. MB4 was identified as TRC. MC was shown to be identical to MB1, which was the hydroxylated product at the methyl group of C4 position of TRC.(ABSTRACT TRUNCATED AT 250 WORDS)