Nitric oxide inhibits oocyte meiotic maturation

Recently, we have found that the nitrate/nitrite concentrations in preovulatory follicles significantly decrease after hCG injection and that inducible nitric oxide synthase (iNOS) plays a main role in the decrease of the intrafollicular nitric oxide (NO) concentration. The purpose of the present study was to investigate the role of NO on oocyte meiotic maturation and to consider the physiological means of the decrease in intrafollicular NO concentration. Immature rats received 15 IU of eCG, and ovaries were removed under ether anesthesia 48 h later. Each ovary was bluntly divided into five or six pieces containing from four to seven preovulatory follicles under the microscope and then incubated with hCG, aminoguanidine (AG; an iNOS inhibitor), or S-nitroso-L-acetyl penicillamine (SNAP; an NO donor) for 5 h. After incubation, preovulatory follicles were punctured, and germinal vesicle breakdown (GVBD) was observed. Also, cGMP concentrations in these follicles were measured. Next, denuded oocytes were recovered from preovulatory follicles at 48 h after injection of 15 IU of eCG and incubated with SNAP with or without ferrous hemoglobin. Every 30 min up to 12 h, GVBD was observed. Both AG and hCG promoted GVBD, and SNAP prevented this effect. In addition, AG decreased intrafollicular cGMP levels, and the concomitant addition of SNAP prevented this decrease. Finally, SNAP dose-dependently inhibited GVBD in denuded oocyte, and this effect of SNAP was reversed by the addition of hemoglobin. We conclude that the iNOS-NO-(cGMP) axis may play an important role in oocyte meiotic maturation.     

Complex II inactivation is lethal in the nematode Caenorhabditis elegans

RNA-mediated interference (RNAi) was employed to systematically inactivate the four subunits of complex II in the mitochondrial electron transport chain. Embryonic lethality was the predominant result of inactivating three subunits (ceSDHB, ceSDHC, and ceSDHD) when using the soaking method to inactivate RNA. The feeding method was employed to deliver dsRNA from the fourth subunit (ceSDHA) to wild-type, mev-1 (mutated in ceSDHC of complex II), and gas-1 animals (mutated in a complex I gene). Survival was reduced only in the mev-1 genetic background, and in an oxygen-dependent fashion. Collectively, these data provide further evidence that compromised complex II integrity can result in sensitivity to oxidative stress.     

Contribution of the Disulfide Bond of the A Subunit to the Action of Escherichia coli Heat-Labile Enterotoxin

Escherichia coli heat-labile enterotoxin (LT) consists of an A subunit and five B subunits. These subunits oligomerize into an assembled holotoxin within the periplasm. Structural analysis of LT has revealed that the A subunit interacts with the B subunit through its carboxy terminus. This indicates that the carboxy-terminal portion of the protein is required for assembly of holotoxin in the periplasm. However, it is not known whether other regions of the A subunit contribute to the assembly. The A subunit constituting the holotoxin contains a disulfide bond between Cys-187 and Cys-199. It has been observed in many proteins that the intramolecular disulfide bond is deeply involved in the function and tertiary structure of the protein. We speculated that the disulfide bond of the A subunit contributes to the assembly in the periplasm, although the bond is not a structural element of the carboxy-terminal portion of the A subunit. We replaced these cysteine residues of the A subunit by oligonucleotide-directed site-specific mutagenesis and analyzed the LTs produced by cells containing the mutant LT genes. The amount of the mutant holotoxin produced was small compared with that of the wild-type strain, indicating that the disulfide bond of the A subunit contributes to the structure which functions as the site of nucleation in the assembly. A reconstitution experiment in vitro supported the notion. Subsequently, we found that the mutant A subunit constituting holotoxin is easily degraded by trypsin and that in cells incubated with mutant LTs, the lag until the intracellular cyclic AMP begins to accumulate is longer than in cells incubated with native LTs. These results might be useful for the analysis of the interaction of LT with target cells at the molecular level.     

Reduced allelopathic inhibition of lettuce (Lactuca sativa) growth caused by velvet bean (Mucuna pruriens) under 3D-clinorotation.

Allelopathy between Mucuna pruriens (velvet bean) and Lactuca sativa (lettuce) was studied under 3D-clinorotation. Growth of both roots and shoots of lettuce seedlings was suppressed by the presence of velvet bean. The degree of suppression was less on the clinostat compared to the normal static earth gravity. L-DOPA (L-3, 4-dihydroxyphenylalanine) is known to be a major substance in allelopathy of velvet bean. Amount of L-DOPA diffused out from a sintered filter paper into agar medium was compared between clinorotation and control group, and found no significant difference. It was concluded that some factors related to release, transport, and sensing phenomena of allelopathic substances may be responsible to the new findings in this study.     

Identification of the cDNA of differentially expressed genes in the transition zone of cucumber seedlings by fluorescent differential display.

Cucumber seedlings have potential to develop two pegs on the transition zone between the hypocotyl and root. Seedlings grown in a horizontal position suppress the development of the peg on the upper side of the transition zone in response to gravity. To understand how the response to gravity suppresses peg formation, we screened cucumber mRNAs to identify the mRNA in the non-peg side that accumulates more than in the peg side. For screening, we determined conditions of fluorescent differential display (FDD). Then, we carried out FDD and found 4 cDNA bands that repeatedly showed stronger intensity in the non-peg side than the peg side. We isolated one of these RT-PCR products. Northern blotting showed the pattern of its mRNA accumulations corresponding to the results of FDD.