A comparative study on the hydroperoxide and thiol specificity of the glutathione peroxidase family and selenoprotein P

Glutathione peroxidase catalyzes the reduction of hydrogen peroxide and organic hydroperoxide by glutathione and functions in the protection of cells against oxidative damage. Glutathione peroxidase exists in several forms that differ in their primary structure and localization. We have also shown that selenoprotein P exhibits a glutathione peroxidase-like activity (Saito, Y., Hayashi, T., Tanaka, A., Watanabe, Y., Suzuki, M., Saito, E., and Takahashi, K. (1999) J. Biol. Chem. 274, 2866-2871). To understand the physiological significance of the diversity among these enzymes, a comparative study on the peroxide substrate specificity of three types of ubiquitous glutathione peroxidase (cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, and extracellular glutathione peroxidase) and of selenoprotein P purified from human origins was done. The specific activities and kinetic parameters against two hydroperoxides (hydrogen peroxide and phosphatidylcholine hydroperoxide) were determined. We next examined the thiol specificity and found that thioredoxin is the preferred electron donor for selenoprotein P. These four enzymes exhibit different peroxide and thiol specificities and collaborate to protect biological molecules from oxidative stress both inside and outside the cells.     

Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase

Adenosie, AMP, ADP and ATP activated adenylate cyclase in pig skin (epidermis) slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phophodiesterase inhibitor, papaverine. But another inhibitor, theophylline, strongly blocked the activation of adenylate cyclase by adenosine and adenine nucleotides. Theophylline apparently competed with adenosine for the cell suface receptor. Like theophylline, the addition of adenine alone caused no accumulation of cyclic AMP, but it significantly inhibited the stimulatory effect of adenosine. Guanosine, or guanine, cytidine, uridine, or thymidine nucleotides has no effect on the accumulation of cyclic AMP. Among other adenine nucleotides was tested, adenosine 5′-monophosphoramidate, but not adenosine 5′-monosulfate, significantly increased cyclic AMP especially with the addition of papaverine. Neither 2′- nor 3′-adenylic acid were effective. Our data indicate that pig epidermis has four specific and independent adenylate cyclase systems for adenosine (and adenine nucleotides), histamine, epinephrine and prostaglandin E.     

Internal Spatiotemporal Population Dynamics of Infection with Three Wolbachia Strains in the Adzuki Bean Beetle, Callosobruchus chinensis (Coleoptera: Bruchidae)

The adzuki bean beetle, Callosobruchus chinensis, is infected with three distinct lineages of endosymbiotic bacteria belonging to the genus Wolbachia, which were designated wBruCon, wBruOri, and wBruAus. In an attempt to understand the mechanisms underlying the infection with these three organisms, the spatiotemporal infection dynamics of the three Wolbachia strains was investigated in detail by using a quantitative PCR technique. During the development of C. chinensis, the wBruCon, wBruOri, and wBruAus infection levels consistently increased but the growth patterns were different. The levels of infection plateaued at the pupal stage at approximately 3 × 10⁸, 2 × 10⁸, and 5 × 10⁷ wsp copy equivalents per insect for wBruCon, wBruOri, and wBruAus, respectively. At the whole-insect level, the population densities of the three Wolbachia types did not show remarkable differences between adult males and females. At the tissue level, however, the total densities and relative levels of the three Wolbachia types varied significantly when different tissues and organs were compared and when the same tissues derived from males and females were compared. The histological data obtained by in situ hybridization and electron microscopy were concordant with the results of quantitative PCR analyses. Based on the histological data and the peculiar Wolbachia composition commonly found in nurse tissues and oocytes, we suggest that the Wolbachia strains are vertically transmitted to oocytes not directly, but by way of nurse tissue. On the basis of our results, we discuss interactions among the three coinfecting Wolbachia types, reproductive strategies of Wolbachia, and factors involved in the different cytoplasmic incompatibility phenotypes.     

The Lipid Phase of Thylakoid and Cytoplasmic Membranes from the Blue-Green Algae (Cyanobacteria), Anacystis nidulans and Anabaena variabilis

The lipid phases of the thylakoid and cytoplasmic membranesfrom the blue-green alga, Anacystis nidulans, were studied bya spin-probe method using 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl.The thylakoid and cytoplasmic membranes of this alga were bothin the liquid crystalline state at growth temperature, and inthe phase separation state at about 0?C. The thylakoid membranesentered the phase separation state at a temperature higher thanthe cytoplasmic membranes. The lipid phase of the thylakoidmembranes from Anabaena variabilis was studied in a similarway, and these membranes were found also to undergo the phasetransition. The temperature for the onset of the phase separationand the fluidity of the membrane lipids of both algae dependedon the growth temperature of the culture. (Received April 9, 1984; Accepted June 1, 1984)     

Identification of cultured and uncultured picocyanobacteria from a mesotrophic freshwater lake based on the partial sequences of 16S rDNA

Eight cultured strains (OK01, OK02, OK03, OK05, OK07, OK08, OK09, and OK10) of picocyanobacteria were isolated from Lake Okutama. Five cyanobacterial DNA fragments (DGGE bands; B4, B5, B6, B7, and B8) were obtained from the lake water samples by denaturing gradient gel electrophoresis (DGGE) after polymerase chain reaction (PCR) amplification of 16S ribosomal genes. To classify the picocyanobacterial strains and the DGGE bands, a partial sequence of 16S rDNA was used. Among seven strains, OK01, OK07, and OK09 were identified as the genus Synechococcus and OK02 and OK05 as the genus Phormidium. OK03 was identified as the genus Oscillatoria and was closely related to B4 (100% homology). B5, B6, B7, and B8 were related to the genus Synechococcus. These results revealed that the picocyanobacteria in the lake are phylogenetically diverse. PCR-DGGE analysis is a useful tool to determine picocyanobacterial community structure in freshwater environments. Received: February 25, 2001 / Accepted: July 27, 2001     

Overexpression of yccL (gnsA) and ydfY (gnsB) Increases Levels of Unsaturated Fatty Acids and Suppresses both the Temperature-Sensitive fabA6 Mutation and Cold-Sensitive secG Null Mutation of Escherichia coli

A multicopy suppressor of the cold-sensitive secG null mutation was isolated. The suppressor contained sfa and yccL, the former of which has been reported to be a multicopy suppressor of the fabA6 mutation carried by a temperature-sensitive unsaturated fatty acid auxotroph. Subcloning of the suppressor gene revealed that yccL, renamed gnsA (secG null mutant suppressor), was responsible for the suppression of both the secG null mutation and the fabA6 mutation. In contrast, the sfa gene did not suppress the fabA6 mutation. The ydfY (gnsB) gene, encoding a protein which is highly similar to GnsA, also suppressed both the secG null mutation and the fabA6 mutation. Although both gnsA and gnsB are linked to cold shock genes, the levels of GnsA and GnsB did not exhibit a cold shock response. A gnsA-gnsB double null mutant grew normally under all conditions examined; thus, the in vivo functions of gnsA and gnsB remain unresolved. However, overexpression of gnsA and gnsB stimulated proOmpA translocation of the secG null mutant at low temperature and caused a significant increase in the unsaturated fatty acid content of phospholipids. Taken together, these results suggest that an increase in membrane fluidity due to the increase in unsaturated fatty acids compensates for the absence of the SecG function, especially at low temperature.     

Variation in photoinhibition among Sasa senanensis, Quercus mongolica, and Acer mono in the understory of a deciduous broad-leaved forest exposed to canopy gaps caused by typhoons

To elucidate mechanisms for tolerating sudden increases in light intensity following canopy gap formation, we investigated susceptibility to photoinhibition in the evergreen clonal plant bamboo, Sasa senanensis, and two deciduous broadleaf woody plants, Quercus mongolica, and Acer mono. We measured pre-dawn photochemical efficiency of photosystem II (F _v /F _m) in plants exposed to canopy gaps and in shade-grown plants through the month following gap formation. Photoinhibition (indicated by decreased F _v /F _m) was smallest in S. senanensis and largest in A. mono. S. senanensis had the highest area-based net CO₂ assimilation rate (A _area) and electron transport rate (ETR) under high light conditions. This species also had the highest leaf mass per area (LMA) and leaf nitrogen content per area (N _area). Higher values of LMA and N _area under shade conditions probably contribute to circumvent photoinhibition through maintenance of a higher ETR capacity. Q. mongolica, a gap-dependent species, had properties intermediate between S. senanensis and A. mono; it appeared less susceptible to photoinhibition than the shade-tolerant A. mono. None of the species examined had increased photosynthetic capacity 1 month after gap formation, indicating that shade-grown leaves were unable to fully acclimate to increased light.     

Evaluation of mutation effects on UGT1A1 activity toward 17beta-estradiol using liquid chromatography-tandem mass spectrometry

Mutations in the gene encoding UDP-glucuronosyltransferase 1A1 (UGT1A1) may reduce the glucuronidation of estradiol, bilirubin, etc. In the present study, we used a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method to assay the activities of recombinant mutated UGT1A1 toward 17beta-estradiol (E2), by determining its glucuronide (E2G) content. Direct evidence for glucuronide formation was provided by E2G-specific ion peaks. The UGT1A1 activities of G71R (exon 1), F83L (exon 1), I322V (exon 2) and G493R (exon 5) mutants were 24, 30, 18 and 0.6% of the normal UGT1A1 activity, respectively. In conclusion, our study showed that LC/MS/MS enabled accurate evaluation of the effects of mutations on recombinant UGT1A1 activity towards E2.     

Contribution of spermatozoal centrosomes to the microtubule-organizing centre in Antarctic minke whale ( Balaenoptera bonaerensis )

Using an interspecies microinsemination assay with bovine oocytes, it was examined whether centrosomes of Antarctic minke whale spermatozoa function as the microtubule-organizing centre (MTOC). Bull and rat spermatozoa were used as positive and negative controls, respectively. Vitrified-warmed bovine mature oocytes were subjected to immunostaining against alpha-tubulin 4-6 h after intracytoplasmic injection (ICSI) of 5 mM dithiothreitol-treated spermatozoa. Aster formation occurred from whale spermatozoa (33%) and bull spermatozoa (33%), but very little from rat spermatozoa (3%). Activation treatment for the microinseminated oocytes with 7% ethanol + 2 mM 6-dimethylaminopurine resulted in a similar proportion of oocytes forming a whale sperm aster (35% vs 27% in the non-treated group; 4 h after ICSI) but a significantly larger aster (ratio of aster diameter to oocyte diameter, 0.57 vs 0.30 in the non-treated group). These results indicate that the centrosome introduced into bovine oocytes by whale spermatozoa contributes to the MTOC and that assembly of the microtubule network is promoted by oocyte activation.