Keikichi Uchida

Modification of a single tryptophan residue in human Cu,Zn-superoxide dismutase by peroxynitrite in the presence of bicarbonate

Human recombinant Cu,Zn-SOD was reacted with peroxynitrite in a reaction mixture containing 150 mM potassium phosphate buffer (pH 7.4) 25 mM sodium bicarbonate, and 0.1 mM diethylenetriamine pentaacetic acid. Disappearance of fluorescence emission at 350 nm, which could be attributed to modification of a single tryptophan residue, was observed in the modified enzyme with a pH optimum of around 8.4. A fluorescence decrease with the same pH optimum was also observed without sodium bicarbonate, but with less efficiency. Amino acid contents of the modified enzyme showed no significant difference in all amino acids except the loss of a single tryptophan residue of the enzyme. The peroxynitrite-modified enzyme showed an increase in optical absorption around 350 nm and 30% reduced enzyme activity based on the copper contents. The modified enzyme showed the same electron paramagnetic resonance spectrum as that of the control enzyme. The modified Cu,Zn-SOD showed a single protein band in sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS--PAGE) and five protein bands in non-denaturing PAGE. From this evidence, we conclude that nitration and/or oxidation of the single tryptophan 32 and partial inactivation of the enzyme activity of Cu,Zn-SOD is caused by a peroxynitrite-carbon dioxide adduct without perturbation of the active site copper integrity.

Induction of Oogenesis in Mosquitoes (Diptera: Culicidae) by Infusion of the Hemocoel with Amino Acids

Abstract As done previously with adult females of Culex pipiens pallens Coquillett, a mixture of 17 amino acids was infused into the hemocoel of females of seven anautogenous and one autogenous mosquito species belonging to three genera. In Culex. p. quinquefasciatus Say, Cx. tritaeniorhynchus Giles, Cx. kyotoensis Yamaguti & LaCasse, Aedes albopictus (Skuse), Armigeres subalbatus (Coquillett), and Cx. p. molestus Forskal, which previously had laid autogenously matured first batch of eggs, ovarian development was stimulated and frequently continued to maturity. In most mosquitoes, the number of mature follicles nearly doubled when the period of infusion was extended from 24 to 48 h. Therefore, the two previously indicated roles of amino acids, one to initiate ovarian development and the other to regulate the number of maturing oocytes, were confirmed in these species. In Cx. halifaxii Theobald and Ae. japonicus (Theobald), however, the frequency of activation and maturation of ovaries was low compared with the other species, indicating that those species may require some factors other than an increase in amino acids for normal ovarian development after a blood meal.

Reproductive activity and survival of Culex pipiens pallens and Culex quinquefasciatus (Diptera: Culicidae) in Japan at high temperature.

Abstract The egg hatchability, insemination, and longevity of Japanese Culex pipiens pallens Coquillett and Japanese Culex quinquefasciatus Say were compared at 25 and 30°C. Egg hatchability was high in Cx. p. pallens at 25°C, but it was very low at 30°C because almost no females were inseminated at this temperature. In Cx. quinquefasciatus, the egg hatchability and insemination rates were very high, even at 30°C. The longevity of adult females and males was generally shorter in Cx. p. pallens than in Cx. quinquefasciatus at both temperatures. Because high temperatures may restrict the spread of Cx. p. pallens, we suggest that even if this species spreads to Okinawa, the possibility of it becoming established is very low.

Balanced amino acid composition essential for infusion-induced egg development in the mosquito (Culex pipiens pallens)

Abstract Amino acid mixtures, in which one or more amino acids were omitted from a basal mixture of 17 amino acids, were infused into the hemocoels of female Culex pipiens pallens mosquitoes. The resulting oogenesis was compared with previous studies of oogenesis induced by the basal mixture to evaluate the oogenic stimulatory effect of individual amino acids. Infusion of solutions deficient in isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan or valine, which are known to be absolutely essential for producing mature eggs, failed even to initiate oogenesis. Removal of arginine (also absolutely essential for egg production), cysteine, histidine or methionine from the basal mixture resulted in decreased activation of oogenesis. Single elimination of other amino acids (alanine, glycine, proline and serine) resulted in high percentages of oogenesis and egg maturation, comparable to the levels produced by basal mixture infusion. The combination of asparagine and glutamine, which were reported to increase in the hemolymph of blood-fed females, can be substituted for asparagine and glutamate in the basal mixture to give a high level of oogenesis. On the other hand, infusion of a mixture comprised of the eight amino acids absolutely essential for egg production showed only a slight stimulation of oogenesis initiation. Single amino acid infusion of isoleucine or threonine also failed to stimulate mosquito oogenesis. Thus, we have concluded that initiation of mosquito oogenesis requires an appropriate balance in increased hemolymph amino acids rather than just one or a few special trigger amino acids.

Mosquito (Culex pipiens pallens) egg development induced by infusion of amino acids into the hemocoel

Abstract A mixture of amino acids was infused into the hemocoel of female Culex pipiens pallens mosquitoes at a very slow rate. We attempted to simulate the natural concentration increase of hemolymph amino acids, in order to determine whether amino acids derived from a blood meal trigger mosquito oogenesis. Infusion of a 7.5% (w/v) amino acid mixture for 14–16 h at a flow rate of 0.083 μl/h (2.0 μl/24h) activated oocytes to develop to intermediary stages (stages IIb-IIIa). The same infusion continued for more than 20 h resulted in egg maturation in most of the treated females. The number of mature eggs was not significantly different among the 14–24 h infusions but showed a proportional increase between the 24 and 48 h infusions. The hemolymph amino acid concentrations of these infused females did not greatly differ from the levels observed in blood-fed females, suggesting that the oogenic stimulatory effect of the infused amino acids was close to the natural process. This indicated that amino acids resulting from blood meal digestion are not only utilized for yolk protein synthesis but also have the potential to initiate and promote mosquito oogenesis. In addition, oocyte growth caused by infusion without any distention of organs or body wall clearly indicates that stretching is not an essential trigger for this event.

Effects of Mating on Oogenesis Induced by Amino Acid Infusion, Amino Acid Feeding, or Blood Feeding in the Mosquito Anopheles stephensi (Diptera: Culicidae)

Abstract Hemocoel infusion of an amino acid mixture at a concentration of 7.5% and 10% (wt:vol) for 24 h rarely activated ovarian development in either mated or unmated females of Anopheles stephensi Liston. Infusion of either concentration mixture for 48 h into unmated females was also scarcely stimulatory, but the same 48 h infusion into mated females resulted in ovarian maturation in 25% (7.5% mixture) or in 78% (10% mixture) of the infused specimens. Similarly, feeding on an amino acid mixture (7.5%) containing sugar induced 44% ovarian maturation among mated females, while only 3% of unmated females showed matured oocytes. The increase of hemolymph amino acid concentration levels may be one of the essential factors for oogenesis of An. stephensi, and mating enhances ovarian development, especially in females ingesting poor meals. However, some additional factor(s) may be included for full development comparable to that induced by blood meals.

Cibarial sensilla and pharyngeal valves in Aedes albopictus (skuse) and Culex pipiens pallens coquillett (Diptera : Culicidae)

Abstract The inner surface of the cibarium was studied with scanning electron microscope. Types and distribution of sensilla are similar to those observed by light microscope in different mosquitoes, except for some minor details. On the dorsal wall are 4 types of sensilla: palatal papillae, campaniform sensilla, dorsal papillae and trichoid sensilla. Except the last-named, they show no sexual or specific differences either in appearance or number. Trichoid sensilla in A. albopictus differ in number between males (2 pairs) and females (3 pairs). Ventral papillae on the ventral wall are hair-like in Aedes and pyriform in Culex . The pharyngeal valves comprise dorsal and ventral valves, derived respectively from the respective walls of the cibarium. The dorsal valve has projections that are digitate in Aedes and lanceolate in Culex . Distribution of these projections differs with sex in Culex , but not in Aedes . The ventral pharyngeal valve in both sexes of Aedes bears finger-like projections; those in females of Culex bear rows of teeth along the hind margin that appears smooth in the males.

Ovarian development induced in decapitated female Culex pipiens pallens mosquitoes by infusion of physiological quantities of 20-hydroxyecdysone together with amino acids.

Infusion of 20-hydroxyecdysone into the hemocoel of unfed decapitated female Culex pipiens pallens mosquitoes, at a very low rate of 500-2000pg per day, often stimulated oögenesis of this species, when the hormone was infused together with amino acids. The hormone alone or amino acids alone showed no such stimulatory effect. Previous reports that using an abdomen ligated immediately after a blood meal for hormone injection reduced the quantity of 20-hydroxyecdysone needed to activate unfed female Aedes aegypti by a few thousand times, are therefore due mainly to a sufficient supply of amino acids from the midgut in the isolated abdomen.

Elimination of protein-food ingested into the crop, and failure of ovarian development in female mosquitoes, Culex pipiens pallens

ABSTRACT. A protein‐food rarely activates oogenesis in female mosquitoes when the protein is mixed with sugar and therefore ingested into the crop. A high occurrence of ovarian development occurs, however, when the protein is stored in the midgut. Examination of female C. pipiens pallens fed on egg albumin containing sugar revealed that the protein, once stored in the crop, was easily eliminated without any absorption and that ovarian development occurred only in those few females which retained some part of the ingested albumin for more than 24 h after feeding. Sugar mixed with the albumin was not detected in any excreta of fed females, suggesting that the sugar was digested irrespective of the protein elimination.

Elimination of protein-food ingested into the crop, and failure of ovarian development in female mosquitoes, Culex pipiens pollens. II. Effect of sugar-free egg albumin.

ABSTRACT. Sugar‐free egg albumin solution was ‘force fed’ to female C.pipiens pallens Coquillett, by first stimulating their labella briefly with 1.0 M sucrose, so that they then took the protein meal into the crop. This protein‐food was nevertheless easily eliminated by the females, totally undigested, so that it failed to activate oogenesis, just as sugared albumin fails to do so also.