Effect of detergents on sterol synthesis in a cell-free system of yeast

In order to obtain information about the reactivity of enzymes in sterol synthesis of yeast, the effects of some detergents were investigated. Among the detergents used, Triton X-100 was found to exert a unique action, and its effect on the incorporation of 14C-labeled acetate, mevalonate, farnesyl pyrophosphate, or S-adenosyl-L-methionine into squalene, 2,3-oxidosqualene, and sterols in a cell-free system was examined. Triton X-100 showed virtually no effect on the enzyme activities in the reactions from acetyl CoA to farnesyl pyrophosphate, but it had a marked effect on reactions from farnesyl pyrophosphate to ergosterol. Evidence was obtained suggesting that Triton X-100 apparently activated squalene synthetase (EC 2.5.1.21) but inhibited squalene epoxidase (EC 1.14.99.7) and delta 24-sterol methyltransferase (EC 2.1.1.41). The activity of epoxidase was protected from the inhibition by increasing the concentration of cell-free extracts or by the prior addition of lecithin liposomes to the reaction mixture. The inhibition of methyltransferase was partially reversed by treatment with Bio-heads SM-2, but that of epoxidase was not reversed by the treatment.     

Effects of dopamine on chemoreflexes in breathing

The effects of intravenous infusion of dopamine (20 microgram.min) on the steady-state ventilatory and carotid chemoreceptor responses to successive levels of isocapnic hypoxia and hyperoxic hypercapnia were investigated in cats anesthetized with alpha-chloralose. Dopamine infusion was followed by a maximal decrease in ventilation in about 20 s. Thereafter, the effect diminished and stabilized. Termination of dopamine infusion was promptly followed by an increase in ventilation. These ventilatory responses were smaller than the corresponding carotid chemoreceptor responses. The steady-state effect of dopamine infusion was to diminish ventilation at all levels of arterial O2 tension, the decrease being greater during hypoxia than that during hyperoxia. Bilateral section of the carotid sinus nerves significantly diminished but did not abolish the inhibitory effect of dopamine on ventilation during hyperoxia. Thus the ventilatory depression due to dopamine infusion is not entirely due to its effect on the carotid chemoreceptors. Dopamine decreased ventilatory responses to successive levels of hypercapnia by the same magnitude without changing the slope of the response curves. The steady-state relationship between chemoreceptor activity and ventilation shows that the ventilatory equivalent for carotid chemoreceptor activity is increased during dopamine infusion because of its greater inhibitory effect on carotid chemoreceptor activity than on ventilation with the decrease of arterial O2 tension.     

Biological control of arrowhead scale,Unaspis yanonensis,by parasitic wasps introduced from the People's Republic of China

In 1980, the authors went to the People's Republic of China in search of the natural enemies of the arrowhead scale,Unaspis yanonensis Kuwana, and succeeded in collecting 2 species of parasitic wasps of this pest namelyPhyscus fulvus Compere & Annecke andAphytis yanonensis DeBach & Rosen. The development, reproduction and longevity ofAphytis yanonensis were studied under constant temperature conditions. The number of generations a year was estimated to be from 10 to 12. Reproduction ofPhyscus fulvus is arrhenotokous, and development from egg to adult required 25 days at 25°C and 33 days at 20°C. The number of generations was estimated at 4 to 5 a year. Both parasites were released in several citrus groves in the Shizuoka Prefecture, of central Japan during June and July, 1981. They successfuly hibernated and each stage of development was found from March to December, 1982. They are considered to be potentially highly effective against the arrowhead scale. We have succeeded in establishing bothAphytis yanonensis andPhyscus fulvus in several districts in the Shizuoka Prefecture.

---

Résumé Les auteurs se rendirent en 1980 en République Populaire de Chine pour entreprendre une recherche sur les ennemis naturels deUnaspis yanonensis Kuwana et découvrirent 2 espèces d'hyménoptères parasites de la cochenille. Les ennemis naturels introduits furentPhyscus fulvus Compere & Annecke etAphytis yanonensis DeBach & Rosen. La croissance, la reproduction et la longévité deA. yanonensis furent étudiées à température constante. Le nombre de générations annuelles a été estimé entre 10 et 12. La reproduction deP. fulvus est de type arrhénotoque et la durée du développement de l'œuf à l'adulte, nécessite 25 jours à 25°C et 33 jours à 20°C. Le nombre de générations annuelles a été estimé à 4 ou 5. Les 2 parasites ont été libérés dans plusieurs plantations d'agrumes de la préfecture de Shizuoka, région centrale du Japon pendant les 2 mois de juin et juillet 1981 et ont hiverné avec succès. Chaque stade de développement a été observé de mars à décembre 1982. Ces parasites sont considérés comme des ennemis naturels hautement efficaces de la cochenille. Nous avons introduit avec succèsAphytis yanonensis etPhyscus fulvus en provenance de Chine dans diverses régions de la préfecture de Shizuoka, partie centrale du Japon.

     

Role of cysteine residues in human NADH-cytochrome b5 reductase studied by site-directed mutagenesis. Cys-273 and Cys-283 are located close to the NADH-binding site but are not catalytically essential

Human NADH-cytochrome b5 reductase (EC 1.6.2.2) contains 4 cyteine residues (Cys-203, -273, -283, and -297). Cys-283 was previously proposed to be involved in NADH binding by chemical modification (Hackett, C. S., Novoa, W. B., Ozols, J., and Strittmatter, P. (1986) J. Biol. Chem. 261, 9854-9857). In the present study the role of cysteines in the enzyme was probed by replacing these residues by Ser, Ala, or Gly employing site-directed mutagenesis and chemical modification. Four mutants, in which 1 of the 4 Cys residues was replaced by Ser, retained comparable kcat and Km values to those of the wild type. All of these mutants were as sensitive as the wild type to treatment with SH modifiers, while a double mutant, C273S/C283S was resistant. Since inhibition by SH modifiers was protected by NADH, Cys-273 and Cys-283 were implicated to be close to the NADH-binding site. C273A and C273A/C283A mutants showed approximately one-fifth of the enzyme-FAD reduction rate of the wild type as revealed by steady-state kinetics and by stopped-flow analysis. Anaerobic titration has shown that reduction and re-oxidation processes including formation of the red semiquinone of these mutants were not significantly altered from those of the wild type. From these results it was concluded that none of the Cys residues of the enzyme are essential in the catalytic reaction, but Cys-273 conserved among the enzymes homologous to NADH-cytochrome b5 reductase homologous to NADH-cytochrome b5 reductase plays role(s) in facilitating the reaction. A difference spectrum with a peak at 317 nm, which was formerly considered to be derived from the interaction between NAD+ and Cys-283 of the reduced enzyme, appeared upon binding of NAD+ not only to the reduced wild type enzyme but also to the C273A/C283A mutant in which both of the Cys residues close to the NADH-binding site were replaced.     

Melanocyte growth factor in normal human skin

Normal human skin is shown to contain melanocyte growth factor (MeGF). We found MeGF activity in extracts of both the epidermal portion of skin and the dermal portion. This activity was completely adsorbed onto heparin beads and eluted by 2.5 M NaCl. In addition, the activity of both extracts was completely blocked by antibodies directed against basic fibroblast growth factor (bFGF). It is suggested that melanocytes in epidermis are supported by bFGF-like MeGF in normal human skin.     

The effect of quercetin on cell cycle progression and growth of human gastric cancer cells

Quercetin, a flavonoid, is found in many plants, including edible fruits and vegetables. We examined the effects on cell growth of human malignant cells derived from the gastrointestinal tract and on cell cycle progression. Quercetin markedly inhibited the growth of human gastric cancer cells and the IC50 value was 32-55 microM. DNA synthesis was suppressed to 14% of the control level by the treatment with 70 microM quercetin for 2 days. Furthermore, quercetin blocked cell progression from the G1 to the S phase.     

Cloning and nucleotide sequence of the ispA gene responsible for farnesyl diphosphate synthase activity in Escherichia coli

The molecular cloning and the determination of the nucleotide sequence of the ispA gene responsible for farnesyl diphosphate (FPP) synthase [EC 2.5.1.1] activity in Escherichia coli are described. E. coli ispA strains have temperature-sensitive FPP synthase, and the defective gene is located at about min 10 on the chromosome. The wild-type ispA gene was subcloned from a lambda phage clone containing the chromosomal fragment around min 10, picked up from the aligned genomic library of Kohara et al. [Kohara, Y., Akiyama, K., & Isono, K. (1987) Cell 50, 495-508]. The cloned gene was identified as the ispA gene by the recovery and amplification of FPP synthase activity in an ispA strain. A 1,452-nucleotide sequence of the cloned fragment was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected molecular weights of 8,951 and 32,158, respectively. A part of the deduced amino acid sequence of ORF-2 showed similarity to the sequences of eucaryotic FPP synthases and of crtE product of a photosynthetic bacterium. The plasmid carrying ORF-2 downstream of the lac promoter complemented the defect of FPP synthase activity of the ispA mutant, showing that the product encoded by ORF-2 is the ispA product. The maxicell analysis indicated that a protein of molecular weight 36,000, approximately consistent with the molecular weight of the deduced ORF-2-encoded protein, is the gene product.