Two Menkes-type atpases supply copper for photosynthesis in Synechocystis PCC 6803.

Synechocystis PCC 6803 contains four genes encoding polypeptides with sequence features of CPx-type ATPases, two of which are now designated pacS and ctaA. We show that CtaA and PacS (but not the related transporters, ZiaA or CoaT) facilitate switching to the use of copper (in plastocyanin) as an alternative to iron (in cytochrome c(6)) for the carriage of electrons within the thylakoid lumen. Disruption of pacS reduced copper tolerance but enhanced silver tolerance, and pacS-mediated restoration of copper tolerance was used to select transformants. Disruption of ctaA caused no change in copper tolerance but reduced the amount of copper cell(-1). In cultures supplemented with 0.2 microm copper, photooxidation of cytochrome c(6) (PetJ) was depressed in wild-type cells but remained elevated in both Synechocystis PCC 6803(ctaA) and Synechocystis PCC 6803(pacS). Conversely, plastocyanin transcripts (petE) were less abundant in both mutants at this [copper]. Synechocystis PCC 6803(ctaA) and Synechocystis PCC 6803(pacS) showed increased iron dependence with impaired growth in deferoxamine mesylate (iron chelator)-containing media. Double mutants also deficient in cytochrome c(6), Synechocystis PCC 6803(petJ,ctaA) and Synechocystis PCC 6803(petJ,pacS), were viable, but the former had increased copper dependence with severely impaired growth in the presence of bathocuproinedisulfonic acid (copper chelator). Analogous transporters are likely to supply copper to plastocyanin in chloroplasts.

The effect of the erg26-1 mutation on the regulation of lipid metabolism in Saccharomyces cerevisiae.

A temperature-sensitive Saccharomyces cerevisiae mutant harboring a lesion in the ERG26 gene has been isolated. ERG26 encodes 4alpha-carboxysterol-C3 dehydrogenase, one of three enzymatic activities required for the conversion of 4,4-dimethylzymosterol to zymosterol. Gas chromatography/mass spectrometry analyses of sterols in this mutant, designated erg26-1, revealed the aberrant accumulation of a 4-methyl-4-carboxy zymosterol intermediate, as well as a novel 4-carboxysterol. Neutral lipid radiolabeling studies showed that erg26-1 cells also harbored defects in the rate of biosynthesis and steady-state levels of mono-, di-, and triglycerides. Phospholipid radiolabeling studies showed defects in the rate of biosynthesis of both phosphatidic acid and phosphatidylinositol. Biochemical studies revealed that microsomes isolated from erg26-1 cells contained greatly reduced 4alpha-carboxysterol-C3 dehydrogenase activity when compared with microsomes from wild type cells. Previous studies have shown that loss of function mutations in either of the fatty acid elongase genes SUR4/ELO3 or FEN1/GNS1/ELO2 can "bypass" the essentiality of certain ERG genes (Ladeveze, V., Marcireau, C., Delourme, D., and Karst, F. (1993) Lipids 28, 907-912; Silve, S., Leplatois, P., Josse, A., Dupuy, P. H., Lanau, C., Kaghad, M., Dhers, C., Picard, C., Rahier, A., Taton, M., Le Fur, G., Caput, D., Ferrara, P., and Loison, G. (1996) Mol. Cell. Biol. 16, 2719-2727). Studies presented here have shown that this sphingolipid-dependent "bypass" mechanism did not suppress the essential requirement for zymosterol biosynthesis. However, studies aimed at understanding the underlying physiology behind the temperature-sensitive growth defect of erg26-1 cells showed that the addition of several antifungal compounds to the growth media of erg26-1 cells could suppress the temperature-sensitive growth defect. Fluorescence microscopic analysis showed that GFP-Erg26p and GFP-Erg27p fusion proteins were localized to the endoplasmic reticulum. Two-hybrid analysis indicated that Erg25p, Erg26p, and Erg27p, which are required for the biosynthesis of zymosterol, form a complex within the cell.

Identification, characterization, and partial purification of 4 alpha-carboxysterol-C3-dehydrogenase/ C4-decarboxylase from Zea mays.

A microsomal preparation from seedlings of Zea mays catalyzed the NAD+-dependent oxidative decarboxylation of several substrates, including 4alpha-carboxy-cholest-7-en-3beta-ol, synthesized according to a new procedure, giving the first in vitro evidence for this enzymatic activity in a higher plant. A GC assay has been developed to detect the Delta7-cholestenone produced and the kinetic parameters of the microsomal system have been established. 4alpha-Carboxysterol decarboxylation shows an exclusive requirement for an oxidized pyridine nucleotide, with NAD+ being more efficient than NADP+. The decarboxylation reaction is independent of molecular oxygen. 4alpha-Carboxysterol-C3-dehydrogenase/C4-decarboxylase (4alpha-CD) is a microsome-bound protein which can be efficiently solubilized by detergents, including Brij W-1 and sodium cholate. The Brij W-1-solubilized enzyme was partially purified 290-fold by a combination of DEAE anion-exchange chromatography, Cibacron blue 3GA-agarose dye chromatography, and gel permeation. The apparent molecular mass of 4alpha-CD in sodium cholate was estimated to be 45 kDa. These results support the contention that demethylation at C4 of plant sterols is composed of two separate processes: an oxygen- and NAD(P)H-dependent oxidation of the 4alpha-methyl group to produce the 4alpha-carboxysterol metabolite (S. Pascal et al., J. Biol. Chem. 268, 11639, 1993) followed by oxygen-independent dehydrogenation/decarboxylation to produce an obligatory 3-ketosteroid.

[Anesthesia using propofol during surgery of strabismus in children. A comparison of two different protocols of induction and maintenance]. / Anesthésie au propofol lors de la chirurgie du strabisme chez l'enfant. Comparaison de deux protocoles différents d'induction et d'entretien.

The purpose of this study is an investigation of two protocols using propofol as induction and maintenance agent in 100 children scheduled for strabismus surgery (4-8 year, ASA I, NYHA I). Protocol I; Propofol 6 mg.kg-1 in 60 s with fentanyl 2 micrograms.kg-1 and vecuronium bromide 0.08 mg.kg-1 for induction, followed by propofol 11 mg.kg-1 for maintenance; Protocol II; Propofol 3 mg.kg-1 in 20 s with fentanyl 3 micrograms.kg-1 for induction, followed by propofol 12 mg.kg-1.h-1 for maintenance. It appears that the use of protocol I offers significant advantages compared with protocol II: a better quality of induction with a lesser incidence of pain during injection of propofol; a better quality of maintenance with very infrequent bradycardia from oculocardiac reflectivity; and a better recovery with a greatly reduced frequency of nausea and vomiting.