Cloning and characterization of an extracellular temperature-labile serine protease gene from Aeromonas hydrophila

Aeromonas virulence is thought to depend on multigenic functions. The gene for an extracellular protease from Aeromonas hydrophila SO2/2 was cloned in Escherichia coli C600-1 by using pIJ860, bifunctional plasmid, as a vector. The gene encodes for a temperature-labile serine protease (P2) with a molecular mass of approx. 68 kDa which is highly inhibited by PMSF. The gene was expressed in Streptomyces lividans 1326 by transforming protoplasts with the original clone pPA2. We were also able to transfer and express the prt P2 gene in Pseudomonas putida by mating experiments. The protein P2 was secreted into the periplasms of both P. putida and E. coli C600-1 being identical in properties to one of the proteases secreted into the culture supernatant by A. hydrophila SO2/2.     

Selection of Ethanol-Tolerant Yeast Hybrids in pH-Regulated Continuous Culture

Hybrids between naturally occurring wine yeast strains and laboratory strains were formed as a method of increasing genetic variability to improve the ethanol tolerance of yeast strains. The hybrids were subjected to competition experiments under continuous culture controlled by pH with increasing ethanol concentrations over a wide range to select the fastest-growing strain at any concentration of ethanol. The continuous culture system was obtained by controlling the dilution rate of a chemostat connected to a pH-meter. The nutrient pump of the chemostat was switched on and off in response to the pH of the culture, which was thereby kept near a critical value (pH_c). Under these conditions, when the medium was supplemented with ethanol, the ethanol concentration of the culture increased with each pulse of dilution. A hybrid strain was selected by this procedure that was more tolerant than any of the highly ethanol-tolerant wine yeast strains at any concentration of ethanol and was able to grow at up to 16% (vol/vol) ethanol. This improvement in ethanol tolerance led to an increase in both the ethanol production rate and the total amount of ethanol produced.     

Pigment patterns in mutants affecting the biosynthesis of pteridines and xanthommatin in Drosophila melanogaster

Eye-color mutants of Drosophila melanogaster have been analyzed for their pigment content and related metabolites. Xanthommatin and dihydroxanthommatin (pigments causing brown eye color) were measured after selective extraction in acidified butanol. Pteridines (pigments causing red eye color) were quantitated after separation of 28 spots by thin-layer chromatography, most of which are pteridines and a few of which are fluorescent metabolites from the xanthommatin pathway. Pigment patterns have been studied in 45 loci. The pteridine pathway ramifies into two double branches giving rise to isoxanthopterin, drosopterins, and biopterin as final products. The regulatory relationship among the branches and the metabolic blockage of the mutants are discussed. The Hn locus is proposed to regulate pteridine synthesis in a step between pyruvoyltetrahydropterin and dihydropterin. The results also indicate that the synthesis and accumulation of xanthommatin in the eyes might be related to the synthesis of pteridines.Support for this work was provided to J.F. in part by a grant from the Ministerio de Universidades e Investigación (Spain) and to F.J.S. by a grant from the Ministerio de Educación y Ciencia (Spain).     

Characterization of the biosynthesis in vivo of α-aminoadipyl-cysteinyl-valine inPenicillium chrysogenum

Summary The parameters affecting the formation in vivo of -aminoadipyl-cysteinyl-valine (ACV), an intermediate in penicillin biosynthesis, have been established in low- and high-penicillin producing strains ofPenicillium chrysogenum. ACV was found both in cell extracts and in the culture broth filtrates. (¹⁴C)valine, -(¹⁴C)aminoadipic acid and (¹⁴C)cysteine were efficiently incorporated into ACV. Formation of ACV was stimulated by phenylacetic acid when added during the growth of the culture. ACV biosynthesis was enhanced when protein synthesis was blocked with cycloheximide or anisomicin. The ACV-synthesising activity of the culture increased between 24 and 48 h of the culture preceeding penicillin biosynthesis, and remained constant thereafter. A decay of ACV-forming activity was observed when de novo protein synthesis was inhibited with cycloheximide. The apparent half-life of the ACV-synthesising enzyme system was 2.5 h.     

The antenna system of Rhodospirillum rubrum. Detection of macromolecular constituents not stainable by Coomassie brilliant blue in solubilized preparations of the B880 complex

A solubilized preparation of the major Rhodospirillum rubrum antenna complex (B880) was obtained by a described procedure and its polypeptide composition was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Only two polypeptides of molecular weights close to 7000 were detected after staining the gels with Coomassie brilliant blue. However, several other constituents could be visualized by silver staining or by an immunochemical method. When the preparation was chromatographed on Sephacryl, some of the resulting fractions exhibited the characteristic B880 absorption spectrum and contained only the two proteins that were detectable with Coomassie brilliant blue. In those fractions the A ₂₈₀/A ₈₈₀ratio was 0.4, which indicated a significant improvement of the bacteriochlorophyll to protein ratio over the unchromatographed preparation (A ₂₈₀/A ₈₈₀=0.7). Other chromatography fractions lacked bacteriochlorophyll and contained a carotenoid which seemed to be bound to protein. The macromolecular constituents present in these latter fractions differed from those associated to the purified B880 complex in their electrophoretic moblities and/or in their staining properties. That suggested the possible existence of a carotenoprotein that did not result from the B880 complex upon loss of bacteriochlorophyll.     

Teratogenic effect of trifluoperazine in rats and mice

Trifluoperazine was administered to pregnant mice and rats by gastric intubation during the period of organogenesis. Dams were treated at doses of 0.5, 5, 50 (mice and rats) and 100 (only rats) mg/kg/day. The drug affected the pregnant weight increment in a dose-related manner in rats but only the 50 mg/kg/day dose level affected the weight gain of mice. The foetal weight was not markedly affected in either species. The drug induced cleft palate and micrognathia in rats but did not produce a teratogenic effect in mice. Trifluoperazine caused abortions in mice and there were significant increases in the number of resorptions with the top dose levels in both species.     

Subcellular localization and glycoprotein nature of the invertase from the fission yeast Schizosaccharomyces pombe

The subcellular localization of the enzyme invertase in Schizosaccharomyces pombe cells, both repressed and derepressed for synthesis of the enzyme, was studied. Most of the invertase was found to be located outside the plasma membrane and only a small percentage was found to be associated to membranes. A substantial portion of the external enzyme remained firmly bound to cell-wall material.All of the invertase recovered in soluble form from cellular extracts reacted with concanavalin A and with the lectin from Bandeiraea simplicifolia seeds, indicating the presence in the enzyme of a carbohydrate moiety which probably contains terminal mannosyl (or structurally related) and galactosyl residues.The possibility of the presence of two different forms of invertase in S. pombe was considered. An intracellular, soluble form of invertase, devoid of carbohydrate, similar to the small invertase of the budding yeast Saccharomyces cerevisiae, was not found in S. pombe. However, the Michaelis constant for sucrose of the enzyme present in repressed cells was smaller than that of the invertase synthesized under derepressing conditions, although this difference could also be the result of a different pattern of glycosylation of the invertase synthesized under different growth conditions.     

Redox interconversion of Escherichia coli glutathione reductase. A study with permeabilized and intact cells

Summary The redox interconversion of Escherichia coli glutathione reductase has been studied both in situ, with permeabilized cells treated with different reductants, and in vivo, with intact cells incubated with compounds known to alter their intracellular redox state.The enzyme from toulene-permeabilized cells was inactivated in situ by NADPH, NADH, dithionite, dithiothreitol, or GSH. The enzyme remained, however, fully active upon incubation with the oxidized forms of such compounds. The inactivation was time-, temperature-, and concentration-dependent; a 50% inactivation was promoted by just 2 M NADPH, while 700 M NADH was required for a similar effect. The enzyme from permeabilized cells was completely protected against redox inactivation by GSSG, and to a lesser extent by dithiothreitol, GSH, and NAD(P)⁺. The inactive enzyme was efficiently reactivated in situ by physiological GSSG concentrations. A significant reactivation was promoted also by GSH, although at concentrations two orders of magnitude below its physiological concentrations. The glutathione reductase from intact E. coli cells was inactivated in vivo by incubation with DL-malate, DL-isocitrate, or higher L-lactate concentrations. The enzyme was protected against redox inactivation and fully reactivated by diamide in a concentration-dependent fashion. Diamide reactivation was not dependent on the synthesis of new protein, thus suggesting that the effect was really a true reactivation and not due to de novo synthesis of active enzyme. The glutathione reductase activity increased significantly after incubation of intact cells with tert-butyl or cumene hydroperoxides, suggesting that the enzyme was partially inactive within such cells. In conclusion, the above results show that both in situ and in vivo the glutathione reductase of Escherichia coli is subjected to a redox interconversion mechanism probably controlled by the intracellular NADPH and GSSG concentrations.     

Zur Mauser spanischer Weiden- und Haussperlinge (Passer hispaniolensis unddomesticus)

Zusammenfassung Die Mauserperiode westspanischer Weidensperlinge(Passer hispaniolensis) und Haussperlinge(P. domesticus) reicht von Ende Juli bis Ende September/Anfang Oktober. Beim Weidensperling endet der Federwechsel im Durchschnitt etwa fünf Tage früher als beim Haussperling. Es gibt keine Geschlechtsunterschiede in der Chronologie der Mauser beim Weidensperling. Ad. beider Arten mausern schneller und synchronisierter als juv., die ihr Gefieder um so rascher erneuern, je später sie mit der Mauser begonnen haben. Die Handschwingenmauser dauert etwa 66 Tage beim Weidensperling und 69 Tage beim Haussperling. Beide Arten brauchen ca. 3 weitere Tage für die Verhornung der 5. und 6. Armschwingen. Die ad. beider Arten und die juv. Weidensperlinge beginnen die Mauser im Durchschnitt am selben Tag (24. Juli), die juv. Haussperlinge später (29. Juli). Der Mauserverlauf und die Beziehungen zwischen den verschiedenen Federreihen sind bei beiden Arten identisch. Die Synchronisation der Mauser ist beim Weidensperling höher. Brut und Mauserperiode überschneiden sich beim Haussperling; beim Weidensperling, bei dem noch kurze Wanderungen gleich nach der Fortpflanzungsperiode und vor der Mauser erfolgen, nicht. Das frühere und höher synchronisierte Mauserende beim Weidensperling scheint eine Anpassung an die stärkere Zugtendenz zu sein.

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On the moult of Spanish Sparrows(Passer hispaniolensis) and House Sparrows(Passer domesticus) in Iberia

Summary The moulting period of Spanish sparrows(Passer hispaniolensis) and House Sparrows(Passer domesticus) in Western Spain extends from late July to late September/early October. House Sparrows finish moulting on average some five days later than Spanish Sparrows. There are no sexual differences in the moulting chronology of adult Spanish Sparrows. Ad. of both species moult faster and better synchronized. The speed of moulting is also higher in later moulting juveniles. The estimated durations of wing feather replacement were 66 days for the Spanish Sparrow and 69 days for the House Sparrow. Some three more days are needed to complete the growth of the 5th and 6th secondary remiges in both species. Adults of both species and juvenile Spanish Sparrows start moulting on average on the same date: 24th July; juvenile House Sparrows start moulting on 29th July. The sequence of moult and the relations between different feather tracts are identical in both species. The synchronization of the moult is higher in the Spanish Sparrow. Breeding and moulting seasons slightly overlap in the House Sparrow, but not in the Spanish Sparrow. In this species the time lapse between both periods allows the birds to wander to suitable areas, where they moult. The earlier ending and higher synchronization of the moult in the Spanish Sparrow is related to its higher migratory tendency.