Biosynthesis of Branched-Chain Amino Acids in Yeast: Correlation of Biochemical Blocks and Genetic Lesions in Leucine Auxotrophs

The three enzymatic steps in the conversion of alpha-ketoisovalerate to alpha-ketoisocaproate were examined in wild-type and in leucine auxotrophic stocks of yeast. Procedures for the reliable assay of each of the enzymatic steps in crude extracts were devised. Crude extracts of the prototrophic haploid stock catalyzed all three enzymatic steps. Examination of a series of leucine auxotrophs permitted a correlation between the three enzymatic steps and the genetic lesions affecting 10 different loci. This examination revealed that a single locus (le-6) affected primarily alpha-isopropylmalate synthetase, the first step in the pathway. Lesions in six loci (le-1, le-4, le-5, le-7, le-8, and le-10) lead primarily to a deficiency in the activity of the second enzyme in the pathway, alpha-isopropylmalate isomerase. Stocks with lesions in three loci (le-2, le-3, and le-9) were primarily blocked in the third step of the pathway, catalyzed by beta-isopropylmalate dehydrogenase. The results with the mutants provide strong evidence that the pathway for leucine biosynthesis proposed by Strassman and his colleagues is the sole significant pathway in yeast.     

Presence of Conjugated 5-Hydroxytryptamine in In Vivo Superfusates of Rat Spinal Cord

The presence of an acid-hydrolyzable conjugate of 5-hydroxytryptamine, presumably 5-hydroxytryptamine-O-sulfate, was demonstrated in in vivo superfusates of rat spinal cord by HPLC with electrochemical detection. In untreated rats, the concentration of the 5-hydroxytryptamine conjugate measured during the basal efflux of 5-hydroxytryptamine did not differ from that measured during the release of 5-hydroxytryptamine evoked by DL-p-chloroamphetamine. Pretreatment of the rats with clorgyline, an inhibitor of the enzyme monoamine oxidase (EC 1.4.3.4), or with probenecid did not alter the concentrations of conjugated 5-hydroxytryptamine measured during the basal efflux of 5-hydroxytryptamine, but did elevate the concentrations of conjugate measured during the evoked release of 5-hydroxytryptamine.     

Notizen über endopolyploide Kerne im Bereich der Samenanlage von Angiospermen

Zusammenfassung Es bestätigt sich die Beobachtung, daß gerade in der Samenanlage nicht nur sehr häufig hohe Polyploidiegrade erreicht werden, sondern daß sich die Struktur in diesen Kernen zumeist sehr wesentlich von der in den diploiden unterscheidet.In den hochendopolyploiden Kernen des mikropylaren und des chalazalen Endospermhaustoriums vonLoasa papaverifolia findet sich eine den pflanzlichen Riesenchromosomen ähnliche Struktur, wobei die Endochromosomen wohl im heterochromatischen Abschnitt, aber nicht — oder nur über einen außerordentlich kurzen Teil — im euchromatischen zusammenhalten.In den entsprechenden Kernen vonPlantago psyllium sind die Endochromosomen stets als mehr oder weniger gewundene Fäden erkennbar, die entweder im Kernraum annähernd gleichmäßig verteilt sind, oder aber selten radiär gebaute Gruppen in der Anzahl 3n bilden; die SAT-Endochromosomen sind stets zu drei radiär gebauten Gruppen vereinigt. Außerdem erfährt die Kernstruktur eine weitere Abwandlung durch die unterschiedliche Dicke der Endochromosomen.Von den 25 Pflanzen, deren Antipodenkerne untersucht wurden, besitzen 17 hochendopolyploide Kerne; bei 11 zeigen sie Ausbildung bemerkenswerter Strukturen, wie Riesenchromosomen, Bündel, lichtmikroskopisch deutliche Spiralen oder lockere Areale, wobei manche nur einen bestimmten Strukturtypus ausbilden, andere dagegen mehrere.In den Kernen des Suspensorhaustoriums vonGagea lutea weisen die als zarte Fäden erkennbaren Endochromosomen in den niedrigen Polyploidiestufen eine leichte Gruppierung auf, während sie sich in den hochendopolyploiden Kernen zu Riesenchromosomen zusammenschließen.     

Über die Histologie der Intumeszenzen und die Cytologie zweierIpomoea-Arten

Zusammenfassung Ipomoea Batatas undIpomoea purpurea besitzen Chromozentrenkerne; bei ersterer entspricht die Chromozentrenzahl mit durchschnittlich 83 ungefähr der Chromosomenzahl (2n=90), bei letzterer ist sie mit durchschnittlich 60 Chromzentren doppelt so groß (2n=30). In den Dauergeweben der Wurzel bzw. der Knolle sowie in der Achse beider Arten finden sich keine Anzeichen von Endopolyploidie; die Gewebe bleiben also diploid. — Die Entwicklung der Intumeszenzen, die ebenfalls diploid bleiben, geht entweder von den Nebenzellen oder ihnen benachbarten Zellen der Epidermis, oder von der subepidermalen Zellschicht aus; im letztgenannten Fall finden sich die ersten Teilungen dann stets unterhalb einer Spaltöffnung. Die Intumeszenzen sind feste, aus der Epidermis und der ersten Mesophyllschicht hervorgegangene Wucherungen, die bei der Batate eine Länge von ungefähr 1,5 mm erreichen, beiIpomoea purpurea var. morning glory sogar 2 mm lang werden. — Der Endodermis ergrünter Achsen vonIpomoea Batatas fehlt einCasparyscher Streifen, bei im Dunkeln getriebenen Sprossen hingegen wird ein solcher ausgebildet.     

Expression of the pro-apoptotic gene gadd153/chop is elevated in liver with aging and sensitizes cells to oxidant injury

Aging is generally accompanied by reduced tolerance to oxidative stress and altered responsiveness to proliferative signals. We have shown that hepatocytes derived from aged rats (24-26 months) exhibit greater sensitivity to H(2)O(2) treatment and reduced proliferation following epidermal growth factor (EGF) treatment than cells of young adult rats (5-6 months). Here we examined the effects of aging and calorie restriction (CR) on expression of the oxidative stress-inducible and pro-apoptotic gene gadd153 (chop) in these hepatocytes, and we investigated its influence on sensitivity to oxidants. We show that aging was associated with elevated expression of gadd153, both basally and in response to H(2)O(2) treatment. CR, which attenuates age-associated declines in stress tolerance, prevented the age-related increase in gadd153 expression. EGF treatment also resulted in gadd153 induction in old cells. This effect was absent in young cells and in old cells of CR rats. gadd153 induction by EGF was reactive oxygen species-dependent and correlated with heightened sensitivity to subsequent H(2)O(2) treatment, suggesting that elevated Gadd153 contributes to the greater sensitivity of EGF-pretreated old cells to oxidative stress. Additional support for this hypothesis was provided by experiments with Rat1 fibroblasts in which conditional expression of Gadd153 conferred increased sensitivity to H(2)O(2). We propose a model whereby the diminished ability of old hepatocytes to overcome an EGF-triggered reactive oxygen species load leads to induction of the proapoptotic gene gadd153, which, in turn, sensitizes the cells to oxidant injury. Our findings point to gadd153 expression levels as an important factor in liver aging.     

Caveolin-induced activation of the phosphatidylinositol 3-kinase/Akt pathway increases arsenite cytotoxicity

The inhibitory effect of caveolin on the cellular response to growth factor stimulation is well established. Given the significant overlap in signaling pathways involved in regulating cell proliferation and stress responsiveness, we hypothesized that caveolin would also affect a cell's ability to respond to environmental stress. Here we investigated the ability of caveolin-1 to modulate the cellular response to sodium arsenite and thereby alter survival of the human cell lines 293 and HeLa. Cells stably transfected with caveolin-1 were found to be much more sensitive to the toxic effects of sodium arsenite than either untransfected parental cells or parental cells transfected with an empty vector. Unexpectedly, the caveolin-overexpressing cells also exhibited a significant activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which additional studies suggested was likely due to decreased neutral sphingomyelinase activity and ceramide synthesis. In contrast to its extensively documented antiapoptotic influence, the elevated activity of Akt appears to be important in sensitizing caveolin-expressing cells to arsenite-induced toxicity, as both pretreatment of cells with the PI3K inhibitor wortmannin and overexpression of a dominant-negative Akt mutant markedly improved the survival of arsenite-treated cells. This death-promoting influence of the PI3K/Akt pathway in caveolin-overexpressing cells appeared not to be unique to sodium arsenite, as wortmannin pretreatment also resulted in increased survival in the presence of H(2)O(2). In summary, our results indicate that caveolin-induced upregulation of the PI3K/Akt signaling pathway, which appears to be a death signal in the presence of arsenite and H(2)O(2), sensitizes cells to environmental stress.