Effects of medium composition on MFα1 promoter-directed secretion of a small protease inhibitor in Saccharomyces cerevisiae batch fermentation

Summary Casaminoacid supplementation of yeast nitrogen base (YNB) medium is widely used in the culture of recombinant yeasts as it enables respiratory growth on YNB and selection for URA3- or TRP1-bearing plasmids. Despite these advantages, this supplementation to YNB medium resulted in marked decreases in cellular productivity for the secretion of a protease inhibitor product in batch fermentation.     

Assay,properties, and regulation of rat ovarian δ-aminolevulinic acid synthetase activity

δ-Aminolevulinic acid synthetase (EC 2.3.1.37) has been detected in homogenates of rat ovaries. Optimal substrate and coenzyme concentrations, and parameters for assay of ovarian δ-aminolevulinic acid synthetase have been determined. Subcellular fractionation studies have shown that enzyme activity is predominantly localized in the mitochondrial fraction. Fasting, which is known to increase enzyme activity in the adrenal and to have no effect on activity in the testis, had no effect on enzyme activity in the ovary. Administration of the hepatic inducer allylisopropylacetamide or the hormone progesterone failed to alter activity of the ovarian enzyme. The activity of the enzyme was significantly increased during the diestrus-1 phase of the estrus cycle, during pregnancy, and by human chorionic gonadotropin at 24 and 48 h, suggesting that ovarian δ-aminolevulinic acid synthetase and the synthesis of heme may be under hormonal control.     

Acute toxicity effects of ibuprofen on behaviour and haematological parameters of African catfish Clarias gariepinus (Burchell, 1822)

Indiscriminate discharge of pharmaceutical waste into the aquatic ecosystem may pose serious health challenges to aquatic biota. The effect of acute exposure to ibuprofen was evaluated using changes in behaviour and haematological parameters under static bio-assay method in Clarias gariepinus. Test specimens were exposed to acute concentrations of ibuprofen (0.28, 0.33, 0.38, 0.43 and 0.48 mg l^?1) for 24, 48, 72 and 96 h durations respectively. Behavioural and phenotypic changes were observed in surviving fish. There were significant (p < 0.05) concentration and duration-dependent increases in erythrocyte (RBC), haemoglobin (Hb), pack cell volume (PCV) and leukocytes (WBC) in treated fish compared to the control. Insignificant decreases (p > 0.05) in mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were observed in treated fish compared to the control. Ibuprofen elicited dose and duration- dependent decrease in neutrophil counts with the decreases being significant (p < 0.05) in the higher doses of 0.43 and 0.48 mg l^?1. Ibuprofen did not elicit any significant changes in monocytes, basophils and eosinophils. Changes observed in this study showed that ibuprofen negatively affected the health of the fish and we recommend that discharge of ibuprofen into the aquatic environment should be monitored and controlled.     

UCS protein function is partially restored in the <Emphasis Type="Italic">Saccharomyces cerevisiae she4</Emphasis> mutant with expression of the human UNC45-GC,but not UNC45-SM

A dedicated UNC45, Cro1, She4 (UCS) domain-containing protein assists in the Hsp90-mediated folding of the myosin head. Only weak sequence conservation exists between the single UCS protein of simple eukaryotes (She4 in budding yeast) and the two UCS proteins of higher organisms (the general cell and striated muscle UNC45s; UNC45-GC and UNC45-SM, respectively). In vertebrates, UNC45-GC facilitates cytoskeletal functions, whereas the 55% identical UNC45-SM assists assembly of the contractile apparatus of cardiac and skeletal muscles. A Saccharomyces cerevisiae she4Δ mutant, totally lacking any UCS protein, was engineered to express as its sole Hsp90 either the Hsp90α or the Hsp90β isoforms of human cytosolic Hsp90. A transient induction of the human UNC45-GC, but not UNC45-SM, could rescue the defective endocytosis in these she4Δ cells at 39 °C, irrespective of whether they possessed Hsp90α or Hsp90β. UNC45-GC-mediated rescue of the localisation of a Myo5-green fluorescent protein (GFP) fusion to cortical patches at 39 °C was more efficient in the yeast containing Hsp90α, though this may relate to more efficient functioning of Hsp90α as compared to Hsp90β in these strains. Furthermore, inducible expression of UNC45-GC, but not UNC45-SM, could partially rescue survival at a more extreme temperature (45 °C) that normally causes she4Δ mutant yeast cells to lyse. The results indicate that UCS protein function has been most conserved—yeast to man—in the UNC45-GC, not UNC45-SM. This may reflect UNC45-GC being the vertebrate UCS protein that assists formation of the actomyosin complexes needed for cytokinesis, cell morphological change, and organelle trafficking—events also facilitated by the myosins in yeast.     

UBI4, the polyubiquitin gene of Saccharomyces cerevisiae , is a heat shock gene that is also subject to catabolite derepression control

 Carbon and nitrogen regulation of UBI4, the stress-inducible polyubiquitin gene of Saccharomyces cerevisiae, was investigated using a UBI4 promoter-LacZ fusion gene (UBI4-LacZ). Expression of this gene in cells grown on different media indicated that the UBI4 promoter is more active during growth on respiratory than on fermentable carbon sources but is not subject to appreciable control by nitrogen catabolite repression. UBI4-LacZ expression was virtually identical in cells having constitutively high (ras2, sra1-13) or constitutively low (ras2) levels of cyclic AMP-dependent protein kinase activity, indicating that this kinase does not exert a major influence on UBI4 expression. Catabolite derepression control of the UBI4 promoter was confirmed by measurements of UBI4-LacZ expression in hap mutant and wild-type strains before and after transfer from glucose to lactate. Mutagenesis of the perfect consensus for HAP2/3/4 complex binding at position −542 resulted in considerable reduction of UBI4 promoter derepression with respiratory adaptation in HAP wild-type cells and abolished the reduced UBI4-LacZ derepression normally seen when aerobic cultures of the hap1 mutant are transferred from glucose to lactate. This HAP2/3/4 binding site is therefore a major element contributing to catabolite derepression of the UBI4 promoter, although data obtained with hap1 mutant cells indicated that HAP1 also contributes to this derepression. The HAP2/3/4 and HAP1 systems are normally found to activate genes for mitochondrial (respiratory) functions. Their involvement in mediating higher activity of the UBI4 promoter during respiratory growth may reflect the contribution of UBI4 expression to tolerance of oxidative stress. Received: 3 June 1996 / Accepted: 20 August 1996     

Interaction of endophyte-infected fescue and heat stress on ovarian function in the beef heifer.

The objective of the experiment was to examine the interaction of endophyte-infected tall fescue and environmental temperature on follicular and luteal development and function in beef heifers. Heifers were fed endophyte-free or endophyte-infected tall fescue seed at thermoneutral or heat stress temperatures (n = 6/treatment) 4 wk before and 3 wk after synchronized ovulation. All heifers were subjected to thermoneutral conditions (19 degrees C, 50% relative humidity) from Days -7 to -2; temperature increased incrementally from Days -1 to 0 and cycled between 25 degrees C and 31 degrees C between Days 1 and 20 for heat-stressed heifers. Serum was collected and ovaries monitored every other day after induced luteolysis between Days 1 and 23 or until ovulation. Size and location of follicles >4 mm and corpora lutea were recorded. Serum concentrations of prolactin were reduced in heat-stressed heifers fed infected seed and both heat stress and infected seed decreased total cholesterol. Rectal temperature and respiration rate were greatest in heifers fed the infected seed when exposed to maximal temperatures. Heat stress led to reduced diameter of the corpus luteum and serum progesterone compared with thermoneutral conditions. Progesterone was reduced more so in heifers fed infected seed. The combination of infected seed and heat stress was associated with reduced diameter of the preovulatory dominant follicle, and consumption of infected seed led to fewer large follicles during the estrous cycle. Both stressors led to reduced serum estradiol. Impaired follicle function may explain reduced pregnancy rates commonly observed in heifers grazing infected tall fescue pasture.     

Context-dependent memory: colour versus odour

An olfactory stimulus and a visual stimulus were employed in a context- dependent memory study using a prose passage as the to-be-remembered item. Ninety-five university students (aged 17-35 years) learned the passage of prose in the presence of one of the stimuli and were then asked to recall the passage with the original context either reinstated or not reinstated. The results revealed a significant context-dependent memory effect for the olfactory cue but not for the visual cue. They demonstrate support for the effectiveness of odours as context cues and it is suggested that context-dependent memory processes may underlie the formation and retrieval of odour-evoked autobiographical memories.