Metabolic Flux Analysis during the Exponential Growth Phase of Saccharomyces cerevisiae in Wine Fermentations

As a consequence of the increase in global average temperature, grapes with the adequate phenolic and aromatic maturity tend to be overripe by the time of harvest, resulting in increased sugar concentrations and imbalanced C/N ratios in fermenting musts. This fact sets obvious additional hurdles in the challenge of obtaining wines with reduced alcohols levels, a new trend in consumer demands. It would therefore be interesting to understand Saccharomyces cerevisiae physiology during the fermentation of must with these altered characteristics. The present study aims to determine the distribution of metabolic fluxes during the yeast exponential growth phase, when both carbon and nitrogen sources are in excess, using continuous cultures. Two different sugar concentrations were studied under two different winemaking temperature conditions. Although consumption and production rates for key metabolites were severely affected by the different experimental conditions studied, the general distribution of fluxes in central carbon metabolism was basically conserved in all cases. It was also observed that temperature and sugar concentration exerted a higher effect on the pentose phosphate pathway and glycerol formation than on glycolysis and ethanol production. Additionally, nitrogen uptake, both quantitatively and qualitatively, was strongly influenced by environmental conditions. This work provides the most complete stoichiometric model used for Metabolic Flux Analysis of S. cerevisiae in wine fermentations employed so far, including the synthesis and release of relevant aroma compounds and could be used in the design of optimal nitrogen supplementation of wine fermentations.     

Photochemical response of the scleractinian coral Stylophora pistillata to some sunscreen ingredients

Coral Reefs - Ultraviolet (UV) filters and preservatives, which are common constituents of sunscreens and other cosmetics, are reported as a threat for coastal coral reef ecosystems; however, few...     

Decreased responsiveness of gluconeogenesis to the modulation by sulfonylureas in hepatocytes isolated from obese (fa/fa) Zucker rats

The influence of the hypoglycemic agent glipizide (0-100 microM) on the rate of gluconeogenesis from lactate, as well as on the levels of fructose 2,6-bisphosphate, has been investigated in hepatocytes isolated from genetically obese (fa/fa) Zucker rats and from their corresponding lean (Fa/-) littermates. As compared to lean rat hepatocytes, liver cells isolated from obese animals showed a lower rate of basal gluconeogenesis (0.9 +/- 0.2 vs 5.4 +/- 0.5 micromol of lactate converted to glucose/g cell x 30 min, n=4) and higher levels of fructose 2,6-bisphosphate (11.5 +/- 1.0 vs 5.9 +/- 0.4 nmol/g cell, n=8-9). In lean rat hepatocytes, the presence of glipizide in the incubation medium caused a dose-dependent inhibition of the rate of lactate conversion to glucose (maximal inhibition=46%; EC50 value=26 microM), and simultaneously raised the cellular content of fructose-2,6-bisphosphate (maximal increment=40%; EC50 value=10 microM). In contrast, in hepatocytes isolated from obese rats, the inhibition of gluconeogenesis and the increment in fructose-2,6-bisphosphate levels elicited by glipizide were significantly reduced (maximal effects of 22 and 13%, respectively). Similarly, the activation of glycogen phosphorylase and the increase in hexose 6-phosphate levels in response to glipizide were less marked in obese rat hepatocytes than in liver cells isolated from lean animals. These results demonstrate that the efficacy of sulfonylureas as inhibitors of hepatic gluconeogenesis is reduced in the genetically obese (fa/fa) Zucker rat.     

Inhibition of acid secretion by the nonsteroidal anti-inflammatory drugs diclofenac and piroxicam in isolated gastric glands: analysis of a multifocal mechanism

In nonstimulated rabbit gastric glands, acetylsalicylic acid (10-500 microM) and indomethacin (3-300 microM) did not significantly modify the basal rate of acid secretion, whereas diclofenac and piroxicam (10-1,000 microM each) caused a marked and dose-dependent inhibitory effect (EC(50) = 138 and 280 microM, respectively). In gastric glands stimulated by histamine (100 microM), diclofenac also reduced the rate of acid formation in a dose-dependent manner. In contrast, acetylsalicylic acid, indomethacin, and piroxicam exerted a biphasic effect; thus low concentrations (3-100 microM) of these three agents significantly increased the rate of histamine-stimulated acid secretion (10-20% over the corresponding control value) by a cAMP-independent mechanism, whereas higher concentrations reduced the rate of acid formation. With respect to underlying biochemical mechanisms that could mediate inhibitory effects of NSAIDs on gastric acid formation, it was observed that both diclofenac and piroxicam, but not acetylsalicylic acid or indomethacin, decreased the glandular content of ATP, inhibited hydrolytic activity of gastric gland microsomal H(+)-K(+)-ATPase, and reduced the rate of H(+)-K(+)-ATPase-dependent proton transport across microsomal membranes in a dose-dependent manner. Furthermore, diclofenac and piroxicam also significantly increased passive permeability of microsomal membranes to protons. In conclusion, our work shows that diclofenac and piroxicam cause a significant reduction in the rate of basal and histamine-stimulated acid formation in isolated rabbit gastric glands at concentrations that can be attained in the gastric lumen of patients treated with these drugs. Mechanisms involved in these inhibitory effects appear to be multifocal and include different steps of stimulus-secretion coupling.     

Modulation of gluconeogenesis by epinephrine in hepatocytes isolated from genetically obese (fa/fa) Zucker rats

The obese (fa/fa) Zucker rat shows an impaired sympathetic tone which is accompanied by an altered thermogenesis and changes in both lipid and carbohydrate metabolism. In this work, we have investigated the regulatory effects of epinephrine on the rate of gluconeogenesis from a mixture of [(14)C]lactate/pyruvate, in hepatocytes isolated from obese (fa/fa) rats and their lean (Fa/-) littermates. Epinephrine caused a dose-dependent stimulation of the rate of [(14)C]glucose formation in both obese and lean rat hepatocytes, the maximal rates being five- and twofold higher than the corresponding basal values (0.50 +/- 0.06 and 1.96 +/- 0.15 micromol of lactate converted to glucose/g of cell x 20 min, respectively). No significant differences were found between the calculated half-maximal effective concentrations (EC(50)) for epinephrine in obese and lean rat liver cells. The stimulation of gluconeogenesis by epinephrine was accompanied by a decrease in the cellular concentration of fructose 2,6-bisphosphate, and an inactivation of both pyruvate kinase and 6-phosphofructo 2-kinase, to similar extents in both types of hepatocytes. Epinephrine also significantly raised the hepatocyte content of cyclic AMP, with about a twofold increase at a saturating concentration of the catecholamine (1 microM), in both lean and obese rat liver cells. However, at suboptimal concentrations of epinephrine, the rise in cyclic AMP levels was significantly less marked in obese than in lean rat hepatocytes. Nevertheless, no significant differences were found in either the affinity or the number of beta-adrenergic receptors, in radioligand binding studies carried out in liver plasma membranes obtained from obese and lean Zucker rats. In conclusion, compared to the corresponding basal values, the response of gluconeogenesis from lactate to the stimulatory effect of epinephrine is higher in obese (fa/fa) than in lean (Fa/-) Zucker rat hepatocytes, with no significant differences in the calculated EC(50) values for this hormone. This occurs in spite of an apparent decreased sensitivity of the adenylate cyclase system to the stimulatory effect of epinephrine in obese rat liver cells.     

Co-composting of hair waste from the tanning industry with de-inking and municipal wastewater sludges

Production of waste hair in the leather manufacturing industry is increasing every year due to the adoption of hair-save unhairing techniques, leaving the tanners with the problem of coping with yet another solid by-product. Numerous potential strategies for hair utilisation have been proposed. However, the use of hair waste as agricultural fertiliser is one of its most promising applications due to the high nitrogen content of hair. Agricultural value of hair can be increased by composting. This paper deals with the composting of hair from the unhairing of bovine hide. Results indicated that hair cannot be either composted on its own or co-composted with de-inking sludge, a chemical complementary co-substrate. However, good results were obtained when co-composted with raw sludge from a municipal wastewater treatment plant at hair:raw sludge weight ratios 1:1, 1:2 and, 1:4 in lab scale and pilot plant scale composters. In all cases, a more stable product was achieved at the end of the process. Composting in the pilot plant composter was effectively monitored using Static Respiration Indices determined at process temperature at sampling (SRI _T ) and at 37°C (SRI₃₇). Notably, SRI _T values were more sensitive to changes in the biological activity. In contrast, Respiratory Quotient (RQ) values were not adequate to follow the development of the process.     

Influence of growth temperature on the production of antibody Fab fragments in different microbes: a host comparative analysis

Microorganisms encounter diverse stress conditions in their native habitats but also during fermentation processes, which have an impact on industrial process performance. These environmental stresses and the physiological reactions they trigger, including changes in the protein folding/secretion machinery, are highly interrelated. Thus, the investigation of environmental factors, which influence protein expression and secretion is still of great importance. Among all the possible stresses, temperature appears particularly important for bioreactor cultivation of recombinant hosts, as reductions of growth temperature have been reported to increase recombinant protein production in various host organisms. Therefore, the impact of temperature on the secretion of proteins with therapeutic interest, exemplified by a model antibody Fab fragment, was analyzed in five different microbial protein production hosts growing under steady-state conditions in carbon-limited chemostat cultivations. Secretory expression of the heterodimeric antibody Fab fragment was successful in all five microbial host systems, namely Saccharomyces cerevisiae, Pichia pastoris, Trichoderma reesei, Escherichia coli and Pseudoalteromonas haloplanktis. In this comparative analysis we show that a reduction of cultivation temperature during growth at constant growth rate had a positive effect on Fab 3H6 production in three of four analyzed microorganisms, indicating common physiological responses, which favor recombinant protein production in prokaryotic as well as eukaryotic microbes.     

Antibiotics threaten wildlife: circulating quinolone residues and disease in Avian scavengers

Antibiotic residues that may be present in carcasses of medicated livestock could pass to and greatly reduce scavenger wildlife populations. We surveyed residues of the quinolones enrofloxacin and its metabolite ciprofloxacin and other antibiotics (amoxicillin and oxytetracycline) in nestling griffon Gyps fulvus, cinereous Aegypius monachus and Egyptian Neophron percnopterus vultures in central Spain. We found high concentrations of antibiotics in the plasma of many nestling cinereous (57%) and Egyptian (40%) vultures. Enrofloxacin and ciprofloxacin were also found in liver samples of all dead cinereous vultures. This is the first report of antibiotic residues in wildlife. We also provide evidence of a direct association between antibiotic residues, primarily quinolones, and severe disease due to bacterial and fungal pathogens. Our results indicate that, by damaging the liver and kidney and through the acquisition and proliferation of pathogens associated with the depletion of lymphoid organs, continuous exposure to antibiotics could increase mortality rates, at least in cinereous vultures. If antibiotics ingested with livestock carrion are clearly implicated in the decline of the vultures in central Spain then it should be considered a primary concern for conservation of their populations.