Role of glutamine synthetase in citric acid fermentation byAspergillus niger

The activity of glutamine synthetase fromAspergillus niger was significantly lowered under conditions of citric acid fermentation. The intracellular pH of the organism as determined by bromophenol blue dye distribution and fluorescein diacetate uptake methods was relatively constant between 6•0–6•5, when the pH of the external medium was varied between 2•3–7•0.Aspergillus niger glutamine synthetase was rapidly inactivated under acidic pH conditions and Mn2+ ions partially protected the enzyme against this inactivation. Mn2+-dependent glutamine synthetase activity was higher at acidic pH (6•0) compared to Mg2+-supported activity. While the concentration of Mg2+ required to optimally activate glutamine synthetase at pH 6•0 was very high (≥ 50 mM), Mn2+ was effective at 4 mM. Higher concentrations of Mn2+ were inhibitory. The inhibition of both Mn2+ and Mg2+-dependent reactions by citrate, 2-oxoglutarate and ATP were probably due to their ability to chelate divalent ions rather than as regulatory molecules. This suggestion was supported by the observation that a metal ion chelator, EDTA also produced similar effects. Of the end-products of the pathway, only histidine, carbamyl phosphate, AMP and ADP inhibitedAspergillus niger glutamine synthetase. The inhibitions were more pronounced when Mn2+ was the metal ion activator and greater inhibition was observed at lower pH values. These results permit us to postulate that glutamine synthesis may be markedly inhibited when the fungus is grown under conditions suitable for citric acid production and this block may result in delinking carbon and nitrogen metabolism leading to acidogenesis.

Effects of gender, and SLCO1B1 and CYP7A1 polymorphisms on plasma bile acids in humans and the pharmacokinetics of therapeutic ursodeoxycholic acid

Bile acids are important steroid-derived molecules essential for fat absorption in the small intestine. They are produced in the liver and secreted into the bile. Bile acids are transported by bile flow to the small intestine, where they aid the digestion of lipids. Most bile acids are reabsorbed in the small intestine and return to the liver through the portal vein. The whole recycling process is referred to as the enterohepatic circulation, during which only a small amount of bile acids are removed from the body via faeces. The enterohepatic circulation of bile acids involves the delicate coordination of a number of bile acid transporters expressed in the liver and the small intestine. Organic anion transporting polypeptide 1B1 (OATP1B1), encoded by the solute carrier organic anion transporter family, member 1B1 (SLCO1B1) gene, mediates the sodium independent hepatocellular uptake of bile acids. Two common SNPs in the SLCO1B1 gene are well known to affect the transport activity of OATP1B1. Moreover, bile acid synthesis is an important elimination route for cholesterol. Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme of bile acid production. The aim of this thesis was to investigate the effects of SLCO1B1 polymorphism on the fasting plasma levels of individual endogenous bile acids and a bile acid synthesis marker, and the pharmacokinetics of exogenously administered ursodeoxycholic acid (UDCA). Furthermore, the effects of CYP7A1 genetic polymorphism and gender on the fasting plasma concentrations of individual endogenous bile acids and the bile acid synthesis marker were evaluated. Firstly, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the determination of bile acids was developed (Study I). A retrospective study examined the effects of SLCO1B1 genetic polymorphism on the fasting plasma concentrations of individual bile acids and a bile acid synthesis marker in 65 healthy subjects (Study II). In another retrospective study with 143 healthy individuals, the effects of CYP7A1 genetic polymorphism and gender as well as SLCO1B1 polymorphism on the fasting plasma levels of individual bile acids and the bile acid synthesis marker were investigated (Study III). The effects of SLCO1B1 polymorphism on the pharmacokinetics of exogenously administered UDCA were evaluated in a prospective genotype panel study including 27 healthy volunteers (Study IV). A robust, sensitive and simple HPLC-MS/MS method was developed for the simultaneous determination of 16 individual bile acids in human plasma. The method validation parameters for all the analytes met the requirements of the FDA (Food and Drug Administration) bioanalytical guidelines. This HPLC-MS/MS method was applied in Studies II-IV. In Study II, the fasting plasma concentrations of several bile acids and the bile acid synthesis marker seemed to be affected by SLCO1B1 genetic polymorphism, but these findings were not replicated in Study III with a larger sample size. Moreover, SLCO1B1 polymorphism had no effect on the pharmacokinetic parameters of exogenously administered UDCA. Furthermore, no consistent association was observed between CYP7A1 genetic polymorphism and the fasting plasma concentrations of individual bile acids or the bile acid synthesis marker. In contrast, gender had a major effect on the fasting plasma concentrations of several bile acids and also total bile acids. In conclusion, gender, but not SLCO1B1 or CYP7A1 polymorphisms, has a major effect on the fasting plasma concentrations of individual bile acids. Moreover, the common genetic polymorphism of CYP7A1 is unlikely to influence the activity of CYP7A1 under normal physiological conditions. OATP1B1 does not play an important role in the in vivo disposition of exogenously administered UDCA.

Effect of support and acidity of catalyst on the direct oxidation of ethylene to acetic acid

The selective oxidation of ethylene to acetic acid was investigated on Pd-acid/support catalyst system. The catalytic activity is influenced strongly by the acidity of the catalyst. The stronger the catalyst acidity the higher the catalytic activity. The nature of the support also influences the activity of the catalyst substantially. The catalyst has highest activity when it exhibits highest acidity on silica.

Exercise training reduces fatty acid availability and improves the insulin sensitivity of glucose metabolism

Aims/hypothesis - It is not known whether the beneficial effects of exercise training on insulin sensitivity are due to changes in hepatic and peripheral insulin sensitivity or whether the changes in insulin sensitivity can be explained by adaptive changes in fatty acid metabolism, changes in visceral fat or changes in liver and muscle triacylglycerol content. We investigated the effects of 6 weeks of supervised exercise in sedentary men on these variables. Subjects and methods - We randomised 17 sedentary overweight male subjects (age 50 ± 2.6 years, BMI 27.6 ± 0.5 kg/m2) to a 6-week exercise programme (n = 10) or control group (n = 7). The insulin sensitivity of palmitic acid production rate (Ra), glycerol Ra, endogenous glucose Ra (EGP), glucose uptake and glucose metabolic clearance rate were measured at 0 and 6 weeks with a two-step hyperinsulinaemic–euglycaemic clamp [step 1, 0.3 (low dose); step 2, 1.5 (high dose) mU kg−1 min−1]. In the exercise group subjects were studied >72 h after the last training session. Liver and skeletal muscle triacylglycerol content was measured by magnetic resonance spectroscopy and visceral adipose tissue by cross-sectional computer tomography scanning. Results - After 6 weeks, fasting glycerol, palmitic acid Ra (p = 0.003, p = 0.042) and NEFA concentration (p = 0.005) were decreased in the exercise group with no change in the control group. The effects of low-dose insulin on EGP and of high-dose insulin on glucose uptake and metabolic clearance rate were enhanced in the exercise group but not in the control group (p = 0.026; p = 0.007 and p = 0.04). There was no change in muscle triacylglycerol and liver fat in either group. Conclusions/interpretation - Decreased availability of circulating NEFA may contribute to the observed improvement in the insulin sensitivity of EGP and glucose uptake following 6 weeks of moderate exercise.

Bioproduction of succinic acid from renewable feedstocks by Actinobacillus succinogenes 130Z

Succinic acid (SA) is a highly versatile building block that is used in a wide range of industrial applications. The biological production of succinic acid has emerged in the last years as an efficient alternative to the chemical production based on fossil fuels. However, in order to fully replace the competing petro-based chemical process from which it has been produced so far, some challenges remain to be surpassed. In particular, one main obstacle would be to reduce its production costs, mostly associated to the use of refined sugars. The present work is focused on the development of a sustainable and cost-e↵ective microbial production process based on cheap and renewable resources, such as agroindustrial wastes. Hence, glycerol and carob pods were identified as promising feedstocks and used as inexpensive carbon sources for the bioproduction of succinic acid by Actinobacillus succinogenes 130Z, one of the best naturally producing strains. Even though glycerol is a highly available carbon source, as by-product of biodiesel production, its consumption by A. succinogenes is impaired due to a redox imbalance during cell growth. However, the use of an external electron acceptor such as dimethylsulfoxide (DMSO) may improve glycerol metabolism and succinic acid production by this strain. As such, DMSO was tested as a co-substrate for glycerol consumption and concentrations of DMSO between 1 and 4% (v/v) greatly promoted glycerol consumption and SA production by this biocatalyst. Aiming at obtaining higher succinic acid yield and production rate, batch and fed-batch experiments were performed under controlled cultivation conditions. Batch experiments resulted in a succinic acid yield on glycerol of 0.95 g SA/g GLY and a production rate of 2.13 g/L.h, with residual production of acetic and formic acids. In fed-batch experiment, the SA production rate reached 2.31 g/L.h, the highest value reported in the literature for A. succinogenes using glycerol as carbon source. DMSO dramatically improved the conversion of glycerol by A. succinogenes and may be used as a co-substrate, opening new perspectives for the use of glycerol by this biocatalyst. Carob pods, highly available in Portugal as a residue from the locust bean gum industry, contain a significant amount of fermentable sugars such as sucrose, glucose and fructose and were also used as substrate for succinic acid production. Sugar extraction from raw and roasted carobs was optimized varying solid/water ratio and extraction time, maximizing sugar recovery while minimizing the extraction of polyphenols. Kinetic studies of glucose, fructose and sucrose consumption by A. succinogenes as individual carbon sources till 30 g/L were first determined to assess possible metabolic diferences. Results showed no significant diferences related to sugar consumption and SA production between the diferent sugars. Carob pods water extracts were then used as carbon source during controlled batch cultivations. (...)

Investigations on the production of long chain fatty acids in Choanephora cucurbitarum (Berk.

The fatty acid composition of the total cellular lipids of Choanephora cucurbitarum incubated for 96 hrs on either glucose-ammonium sulfate or malt-weast extract media was determined. The major fatty acids were palmitic, palmitoleic, stearic and linoleic acids. The saturated fatty acid possessing the longest acyl chain was stearate (C 18:0). The presence of glutamic acid (2.0 x 10-1% or 1.36 x la-2M) in either of the above growth media resulted in increase in percent of 1f-linolenic acid, decrease in percent of linoleic ~iCid and appearance of a new series of fatty acid> C ~8 e.g. C ",,,,'V' C2k:O, C26,O. The addition of glutamic acid had no effect on the lipid yield but slightly decreased the degree of unsaturation. Compounds which duplicated the effect of glutamic acid were acetate, malate, citrate, succinate, 0( -ketoglutarate, prOline, -y -aminobutyric acid and glucose (3%) but not aspartic acid or alanine. ~o correlation was found between glutamic acid pool concentration and the presence in the growth medium of those compounds which stimulate long chain fatty acid production. Four hours of incubation with 27 JJ 1-1 glutamate supported the production of long chain fatty acids. This stimulation is inhibited if 272 .u M isophthalic acid is added with 27 AJ M glutamate. But, long chain fatty acids were detected when 27 JJ M eX -ketoglutarate is also present in the incubation mixture. Five hours of incubation with 100 ,Mg/ml of cycloheximide resulted in over 9CY/o inhibition of cytoplasmic :protein synthesise Glutamate (27 .uM) enhanced the synthesis of long chain fatty acids under these conditions. These findings are discussed in an attempt to provide a plausible explanation COmmon to compounds that support the production of long chain fatty acids.

Study of new yeast strains as novel starter cultures for Riesling icewine production

Icewine is a sweet dessert wine fermented from the juice of grapes naturally frozen on the vine. The production of Icewine faces many challenges such as sluggish fermentation, which often yields wines with low ethanol, and an accumulation of high concentration of volatile acidity, mainly in the form of acetic acid. This project investigated three new yeast strains as novel starter cultures for Icewine fermentation with particular emphasis on reducing acetic acid production: a naturally occurring strain of S. bayanus/S. pastorianus isolated from Icewine grapes, and two hybrids between S. cerevisiae and S. bayanus, AWRI 1571 and AWRI 1572. These strains were evaluated for sugar consumption patterns and metabolic production of ethanol, glycerol and acetic acid, and were compared to the performance of a standard commercial wine yeast KI-VI116. The ITS rONA region of the two A WRI crosses was also analyzed during fermentations to assess their genomic stability. Icewine fermentations were performed in sterile filtered juice, in the absence of indigenous microflora, and also in unfiltered juice in order to mirror commercial wine making practices. The hybrid A WRI 1572 was found to be a promising candidate as a novel starter culture for Icewine production. I t produced 10.3 % v/v of ethanol in sterile Riesling Icewine fermentations and 11.2 % v/v in the unfiltered ones within a reasonable fermentation time (39 days). Its acetic acid production per gram sugar consumed was approximately 30% lower in comparison with commercial wine yeast K I -V 1116 under both sterile filtered and unfiltered fermentations. The natural isolate S. bayanus/S. pastorianus and AWRI 1571 did not appear to be suitable for commercial Icewine production. They reached the target ethanol concentration of approximately 10 % v/v in 39 day fermentations and also produced less acetic acid as a function of both time and sugar consumed in sterile fermentations compared to KI-V1116. However, in unfiltered fermentations, both of them failed to produce the target concentration of ethanol and accumulated high concentration of acetic acid. Both A WRI crosses displayed higher loss of or reduced copies in ITS rDNA region from the S. bayanus parent compared to the S. cerevisiae parent; however, these genomic losses could not be related to the metabolic profile.

Kinetics of autocatalytic acid hydrolysis of cellulose with crystalline and amorphous fractions

The acid hydrolysis of cellulose with crystalline and amorphous fractions is analyzed on the basis of autocatalytic model with a positive feedback of acid production from the degraded biopolymer. In the condition of low acid rate production compared with hydrolysis rate, both fraction of cellulose decrease exponentially with linear and cubic time dependence, and the normalized number of scissions per cellulose chain follows a sigmoid behavior with reaction time. The model predicts that self generated acidic compounds from cellulose accelerate the degradation of the biopolymer. However, if the acidic compounds produced are volatile species, then their release under low pressure will reduce the global rate of degradation of cellulose toward its intrinsic rate value determined by the residual acid catalyst present in the starting material.

Suppression by ABA of salicylic acid and lignin accumulation and the expression of multiple genes, in Arabidopsis infected with Pseudomonas syringae pv. tomato

Abscisic acid (ABA) has been implicated in determining the outcome of interactions between many plants and their pathogens. We had previously shown that increased concentrations of ABA within leaves of Arabidopsis induced susceptibility towards an avirulent strain of Pseudomonas syringae pathovar (pv.) tomato. We now show that ABA induces susceptibility via suppression of the accumulation of components crucial for a resistance response. Lignin and salicylic acid concentrations in leaves were increased during a resistant interaction but reduced when plants were treated with ABA. The reduction in lignin and salicylic acid production was independent of the development of the hypersensitive response (HR), indicating that, in this host-pathogen system, HR is not required for resistance. Genome-wide gene expression analysis using microarrays showed that treatment with ABA suppressed the expression of many defence-related genes, including those important for phenylpropanoid biosynthesis and those encoding resistance-related proteins. Together, these results show that resistance induction in Arabidopsis to an avirulent strain of P. syringae pv. tomato is regulated by ABA.

The whole-body fatty acid balance method : examples of its potential for feed efficiency and product quality optimisation in fish and poultry

Dietary lipids and fatty acids are not only fundamental in determining animal performance, but also determine the eating qualities of animal products. Several methods have been used to quantify fatty acid metabolism but most involve expensive in vitro approaches that are not suitable for most laboratories. Furthermore, there is considerable variation between methods with regard to enzyme activity, which makes comparison of results between studies difficult. The recently developed whole-body fatty acid balance method (WBFABM) is a simple and reliable in vivo method for assessing fatty acid metabolism, including rates of liponeogenesis and de novo fatty acid production, β-oxidation of fatty acids and bioconversion (elongation and desaturation) of fatty acids to long-chain polyunsaturated fatty acids. Initially developed for implementation with a fish model, the WBFABM has proven to be a simple and effective method that can be used in any laboratory equipped with a gas chromatography unit. Since its development, it has been used in several farmed finfish feeding trials and in broiler chicken feeding trials. The WBFABM is currently used at research institutions worldwide and its use is increasing in popularity among animal scientists. With this method, it is possible to track the fate of individual dietary fatty acids within the body. The WBFABM could contribute significantly to information generated by animal feeding trials.

Hypochlorous acid inhibition by acetoacetate: Implications on neutrophil functions

Type-1 diabetic patients experience hyperketonemia caused by an increase in fatty acid metabolism. Thus, the aim of this study was to measure the effect of ketone bodies as suppressors of oxidizing species produced by stimulated neutrophils. Both acetoacetate and 3-hydroxybutyrate have suppressive effect on the respiratory burst measured by luminol-enhanced chemiluminescence. Through measurements of hypochlorous acid production, using neutrophils or the myeloperoxidase/H2O2/Cl- system, it was found that acetoacetate but not 3-hydroxybutyrate is able to inhibit the generation of this antimicrobial oxidant. The superoxide anion scavenging properties were confirmed by ferricytochrome C reduction and lucigenin-enhanced chemiluminescence assays. However, ketone bodies did not alter the rate of oxygen uptake by stimulated neutrophils, measured with an oxygen electrode. A strong inhibition of the expression of the cytokine IL-8 by cultured neutrophils was also observed; this is discussed with reference to the antioxidant-like property of acetoacetate. © 2004 Pharmaceutical Society of Japan.

Screening of wild type Streptomyces isolates able to overproduce clavulanic acid

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Caffeic Acid Phenethyl Ester: Consequences of Its Hydrophobicity in the Oxidative Functions and Cytokine Release by Leukocytes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Culture medium of diluted skimmed milk for the production of nisin in batch cultivations

Nisin is a promising alternative to chemical preservatives for use as a natural biopreservative in foods. This bacteriocin has also potential biomedical applications. Lactic acid bacteria are commonly cultivated in expensive standard complex media. We have evaluated the cell growth and nisin production of Lactococcus lactis in a low-cost natural medium consisting of diluted skimmed milk in a 2-L bioreactor. The assays were performed at 30 degrees C for 56 h, at varying agitation speeds and airflow rates: (1) 200 rpm (no airflow, and airflow at 0.5, 1.0 and 2.0 L/min); (2) 100 rpm (no airflow, and airflow at 0.5 L/min). Nisin activity was evaluated using agar diffusion assays. The highest nisin concentration, 49.88 mg/L (3.3 log AU/mL or 1,995.29 AU/mL), was obtained at 16 h of culture, 200 rpm and no airflow (k(L)a = 5.29 x 10(-3)). These results show that a cultivation medium composed of diluted skimmed milk supports cell growth to facilitate nisin biosynthesis.

ADP-ribosylation factor and phosphatidic acid levels in Golgi membranes during budding of coatomer-coated vesicles

The finding that ADP-ribosylation factor (ARF) can activate phospholipase D has led to debate as to whether ARF recruits coat proteins through direct binding or indirectly by catalytically increasing phosphatidic acid production. Here we test critical aspects of these hypotheses. We find that Golgi membrane phosphatidic acid levels do not rise—in fact they decline—during cell-free budding reactions. We confirm that the level of membrane-bound ARF can be substantially reduced without compromising coat assembly [Ktistakis, N. T., Brown, H. A., Waters, M. G., Sternweis, P. C. & Roth, M. G. (1996) J. Cell Biol. 134, 295–306], but find that under all conditions, ARF is present on the Golgi membrane in molar excess over bound coatomer. These results do not support the possibility that the activation of coat assembly by ARF is purely catalytic, and they are consistent with ARF forming direct interactions with coatomer. We suggest that ARF, like many other G proteins, is a multifunctional protein with roles in trafficking and phospholipid signaling.