The anti-obesity effects exerted by different fractions of Artemisia sphaerocephala Krasch polysaccharide in diet-induced obese mice.

Artemisia sphaerocephala Krasch polysaccharide (ASKP) consists of two main fractions, 60P (molecular weight at 551 kDa) and 60S (molecular weight at 39 kDa). The anti-obesity effects of ASKP and its two fractions were investigated in high-fat-diet-fed mice and showed similar capability in efficiently preventing the development of obesity. The final body weight and body weight gain of obesity mice model were reduced by 12.44% and 35.33% by ASKP, 10.63% and 34.35% by 60P, and 7.82% and 20.04% by 60S. They also showed similar efficiency to ameliorate dyslipidemia, systematic inflammation, and gut dysbiosis. The colonic genes of barrier integrity were significantly upregulated and the genes of hepatic lipid metabolism and that of colonic inflammatory response were suppressed. They attenuated the gut dysbiosis in obese mice, such as the significant enrichment of beneficial genera (Bifidobacterium and Olsenella) and suppression of harmful ones (Mucispirillum and Helicobacter). Significant enrichment of carbohydrate metabolism associated with the promotion of short-chain fatty acid production and decrease of the metabolisms related to obesity and gut dysbiosis (valine, leucine, and isoleucine biosynthesis, and nitrogen metabolism) were also observed by the administration of ASKP, 60P, and 60S. Overall, these polysaccharides showed potential in acting as prebiotics in preventing high-fat-diet-induced obesity.

Evaluation of in situ valine production by Bacillus subtilis in young pigs.

Mutants of Bacillus subtilis can be developed to overproduce Val in vitro. It was hypothesized that addition of Bacillus subtilis mutants to pig diets can be a strategy to supply the animal with Val. The objective was to investigate the effect of Bacillus subtilis mutants on growth performance and blood amino acid (AA) concentrations when fed to piglets. Experiment 1 included 18 pigs (15.0±1.1 kg) fed one of three diets containing either 0.63 or 0.69 standardized ileal digestible (SID) Val : Lys, or 0.63 SID Val : Lys supplemented with a Bacillus subtilis mutant (mutant 1). Blood samples were obtained 0.5 h before feeding and at 1, 2, 3, 4, 5 and 6 h after feeding and analyzed for AAs. In Experiment 2, 80 piglets (9.1±1.1 kg) were fed one of four diets containing 0.63 or 0.67 SID Val : Lys, or 0.63 SID Val : Lys supplemented with another Bacillus subtilis mutant (mutant 2) or its parent wild type. Average daily feed intake, daily weight gain and feed conversion ratio were measured on days 7, 14 and 21. On day 17, blood samples were taken and analyzed for AAs. On days 24 to 26, six pigs from each dietary treatment were fitted with a permanent jugular vein catheter, and blood samples were taken for AA analysis 0.5 h before feeding and at 1, 2, 3, 4, 5 and 6 h after feeding. In experiment 1, Bacillus subtilis mutant 1 tended (P<0.10) to increase the plasma levels of Val at 2 and 3 h post-feeding, but this was not confirmed in Experiment 2. In Experiment 2, Bacillus subtilis mutant 2 and the wild type did not result in a growth performance different from the negative and positive controls. In conclusion, results obtained with the mutant strains of Bacillus subtilis were not better than results obtained with the wild-type strain, and for both strains, the results were not different than the negative control.

Integrative drug efficacy assessment of Danggui and European Danggui using NMR-based metabolomics.

Danggui (DG) is a commonly used herbal drug in traditional Chinese medicine, and usually adulterated with European Danggui (EDG) due to the increasing demand. In present study, global metabolic profiling with NMR coupled with integrative drug efficacy evaluation methods was performed to compare and discover underlying blood-enriching regulation mechanisms of DG and EDG on blood deficiency rats induced by acetyl phenylhydrazine (APH). Totally, the contents of 12 key metabolites in serum and 4 in urine of DG group, 7 in serum and 4 in urine of EDG group were significantly reversed in comparison with model group. DG was more effective than EDG as revealed by the relative distance, efficacy index and similarity analysis. The metabolism pathways analysis showed that the better effect of DG maybe related with the regulatory effect on valine, leucine and isoleucine biosynthesis, synthesis and degradation of ketone bodies, glycine, serine and threonine metabolism, as well as nicotinate and nicotinamide metabolism. The results presented here showed that metabolomic coupled with efficacy index and similarity analysis made it possible to disclose the subtle biological difference between DG and EDG, which highlight the potential of metabolomic approach to quantitatively compare the pharmacological effect of the herbal drugs.

Trace element associated reduction of norleucine and norvaline accumulation during oxygen limitation in a recombinant Escherichia coli fermentation.

BACKGROUND: Norleucine and norvaline belong to a group of non-canonical amino acids which are synthesized as byproducts in the branched chain amino acid metabolism of Escherichia coli. The earlier observed misincorporation of these rare amino acids into recombinant proteins has attracted increasing attention due to the rising use of protein based biopharmaceuticals in clinical application. Experimental data revealed pyruvate overflow inducing conditions, which typically occur in oxygen limited zones of large-scale fermentations as a major reason leading to norvaline and norleucine synthesis during E. coli cultivation. Previous approaches to suppress misincorporation of norleucine and norvaline considered growth media supplementation with the relevant canonical isostructural compounds, but no research was performed on the impact of the overflow metabolism related trace elements molybdenum, nickel and selenium. These elements form essential parts of the formate hydrogen lyase (FHL) metalloprotein complex, which is a key enzyme of anaerobic pyruvate metabolism in E. coli and could therefore represent a crucial connection to the pyruvate accumulation associated biosynthesis of rare amino acids. RESULTS: In this study, the trace element associated response of recombinant antibody producing E. coli to oxygen limitation at high glucose concentration with a special focus on non-canonical amino acids was analysed. During fed-batch cultivation with provoked oxygen limitation and glucose excess norleucine and norvaline were only accumulated in the absence of molybdenum, nickel and selenium. In contrast, the trace element supplemented stress fermentation showed significantly reduced concentrations of these rare amino acids and the major signature fermentation product formate, supporting the correlation between a functional formate hydrogen lyase complex and low unspecific amino acid synthesis under oxygen limitation at high glucose concentration. CONCLUSIONS: The formation of norleucine and norvaline by recombinant E. coli during cultivation with provoked oxygen limitation and glucose excess can be reduced to levels at the detection limit by adding the trace elements molybdenum, selenium and nickel to the fermentation medium. Even under the metabolic burden during induction phase the physiologically available concentrations of non-canonical amino acids remained low. Since our results allow facile process changes that can be easily implemented to avoid the undesirable accumulation of norleucine and norvaline, we consider this study highly interesting for improved process development in E. coli based recombinant drug production and the future development of possible mechanisms to reduce misincorporation events into protein based biopharmaceuticals.

Elaborate transcription regulation of the Bacillus subtilis ilv-leu operon involved in the biosynthesis of branched-chain amino acids through global regulators of CcpA, CodY and TnrA.

The Bacillus subtilis ilv-leu operon involved in the biosynthesis of branched-chain amino acids is under negative regulation mediated by TnrA and CodY, which recognize and bind to their respective cis-elements located upstream of the ilv-leu promoter. This operon is known to be under CcpA-dependent positive regulation. We have currently identified a catabolite-responsive element (cre) for this positive regulation (bases -96 to -82; +1 is the ilv-leu transcription initiation base) by means of DNase I-footprinting in vitro, and deletion and base-substitution analyses of cre. Under nitrogen-rich growth conditions in glucose-minimal medium supplemented with glutamine and amino acids, CcpA and CodY exerted positive and negative regulation of ilv-leu, respectively, but TnrA did not function. Moreover, CcpA and CodY were able to function without their counteracting regulation of each other, although the CcpA-dependent positive regulation did not overcome the CodY-dependent negative regulation. Furthermore, under nitrogen-limited conditions in glucose-minimal medium with glutamate as the sole nitrogen source, CcpA and TnrA exerted positive and negative regulation, respectively, but CodY did not function. This CcpA-dependent positive regulation occurred without the TnrA-dependent negative regulation. However, the TnrA-dependent negative regulation did not occur without the CcpA-dependent positive regulation, raising the possibility that this negative regulation might decrease the CcpA-dependent positive regulation. The physiological role of this elaborate transcription regulation of the B. subtilis ilv-leu operon in overall metabolic regulation in this organism is discussed.

Formation of N-(2-hydroxyethyl)valine in human hemoglobin-effect of lifestyle factors.

The formation of N-(2-hydroxyethyl)valine (HEV) in hemoglobin has been considered as a biomarker to assess exogenous and endogenous exposures to ethylene oxide (EO) and/or ethylene (ET). Factors associated with daily exposures to such compounds might significantly affect the formation of HEV. Tobacco smoke containing EO elicited a significant increase in the levels of HEV amongst smokers, although other factors related to lifestyles may warrant further studies. The objective of this study was to specifically analyze HEV using a modified Edman degradation technique in order to study the association between lifestyle related factors (smoking, second-hand smoke exposure, tea and alcohol consumption) and HEV formation in vivo. Total of 148 Taiwanese volunteers with no history of occupational exposure to either EO or ET were recruited in this study. The HEV levels for smokers (204 +/- 151 pmol HEV/g globin, n = 70 ) were greater than those for non-smokers (57 +/- 46 pmol HEV/g globin, n = 78), HEV level increasing with the number of cigarettes smoked by subjects per day with a rate of 8.8 pmol HEV/g globin per cigarettes per day. Further analysis revealed that the rate of HEV formation in our study subjects was significantly associated with the number of daily cigarettes smoked (P < 0.001), but was not associated with tea or alcohol consumption, second-hand smoke exposure, subject age, or subject gender. These results suggest that the significantly higher levels of HEV for smokers than for non-smokers were mainly due to subject exposure to EO contained in cigarette smoke.

Acetohydroxy acid synthase I is required for isoleucine and valine biosynthesis by Salmonella typhimurium LT2 during growth on acetate or long-chain fatty acids.

Salmonella typhimurium LT2 normally expresses two acetohydroxy acid synthases (AHAS I and AHAS II). The function of AHAS I in this organism was unclear, since AHAS I-deficient (ilvBN) mutants of LT2 grew well on glucose or succinate minimal media, whereas AHAS II-deficient (ilvGM) mutants requried isoleucine for normal growth on glucose minimal media. We report that AHAS I-deficient mutants of S. typhimurium required isoleucine and valine for growth on acetate or oleate minimal media, whereas AHAS II-deficient mutants were able to grow on these media without isoleucine supplementation.

Valine production from hydrocarbon by Micrococcus varians.

A bacterium isolated from Assam (India) soil was found to accumulate L-valine in the growth medium and was identified as Micrococcus varians. The strain grew and accumulated valine in a purely synthetic medium, but supplementation with either casamino acids or yeast extract or with both, improved the yield. The entire fermentation period could be divided into a growth phase and a production (phase which could be prolonged by adjustment of pH to neutral range. Among the different hydrocarbon and nitrogen sources tested straight run gas-oil and ammonium sulphate, respectively, were found most suitable. Antibiotics inhibited growth but stimulated extracellular valine accumulation. Vitamins stimulated growth and valine yield and an inoculum level of 10% was found to be optimal. The yield of L-valine under optimal conditions was 2.95 g/L.

Production of valine by a Bacillus sp.

A bacterium isolated from Burdwan (India) soil was found to accumulate L-valine in the growth medium and identified to be a strain of Bacillus subtilis. The strain is able to grow and accumulate valine in a purely synthetic medium, but supplementation of the synthetic medium with either Casamino acids or yeast extract or with both, significantly improves the yield. The entire fermentation period can be divided into a growth phase and a production phase, which can be prolonged by adjustment of pH to the neutral range. Among the different carbon and nitrogen sources tested glucose at 8.5% and L-glutamic acid at 0.8%, respectively, were found most suitable. Cane sugar molasses tested as a substitute for glucose significantly stimulated growth but valine production was less. Different vitamins tested stimulated growth and valine yield and an inoculum level of 10% (v/v) of the medium was found to be optimal. The yield of valine under optimal conditions was found to be 4.53 g per litre of the medium. Valine has been isolated in crystalline form from the fermented broth by ion exchange resin chromatography and found to be a pure sample of the L-isomer.

Enzymes of the isoleucine-valine pathway in Acinetobacter.

Regulation of four of the enzymes required for isoleucine and valine biosynthesis in Acinetobacter was studied. A three- to fourfold derepression of acetohydroxyacid synthetase was routinely observed in two different wild-type strains when grown in minimal medium relative to cells grown in minimal medium supplemented with leucine, valine, and isoleucine. A similar degree of synthetase derepression was observed in appropriately grown isoleucine or leucine auxotrophs. No significant derepression of threonine deaminase or transaminase B occurred in either wild-type or mutant cells grown under a variety of conditions. Three amino acid analogues were tested with wild-type cells; except for a two- to threefold derepression of dihydroxyacid dehydrase when high concentrations of aminobutyric acid were added to the medium, essentially the same results were obtained. Experiments showed that threonine deaminase is subject to feedback inhibition by isoleucine and that valine reverses this inhibition. Cooperative effects in threonine deaminase were demonstrated with crude extracts. The data indicate that the synthesis of isoleucine and valine in Acinetobacter is regulated by repression control of acetohydroxyacid synthetase and feedback inhibition of threonine deaminase and acetohydroxyacid synthetase.