Major Metabolic Product Research Articles

Rarimicrobium hominis gen. nov., sp. nov., representing the fifth genus in the phylum Synergistetes that includes human clinical isolates.

Five human clinical isolates of an unknown, strictly anaerobic, slow-growing, Gram-stain-negative, rod-shaped micro-organism were subjected to a polyphasic taxonomic study. Comparative 16S rRNA gene sequence-based phylogeny showed that the isolates grouped in a clade that included members of the genera Pyramidobacter, Jonquetella, and Dethiosulfovibrio; the type strain of Pyramidobacter piscolens was the closest relative with 91.5-91.7 % 16S rRNA gene sequence similarity. The novel strains were mainly asaccharolytic and unreactive in most conventional biochemical tests. Major metabolic end products in trypticase/glucose/yeast extract broth were acetic acid and propionic acid and the major cellular fatty acids were C13 : 0 and C16 : 0, each of which could be used to differentiate the strains from P. piscolens. The DNA G+C content based on whole genome sequencing for the reference strain 22-5-S 12D6FAA was 57 mol%. Based on these data, a new genus, Rarimicrobium gen. nov., is proposed with one novel species, Rarimicrobium hominis sp. nov., named after the exclusive and rare finding of the taxon in human samples. Rarimicrobium is the fifth genus of the 14 currently characterized in the phylum Synergistetes and the third one in subdivision B that includes human isolates. The type strain of Rarimicrobium hominis is ADV70T ( = LMG 28163T = CCUG 65426T).

Bugs, genes, fatty acids, and serotonin: Unraveling inflammatory bowel disease?

The annual incidence of the inflammatory bowel diseases (IBDs) ulcerative colitis and Crohn’s disease has increased at an alarming rate. Although the specific pathophysiology underlying IBD continues to be elusive, it is hypothesized that IBD results from an aberrant and persistent immune response directed against microbes or their products in the gut, facilitated by the genetic susceptibility of the host and intrinsic alterations in mucosal barrier function. In this review, we will describe advances in the understanding of how the interaction of host genetics and the intestinal microbiome contribute to the pathogenesis of IBD, with a focus on bacterial metabolites such as short chain fatty acids (SCFAs) as possible key signaling molecules. In particular, we will describe alterations of the intestinal microbiota in IBD, focusing on how genetic loci affect the gut microbial phylogenetic distribution and the production of their major microbial metabolic product, SCFAs. We then describe how enteroendocrine cells and myenteric nerves express SCFA receptors that integrate networks such as the cholinergic and serotonergic neural systems and the glucagon-like peptide hormonal pathway, to modulate gut inflammation, permeability, and growth as part of an integrated model of IBD pathogenesis. Through this integrative approach, we hope that novel hypotheses will emerge that will be tested in reductionist, hypothesis-driven studies in order to examine the interrelationship of these systems in the hope of better understanding IBD pathogenesis and to inform novel therapies.

Faecalibaculum rodentium gen. nov., sp. nov., isolated from the faeces of a laboratory mouse.

A novel strictly anaerobic strain, ALO17(T), was isolated from mouse faeces and found to produce lactic acid as a major metabolic end product. The isolate was observed to be Gram-stain positive, non-motile, non-spore forming small rods, oxidase and catalase negative, and to form cream-coloured colonies on DSM 104 agar plates. The NaCl range for growth was determined to be 0-2% (w/v). The isolate was found to grow optimally at 37°C, with 0.5% (w/v) NaCl and at pH 7. The cell wall hydrolysates were found to contain ribose as a major sugar. The genomic DNA G+C content was determined to be 52.3mol%. A phylogenetic analysis of the 16S rRNA gene sequence revealed that Holdemanella biformis DSM 3989(T), Faecalicoccus pleomorphus ATCC 29734(T), Faecalitalea cylindroides ATCC 27803(T), and Allobaculum stercoricanis DSM 13633(T) are closely related to the isolate (87.4, 87.3, 86.9 and 86.9% sequence similarity), respectively. The major cellular fatty acids (>10%) of the isolate were identified as C18:1 cis 9 FAME (36.9%), C16:0 FAME (33.7%) and C18:0 FAME (13.2%). In contrast to the tested reference strains, C20:0 FAME (4.0%) was detected only in strain ALO17(T) whilst C16:0 DMA was absent. The isolate also differed in its substrate oxidation profiles from the reference strains by being positive for D-melibiose and stachyose but negative for N-acetyl-D-galactosamine and 3-methyl-D-glucose. On the basis of polyphasic taxonomic evidence from this study, the isolate is concluded to belong to a novel genus within the family Erysipelothricaceae. We propose the name Faecalibaculum rodentium gen. nov., sp. nov. to accommodate strain ALO17(T) (=KCTC 15484(T)=JCM 30274(T)) as the type strain.

Estrogenicity and androgenicity screening of PCB sulfate monoesters in human breast cancer MCF-7 cells.

Recent studies identified polychlorinated biphenyl (PCB) sulfate esters as a major product of PCB metabolism. Since hydroxy-PCBs (HO-PCBs), the immediate precursors of PCB sulfates and important contributors to PCB toxicity, were shown to have estrogenic activity, we investigated the estrogenicity/androgenicty of a series of PCB sulfate metabolites. We synthesized the five possible structural sulfate monoester metabolites of PCB 3, a congener shown to be biotransformed to sulfates, a sulfate ester of the paint-specific congener PCB 11, and sulfate monoesters of two HO-PCBs reported to interact with sulfotransferases (PCB 39, no ortho chlorines, and PCB 53, 3 ortho chlorines). We tested these PCB sulfates and 4'-HO-PCB 3 as positive control for estrogenic, androgenic, anti-estrogenic, and anti-androgenic activity in the E- and A-screen with human breast cancer MCF7-derived cells at 100 μM-1 pM concentrations. Only 4'-HO-PCB 3 was highly cytotoxic at 100 μM. We observed structure-activity relationships: compounds with a sulfate group in the chlorine-containing ring of PCB 3 (2PCB 3 and 3PCB 3 sulfate) showed no interaction with the estrogen (ER) and androgen (AR) receptor. The 4'-HO-PCB 3 and its sulfate ester had the highest estrogenic effect, but at 100-fold different concentrations, i.e., 1 and 100 μM, respectively. Four of the PCB sulfates were estrogenic (2'PCB 3, 4'PCB 3, 4'PCB 39, and 4'PCB 53 sulfates; at 100 μM). These sulfates and 3'PCB 3 sulfate also exhibited anti-estrogenic activity, but at nM and pM concentrations. The 4'PCB 3 sulfate (para-para' substituted) had the strongest androgenic activity, followed by 3'PCB 3, 4'PCB 53, 4PCB11, and 4PCB 39 sulfates and the 4'HO-PCB 3. In contrast, anti-androgenicity was only observed with the two compounds that have the sulfate group in ortho- or meta- position in the second ring (2'PCB 3 and 3'PCB 3 sulfate). No dose-response was observed in any screen, but, with exception of estrogenic activity (only seen at 100 μM), endocrine activity was often displayed at several concentrations and even at 1 pM concentration. These data suggest that sulfation of HO-PCBs is indeed reducing their cytotoxicity and estrogenicity, but may produce other endocrine disruptive activities at very low concentrations.

5'-O-Alkylpyridoxamines: Lipophilic Analogues of Pyridoxamine Are Potent Scavengers of 1,2-Dicarbonyls.

Pyridoxamine (PM) is a prospective drug for the treatment of diabetic complications. In order to make zwitterionic PM more lipophilic and improve its tissue distribution, PM derivatives containing medium length alkyl groups on the hydroxymethyl side chain were prepared. The synthesis of these alkylpyridoxamines (alkyl-PMs) starting from pyridoxine offers high yields and is amenable to bulk preparations. Interestingly, alkyl-PMs were found to react with methylglyoxal (MGO), a major toxic product of glucose metabolism and autoxidation, several orders of magnitude faster than PM. This suggests the formation of nonionic pyrido-1,3-oxazine as the key step in the reaction of PM with MGO. Since the primary target of MGO in proteins is the guanidine side chain of arginine, alkyl-PMs were shown to be more effective than PM in reducing the modification of N-α-benzoylarginine by MGO. Alkyl-PMs in the presence of MGO also protected the enzymatic activity of lysozyme that contains several arginine residues next to its active site. Alkyl-PMs can be expected to trap MGO and other toxic 1,2-carbonyl compounds more effectively than PM, especially in lipophilic tissue environments, thus protecting macromolecules from functional damage. This suggests potential therapeutic uses for alkyl-PMs in diabetes and other diseases characterized by the elevated levels of toxic dicarbonyl compounds.

Acetoanaerobium pronyense sp. nov., an anaerobic alkaliphilic bacterium isolated from a carbonate chimney of the Prony Hydrothermal Field (New Caledonia).

A novel anaerobic bacterial strain, ST07-YET, was isolated from a carbonate chimney of the Prony Hydrothermal Field (PHF) in New Caledonia. Cells were Gram-stain-positive, straight rods (0.7-0.8 × 3.0-5.0 μm) and motile by means of lateral flagella. Strain ST07-YET was mesophilic (optimum 35 °C), moderately alkaliphilic and halotolerant (optimum pH 8.7 and 5 g l- 1 NaCl). Elemental sulfur, sulfate, thiosulfate, sulfite, nitrate and nitrite were not used as terminal electron acceptors. Yeast extract, peptone, tryptone, Casamino acids, crotonate, pyruvate, galactose, maltose, sucrose, ribose, trehalose and glucose were used as carbon sources. Glucose fermentation led to acetate, H2 and CO2 formation. Arginine, serine, histidine, lysine, methionine and cysteine improved growth, but the Stickland reaction was negative for the combinations of amino acids tested. The major metabolic products from yeast extract fermentation were H2, CO2, acetate, butyrate, isobutyrate, isovalerate and propionate. The predominant cellular fatty acids were C16 : 0, C16 : 1cis9, C14 : 0 and C16 : 1cis7 (>5 % of total fatty acids). The G+C content of the genomic DNA was 32.9 mol%. Phylogenetic analysis revealed that strain ST07-YET was most closely related to Clostridium sticklandii DSM 519T and Acetoanaerobium noterae NOT-3T (96.7 % and 96.8 % 16S rRNA gene sequence similarity, respectively). On the basis of phylogenetic, chemotaxonomic and physiological properties, strain ST07-YET is proposed to represent a novel species of the genus Acetoanaerobium (order Clostridiales, phylum Firmicutes) with the name Acetoanaerobium pronyense sp. nov. The type strain is ST07-YET ( = DSM 27512T = JCM 19400T).

Use of Bacillus thuringiensis supernatant from a fermentation process to improve bioremediation of chlorpyrifos in contaminated soils

The aim of this research was to investigate the potential of a nutrient-rich organic waste, namely the cell-free supernatant of Bacillus thuringiensis (BtS) gathered from fermentation, as a biostimulating agent to improve and sustain microbial populations and their enzymatic activities, thereby assisting in the bioremediation of chlorpyrifos-contaminated soil at a high dose (70 mg kg−1). Experiments were performed for up to 80 d. Chlorpyrifos degradation and its major metabolic product, 3,5,6-trichloro-2-pyridinol (TCP), were quantified by high-performance liquid chromatography (HPLC); total microbial populations were enumerated by direct counts in specific medium; and fluorescein diacetate (FDA) hydrolysis was measured as an index of soil microbial activity. Throughout the experiment, there was higher chlorpyrifos degradation in soil supplemented with BtS (83.1%) as compared to non-supplemented soil. TCP formation and degradation occurred in all soils, but the greatest degradation (30.34%) was observed in soil supplemented with BtS. The total microbial populations were significantly improved by supplementation with BtS. The application of chlorpyrifos to soil inhibited the enzymatic activity; however, this negative effect was counteracted by BtS, inducing an increase of approximately 16% in FDA hydrolysis. These results demonstrate the potential of B. thuringiensis supernatant as a suitable biostimulation agent for enhancing chlorpyrifos and TCP biodegradation in chlorpyrifos-contaminated soils.

Metabolite-sensing receptors GPR43 and GPR109A facilitate dietary fibre-induced gut homeostasis through regulation of the inflammasome.

Diet and the gut microbiota may underpin numerous human diseases. A major metabolic product of commensal bacteria are short-chain fatty acids (SCFAs) that derive from fermentation of dietary fibre. Here we show that diets deficient or low in fibre exacerbate colitis development, while very high intake of dietary fibre or the SCFA acetate protects against colitis. SCFAs binding to the 'metabolite-sensing' receptors GPR43 and GPR109A in non-haematopoietic cells mediate these protective effects. The inflammasome pathway has hitherto been reported as a principal pathway promoting gut epithelial integrity. SCFAs binding to GPR43 on colonic epithelial cells stimulates K(+) efflux and hyperpolarization, which lead to NLRP3 inflammasome activation. Dietary fibre also shapes gut bacterial ecology, resulting in bacterial species that are more effective for inflammasome activation. SCFAs and metabolite receptors thus explain health benefits of dietary fibre, and how metabolite signals feed through to a major pathway for gut homeostasis.

Clinical pharmacology of theophylline in preterm infants: effects, metabolism and pharmacokinetics.

Recurrent apnea is common in preterm infants with consequent episodes of loss of effective breathing and the bronchodilator theophylline prevents apnea and reduces the number of apneic attacks. This drug also reduces hypoxaemic episodes. Theophylline acts on the lungs, kidneys and brain. Theophylline inhibits solute reabsorption in various segments of the nephron and a marked diuresis which occurs immediately after the administration of theophylline. This drug ameliorates kidney dysfunction and prophylaxis given early after birth, preventing vasomotor nephropathy. Theophylline reduces brain activity and reduces the spontaneous activity transients and alters the sleep-wake state in pre-term infants. Theophylline is extensively metabolized in premature infants and its major metabolic product is caffeine. The demethylation pathway occurring predominantly in adults is substituted by N-methylation to caffeine in premature infants. The halflife of theophylline is 5-fold longer in neonates than in adults and reaches the adult value at the age of 55 weeks. Theophylline may be administered trans-cutaneously by applying this drug to the back or abdomen of the infants and the mean fractional absorbance at 30 hours is 0.25. Theophylline is present in saliva and the concentration of this drug in saliva is similar to that in plasma, saliva may be used to monitor theophylline concentration. In conclusion, theophylline is a useful drug to treat apnea and ameliorate kidney dysfunction.

Direct fermentation of potato starch and potato residues to lactic acid by Geobacillus stearothermophilus under non-sterile conditions.

BACKGROUNDLactic acid is an important biorefinery platform chemical. The use of thermophilic amylolytic microorganisms to produce lactic acid by fermentation constitutes an efficient strategy to reduce operating costs, including raw materials and sterilization costs.RESULTSA process for the thermophilic production of lactic acid by Geobacillus stearothermophilus directly from potato starch was characterized and optimized. Geobacillus stearothermophilus DSM 494 was selected out of 12 strains screened for amylolytic activity and the ability to form lactic acid as the major product of the anaerobic metabolism. In total more than 30 batches at 3–l scale were run at 60 °C under non-sterile conditions. The process developed produced 37 g L−1 optically pure (98%) L-lactic acid in 20 h from 50 g L−1 raw potato starch. As co-metabolites smaller amounts (<7% w/v) of acetate, formate and ethanol were formed. Yields of lactic acid increased from 66% to 81% when potato residues from food processing were used as a starchy substrate in place of raw potato starch.CONCLUSIONSPotato starch and residues were successfully converted to lactic acid by G. stearothermophilus. The process described in this study provides major benefits in industrial applications and for the valorization of starch-rich waste streams. © 2015 The Authors.Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Inhibition of 12/15-Lipoxygenase Reduces Renal Inflammation and Injury in Streptozotocin-Induced Diabetic Mice.

Previous studies suggest that 12/15 lipoxygenase (12/15-LO) is implicated in diabetic vascular complications. We hypothesize that 12/15-LO inhibition attenuates renal inflammation and injury in streptozotocin-induced diabetes. Diabetes was induced in wild-type C57BL/6J (WT) and 12/15-LO deficient mice using streptozotocin. Additionally, four groups of WT mice were also used; control non diabetic, diabetic, diabetic treated with the 12/15-LO inhibitor baicalein for 10 weeks and diabetic treated with baicalein only for the last 4 weeks of the experiment. After 10 weeks of induction of diabetes with streptozotocin, WT diabetic mice exhibited marked elevation in proteinuria together with elevation in the excretion levels of thiobarbituric acid reactive substance (TBARs), a marker of oxidative stress, and monocyte chemoattractant protein-1 (MCP-1), a marker of inflammation and these changes were significantly reduced in 12/15-LO deficient diabetic mice (P<0.05). Similarly, pharmacological inhibition of 12/15-LO with baicalein prevented the elevation in renal 12-HETE production, the major murine metabolic product of 12/15-LO, in diabetic mice, and this effect was associated with decreased proteinuria, TBARs excretion and renal collagen deposition compared to untreated diabetic mice. Interestingly, the protective effects of baicalein were not noticed when only administered in the last 4 weeks of diabetes compared to untreated diabetic mice. WT diabetic mice displayed elevation in renal interleukin-6 (IL-6) levels and these changes were only reduced in diabetic mice treated with baicalein for 10 weeks (P<0.05). The anti-inflammatory effects of baicalein or 12/15-LO deficiency were further confirmed in lipopolysaccharide (LPS)-induced acute renal inflammation as inhibition of 12/15-LO reduced the elevation in renal soluble epoxide hydrolase expression in LPS-injected mice. These results suggest that increased 12/15-LO activity and 12-HETE production contribute to the elevation of renal oxidative stress, inflammation and injury in streptozotocin-induced diabetic mice.

Stereoselective suppressive effects of protopanaxadiol epimers on UV-B-induced reactive oxygen species and matrix metalloproteinase-2 in human dermal keratinocytes.

This study aimed to assess the skin-related anti-photoaging activities of the 2 epimeric forms of protopanaxadiol (PPD), 20(S)-PPD and 20(R)-PPD, in cultured human keratinocytes (HaCaT cells). The anti-photoaging activity was evaluated by analyzing the levels of reactive oxygen species (ROS) and matrix metalloproteinases (MMPs), as well as cell viability for HaCaT cells under UV-B irradiation. The activities for MMP-2 and -1 in conditioned medium were determined using gelatin zymography, and MMP-2 protein in the conditioned medium was detected using Western blot analysis. 20(S)-PPD, but not 20(R)-PPD, suppressed UV-B-induced ROS elevation. Neither of the epimers, at the concentrations used, exhibited cytotoxicity, irrespective of UV-B irradiation. 20(S)-PPD, but not 20(R)-PPD, exhibited an inhibitory effect on UV-B-induced MMP-2 activity and expression in HaCaT cells. In brief, only 20(S)-PPD, a major metabolic product of PPD-type ginsenosides, inhibits UV-B-induced ROS and MMP-2 elevation, implying its stereospecific anti-photoaging activity on the skin.

Faecalicoccus acidiformans gen. nov., sp. nov., isolated from the chicken caecum, and reclassification of Streptococcus pleomorphus (Barnes et al. 1977), Eubacterium biforme (Eggerth 1935) and Eubacterium cylindroides (Cato et al. 1974) as Faecalicoccus pleomorphus comb. nov., Holdemanella biformis gen. nov., comb. nov. and Faecalitalea cylindroides gen. nov., comb. nov., respectively, within the family Erysipelotrichaceae.

Strains LMG 27428(T) and LMG 27427 were isolated from the caecal content of a chicken and produced butyric, lactic and formic acids as major metabolic end products. The genomic DNA G+C contents of strains LMG 27428(T) and LMG 27427 were 40.4 and 38.8 mol%. On the basis of 16S rRNA gene sequence similarity, both strains were most closely related to the generically misclassified Streptococcus pleomorphus ATCC 29734(T). Strain LMG 27428(T) could be distinguished from S. pleomorphus ATCC 29734(T) based on production of more lactic acid and less formic acid in M2GSC medium, a higher DNA G+C content and the absence of activities of acid phosphatase and leucine, arginine, leucyl glycine, pyroglutamic acid, glycine and histidine arylamidases, while strain LMG 27428 was biochemically indistinguishable from S. pleomorphus ATCC 29734(T). The novel genus Faecalicoccus gen. nov. within the family Erysipelotrichaceae is proposed to accommodate strains LMG 27428(T) and LMG 27427. Strain LMG 27428(T) ( =DSM 26963(T)) is the type strain of Faecalicoccus acidiformans sp. nov., and strain LMG 27427 ( =DSM 26962) is a strain of Faecalicoccus pleomorphus comb. nov. (type strain LMG 17756(T) =ATCC 29734(T) =DSM 20574(T)). Furthermore, the nearest phylogenetic neighbours of the genus Faecalicoccus are the generically misclassified Eubacterium cylindroides DSM 3983(T) (94.4% 16S rRNA gene sequence similarity to strain LMG 27428(T)) and Eubacterium biforme DSM 3989(T) (92.7% 16S rRNA gene sequence similarity to strain LMG 27428(T)). We present genotypic and phenotypic data that allow the differentiation of each of these taxa and propose to reclassify these generically misnamed species of the genus Eubacterium formally as Faecalitalea cylindroides gen. nov., comb. nov. and Holdemanella biformis gen. nov., comb. nov., respectively. The type strain of Faecalitalea cylindroides is DSM 3983(T) =ATCC 27803(T) =JCM 10261(T) and that of Holdemanella biformis is DSM 3989(T) =ATCC 27806(T) =CCUG 28091(T).

Modulation of GR activity does not affect the in vitro metabolism of cortisol by rainbow trout ovarian follicles.

The goal of the study was to determine whether the metabolic clearance of cortisol from rainbow trout (Oncorhynchus mykiss) ovarian follicles is affected by the level of ovarian steroidogenesis, and whether it involves the activation of glucocorticoid receptors (GRs). Ovarian follicles were incubated in vitro; the adenylate cyclase activator, forskolin, was used to stimulate ovarian steroidogenesis, and the modulation of GR activity was brought about using GR agonists (cortisol and dexamethasone) or the GR antagonist, mifepristone (RU486). The follicles were co-incubated with [2, 4, 6, 7 (3)H] cortisol, and the tritium-labelled steroid products were separated by HPLC. In addition, the rates of expression of genes encoding for the two forms of GR (gr1 and gr2) were measured. Cortisone, cortisol sulphate, and cortisone sulphate were the major glucocorticoid products of cortisol metabolism, indicative of the action of 11β-hydroxysteroid dehydrogenase and glucocorticoid sulphotransferase in the follicular cells. There were no effects of RU486 or forskolin on the rates of [(3)H]cortisol metabolism suggesting that cortisol metabolism by ovarian follicles was independent of GR activation, and not influenced by increased activation of gonadal reproductive steroidogenesis.

Oribacterium parvum sp. nov. and Oribacterium asaccharolyticum sp. nov., obligately anaerobic bacteria from the human oral cavity, and emended description of the genus Oribacterium

Three strictly anaerobic, Gram-positive, non-spore-forming, rod-shaped, motile bacteria, designated strains ACB1(T), ACB7(T) and ACB8, were isolated from human subgingival dental plaque. All strains required yeast extract for growth. Strains ACB1(T) and ACB8 were able to grow on glucose, lactose, maltose, maltodextrin and raffinose; strain ACB7(T) grew weakly on sucrose only. The growth temperature range was 30-42 °C with optimum growth at 37 °C. Major metabolic fermentation end products of strain ACB1(T) were acetate and lactate; the only product of strains ACB7(T) and ACB8 was acetate. Major fatty acids of strain ACB1(T) were C(14 : 0), C(16 : 0), C(16 : 1)ω7c dimethyl aldehyde (DMA) and C(18 : 1)ω7c DMA. Major fatty acids of strain ACB7(T) were C(12 : 0), C(14 : 0), C(16 : 0), C(16 : 1)ω7c and C(16 : 1)ω7c DMA. The hydrolysate of the peptidoglycan contained meso-diaminopimelic acid, indicating peptidoglycan type A1γ. Genomic DNA G+C content varied from 42 to 43.3% between strains. According to 16S rRNA gene sequence phylogeny, strains ACB1(T), ACB8 and ACB7(T) formed two separate branches within the genus Oribacterium, with 98.1-98.6% sequence similarity to the type strain of the type species, Oribacterium sinus. Predicted DNA-DNA hybridization values between strains ACB1(T), ACB8, ACB7(T) and O. sinus F0268 were <70%. Based on distinct genotypic and phenotypic characteristics, strains ACB1(T) and ACB8, and strain ACB7(T) are considered to represent two distinct species of the genus Oribacterium, for which the names Oribacterium parvum sp. nov. and Oribacterium asaccharolyticum sp. nov. are proposed. The type strains are ACB1(T) ( = DSM 24637(T) = HM-481(T) = ATCC BAA-2638(T)) and ACB7(T) ( = DSM 24638(T) = HM-482(T) = ATCC BAA-2639(T)), respectively.

The dual effects of nitrite on hemoglobin-dependent redox reactions

Evidence to support the role of heme proteins-dependent reactions as major inducers of oxidative damage is increasingly present. Nitrite (NO2−) is one of the major end products of NO metabolism, and from the daily consumption. Although the biological significance of heme proteins/NO2−-mediated protein tyrosine nitration is a subject of great interest, the important roles of NO2− on heme proteins-dependent redox reactions have been greatly underestimated. In this study, we investigated the influence of NO2− on met-hemoglobin (Hb)-dependent oxidative and nitrative stress. It was found that NO2− effectively reduced cytotoxic ferryl intermediate back to ferric Hb in a biphasic kinetic reaction. However, the presence of NO2− surprisingly exerted pro-oxidant effect on Hb–H2O2-induced protein (bovine serum albumin, enolase) oxidation at low concentrations and enhanced the loss of HepG2 cell viability. In the reduction of ferryl Hb to ferric state, NO2− was decreased and oxidized to a nitrating agent NO2, Tyr12 and Tyr191 in enolase were subsequently nitrated. In contrast to the frequently inhibitive effect of nitrotyrosine, NO2−-triggered tyrosine nitration might play an important role in enolase activation. These data provided novel evidence that the dietary intake and potential therapeutic application of NO2− would possess anti- and pro-oxidant activities through interfering in hemoglobin-dependent redox reactions. Besides the classic role in protein tyrosine nitration, the dual effects on hemoglobin-triggered oxidative stress may provide new insights into the physiological and toxicological implications of NO2− with heme proteins.

Nitrogen metabolism and gas exchange parameters associated with zinc stress in tobacco expressing an ipt gene for cytokinin synthesis

Increased endogenous plant cytokinin (CK) content through transformation with an isopentyl transferase (ipt) gene has been associated with improved plant stress tolerance. The impact of zinc (tested levels Zn1=250, Zn2=500, Zn3=750mgkg(-1)soil) on gas exchange parameters (net photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO2 concentration) and nitrogen utilization by plants resulted in changes of free amino acid concentrations (glutamic acid, glutamine, asparagine, aspartate, glycine, serine, cystein) and differed for transformed and non-transformed tobacco plants. For pot experiments, tobacco plants (Nicotiana tabacum L., cv. Wisconsin 38) transformed with a construct consisting of SAG12 promoter fused with the ipt gene for cytokinin synthesis (SAG plants) and its wild type (WT plants as a control) were used. Physiological analyses confirmed that SAG plants had improved zinc tolerance compared with the WT plants. The enhanced Zn tolerance of SAG plants was associated with the maintenance of accumulation of amino acids and with lower declines of photosynthetic and transpiration rates. In comparison to WT plants, SAG plants exposed to the highest Zn concentration accumulated lower concentrations of asparagine, which is a major metabolic product during senescence.

The role of lactate on the immunomodulatory properties of the nonbacterial fraction of kefir

The identification of components responsible for the bioactive properties of functional foods is of central interest in the food industry. In particular, fermented dairy products that are of a health benefit to the consumer may exert those salutary effects through the constituent microorganisms per se and/or through other bioactive components. Kefir is a beverage obtained by the fermentation of milk with kefir grains containing different lactic- and acetic-acid bacteria plus yeasts. We studied the immunomodulatory capacity of the nonbacterial fraction of kefir through approaches involving biochemistry and cell biology. Lactate, a major microbial metabolic product, was identified as the component responsible for the modulation of certain innate immune epithelial response: At the concentrations found in kefir-fermented milk, lactate inhibits the activation of intestinal epithelial cells triggered by interleukin-1β, tumor necrosis factor-α, or flagellin. Lactate treatment furthermore abrogates NF-κB signaling in the cells, whose action could be responsible for the observed modulation of the inflammatory response. These findings provide a new perspective in the analysis of the biologic properties of kefir-fermented-milk products.

Nonsteroidal Anti-Inflammatory Drugs and the Heart

Aspirin has been on the market for 115 years. Beginning with the marketing of indomethacin for the treatment of rheumatoid arthritis in 1963, at least 20 other nonsteroidal anti-inflammatory drugs (NSAIDs) with aspirin-like actions have been developed over the past 50 years,1 culminating with the introduction of a new class of selective inhibitors of cyclooxygenase (COX)-2, the coxibs, approximately 15 years ago.2 The NSAIDs represent the single most crowded family of drugs sharing the same therapeutic activities and mechanism of action, perhaps reflecting the unmet therapeutic need in the area of pain management and the large interindividual variability in response to these agents. NSAIDs provide symptomatic relief of pain and inflammation associated with a variety of human disorders, including the rheumatic diseases. Their shared therapeutic actions (ie, analgesic, anti-inflammatory, and antipyretic) are usually accompanied by mechanism-based adverse effects that can, at least in part, be attenuated as a function of individual pharmacokinetic or pharmacodynamic properties.1 Nuances in the tolerability of different NSAIDs had been described in the precoxib era on the basis of clinical trials of a few hundred patients treated for up to a few months with soft end points. More recently, however, important differences in safety have been demonstrated in head-to-head randomized comparisons of individual coxibs and 1 or more traditional NSAIDs that involved tens of thousands of patients treated for up to a few years with hard end points. Even more significantly, the interpretation of the effects of NSAIDs has been greatly enhanced by the availability, for the first time, of large, placebo-controlled trials that aimed to assess the potential for chemoprevention of colorectal cancer by rofecoxib or celecoxib. As a result of these developments, the whole field of NSAIDs has been illuminated during the past 15 years. Although epidemiological studies had previously associated …

Improvement of cellulose catabolism in Clostridium cellulolyticum by sporulation abolishment and carbon alleviation

BackgroundClostridium cellulolyticum can degrade lignocellulosic biomass, and ferment the soluble sugars to produce valuable chemicals such as lactate, acetate, ethanol and hydrogen. However, the cellulose utilization efficiency of C. cellulolyticum still remains very low, impeding its application in consolidated bioprocessing for biofuels production. In this study, two metabolic engineering strategies were exploited to improve cellulose utilization efficiency, including sporulation abolishment and carbon overload alleviation.ResultsThe spo0A gene at locus Ccel_1894, which encodes a master sporulation regulator was inactivated. The spo0A mutant abolished the sporulation ability. In a high concentration of cellulose (50 g/l), the performance of the spo0A mutant increased dramatically in terms of maximum growth, final concentrations of three major metabolic products, and cellulose catabolism. The microarray and gas chromatography–mass spectrometry (GC-MS) analyses showed that the valine, leucine and isoleucine biosynthesis pathways were up-regulated in the spo0A mutant. Based on this information, a partial isobutanol producing pathway modified from valine biosynthesis was introduced into C. cellulolyticum strains to further increase cellulose consumption by alleviating excessive carbon load. The introduction of this synthetic pathway to the wild-type strain improved cellulose consumption from 17.6 g/l to 28.7 g/l with a production of 0.42 g/l isobutanol in the 50 g/l cellulose medium. However, the spo0A mutant strain did not appreciably benefit from introduction of this synthetic pathway and the cellulose utilization efficiency did not further increase. A technical highlight in this study was that an in vivo promoter strength evaluation protocol was developed using anaerobic fluorescent protein and flow cytometry for C. cellulolyticum.ConclusionsIn this study, we inactivated the spo0A gene and introduced a heterologous synthetic pathway to manipulate the stress response to heavy carbon load and accumulation of metabolic products. These findings provide new perspectives to enhance the ability of cellulolytic bacteria to produce biofuels and biocommodities with high efficiency and at low cost directly from lignocellulosic biomass.