Butyric acid sensitizes Vero cells to ricin-induced apoptosis via accelerated activation of multiple signal transduction pathways

We found that the treatment with 1 mM butyric acid for 2 days renders Vero cells highly sensitive to ricin-induced apoptosis reflected by cytolysis concomitant with apoptotic cellular and nuclear morphological changes, DNA fragmentation, and increase in caspase-3 like activity, whereas butyric acid alone had no cytotoxic effect on Vero cells. During the treatment with butyric acid, gradual increase in alkaline phosphatase activity, an indicator for butyric acid-induced differentiation, was observed in Vero cells. Although the potency of ricin-mediated protein synthesis was increased in butyric acid-treated Vero cells as compared to untreated cells, the binding and internalization of ricin to the cells were not much affected. Furthermore, DNA fragmentation caused by other protein synthesis inhibitors such as diphtheria toxin and anisomysin were also highly potentiated in butyric acid-treated Vero cells, whereas the potencies of these toxins to inhibit the protein synthesis were not affected by butyric acid treatment. These results suggest that the apoptosis signaling pathway, which may be triggered by cytotoxic stress response caused by toxins, is sensitized in butyric acid-treated cells, while the pathways leading to the protein synthesis inhibition by these toxins are relatively unchanged. No significant differences in the expression levels of p21, p53, and Bcl-2 proteins were observed between butyric acid-treated and untreated Vero cells. The treatment with ricin resulted in the activation of p38 MAP kinase, and this activation occurred on an accelerated time schedule in butyric acid-treated Vero cells than in untreated cells. The specific inhibitor of p38 MAP kinase SB203580 showed a partial inhibitory effect on ricin-induced apoptosis in control Vero cells, but it was less effective in butyric acid-treated Vero cells. Taken together, our results suggest that butyric acid-treatment may result in sensitization of multiple intracellular signal transduction pathways including apoptotic signaling pathways and p38 MAP kinase pathway.     

Dissimilation of methionine by fungi

Soil fungi that attacked methionine required a utilizable source of energy such as glucose for growth. This is an example of co-dissimilation. Experiments with one of the fungi, representative of the group, are reported. In the absence of glucose, pregrown mycelium, even when depleted of energy reserves, oxidatively deaminated methionine with accumulation of α-keto-γ-methyl mercapto butyric acid and α-hydroxy-γ-methyl mercapto butyric acid. When glucose was provided, all of the sulfur of methionine was released as methanethiol, part of which was oxidized to dimethyl disulfide. No sulfate, sulfide, or hydrosulfide products were detected. Evidence was obtained that deaminase and demethiolase were constitutive. Deamination preceded demethiolation and α-keto butyric acid accumulated as a product of the two reactions. Other carbon residues were α-hydroxy butyric acid and α-amino butyric acid. Inability of the fungus to metabolize α-keto butyrate was responsible for its inability to utilize methionine as a source of carbon and energy. Several other fungi isolated from soil grew on α-amino butyrate but could not grow on methionine owing to inability to demethiolate it.     

Retrograde transport from the Golgi complex to the ER of both Shiga toxin and the nontoxic Shiga B-fragment is regulated by butyric acid and cAMP

Endocytosed Shiga toxin is transported from the Golgi complex to the endoplasmic reticulum in butyric acid-treated A431 cells. We here examine the extent of this retrograde transport and its regulation. The short B fragment of Shiga toxin is sufficient for transport to the ER. The B fragment of cholera toxin, which also binds to glycolipids, is transported to all the Golgi cisterns, but cannot be localized in the ER even after butyric acid treatment. Under all conditions the toxic protein ricin was found predominantly in the trans-Golgi network. There is no transport of endocytosed fluid to the Golgi apparatus or to the ER even after butyric acid treatment and in the presence of Shiga toxin, indicating that transport to the ER, through the trans-Golgi network and the cisterns of the Golgi apparatus, involves several sorting stations. Since Shiga toxin receptors (Gb3) in butyric acid- treated A431 cells seem to have a ceramide moiety with longer fatty acids than in untreated cells, the possibility exists that fatty acid composition of the receptor is important for sorting to the ER. Both retrograde transport and intoxication with Shiga toxin can also be induced by cAMP, supporting the idea that retrograde transport from the Golgi to the ER is required for intoxication. The data suggest that transport to the ER in cells in situ may depend on fatty acid composition and is regulated by physiological signals.     

Effects of butyric and acetic acids on acetone-butanol formation by Clostridium acetobutylicum

The actions of butyric and acetic acids on acetone-butanol fermentation are investigated. Production of butyric and acetic acids are controlled by the extracellular concentrations of both acids: acetic acid added to the medium inhibits its own formation but has no effect on butyric acid formation, and added butyric acid inhibits its own formation but not that of acetic acid. The ratio of end metabolites depends upon acetic and butyric acid quantities excreted during the fermentation. In contrast to acetic acid, which specifically increases acetone formation, butyric acid increases both acetone and butanol formations. Acetate and butyrate kinase activities were also examined. Both increase at the start of fermentation and decrease when solvents appear in the medium. Coenzyme A transferase activity is weak in the acidogenic phase and markedly increases in the solvent phase. Acetic and butyric acids appear to be co-substrates. On the basis of these results, a mechanism of acetic and butyric acid pathways, coupled to solvent formation by C. acetobutylicum glucose fermentation is proposed.     

Cellular events involved in butyric acid-induced T cell apoptosis

We have previously demonstrated that butyric acid induces cytotoxicity and apoptosis of murine thymocytes, splenic T cells, and human Jurkat T cells. Therefore, to determine the apoptotic signaling pathway induced by butyric acid, we investigated the contribution of reactive oxygen species (ROS), mitochondria, ceramide, and mitogen-activated protein kinases in butyric acid-induced human Jurkat cell apoptosis. After exposure of cells to butyric acid, a pronounced accumulation of ROS was seen. Pretreatment of cells with the antioxidant N-acetyl-cysteine or 3-aminobenzamide attenuated butyric acid-induced apoptosis through a reduction of ROS generation. Cytochrome c, apoptosis-inducing factor, and second mitochondria-derived activator of caspases protein release from mitochondria into the cytosol were detected shortly after butyric acid treatment. Exposure of cells to butyric acid resulted in an increase in cellular ceramide in a time-dependent fashion. In addition, butyric acid-induced apoptosis was inhibited by DL-threo-dihidrosphingosine, a potent inhibitor of sphingosine kinase. Using anti-extracellular signal-regulated kinase (ERK), anti-c-Jun N-terminal kinase (JNK), and anti-p38 phosphospecific Abs, we showed a decrease in ERK, but not in JNK and p38 phosphorylation after treatment of cells with butyric acid. Pretreatment of cells with the JNK inhibitor SP600125 attenuated the effect of butyric acid on apoptosis, whereas no effect was seen with the p38 inhibitor SB202190 or the ERK inhibitor PD98059. Taken together, our results indicate that butyric acid-induced T cell apoptosis is mediated by ceramide production, ROS synthesis in mitochondria, and JNK activation in the mitogen-activated protein kinase cascade. Finally, these results were further substantiated by the expression profile of butyric acid-treated Jurkat cells obtained by means of cDNA array.     

Buffering as a means for increasing growth and butanol production byClostridium acetobutylicum

Summary The objective of this work was to optimize butanol formation in the acetone-butanol-ethanol (ABE) fermentation by examining the level of buffering as it affects the dissociation of butyric acid to the less toxic butyrate anion. Experiments were carried out in batch culture using chemically defined (P2) or complex media containing various buffering agents. These included salts of acetate, citrate, phosphate, nitrate, or bicarbonate, representing a range of pK _a values and buffering capacities. Growth in highly buffered medium was found to increase the stationary phase cell density, carbohydrate utilization, and the final butanol concentration. At higher levels of buffering, increased growth and elevated concentrations of butyric acid were required to initiate solventogenesis, suggesting the involvement of a critical threshold level of undissociated butyric acid.     

THE EFFECTS OF AMINO ACIDS UPON THE DEVELOPMENT OF EXCISED FLORAL BUDS OF AQUILEGIA

Young excised floral buds of Aquilegia were grown on a chemically defined medium containing various concentrations of single amino acids or mixtures of amino acids. γ-Amino butyric acid significantly promoted floral development through the initiation and differentiation of carpels. These floral organs were generally absent on the basal medium. Alanine, glutamic acid, and aspartic acid had no effect upon floral development. All other amino acids were either ineffective at lower concentrations and inhibitory at higher concentrations or were inhibitory at all concentrations. Casein hydrolysate and a mixture of amino acids found in coconut milk were ineffective. The addition of both γ-amino butyric acid and alanine to the basal medium promoted development approaching that achieved on the coconut-milk medium. However, further growth factors appear to be required before development on coconut-milk medium is equalled or exceeded.     

Increased susceptibility to tumor necrosis factor-alpha in butyric acid-induced apoptosis is caused by downregulation of cFLIP expression in Jurkat T cells

Butyric acid is one of the major extracellular metabolites of periodontopathic Gram-negative bacteria. We previously demonstrated that butyric acid induced apoptosis in human T cells. In the present study, we examined the interaction between butyric acid and TNF-alpha in Jurkat T-cell apoptosis. Simultaneous treatment with TNF-alpha enhanced butyric acid-induced apoptosis by promoting caspase activity more than was achieved by either reagent alone. We examined which genes were associated with the increased susceptibility to TNF-alpha caused by butyric acid, and revealed that expression of cFLIP decreased with increased concentrations of butyric acid. Furthermore, exogenous expression of cFLIP protein suppressed the enhancing effect by TNF-alpha in the apoptosis. These results suggest that butyric acid downregulates cFLIP expression and increases the susceptibility to TNF-alpha by activating caspases via the death receptor signal.     

Increased susceptibility to tumor necrosis factor-α in butyric acid-induced apoptosis is caused by downregulation of cFLIP expression in Jurkat T cells

Butyric acid is one of the major extracellular metabolites of periodontopathic Gram-negative bacteria. We previously demonstrated that butyric acid induced apoptosis in human T cells. In the present study, we examined the interaction between butyric acid and TNF-α in Jurkat T-cell apoptosis. Simultaneous treatment with TNF-α enhanced butyric acid-induced apoptosis by promoting caspase activity more than was achieved by either reagent alone. We examined which genes were associated with the increased susceptibility to TNF-α caused by butyric acid, and revealed that expression of cFLIP decreased with increased concentrations of butyric acid. Furthermore, exogenous expression of cFLIP protein suppressed the enhancing effect by TNF-α in the apoptosis. These results suggest that butyric acid downregulates cFLIP expression and increases the susceptibility to TNF-α by activating caspases via the death receptor signal.     

Determination of PHB in activated sludge by a gas chromatographic method

Summary In this study the gas chromatographic method of Poly-β-hydroxy butyric acid determination of Braunegg et al. (1978), was optimised for activated sludge samples. The poly-β-hydroxy butyric acid was extracted and quantified gravimetrically to confirm the accuracy of the gas chromatographic method. The authenticity of the extracted material was confirmed by several methods. It was also confirmed that the mixed liquor of an activated sludge process did not interfere with the esterification. The sample size required was 25 ml of mix liquor, or 50 mg of freeze-dried sludge.     

Butyric acid production from red algae by a newly isolated <Emphasis Type="Italic">Clostridium</Emphasis> sp. S1

Objective

To produce butyric acid from red algae such as Gelidium amansii in which galactose is a main carbohydrate, microorganisms utilizing galactose and tolerating inhibitors in hydrolysis including levulinic acid and 5-hydroxymethylfurfural (HMF) are required.

Results

A newly isolated bacterium, Clostridium sp. S1 produced butyric acid not only from galactose as the sole carbon source but also from a mixture of galactose and glucose through simultaneous utilization. Notably, Clostridium sp. S1 produced butyric acid and a small amount of acetic acid with the butyrate:acetate ratio of 45.4:1 and it even converted acetate to butyric acid. Clostridium sp. S1 tolerated 0.5–2 g levulinic acid/l and recovered from HMF inhibition at 0.6–2.5 g/l, resulting in 85–92 % butyric acid concentration of the control culture. When acid-pretreated G. amansii hydrolysate was used, Clostridium sp. S1 produced 4.83 g butyric acid/l from 10 g galactose/l and 1 g glucose/l.

Conclusion

Clostridium sp. S1 produces butyric acid from red algae due to its characteristics in sugar utilization and tolerance to inhibitors, demonstrating its advantage as a red algae-utilizing microorganism.

     

Influence of growth factor supplements on butyric acid production from sucrose byClostridium butyricum

During batch fermentation of sucrose to butyric acid byClostridium butyricum the effect of growth factor supplementation was determined: addition of yeast extract (5 g/L) stimulated most. Using biotin as the sole growth factor, average productivity was definitely lower. Beet molasses as a combined source of carbon and growth factor were effective only at a high concentration (150 g/L). The optimal butyric acid production (45 g/L, yield 45%) was achieved with sucrose concentration of 100 g/L in a medium supplemented with yeast extract (5 g/L). It represents an average productivity of 0.90 gL⁻¹ h⁻¹ and relative butyric acid concentration of 91%.     

Enhanced butyric acid tolerance and bioproduction by Clostridium tyrobutyricum immobilized in a fibrous bed bioreactor

Repeated fed‐batch fermentation of glucose by Clostridium tyrobutyricum immobilized in a fibrous bed bioreactor (FBB) was successfully employed to produce butyric acid at a high final concentration as well as to adapt a butyric‐acid‐tolerant strain. At the end of the eighth fed‐batch fermentation, the butyric acid concentration reached 86.9 ± 2.17 g/L, which to our knowledge is the highest butyric acid concentration ever produced in the traditional fermentation process. To understand the mechanism and factors contributing to the improved butyric acid production and enhanced acid tolerance, adapted strains were harvested from the FBB and characterized for their physiological properties, including specific growth rate, acid‐forming enzymes, intracellular pH, membrane‐bound ATPase and cell morphology. Compared with the original culture used to seed the bioreactor, the adapted culture showed significantly reduced inhibition effects of butyric acid on specific growth rate, cellular activities of butyric‐acid‐forming enzyme phosphotransbutyrylase (PTB) and ATPase, together with elevated intracellular pH, and elongated rod morphology. Biotechnol. Bioeng. 2011; 108:31–40. © 2010 Wiley Periodicals, Inc.     

Production of butyric acid by a cellulolytic actinobacterium Thermobifida fusca on cellulose

Thermobifida fusca not only produces cellulases, hemicellulases and xylanases, but also excretes butyric acid. In order to achieve a high yield of butyric acid, the effect of different carbon sources: mannose, xylose, lactose, cellobiose, glucose, sucrose and acetates, on butyric acid production was studied. The highest yield of butyric acid was 0.67 g/g C (g-butyric acid/g-carbon input) on cellobiose. The best stir speed and aeration rate for butyric acid production were found to be 400 rpm and 2 vvm in a 5-L fermentor. The maximum titer of 2.1 g/L butyric acid was achieved on 9.66 g/L cellulose. In order to test the production of butyric acid on lignocellulosic biomass, corn stover was used as the substrate, on which there was 2.37 g/L butyric acid produced under the optimized conditions. In addition, butyric acid synthesis pathway was identified involving five genes that catalyzed reactions from acetyl-CoA to butanoyl-CoA in T. fusca.     

生物技术生产丁酸的研究进展

丁酸作为一种重要的化工原料,已经广泛应用于食品添加剂与医药等领域。目前,工业上生产丁酸主要是从石油中提取有机化合物进行化学合成。与有机化合物合成法相比,微生物发酵产丁酸的优势有：所用的原料来源非常广,发酵过程低能耗,不污染环境,而且可以持续添加原料发酵生产丁酸。因此,通过生物技术发酵生产丁酸越来越受到人们的重视。介绍了丁酸的性质、产丁酸菌株的特点、微生物发酵产丁酸的细胞代谢途径及其调控、发酵法生产丁酸的工艺运行方式和产丁酸菌株及其代谢产物的生理功能这五部分内容,以期为今后开展发酵法产丁酸的微生物基因工程改造以及生产工艺的优化提供参考。     

Optimization of redox reactions employing whole cell biocatalysis

The ability of Saccharomyces cerevisiae to catalyse the reduction reaction of carboxylic acids into alcohols is described. Earlier reports have led to the characterization of the reduction of carbonyl groups into alcohols mediated by the enzyme alcohol dehydrogenase. We investigated the ability of this organism to catalyse the said conversion using the carboxylic acids, acetic acid and butyric acid. In the absence of any previous characterization, whole cell catalysis proved effective. The uptake of these acids from the medium was estimated using a plate assay method involving litmus-agar. The plate assay was found to be a convenient and extremely adaptable method for quantitation of acids in organic as well as aqueous medium. The comparison of existing paradigms in pure protein catalysis with whole cells catalysis proved anomalous. We report that it is solvent toxicity rather than hydrophobic index that correlates with the activity observed in non-aqueous conditions for whole cell biocatalysis. Reduction of acetic acid as well as butyric acid occurred, with efficiency of reaction with butyric acid being marginally higher. The reduction therefore occurs for both the short chain carboxylic acids used in this study. We therefore illustrate the reduction route of acids into alcohols and propose a model two-step pathway for the reaction. Process optimization may be further attempted to enhance the presently moderate reaction efficiencies. Steps made in the direction by studying the pH dependency and use of sacrificial substrate have yielded encouraging results.     

A novel isolate of <Emphasis Type="Italic">Clostridium butyricum</Emphasis> for efficient butyric acid production by xylose fermentation

Bacterial fermentation of lignocellulose has been regarded as a sustainable approach to butyric acid production. However, the yield of butyric acid is hindered by the conversion efficiency of hydrolysate xylose. A mesophilic alkaline-tolerant strain designated as Clostridium butyricum B10 was isolated by xylose fermentation with acetic and butyric acids as the principal liquid products. To enhance butyric acid production, performance of the strain in batch fermentation was evaluated with various temperatures (20–47 °C), initial pH (5.0–10.0), and xylose concentration (6–20 g/L). The results showed that the optimal temperature, initial pH, and xylose concentration for butyric acid production were 37 °C, 9.0, and 8.00 g/L, respectively. Under the optimal condition, the yield and specific yield of butyric acid reached about 2.58 g/L and 0.36 g/g xylose, respectively, with 75.00% butyric acid in the total volatile fatty acids. As renewable energy, hydrogen was also collected from the xylose fermentation with a yield of about 73.86 mmol/L. The kinetics of growth and product formation indicated that the maximal cell growth rate (μ _m ) and the specific butyric acid yield were 0.1466 h^?1 and 3.6274 g/g cell (dry weight), respectively. The better performance in xylose fermentation showed C. butyricum B10 a potential application in efficient butyric acid production from lignocellulose.     

Efficient production of butyric acid from Jerusalem artichoke by immobilized Clostridium tyrobutyricum in a fibrous-bed bioreactor

Butyric acid is an important specialty chemical with wide industrial applications. The feasible large-scale fermentation for the economical production of butyric acid requires low-cost substrate and efficient process. In the present study, butyric acid production by immobilized Clostridium tyrobutyricum was successfully performed in a fibrous-bed bioreactor using Jerusalem artichoke as the substrate. Repeated-batch fermentation was carried out to produce butyric acid with a high butyrate yield (0.44 g/g), high productivity (2.75 g/L/h) and a butyrate concentration of 27.5 g/L. Furthermore, fed-batch fermentation using sulfuric acid pretreated Jerusalem artichoke hydrolysate resulted in a high butyric acid concentration of 60.4 g/L, with the yield of 0.38 g/g and the selectivity of ∼85.1 (85.1 g butyric acid/g acetic acid). Thus, the production of butyric acid from Jerusalem artichoke on a commercial scale could be achieved based on the system developed in this work.     

Assessment of toxicity of volatile fatty acids to Photobacterium phosphoreum

The toxicity of four volatile fatty acids (VFAs) as anaerobic digestion (AD) intermediates was investigated at pH 7. Photobacterium phosphoreum T3 was used as an indicator organism. Binary, ternary and mixtures of AD intermediates were designated by letters A (acetic acid + propionic acid), B (acetic acid + butyric acid), C (acetic acid + ethanol), D (propionic acid + butyric acid), E (propionic acid + ethanol), F (butyric acid + ethanol), G (acetic acid + propionic acid + butyric acid), H (acetic acid + propionic acid + ethanol), I (acetic acid + butyric acid+ ethanol), J (propionic acid + butyric acid + ethanol) and K (acetic acid + propionic acid + butyric acid + ethanol) to assess the toxicity through equitoxic mixing ratio method. The IC₅₀ values of acetic acid, propionic acid, butyric acid and ethanol were 9.812, 7.76, 6.717 and 17.33 g/L respectively, displaying toxicity order of: butyric acid > propionic acid > acetic acid > ethanol being additive in nature. The toxic effects of four VFAs could be designated as synergistic and one additive in nature.     

丁酸菌与老年肠道功能的相关性

丁酸菌是一种专性厌氧芽胞杆菌,是以丁酸为主要代谢产物的益生菌,可定植于人体肠道,在某种程度上与乳酸菌有协同作用,可抑制人肠道内有害菌的生长、促进营养物质的吸收、改善肠道功能等。本研究主要对丁酸菌在临床中的应用研究进展进行综述,尤其是对丁酸菌与老年人肠道功能的相关性进行阐述,并展望丁酸菌对老年慢性疾病如糖尿病、心血管疾病、老年痴呆、风湿性疾病及骨质疏松症防治的研究未来。