Effect of Butyrate on Collagen Expression, Cell Viability, Cell Cycle Progression and Related Proteins Expression of MG-63 Osteoblastic Cells.
ABSTRACT: AIMS:Butyric acid is one major metabolic product generated by anaerobic Gram-negative bacteria of periodontal and root canal infection. Butyric acid affects the activity of periodontal cells such as osteoblasts. The purposes of this study were to investigate the effects of butyrate on MG-63 osteoblasts. METHODS:MG-63 cells were exposed to butyrate and cell viability was estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The mRNA and protein expression of type I collagen and cell cycle-related proteins were measured by reverse-transcriptase polymerase chain reaction (RT-PCR), western blotting or immunofluorescent staining. Cellular production of reactive oxygen species (ROS) was analyzed by 2',7'-dichlorofluorescein (DCF) fluorescence flow cytometry. RESULTS:Exposure to butyrate suppressed cell proliferation, and induced G2/M (8 and 16 mM) cell cycle arrest of MG-63 cells. Some cell apoptosis was noted. The mRNA expression of cdc2 and cyclin-B1 decreased after exposure to butyrate. The protein expression of type I collagen, cdc2 and cyclin B1 were decreased, whereas the expression of p21, p27 and p57 was stimulated. Under the treatment of butyrate, ROS production in MG-63 cells markedly increased. CONCLUSIONS:The secretion of butyric acid by periodontal and root canal microorganisms may inhibit bone cell growth and matrix turnover. This is possibly due to induction of cell cycle arrest and ROS generation and inhibition of collagen expression. These results suggest the involvement of butyric acid in the pathogenesis of periodontal and periapical tissue destruction by impairing bone healing responses.
Project description:BACKGROUND:Short-chain fatty acids (SCFAs) alteration have been reported in irritable bowel syndrome (IBS), but the results are conflicting. Our study aims to explore the alteration of SCFAs in patients with diarrhea-predominant IBS (IBS-D) and their potential role in the occurrence and development of IBS. METHODS:We recruited patients with IBS-D defined by Rome IV criteria and age-and-gender matched healthy controls (HCs). A headspace solid-phase microextraction gas chromatography-mass spectrometric (HS-SPME-GC-MS) method was developed for the analysis of acetic, propionic and butyric acid in feces and serum. RESULTS:Compared with HCs, the levels of the serum propionate (2.957?±?0.157 vs 2.843?±?0.098?mmol/L, P?=?0.012) and butyrate (2.798?±?0.126 vs 2.697?±?0.077?mmol/L, P?=?0.012) were significantly higher in IBS-D group. No significant differences were found among two groups with regard to the concentration of fecal acetate (4.953?±?1.065 vs 4.774?±?1.465?mg/g, P?=?0.679), propionate (6.342?±?1.005 vs 6.282?±?1.077?mg/g, P?=?0.868) and butyrate (2.984?±?0.512 vs 3.071?±?0.447?mg/g, P?=?0.607). CONCLUSIONS:Metabolites of gut microbiota, the propionic and butyric acid, are increased in patients with IBS-D in serum but not in feces. It suggests that propionic and butyric acid might be associated with the occurrence and development of IBS.
Project description:Background:Several anaerobic bacteria produce butyric acid, a commodity chemical with use in chemical, pharmaceutical, food and feed industries, using complex media with acetate as a co-product. Butyrate titer of various recombinant Escherichia coli did not exceed 10 g l-1 in batch fermentations in any of the media tested. Results:A recombinant E. coli (strain LW393) that produced butyrate as the major fermentation product was constructed with genes from E. coli, Clostridium acetobutylicum and Treponema denticola. Strain LW393 produced 323 ± 6 mM (28.4 ± 0.4 g l-1) butyric acid in batch fermentations in mineral salt medium with glucose as C source at a yield of 0.37 ± 0.01 g (g glucose consumed)-1. Butyrate accounted for 90% of the total products produced by the culture. Supplementing this medium with yeast extract further increased butyric acid titer to 375 ± 4 mM. Average volumetric productivity of butyrate with xylose as C source was 0.89 ± 0.07 g l-1 h-1. Conclusions:The butyrate titer reported in this study is about 2.5-3-times higher than the values reported for other recombinant E. coli and this is achieved in mineral salt medium with an expectation of lower purification and production cost of butyrate.
Project description:Clostridium tyrobutyricum is a Gram-positive anaerobic bacterium that efficiently produces butyric acid and is considered a promising host for anaerobic production of bulk chemicals. Due to limited knowledge on the genetic and metabolic characteristics of this strain, however, little progress has been made in metabolic engineering of this strain. Here we report the complete genome sequence of C. tyrobutyricum KCTC 5387 (ATCC 25755), which consists of a 3.07-Mbp chromosome and a 63-kbp plasmid. The results of genomic analyses suggested that C. tyrobutyricum produces butyrate from butyryl-coenzyme A (butyryl-CoA) through acetate reassimilation by CoA transferase, differently from Clostridium acetobutylicum, which uses the phosphotransbutyrylase-butyrate kinase pathway; this was validated by reverse transcription-PCR (RT-PCR) of related genes, protein expression levels, in vitro CoA transferase assay, and fed-batch fermentation. In addition, the changes in protein expression levels during the course of batch fermentations on glucose were examined by shotgun proteomics. Unlike C. acetobutylicum, the expression levels of proteins involved in glycolytic and fermentative pathways in C. tyrobutyricum did not decrease even at the stationary phase. Proteins related to energy conservation mechanisms, including Rnf complex, NfnAB, and pyruvate-phosphate dikinase that are absent in C. acetobutylicum, were identified. Such features explain why this organism can produce butyric acid to a much higher titer and better tolerate toxic metabolites. This study presenting the complete genome sequence, global protein expression profiles, and genome-based metabolic characteristics during the batch fermentation of C. tyrobutyricum will be valuable in designing strategies for metabolic engineering of this strain.Bio-based production of chemicals from renewable biomass has become increasingly important due to our concerns on climate change and other environmental problems. C. tyrobutyricum has been used for efficient butyric acid production. In order to further increase the performance and expand the capabilities of this strain toward production of other chemicals, metabolic engineering needs to be performed. For this, better understanding on the metabolic and physiological characteristics of this bacterium at the genome level is needed. This work reporting the results of complete genomic and proteomic analyses together with new insights on butyric acid biosynthetic pathway and energy conservation will allow development of strategies for metabolic engineering of C. tyrobutyricum for the bio-based production of various chemicals in addition to butyric acid.
Project description:1. Interactions in the rates of consumption of acetate, propionate and butyrate in sheep liver mitochondria were examined in the presence and absence of l-malate and alpha-oxoglutarate. 2. Acetate was not consumed in absence of ancillary substrate but utilization of acetate (7.2nmol/min per mg of protein) occurred in the presence of alpha-oxoglutarate. This consumption was abolished by propionate or butyrate but the presence of acetate did not affect consumption of propionate or butyrate. 3. Propionate consumption (10.1nmol/min per mg of protein) was unaffected by malate but was stimulated by 63% by butyrate or by 180% by alpha-oxoglutarate. 4. Butyrate consumption (3.3nmol/min per mg of protein) was stimulated by 117% by malate, by 151% by propionate and by 310% by alpha-oxoglutarate. 5. In the absence of ancillary substrates the maximum rate of total volatile fatty acid utilization (24.7nmol/min per mg of protein) occurred with a mixture of propionate and butyrate. When both propionate and butyrate were present total consumption was not affected by malate but was stimulated by 24% by alpha-oxoglutarate. With alpha-oxoglutarate present, propionate and butyrate each decreased the other's consumption by about 26%, but the total utilization was the greatest observed. 6. The inhibition of acetate consumption by propionate or butyrate is unexplained, but the remaining effects are consistent with an interaction of propionate and butyrate through oxaloacetate together with a general limitation imposed by a need for GTP to rephosphorylate AMP formed during activation of the volatile fatty acids.
Project description:BACKGROUND:Currently, the most promising microorganism used for the bio-production of butyric acid is Clostridium tyrobutyricum ATCC 25755T; however, it is unable to use sucrose as a sole carbon source. Consequently, a newly isolated strain, Bacillus sp. SGP1, that was found to produce a levansucrase enzyme, which hydrolyzes sucrose into fructose and glucose, was used in a co-culture with this strain, permitting C. tyrobutyricum ATCC 25755T to ferment sucrose to butyric acid. RESULTS:B. sp. SGP1 alone did not show any butyric acid production and the main metabolite produced was lactic acid. This allowed C. tyrobutyricum ATCC 25755T to utilize the monosaccharides resulting from the activity of levansucrase together with the lactic acid produced by B. sp. SGP1 to generate butyric acid, which was the main fermentative product within the co-culture. Furthermore, the final acetic acid concentration in the co-culture was significantly lower when compared with pure C. tyrobutyricum ATCC 25755T cultures grown on glucose. In fed-batch fermentations, the optimum conditions for the production of butyric acid were around pH 5.50 and a temperature of 37°C. Under these conditions, the final butyrate concentration was 34.2±1.8 g/L with yields of 0.35±0.03 g butyrate/g sucrose and maximum productivity of 0.3±0.04 g/L/h. CONCLUSIONS:Using this co-culture, sucrose can be utilized as a carbon source for butyric acid production at a relatively high yield. In addition, this co-culture offers also the benefit of a greater selectivity, with butyric acid constituting 92.8% of the acids when the fermentation was terminated.
Project description:The cysteine-rich domain of the haemorrhagic metalloproteinase atrolysin A was shown to inhibit collagen-stimulated platelet aggregation and to interact with MG-63 osteosarcoma cells via integrin alpha2beta1 to inhibit adhesion to collagen I. In addition, we demonstrate by solid-phase binding assays that atrolysin A binds to collagen I and to vWF (von Willebrand factor) via exosites in the cysteine-rich domain. Interestingly, the binding site of the cysteine-rich domain on collagen I is distinct from the cell adhesion site, since the incubation of collagen-I-coated plates with the cysteine-rich domain did not prevent the adhesion of MG-63 cells to collagen. Finally, we show by surface plasmon resonance (BIAcore) analyses that the cysteine-rich domain can block vWF binding to collagen I as well as the binding of collagen I to vWF. Taken together, these results indicate that this domain may function as a cell-surface-receptor-binding site and/or a substrate recognition exosite and may thus play a role in the pathologies associated with atrolysin A.
Project description:Using real-time RT-PCR and Western blot analysis in bovine kidney epithelial cells, we systematically investigated the effects of butyrate on patterns of gene expression relevant to DNA replication apparatus. The real-time PCR and Western blot data generally confirmed previously reported microarray data. Of the five genes tested by quantitative RT-PCR, CDKN1A (p21(waf1)) was up regulated, CDC2/cdk1, MCM6, ORC1L were down regulated, while ORC3L expression remained unchanged following butyrate treatment. Also consistent with RT-PCR results, Western blot analysis confirmed that butyrate up-regulated cyclin-kinase inhibitor p21(waf1) in a does-dependent manner. In contrast, butyrate treatment had no effect on the expression of ERK 1/2 proteins. Also consistent with mRNA results, ORC1 and MCM3 proteins were down-regulated by butyrate treatment, while ORC2 protein remained unchanged. The present results suggest that ORC1, not ORC2 or ORC3, along with MCM proteins play a critical role in regulating the initiation of DNA replication and cell cycle progression in MDBK cells and are targets of butyrate regulation.
Project description:The effects and underlying mechanisms of butyrate and butyrate+niacin on apoptosis in sheep rumen epithelial cells were investigated. Cells were exposed to butyrate (0-140?mM) for 6?h. A low concentration (20?mM) of butyrate increased cell viability and promoted growth whereas high concentrations (40-140?mM) inhibited proliferation. Cells were then cocultured with 120?mM butyrate and niacin (0-100?mM) for 6?h. Niacin addition attenuated butyrate-induced cellular damage and promoted proliferation at 20-80?mM; 40?mM presented the optimal effect. Higher concentrations (100?mM) of niacin resulted in low cell viability. Subsequent experiments confirmed that 120?mM butyrate increased intracellular reactive oxygen species (ROS) production and reduced the intracellular total antioxidant capacity (T-AOC) versus the untreated control. Compared with 120?mM butyrate, cotreatment with 40?mM niacin significantly reduced the intracellular ROS content and increased the intracellular T-AOC. Flow cytometry analysis revealed that 120?mM butyrate increased the proportion of apoptotic cells by 17.8% versus the untreated control, and 120?mM butyrate+40?mM niacin treatment reduced the proportion of apoptotic cells by 28.6% and 39.4% versus the untreated control and butyrate treatment, respectively. Treatment with 120?mM butyrate increased caspase-9 and p53 mRNA levels and decreased the expression of Bcl-2 and Bax, and the Bcl-2/Bax ratio versus the untreated control. Treatment with 120?mM butyrate+40?mM niacin downregulated the expression of caspase-3 and p53 and increased the expression of Bcl-2 and Bax versus butyrate treatment alone but had no effect on the Bcl-2/Bax ratio. Thus, high concentrations of butyrate may induce rumen epithelial cell apoptosis by increasing oxidative stress and inducing caspase-9 and p53 expression. Cotreatment with niacin regulates apoptosis-related gene expression by reducing intracellular ROS production and DNA damage and downregulating caspase-3 and p53 expressions to protect rumen epithelial cells against butyrate-induced apoptosis.
Project description:Butyrate-producing bacteria can biosynthesize butyrate and alleviate inflammatory diseases. However, few studies have reported that the genus Collinsella has the ability to produce butyric acid. Here, our study depicts a Collinsella strain, which is a rod-shaped obligate anaerobe that is able to produce butyric acid. This microorganism was isolated from a human gut, and the optimal growth conditions were found to be 37 °C on PYG medium with pH 6.5. The 16S rRNA gene sequence demonstrated that this microorganism shared 99.93% similarity with C. aerofaciens ATCC 25986T, which was higher than the threshold (98.65%) for differentiating two species. Digital DNA?DNA hybridization and average nucleotide identity values also supported that this microorganism belonged to the species C. aerofaciens. Distinct phenotypic characteristics between TF06-26 and the type strain of C. aerofaciens, such as the fermentation of D-lactose, D-fructose and D-maltose, positive growth under pH 5 and 0.2% (w/v) cholate, suggested this strain was a novel subspecies. Comparative genome analysis revealed that butyric acid kinase and phosphate butyryltransferase enzymes were coded exclusively by this strain, indicating a specific butyric acid-producing function of this C. aerofaciens subspecies within the genus Collinsella. Thus, Collinsella aerofaciens subsp. shenzhenensis subsp. nov. was proposed, with set strain TF06-26T (=CGMCC 1.5216T = DSM 105138T) as the type strain.
Project description:Extractive fermentation with the removal of carboxylic acid requires low pH conditions because acids are better partitioned into the solvent phase at low pH values. However, this requirement conflicts with the optimal near-neutral pH conditions for microbial growth.CO2 pressurization was used, instead of the addition of chemicals, to decrease pH for the extraction of butyric acid, a fermentation product of Clostridium tyrobutyricum, and butyl butyrate was selected as an extractant. CO2 pressurization (50 bar) improved the extraction efficiency of butyric acid from a solution at pH 6, yielding a distribution coefficient (D) 0.42. In situ removal of butyric acid during fermentation increased the production of butyric acid by up to 4.10 g/L h, an almost twofold increase over control without the use of an extraction process.In situ extraction of butyric acid using temporal CO2 pressurization may be applied to an integrated downstream catalytic process for upgrading butyric acid to value-added chemicals in an organic solvent.