Abstract  The capacity of the rumen microbiota to produce volatile fatty acids (VFAs) has important implications in animal well-being and production. We investigated temporal changes of the rumen microbiota in response to butyrate infusion using pyrosequencing of the 16S rRNA gene. Twenty one phyla were identified in the rumen microbiota of dairy cows. The rumen microbiota harbored 54.5±6.1 genera (mean ± SD) and 127.3±4.4 operational taxonomic units (OTUs), respectively. However, the core microbiome comprised of 26 genera and 82 OTUs. Butyrate infusion altered molar percentages of 3 major VFAs. Butyrate perturbation had a profound impact on the rumen microbial composition. A 72 h-infusion led to a significant change in the numbers of sequence reads derived from 4 phyla, including 2 most abundant phyla, Bacteroidetes and Firmicutes. As many as 19 genera and 43 OTUs were significantly impacted by butyrate infusion. Elevated butyrate levels in the rumen seemingly had a stimulating effect on butyrate-producing bacteria populations. The resilience of the rumen microbial ecosystem was evident as the abundance of the microorganisms returned to their pre-disturbed status after infusion withdrawal. Our findings provide insight into perturbation dynamics of the rumen microbial ecosystem and should guide efforts in formulating optimal uses of probiotic bacteria treating human diseases.

Abstract  Colorectal cancer is a leading cause of morbidity and mortality worldwide, and its incidence has been increasing in recent years. The role of epigenetic modifications, including DNA methylation and histone modifications, has only recently been investigated. In this study, the effects of epigenetic agents such as folic acid (FA) and sodium butyrate (NaBu) on the development of colorectal cancer induced by 1,2-dimethylhydrazine (DMH) using ICR mice was examined. Of the mice treated in a chemopreventive manner with epigenetic agents, FA and NaBu, 15-50% developed colorectal cancer at 24 weeks compared with a 95% incidence of colorectal cancer in DMH-treated control mice. Folate deficiency can alter cytosine methylation in DNA leading to inappropriate activation of the proto-oncogene c-myc. We detected lower levels of p21(WAF1) gene expression in colorectal cancer samples, as well as significantly lower levels of acetylated histone H3, compared with samples from corresponding normal colorectal mucosa. In contrast, administration of NaBu increased levels of p21(WAF1) mRNA and p21(WAF1) protein, and was associated with an accumulation of histone acetylation. In summary, our results show that FA and NaBu reduce the incidence of colorectal cancer induced by DMH-induced in ICR mice, and therefore we hypothesize that targeting epigenetic targets should be further investigated for the prevention of colorectal cancer in humans.

Abstract  It was studied the use of butyric acid (BA) and phytase in growing pigs diets, varying calcium level. The experiment was divided into two periods of time, of 17 days, with 3 days of adaptation and 14 of measurements. In each period, it was used 16 barrows weighing 24.6 +/- 0.7kg in the first and 43.2 +/- 1.77kg in the second. Diets were different in calcium level (0.5 or 0.72%), AB (0 or 0.3% sodium butyrate 84%) and phytase (0 or 500FTU kg(-1) phytase of bacterial origin derived from Escherichia coli). The experimental design was in randomized blocks (periods), decomposed in 2x2x2 factorial, with four replications. It was evaluated the aarent digestibility of nutrients, gross energy and balance of Ca and P. AB improved crude protein digestibility, but individually or in combination with phytase did not increase minerals retention. Phytase increased P retention by reducing urinary and fecal excretion. The lowest diet calcium level provided higher Ca retention and lower P retention as a result of increased P excretion in urine (P<0.0001). Despite improving protein digestibility, AB did not increase mineral retention and didn't have an additive effect with the use of phytase, while phytase had positive effect on retention of P.

Abstract  Cyclo-oxygenase (COX) profile predicts prognosis of gastric cancer; COX-2 positive tumors are more often aggressive, and COX-2 suppression is protective against gastric cancer. In contrast, COX-1 suppression is harmful to the intestinal mucosa. The COX-1, COX-2, and COX-1ir expression profiles were measured with real-time PCR in primary (AGS) and metastatic (NCI-N87) gastric adenocarcinoma cell lines treated with butyrate, hyperosmolar medium, and, in the case of NCI-N87, cell-free supernatants of probiotic bacteria Lactobacillus acidophilus 74-2 and Bifidobacterium lactis 420. The cell lines showed differences in the profile when treated with either hyperosmolar medium or butyrate. In NCI-N87 COX-2 expression was higher but only COX-1 expression was significantly upregulated by butyrate. Similarly to butyrate, the cell-free supernatant of L. acidophilus 74-2 upregulated COX-1, while COX-2 expression remained unchanged. COX-1ir, including COX-3, was upregulated by probiotics and osmotic stress. In conclusion, consumption of L. acidophilus 74-2 could be beneficial for the expression of cytoprotective COX-1.

Abstract  This study aimed to evaluate the effects of oat β-glucan in combination with low- and high-dietary calcium phosphate (CaP) content on gastrointestinal bacterial microbiota, prevalence of butyrate-production pathway genes and fermentation end-products in 32 weaned pigs allocated to four diets: a cornstarch-casein-based diet with low [65% of the calcium (Ca) and phosphorous (P) requirement] and high CaP content (125% and 115% of the Ca and P requirement, respectively); and low and high CaP diets supplemented with 8.95% of oat β-glucan concentrate. Pigs were slaughtered after 14 days, and digesta were collected for quantitative PCR analysis, and quantification of short-chain fatty acids and lactate. The high CaP content reduced gastric lactate and streptococci and propionate in the large intestine. Oat β-glucan distinctly raised gastric bacterial numbers, and colonic lactobacilli and bifidobacteria. Although not reflected by gene copies of butyrate-production pathway genes, oat β-glucan also increased gastric, caecal and colonic butyrate concentrations, which may be favourable for intestinal development in weaned pigs. Thus, a high CaP content negatively affected the intestinal abundance of certain fermentation end-products, whereas oat β-glucan generally enhanced bacterial numbers and activity. The results emphasize the importance of the stomach for bacterial metabolism of oat β-glucan in weaned pigs.

Abstract  Butyrate is the preferred energy source for colonocytes and has an important role in gut health; in contrast, accumulation of high concentrations of lactate is detrimental to gut health. The major butyrate-producing bacterial species in the human colon belong to the Firmicutes. Eubacterium hallii and a new species, Anaerostipes coli SS2/1, members of clostridial cluster XIVa, are able to utilize lactate and acetate via the butyryl CoA : acetate CoA transferase route, the main metabolic pathway for butyrate synthesis in the human colon. Here we provide a mathematical model to analyse the production of butyrate by lactate-utilizing bacteria from the human colon. The model is an aggregated representation of the fermentation pathway. The parameters of the model were estimated using total least squares and maximum likelihood, based on in vitro experimental data with E. hallii L2-7 and A. coli SS2/1. The findings of the mathematical model adequately match those from the bacterial batch culture experiments. Such an in silico approach should provide insight into carbohydrate fermentation and short-chain fatty acid cross-feeding by dominant species of the human colonic microbiota.

Abstract  An experiment was conducted to study the effect of graded levels of butyric acid (butyrate) on performance, gastrointestinal tract health and carcass characteristics in young broiler chickens. Control starter (0-3 wk) and finisher (4-5 wk) diets were formulated to contain 2,900 kcal ME/kg and 22% CP, and 3,000 kcal ME/kg and 20% CP, respectively Subsequently, four other experimental diets were formulated to contain 0.05% antibiotic (furazolidone) or 0.2, 0.4 and 0.6% butyric acid. Each diet was fed at random to 8 replicates of 6 chicks each throughout the experimental period (0-5 wk). The results showed that 0.4% butyrate in the diet was similar to antibiotic in maintaining body weight gain and reducing E. coli numbers but superior for feed conversion ratio. No added advantage on these parameters was obtained by enhancing the concentration of butyrate from 0.4 to 0.6% in the diet. Feed intake and mortality were not influenced by the dietary treatments. A reduction in pH of the uer GI tract (crop, proventiculus and gizzard) was observed by inclusion of butyrate in the diets of broilers compared to either control or antibiotic-fed group. Butyrate at 0.4% was more effective in reducing the pH than 0.2% butyrate. Within the lower GI tract, 0.4 and 0.6% butyrate was effective in lowering pH in the duodenum, but no effect was found in either the jejunum or ileum. The villus length and crypt depth in the duodenum increased significantly in all the butyrate treated diets irrespective of the level tested. Carcass yield was higher and abdominal fat content was lower significantly in all the butyrate treatment groups compared to the control or antibiotic group. From these findings, it is concluded that 0.4% butyric acid supplementation maintained performance, intestinal tract health, and villi development and carcass quality in broiler chickens.

Abstract  Butyrate, one of the SCFA, promotes the development of the intestinal barrier. However, the molecular mechanisms underlying the butyrate regulation of the intestinal barrier are unknown. To test the hypothesis that the effect of butyrate on the intestinal barrier is mediated by the regulation of the assembly of tight junctions involving the activation of the AMP-activated protein kinase (AMPK), we determined the effect of butyrate on the intestinal barrier by measuring the transepithelial electrical resistance (TER) and inulin permeability in a Caco-2 cell monolayer model. We further used a calcium switch assay to study the assembly of epithelial tight junctions and determined the effect of butyrate on the assembly of epithelial tight junctions and AMPK activity. We demonstrated that the butyrate treatment increased AMPK activity and accelerated the assembly of tight junctions as shown by the reorganization of tight junction proteins, as well as the development of TER. AMPK activity was also upregulated by butyrate during calcium switch-induced tight junction assembly. Compound C, a specific AMPK inhibitor, inhibited the butyrate-induced activation of AMPK. The facilitating effect of butyrate on the increases in TER in standard culture media, as well as after calcium switch, was abolished by compound C. We conclude that butyrate enhances the intestinal barrier by regulating the assembly of tight junctions. This dynamic process is mediated by the activation of AMPK. These results suggest an intriguing link between SCFA and the intracellular energy sensor for the development of the intestinal barrier.

Abstract  Production of short-chain fatty acids (SCFA) in the intestinal lumen may play an important role in the maintenance of the intestinal barrier. However, overproduction/accumulation of SCFA in the bowel may be toxic to the intestinal mucosa and has been hypothesized to play a role in the pathogenesis of neonatal necrotizing enterocolitis (NEC). By using a Caco-2 cell monolayer model of intestinal barrier, we report here that the effect of butyrate on the intestinal barrier is paradoxical. Butyrate at a low concentration (2 mM) promotes intestinal barrier function as measured by a significant increase in transepithelial electrical resistance (TER) and a significant decrease in inulin permeability. Butyrate at a high concentration (8 mM) reduces TER and increases inulin permeability significantly. Butyrate induces apoptosis and reduces the number of viable Caco-2 cells in a dose-dependent manner. Intestinal barrier function impairment induced by high concentrations of butyrate is most likely related to butyrate-induced cytotoxicity due to apoptosis. We conclude that the effect of butyrate on the intestinal barrier is paradoxical; i.e. whereas low concentrations of butyrate may be beneficial in promoting intestinal barrier function, excessive butyrate may induce severe intestinal epithelial cell apoptosis and disrupt intestinal barrier.

Abstract  Histone deacetylase inhibitors (HDACis) have shown significant antiproliferative and apoptotic properties in various types of cancer cells, including prostate cancer cells, and are therefore being evaluated as a treatment modality. However, the mechanism by which sodium butyrate (SB) induces apoptosis is not completely understood. We focused on SB which exists in the intestine and is therefore expected to have less adverse effects. In this study, three prostate cancer cell lines (LNCaP, DU145 and PC-3) were treated in vitro with different concentrations of SB. Cell proliferation was studied by the XTT assay; cell cycle analysis and induction of apoptosis were studied by laser scanning cytometry. Western blot analysis was used to study p21, p27, CDK2, CDK4, CDK6, caspase-3, caspase-7, Fas, FADD, TRADD, Bcl-2 and Bax protein expression. SB inhibited cell growth and induced apoptosis in a concentration-dependent manner in human prostate cancer cells (LNCaP, DU145 and PC-3). Western blot analysis showed dose-dependent increases of p21 levels in DU145 and PC-3 cells, and dose-dependent decreases of CDK2, CDK4, CDK6 and procaspase-3 protein levels in all three prostate cancer cell lines. Bcl-xL was significantly down-regulated in DU145 cells, and Bcl-2 was significantly down-regulated in PC-3 and LNCaP cells. No significant changes were observed in procaspase-7, TRADD and Bax expression, although slight decreases in Fas and FADD expression were seen in all three prostate cancer cell lines. Analysis of cell morphology using laser scanning microscopy detected condensed and fragmented nuclei. In conclusion, SB induces G1 and G2 arrest by increasing p21 expression resulting in CDK2, CDK4 and CDK6 down-regulation. SB potently induced apoptosis, which was accompanied by DNA fragmentation, down-regulated Bcl-2 in LNCaP and PC-3 cells, Bcl-xL in DU145 cells, and down-regulated procaspase-3, but not procaspase-7, in these human prostate cancer cell lines. These results suggest that SB may serve as a new modality for the treatment of hormone refractory prostate cancer.

Abstract  BACKGROUND: Colorectal cancer is the second cause of death for tumour worldwide. Among the risk factors for this disease the dietary habits seem to have a pivotal role. An elevated intake of fats causes a high release in the gut lumen of bile acids that are positively correlated with colorectal cancer, since they act as detergents and proliferation promoters. Recently, it was evidenced that bile acids can also be able to induce DNA damage.

AIM OF THE STUDY: In this study the genotoxicity of deoxycholic acid (DCA) and chenodeoxycholic acid CDCA) has been evaluated in human normal colonocytes derived from 60 colon biopsies and in tumour cells. The involvement of reactive oxygen species (ROS) and the oxidative DNA damage was assessed. In addition, the protective effect exerted by both two well-known antioxidants commonly present in the diet, beta-carotene and alpha-tocopherol, and butyrate which is known to be involved in the regulation of several cellular functions, has also been tested.

METHODS: The DNA damage was evaluated by the "comet assay" or single cell gel electrophoresis (SCGE) both in its conventional use and by the Endonuclease III modified method, which allow to detect the presence of oxidized pyrimidines.

RESULTS: Bile acids (CDA and CDCA) resulted genotoxic on both normal and tumour human colon cells. The inclusion of the endonuclease III digestion step in the comet assay demonstrated that bile acids induced an oxidative DNA damage. In addition, treatment of colonocytes with bile acids in the presence of the antioxidants (beta-carotene, alpha-tocopherol) and Na-butyrate caused a reduction of DNA damage.

CONCLUSION: Our results suggest that bile acids may be involved in the tumour initiation by inducing a DNA oxidative damage, and so add further evidences to the preventive properties of antioxidants present in the Mediterranean diet.

Abstract  Epigenetic mechanisms of histone acetylation/deacetylation play an important role in the regulation of gene expression associated with the cell cycle and apoptosis. Recently, sodium butyrate, a histone deacetylase (HDAC) inhibitor, has been shown to exhibit anticancer effects via differentiation and apoptosis of cancer cells. Sodium butyrate may be a potential anticancer chemotherapeutic drug; however, the precise mechanism underlying the anticancer effects of sodium butyrate has not been clearly elucidated. In the present study, we investigated the role of death-associated protein kinase (DAPK) on the apoptosis of human gastric cancer cells induced by sodium butyrate. We observed that sodium butyrate induced apoptosis in human gastric cancer cells. Treatment with the HDAC inhibitor sodium butyrate increased the expression of caspase-3 and DAPK1/2 genes but decreased the expression of Bcl-2 in human gastric cancer cells. The expression of DAPK3, p53 and p21 were not altered by sodium butyrate treatment. Analysis of the general expression patterns revealed that sodium butyrate increased the expression of DAPK1/2 but decreased the expression of FAK and induced changes in the proliferation of apoptosis-related genes in human gastric cancer cells. These data suggest that DAPK expression prompts apoptosis by reducing the FAK protein level in sodium butyrate-induced apoptosis of human gastric cancer cells.

Abstract  Activation of Wnt signaling without mutation of β-catenin or APC occurs frequently in human gastric cancers. Secreted frizzled-related protein (SFRP), a negative modulator of the Wnt signaling pathway, are frequently inactivated in human gastric cancers. Inhibition of SFRP gene expression may account for the Wnt/β-catenin activation in human gastric cancer. However, the molecular mechanisms of silencing of SFRP genes are not fully understood. Sodium butyrate, a histone deacetylase (HDAC) inhibitor, is known to exhibit anti-cancer effects partly through the differentiation of various cancer cells. In the present study, we investigated: i) the relationship between the silencing of SFRP genes and Wnt signaling; ii) the mechanism of sodium butyrate mediated epigenetic regulation of SFRPs expression in human gastric cancer. We observed that nuclear β-catenin was significantly increased in gastric cancer tissues as compared to adjacent non-cancerous tissues. Nuclear β-catenin accumulation and SFRP promoter methylation in human gastric cancer cells were noted. Treatment with the DNA methyltransferase inhibitor, 5'-Aza-2-deoxycytidine (5'-Aza-dC) rapidly restored SFRPs expression. Sodium butyrate (NaB) induced demethylation and histone modification at the promoter region of SFRP1/2 restoring the SFRP expression in human gastric cancer cells. Analysis of general expression revealed that overexpression of SFRPs repressed Wnt target gene expression and induced changes in the proliferation and apoptosis related genes in human gastric cancer cells. These data suggest that aberrant epigenetic modification of SFRP genes is one of the major mechanisms by which Wnt signaling is activated in human gastric cancer cells and sodium butyrate may modulate the SFRP1/2 expression through histone modification and promoter demethylation causing anti-tumor effects.

Abstract  An experiment on ROSS 308 male broilers was conducted to evaluate probiotic (Primalac), prebiotic (Fermacto) and butyric acid glycerides (Baby C(4)) on broiler performance and serum composition. Seven hundred and four day-old broilers were randomly distributed in a 2 x 2 x 2 factorial arrangement with two levels of probiotic, prebiotic and butyric acid glycerides. Eight treatments with four replicates each with 22 birds per pen were used. Three-way interaction between dietary treatments were observed for final body weight (P<0.05), feed intake (P<0.01) and feed conversion ratio (P<0.01) in the experiment. Body weight, feed intake and feed conversion ratio between sulementary treatments and control group were significantly different (P<0.01). Three-way interaction between dietary treatments were observed for total cholesterol (P<0.05), LDL (P<0.01), cholesterol/HDL ratio and HDL/LDL ratio (P<0.01) in the experiment. Total cholesterol concentration, LDL, HDL/LDL ratio and cholesterol/HDL ratio between supplementary treatments and control group were significantly different (P<0.05 and P<0.01). Serum triglyceride, HDL and VLDL cholesterol concentrations were not significantly different among dietary treatment when compared to control group (P>0.05). In conclusion, dietary supplementation improved the body weight, feed conversion ratio, HDL/LDL ratio and decreased feed intake, total cholesterol, LDL cholesterol and cholesterol/HDL ratio in the serum of broiler chickens.

Abstract  Colorectal cancer is one of the most common cancers in developed countries, and its incidence is negatively associated with high dietary fiber intake. Butyrate, a short-chain fatty acid fermentation by-product of fiber induces cell maturation with the promotion of growth arrest, differentiation, and/or apoptosis of cancer cells. The stimulation of cell maturation by butyrate in colonic cancer cells follows a temporal progression from the early phase of growth arrest to the activation of apoptotic cascades. Previously we performed two-dimensional DIGE to identify differentially expressed proteins induced by 24-h butyrate treatment of HCT-116 colorectal cancer cells. Herein we used quantitative proteomics approaches using iTRAQ (isobaric tags for relative and absolute quantitation), a stable isotope labeling methodology that enables multiplexing of four samples, for a temporal study of HCT-116 cells treated with butyrate. In addition, cleavable ICAT, which selectively tags cysteine-containing proteins, was also used, and the results complemented those obtained from the iTRAQ strategy. Selected protein targets were validated by real time PCR and Western blotting. A model is proposed to illustrate our findings from this temporal analysis of the butyrate-responsive proteome that uncovered several integrated cellular processes and pathways involved in growth arrest, apoptosis, and metastasis. These signature clusters of butyrate-regulated pathways are potential targets for novel chemopreventive and therapeutic drugs for treatment of colorectal cancer.

Abstract  All-trans retinoic acid (ATRA) and sodium butyrate (SB) have shown growth-inhibitory and differentiation-inducing properties to tumor cells when used as single agents or in combination, but the exact molecular mechanism still remains to be determined. In order to determine the mechanism of the synergy in treatment with RA and SB, we evaluated the growth inhibition capability of ATRA and SB, alone or in combination, in human oral squamous carcinoma cell lines SCC-1 and SCC-9, and identified the expression of cell cycle-related genes. ATRA and SB inhibited cell growth and induced cell cycle G1 arrest. The inhibition effect was more pronounced with SB than with ATRA (p = 0.000). There were interactions between ATRA and SB (p = 0.000). Consistent with the inhibition effect and G1 arrest, ATRA and SB, alone or in combination, induced the expression of G1 phase markers cyclin-dependent kinase (CDK) 6, p21, and p27; inhibited the expression of S-G2 phase proteins CDK2; and decreased Rb phosphorylation. Cyclin D1 expression was increased in the SB- and ATRA + SB-treated groups, but inhibited in the ATRA-treated group. Cyclin B1 and cyclin E expression was slightly decreased in the SB- and ATRA + SB-treated groups, but did not change in the ATRA-treated group. These results indicate that the growth inhibition and G1 arrest of oral squamous carcinoma cells in response to ATRA and/or SB correlates with the induction of G1 phase cell cycle regulatory proteins CDK6, p21, and p27 and the inhibition of S-G2 phase cell cycle regulatory protein CDK2.

Abstract  To examine the relationship between apoptosis accompanying differentiation and sphingolipid-metabolism, CaCo-2 cells were used as a model of human intestinal epithelial cells and the variation in cellular Cer/GlcCer-content and related enzyme activities during butyrate-induced differentiation were investigated. The simultaneous administration of PDMP as a GlcCer synthase inhibitor caused a significant increase in the amount of Cers, especially palmitoyl-Cer. Butyrate caused an increase in the amount of GlcCers, especially alpha-hydroxy fatty acid-GlcCers, and in cellular GlcCer synthase activity. Cellular Cer content related to apoptosis was mainly regulated by the GlcCer synthase-based metabolism of Cers.

Abstract  This review emphasises the fact that studies of acetone-butanol-ethanol (ABE) fermentation by solventogenic clostridia cannot be limited to research on the strain Clostridium acetobutylicum ATCC 824. Various 1-butanol producing species of the genus Clostridium, which differ in their patterns of product formation and abilities to ferment particular carbohydrates or glycerol, are described. Special attention is devoted to species and strains that do not produce acetone naturally and to the utilisation of lactose, inulin, glycerol and mixtures of pentose and hexose carbohydrates. Furthermore, process-mapping tools based on different principles, including flow cytometry, DNA microarray analysis, mass spectrometry, Raman microscopy, FT-IR spectroscopy and anisotropy of electrical polarisability, which might facilitate fermentation control and a deeper understanding of ABE fermentation, are introduced. At present, the methods with the greatest potential are flow cytometry and transcriptome analysis. Flow cytometry can be used to visualise and capture cells within clostridial populations as they progress through the normal cell cycle, in which symmetric and asymmetric cell division phases alternate. Cell viability of a population of Clostridium pasteurianum NRRL B-598 was determined by flow cytometry. Transcriptome analysis has been used in various studies including the detection of genes expressed in solventogenic phase, at sporulation, in the stress response, to compare expression patterns of different strains or parent and mutant strains, for studies of catabolite repression, and for the detection of genes involved in the transport and metabolism of 11 different carbohydrates. Interestingly, the results of transcriptome analysis also challenge our earlier understanding of the role of the Spo0A regulator in initiation of solventogenesis in C. acetobutylicum ATCC 824. Lastly, the review describes other significant recent discoveries, including the deleterious effects of intracellular formic acid accumulation in C. acetobutylicum DSM 1731 cells on the metabolic switch from acidogenesis to solventogenesis and the development of a high-cell density continuous system using Clostridium saccharoperbutylacetonicum N1-4, in which 1-butanol productivity of 7.99g/L/h was reached.

Abstract  Experimental and epidemiological evidence supports the idea that dietary fat and fiber influence colon carcinogenesis. Particularly, their components, n-3 polyunsaturated fatty acids (PUFAs) and butyrate, have been proven to exhibit beneficial effects on colon epithelial cell metabolism, signaling, and kinetics, thus preventing colon inflammation and cancer. Moreover, these effects may be strengthened by PUFA and butyrate combination. It appears that administration of these compounds might be a relatively nontoxic form of supportive therapy improving cancer treatment outcomes and slowing down or preventing recurrence of certain types of cancer. However, their efficient application has to be based on solid scientific evidence of their mechanisms of action from the molecular and cellular to the organismal level. In this review, we emphasize the role of lipids and their metabolism during tumor development, describe some important mechanisms considering cellular and molecular levels of PUFA and butyrate action in colon epithelial cells, and particularly focus on the interaction of their metabolism and the signaling pathways with respect to the differences in response of normal and cancer colon cells.

Abstract  Malignant transformation of mammalian cells with ras family oncogenes results in dramatic changes in cellular architecture and growth traits. The generation of flat revertants of v-K-ras-transformed renal cells by exposure to the histone deacetylase inhibitor sodium butyrate (NaB) was previously found to be dependent on transcriptional activation of the PAI-1 (SERPINE1) gene (encoding the type-1 inhibitor of urokinase and tissue-type plasminogen activators). NaB-initiated PAI-1 expression preceded induced cell spreading and entry into G(1) arrest. To assess the relevance of PAI-1 induction to growth arrest in this cell system more critically, two complementary approaches were used. The addition of a stable, long half-life, recombinant PAI-1 mutant to PAI-1-deficient v-K-ras-/c-Ha-ras-transformants or to PAI-1 functionally null, NaB-resistant, 4HH cells (engineered by antisense knockdown of PAI-1 mRNA transcripts) resulted in marked cytostasis in the absence of NaB. The transfection of ras-transformed cells with the Rc/CMVPAI expression construct, moreover, significantly elevated constitutive PAI-1 synthesis (10- to 20-fold) with a concomitant reduction in proliferative rate. These data suggest that high-level PAI-1 expression suppresses growth of chronic ras-oncogene transformed cells and is likely a major cytostatic effector of NaB exposure.

Abstract  We propose comparing angiogenic effects of butyric acid (BA)-impregnated suture vs control suture on an aged tendon model. Twenty-four 3-year-old rabbits underwent bilateral Achilles tendon exposure. BA-impregnated orthopedic suture was sutured into one side, and a control orthopedic suture into the contralateral side similarly. The rabbits were sacrificed at 7, 30, and 45 days and the tendons harvested for gross, histologic, and biochemical study. Histologically, there was increased vascularity/cell migration at all time points in the BA-treated tendons; proteoglycan expression (ie, safranin O staining) increased at 30 and 45 days. DNA concentration was significantly (P = .05) higher in the BA-treated tendon group relative to the control group at 7 days but was unchanged at 30 and 45 days. Similarly, messenger RNA (mRNA) expression of vascular endothelial growth factor (VEGF) was significantly (P = .05) higher in the BA-treated tendon at 7 days. A trend (P = .12) for higher expression in the BA group also was found at 30 days.

Abstract  Clostridium tyrobutyricum ATCC 25755, a butyric acid producing bacterium, has been engineered to overexpress aldehyde/alcohol dehydrogenase 2 (adhE2, Genebank no. AF321779) from Clostridium acetobutylicum ATCC 824, which converts butyryl-CoA to butanol, under the control of native thiolase (thl) promoter. Butanol titer of 1.1g/L was obtained in C. tyrobutyricum overexpressing adhE2. The effects of inactivating acetate kinase (ack) and phosphotransbutyrylase (ptb) genes in the host on butanol production were then studied. A high C4/C2 product ratio of 10.6 (mol/mol) was obtained in ack knockout mutant, whereas a low C4/C2 product ratio of 1.4 (mol/mol) was obtained in ptb knockout mutant, confirming that ack and ptb genes play important roles in controlling metabolic flux distribution in C. tyrobutyricum. The highest butanol titer of 10.0g/L and butanol yield of 27.0% (w/w, 66% of theoretical yield) were achieved from glucose in the ack knockout mutant overexpressing adhE2. When a more reduced substrate mannitol was used, the butanol titer reached 16.0 g/L with 30.6% (w/w) yield (75% theoretical yield). Moreover, C. tyrobutyricum showed good butanol tolerance, with >80% and ∼60% relative growth rate at 1.0% and 1.5% (v/v) butanol. These results suggest that C. tyrobutyricum is a promising heterologous host for n-butanol production from renewable biomass.

Abstract  BACKGROUND: n-Butanol is a promising emerging biofuel, and recent metabolic engineering efforts have demonstrated the use of several microbial hosts for its production. However, most organisms have very low tolerance to n-butanol (up to 2% (v/v)), limiting the economic viability of this biofuel. The rational engineering of more robust n-butanol production hosts relies upon understanding the mechanisms involved in tolerance. However, the existing knowledge of genes involved in n-butanol tolerance is limited. The goal of this study is therefore to identify E. coli genes that are involved in n-butanol tolerance.

METHODOLOGY/PRINCIPAL FINDINGS: Using a genomic library enrichment strategy, we identified approximately 270 genes that were enriched or depleted in n-butanol challenge. The effects of these candidate genes on n-butanol tolerance were experimentally determined using overexpression or deletion libraries. Among the 55 enriched genes tested, 11 were experimentally shown to confer enhanced tolerance to n-butanol when overexpressed compared to the wild-type. Among the 84 depleted genes tested, three conferred increased n-butanol resistance when deleted. The overexpressed genes that conferred the largest increase in n-butanol tolerance were related to iron transport and metabolism, entC and feoA, which increased the n-butanol tolerance by 32.8±4.0% and 49.1±3.3%, respectively. The deleted gene that resulted in the largest increase in resistance to n-butanol was astE, which enhanced n-butanol tolerance by 48.7±6.3%.

CONCLUSIONS/SIGNIFICANCE: We identified and experimentally verified 14 genes that decreased the inhibitory effect of n-butanol tolerance on E. coli. From the data, we were able to expand the current knowledge on the genes involved in n-butanol tolerance; the results suggest that an increased iron transport and metabolism and decreased acid resistance may enhance n-butanol tolerance. The genes and mechanisms identified in this study will be helpful in the rational engineering of more robust biofuel producers.

Abstract  The aim of this study was to determine the changes of short chain fatty acids (SCFAs) in faeces of inflammatory bowel disease (IBD) patients compared to healthy subjects. SCFAs such as pyruvic, lactic, formic, acetic, propionic, isobutyric and butyric acids were analyzed by using high performance liquid chromatography (HPLC). This study showed that the level of acetic, 162.0 micromol/g wet faeces, butyric, 86.9 micromol/g wet faeces, and propionic acids, 65.6 micromol/g wet faeces, decreased remarkably in IBD faecal samples when compared with that of healthy individuals, 209.7, 176.0, and 93.3 micromol/g wet faeces respectively. On the contrary, lactic and pyruvic acids showed higher levels in faecal samples of IBD than in healthy subjects. In the context of butyric acid level, this study also found that the molar ratio of butyric acid was higher than propionic acid in both faecal samples. This might be due to the high intake of starch from rice among Malaysian population. It was concluded that the level of SCFAs differ remarkably between faecal samples in healthy subjects and that in IBD patients providing evidence that SCFAs more likely play an important role in the pathogenesis of IBD.