Secretion of 2,3-dihydroxyisovalerate as a limiting factor for isobutanol production in Saccharomyces cerevisiae.

Isobutanol is a superior biofuel compared to ethanol, and it is naturally produced by yeasts. Previously, Saccharomyces cerevisiae has been genetically engineered to improve isobutanol production. We found that yeast cells engineered for a cytosolic isobutanol biosynthesis secrete large amounts of the intermediate 2,3-dihydroxyisovalerate (DIV). This indicates that the enzyme dihydroxyacid dehydratase (Ilv3) is limiting the isobutanol pathway and/or yeast exhibit effective transport systems for the secretion of the intermediate, competing with isobutanol synthesis. Moreover, we found that DIV cannot be taken up by the cells again. To identify the responsible transporters, microarray analysis was performed with a DIV producing strain compared to a wild type. Altogether, 19 genes encoding putative transporters were upregulated under DIV-producing conditions. Thirteen of these were deleted together with five homologous genes. A yro2 mrh1 deletion strain showed reduced DIV secretion, while a hxt5 deletion mutant showed increased isobutanol production. However, a strain deleted for all the 18 genes secreted even slightly increased amounts of the intermediates and less isobutanol. The lactate transporter Jen1 turned out to transport the intermediate 2-ketoisovalerate, but not DIV. The results suggest that the transport of DIV is a rather complex process and several unspecific transporters seem to be involved.

Identification of a methioninase inhibitor, myrsinoic acid B, from Myrsine seguinii Lév., and its inhibitory activities.

A methioninase inhibitor from Myrsine seguinii was purified and identified as myrsinoic acid B. Its inhibitory activities as to crude methioninase from periodontal bacteria such as Fusobacterium nucleatum, Porphyromonas gingivalis, and Treponema denticola were determined. The IC50 values were 10.5, 82.4, and 30.3 microM respectively.

PPARalpha agonists inhibit nitric oxide production by enhancing iNOS degradation in LPS-treated macrophages.

BACKGROUND AND PURPOSE: Nitric oxide (NO) production through the inducible nitric oxide synthase (iNOS) pathway is increased in response to pro-inflammatory cytokines and bacterial products. In inflammation, NO has pro-inflammatory and regulatory effects. Peroxisome proliferator-activated receptors (PPARs), members of the nuclear steroid receptor superfamily, regulate not only metabolic but also inflammatory processes. The aim of the present study was to investigate the role of PPARalpha in the regulation of NO production and iNOS expression in activated macrophages. EXPERIMENTAL APPROACH: The effects of PPARalpha agonists were investigated on iNOS mRNA and protein expression, on NO production and on the activation of transcription factors NF-kappaB and STAT1 in J774 murine macrophages exposed to bacterial lipopolysaccharide (LPS). KEY RESULTS: PPARalpha agonists GW7647 and WY14643 reduced LPS-induced NO production in a dose-dependent manner as measured by the accumulation of nitrite into the culture medium. However, PPARalpha agonists did not alter LPS-induced iNOS mRNA expression or activation of NF-kappaB or STAT1 which are important transcription factors for iNOS. Nevertheless, iNOS protein levels were reduced by PPARalpha agonists in a time-dependent manner. The reduction was markedly greater after 24 h incubation than after 8 h incubation. Treatment with the proteasome inhibitors, lactacystin or MG132, reversed the decrease in iNOS protein levels caused by PPARalpha agonists. CONCLUSIONS AND IMPLICATIONS: The results suggest that PPARalpha agonists reduce LPS-induced iNOS expression and NO production in macrophages by enhancing iNOS protein degradation through the proteasome pathway. The results offer an additional mechanism underlying the anti-inflammatory effects of PPARalpha agonists.

Antagonistic effects of sodium butyrate and N-(4-hydroxyphenyl)-retinamide on prostate cancer.

Butyrates and retinoids are promising antineoplastic agents. Here we analyzed effects of sodium butyrate and N-(4-hydroxyphenyl)-retinamide (4-HPR) on prostate cancer cells as monotherapy or in combination in vitro and in vivo. Sodium butyrate and 4-HPR induced concentration-dependent growth inhibition in prostate cancer cells in vitro. The isobologram analysis revealed that sodium butyrate and 4-HPR administered together antagonize effects of each other. For the in vivo studies, a water-soluble complex (4-HPR with a cyclodextrin) was created. A single dose of sodium butyrate and 4-HPR showed a peak level in chicken plasma within 30 minutes. Both compounds induced inhibition of proliferation and apoptosis in xenografts of the chicken chorioallantoic membrane. Analysis of the cytotoxic effects of the drugs used in combination demonstrated an antagonistic effect on inhibition of proliferation and on induction of apoptosis. Prolonged jun N-terminal kinase phosphorylation induced by sodium butyrate and 4-HPR was strongly attenuated when both compounds were used in combination. Both compounds induced inhibition of NF-kappaB. This effect was strongly antagonized in LNCaP cells when the compounds were used in combination. These results indicate that combinational therapies have to be carefully investigated due to potential antagonistic effects in the clinical setting despite promising results of a monotherapy.

DNA mismatch repair status may influence anti-neoplastic effects of butyrate.

HNPCC (hereditary non-polyposis colon cancer) is an autosomal-dominant disorder characterized by early-onset CRC (colorectal cancer). HNPCC is most often associated with mutations in the MMR (mismatch repair) genes hMLH1, hMSH2, hMSH6 or hPMS2. The mutator phenotype of a defective MMR system is MSI (microsatellite instability), which also occurs in approx. 15-25% of sporadic CRC cases, where it is associated with the hypermethylation of the promoter region of hMLH1. Dietary factors, including excessive alcohol consumption, ingestion of red meat and low folate intake, may increase the risk of MSI high tumour development. In contrast, aspirin may suppress MSI in MMR-deficient CRC cell lines. Butyrate, a short-chain-fatty-acid end product of carbohydrate fermentation in the colon, shares a number of anti-neoplastic properties with aspirin, including inhibiting proliferation and inducing apoptosis of CRC cells. Recent in vitro studies suggest that physiological concentrations of butyrate (0.5-2 mM) may have more potent anti-neoplastic effects in CRC cell lines deficient in MMR, but mechanisms for such a differential response remain to be established.

Ras/MAP kinase pathways are involved in Ras specific apoptosis induced by sodium butyrate.

Histone deacetylase inhibitors such as TSA, SAHA, and NaBu etc. are prospective cancer therapeutics of growing interest. Here, we demonstrated that oncogenic ras-transformed rat liver epithelial (WB-ras) cells were specifically undergone apoptosis by 48 h treatment of NaBu. During this, inhibition of ras proteins, especially farnesylated form of ras, and down-regulation of ERK1/2 were observed, which suggest ras/raf/MEK/ERK down-regulation, while p38 MAP kinase was maintained up-regulated. In addition, up-regulation of pro-apoptotic proteins such as p53 and p21CIP1/WAF1, and down-regulation of cell cycle regulator/anti-apoptotic proteins such as cdk2, -4 and phosphorylated Akt were observed concurrently with an increase in apoptotic cell portion. A phosphatase inhibitor, sodium orthovanadate (SOV), efficiently blocked apoptosis and restored responsible proteins for each phenomenon including ERK1/2 while SB203580, a specific p38 MAP kinase inhibitor, showed minor effect on them. Thus, ras/ERK signaling pathway can be considered in chemotherapeutic strategies of NaBu regardless of its inhibitory action on histone deacetylase.

Interleukin-1beta converting enzyme subfamily inhibitors prevent induction of CD86 molecules by butyrate through a CREB-dependent mechanism in HL60 cells.

To investigate the underlying mechanism for induction of CD86 molecules, we analysed the ability of the histone deacetylase (HDAC) inhibitor, sodium butyrate (NaB), to induce CD86 at the transcriptional level in HL60 cells. Our studies showed that the expression of CD86 on the cell surface was increased by 24 hr of NaB treatment, and the enhancement of CD86 mRNA expression was observed by real-time polymerase chain reaction. When we measured NF-kappaB binding activity, significant activity was induced upon NaB stimulation, which was suppressed by the addition of pyrrolidine dithiocarbamate. Butyrate also induced phosphorylated cAMP response element-binding protein (CREB), which bound to cAMP-responsive elements. Dibutyryl (db) -cAMP induced active CREB and increased the levels of CD86 by 24 hr. These observations indicated that NF-kappaB and/or CREB are crucial for butyrate-dependent activation of CD86 gene expression. We examined the inhibitory effects of various caspase inhibitors on the expression of CD86 in cells treated with NaB, because NaB also induced apoptosis with slow kinetics. Intriguingly, our results demonstrated that inhibitors of the interleukin-1beta converting enzyme subfamily (caspase-1, -4, -5 and -13) blocked the butyrate-induced increase in level of CD86. These inhibitors interfered with CD86 gene transcription in the presence of activated NF-kappaB, whereas phosphorylated CREB was down-regulated in the reactions where these inhibitors were added to inhibit CD86 gene expression. These results suggested that butyrate not only acetylates histones on the CD86 promoter through the suppression of HDAC activity, but that butyrate also regulates CREB-mediated transcription, possibly through the caspase activities triggered by NaB.

Inhibition of sodium butyrate-induced apoptosis in recombinant Chinese hamster ovary cells by constitutively expressing antisense RNA of caspase-3.

Sodium butyrate (NaBu) can enhance the expression of genes controlled by some of the mammalian promoters, but it can also inhibit cell growth and induce cellular apoptosis. Thus, the beneficial effect of using a higher concentration of NaBu on a foreign protein expression is compromised by its cytotoxic effect on cell growth. To overcome this cytotoxic effect of NaBu, the expression vector of antisense RNA of caspase-3 was constructed and transfected to recombinant Chinese hamster ovary (rCHO) cells producing a humanized antibody. Using this antisense RNA strategy, rCHO cells (B3) producing a low level of caspase-3 proenzyme were established. When batch cultures of both B3 cells and control cells transfected with antisense RNA-deficient plasmid were performed in the absence of NaBu, both cells showed similar profiles of cell growth and antibody production. Compared with control cell culture, under the condition of 5 mM NaBu addition at the exponential growth phase, expression of antisense RNA of caspase-3 significantly suppressed the NaBu-induced apoptosis of B3 cells and extended culture longevity by >2 days if the culture was terminated at cell viability of 50%. However, compared with control cell culture, the final antibody concentration of B3 cell culture was not increased in the presence of NaBu, which may be due to the loss of cellular metabolic capability resulted from the depolarization of mitochondrial membrane. Taken together, this study suggests that, although expression of antisense RNA of caspase-3 does not improve antibody productivity of rCHO cells, it can suppress NaBu-induced apoptotic cell death of rCHO cells and thereby may reduce problems associated with cellular disintegration.

Ligand-independent activation of the androgen receptor by the differentiation agent butyrate in human prostate cancer cells.

Androgens are potent differentiation agents that regulate prostate-specific antigen (PSA) gene expression via the androgen receptor (AR) that binds to androgen response elements (AREs) on the PSA gene to initiate transcription. However, in the absence of androgens, PSA gene expression can become elevated. This suggests that either the AR can be activated in the absence of androgen to elevate PSA gene expression through AREs on the PSA gene or that another transcription factor acting on the PSA promoter is stimulated. We have previously shown in vivo that butyrate, a differentiation agent that causes cell cycle arrest, increases serum PSA levels in castrated animals. Therefore, to determine the mechanism of butyrate induction of PSA, we used the LNCaP human prostate cancer cell line. Northern analyses and transfection experiments using a PSA reporter plasmid demonstrated induction of PSA gene expression by butyrate in LNCaP cells. Application of the antiandrogen bicalutamide blocked the induction of PSA mRNA by butyrate, suggesting a mechanism dependent on the AR. Consistent with this conclusion, electromobility shift assays showed increased AR-ARE complex formation with nuclear extracts from butyrate-treated cells. In addition, other reporter gene constructs that contain AREs were also induced by butyrate. Western blot analysis showed an increase in nuclear levels of AR protein in cells exposed to butyrate, whereas whole cell levels remained unchanged, suggesting that butyrate causes nuclear translocation of the AR. Thus, the differentiation agent butyrate causes ligand-independent activation of the AR to increase expression of the differentiation marker PSA in human prostate cancer cells.

Bile acids reduce the apoptosis-inducing effects of sodium butyrate on human colon adenoma (AA/C1) cells: implications for colon carcinogenesis.

Butyrate is produced in the colon by fermentation of dietary fibre and induces apoptosis in colon adenoma and cancer cell lines, which may contribute to the protective effect of a high fibre diet against colorectal cancer (CRC). However, butyrate is present in the colon together with unconjugated bile acids, which are tumour promoters in the colon. We show here that bile acids deoxycholate (DCA) and chenodeoxycholate (CDCA), at levels present in the colon, gave a modest increase in cell proliferation and decreased spontaneous apoptosis in AA/C1 adenoma cells. Bile acids significantly inhibited the induction of apoptosis by butyrate in AA/C1 cells. However, the survival-inducing effects of bile acids on AA/C1 cells could be overcome by increasing the concentration of sodium butyrate. These results suggest that dysregulation of apoptosis in colonic epithelial cells by dietary factors is a key factor in the pathophysiology of CRC.

N-acetylcysteine increases the biosynthesis of recombinant EPO in apoptotic Chinese hamster ovary cells.

Sodium butyrate (NaBu) is known to enhance the rate of biosynthesis of recombinant proteins in Chinese hamster ovary cells (CHO). Here we demonstrate that supplementation with NaBu during rapid growth brings about abrupt death of the cells. The death of the cells is due to apoptosis, as assessed by intranucleosomal DNA fragmentation. The promotion of apoptotic death of the cells could be partially blocked by treatment with the well-known antioxidant, N-acetylcysteine (NAC). Strikingly, the NAC treatment enhanced the production of recombinant EPO two-fold compared with that of the culture without NAC supplementation. These results showed that NaBu treatment supplemented with NAC not only inhibits apoptosis, but also exerts a synergistic effect on the biosynthesis of recombinant EPO.

Tyrosine kinase inhibitors reverse butyrate stimulation of human Caco-2 intestinal epithelial cell alkaline phosphatase but not butyrate promotion of dipeptidyl dipeptidase.

Short chain fatty acids such as sodium butyrate are concentrated in the colonic lumen and may protect against colon carcinogenesis by maintaining colonocytic differentiation, but the mechanisms by which they act are not fully understood. It has recently been suggested that short chain fatty acids modulate cellular tyrosine kinase activity in addition to altering chromatin structure via regulation of histone acetylation and DNA methylation. Therefore, the authors evaluated the influence of tyrosine kinase inhibition on the effects of 10 mM butyrate on human Caco-2 intestinal epithelial differentiation, using alkaline phosphatase and dipeptidyl dipeptidase specific activity as markers of differentiation, and two tyrosine kinase inhibitors, of different mechanisms of action and different effects on Caco-2 brush border enzyme specific activity, to block tyrosine kinase activity. As expected, butyrate stimulated both alkaline phosphatase and dipeptidyl dipeptidase specific activity. The tyrosine kinase inhibitors prevented, and indeed one inhibitor reversed the effects of butyrate on alkaline phosphatase specific activity. However, tyrosine kinase inhibition did not prevent butyrate stimulation of dipeptidyl dipeptidase specific activity. Different pathways are likely to regulate the effects of butyrate on expression of these two brush border enzymes. Butyrate stimulation of alkaline phosphatase, but not dipeptidyl dipeptidase, may involve tyrosine phosphorylation signaling.

Deoxycholate inhibits in vivo butyrate-mediated BrDU labeling of the colonic crypt.

The short-chain fatty acid butyrate (NaBu) selectively increases colonic crypt base proliferation and inhibits "premalignant" crypt surface hyperproliferation while the secondary bile acid deoxycholate (DCA) induces surface hyperproliferation, in vitro. We hypothesized that NaBu and DCA have similar selective and antagonistic effects on the colonic crypt proliferative pattern, in vivo. Fifty-six adult SD rats underwent surgical isolation of the colon and 24-hr intraluminal instillation with physiological (10 mM) and pharmacological (25 mM) levels of butyrate alone or combined with a physiological DCA level (5 microM). Bromodeoxyuridine-labeling indices (LI) were determined as labeled cells divided by total cells, for the whole crypt and five crypt compartments from base to surface. Treatment with NaBu increased total LI when compared to NaCl. This effect was significant only at the crypt base. Both doses of NaBu resulted in similar LI with no further response at the higher concentration. In contrast to prior in vitro studies, DCA alone at this concentration did not affect LI, but when combined with NaBu, DCA inhibited the effects of NaBu at the crypt base and surface. The conclusions are: (1) the in vivo proliferative effects of NaBu are selective to the crypt base, (2) an in vivo low physiological DCA level does not promote crypt surface hyperproliferation but does inhibit butyrate's proliferative effect, and (3) NaBu and DCA interact in a complex and antagonistic manner to selectively modulate crypt base and surface proliferation, in the rat colon, in vivo. These findings may have clinical relevance since colonic levels of NaBu and DCA are affected by diet.

AML1a but not AML1b inhibits erythroid differentiation induced by sodium butyrate and enhances the megakaryocytic differentiation of K562 leukemia cells.

AML1 may play a role in growth and differentiation of cells along erythroid and/or megakaryocytic lineages, because a significant level of the AML1 gene is expressed in these cells. We overexpressed AML1a (without the transcription-activating domain) and AML1b (with the domain) proteins in K562 leukemia cells, which can be induced to differentiate into hemoglobin-producing cells and megakaryocytes. The AML1a-transfected K562 cells had a reduced capacity to differentiate in the presence of sodium n-butyrate but not in the presence of other inducers, such as hemin, 1-beta-D-arabinofuranosylcytosine, and herbimycin A. The AML1 antisense oligodeoxynucleotide but not the sense oligomer recovered its differentiation-inducing capacity in the presence of butyrate. On the other hand, AML1b conferred a similar differentiation-inducing capacity upon K562 cells transfected with vector alone. AML1a expression was associated with enhanced sensitivity to megakaryocytic differentiation induced by phorbol ester. These results provide evidence that AML1 proteins play a role in erythroid and megakaryocytic differentiation.

Effect of HIV type 1 Tat protein on butyric acid-induced differentiation in a hematopoietic progenitor cell line.

The trans-activator protein (Tat) of HIV-1 plays an important role in viral pathogenesis. Since Tat has been shown to alter expression of a number of host cellular genes, we have investigated the role of Tat in modulating gene expression and differentiation in hematopoietic progenitor cells. Tat protein was introduced in K562 cells, a human hematopoietic progenitor cell line, by either scrape-loading onto HeLa (HL)-tat cells or direct electroporation of an affinity-purified glutathione S-transferase (GST)-Tat fusion protein. Under these conditions, butyric acid-induced hemoglobin production in K562 cells was suppressed by 65 and 52%, respectively. However, coculturing with wild-type HeLa cells or electroporation with the control GST protein did not decrease hemoglobin production. To confirm the presence of bioactive Tat protein within K562 cells, the cells were transiently transfected with a pHIV/LTR-CAT prior to the introduction of Tat. A 30- to 40-fold induction in CAT gene expression was observed in the transfected K562 cells, which were either cocultured with HL-tat or were electroporated with GST-Tat. Simultaneous transient transfection of K562 cells with a TAR expression plasmid, to compete for the availability of Tat protein, significantly downregulated the HIV LTR trans-activation by Tat. In addition, overexpression of the TAR RNAs in K562 cells was able to downregulate the suppressive effect of Tat on butyric acid-induced differentiation. RT-PCR analysis of the total RNAs isolated from these cells demonstrated that Tat protein suppressed the butyric acid-induced gamma-globin gene expression by an average of 54% without affecting the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs. These data indicate that the viral Tat protein plays a significant role in abrogating erythroid differentiation in K562 cells.

Colonic mucin synthesis is increased by sodium butyrate.

The effects of sodium butyrate and sodium bromo-octanoate (an inhibitor of beta oxidation) on colonic mucus glycoprotein (mucin) synthesis have been assessed using tissue from colonic resection samples. Epithelial biopsy specimens were incubated for 16 hours in RPMI 1640 with glutamine, supplemented with 10% fetal calf serum and N-acetyl-[3H]-glucosamine ([3H]-Glc NAc), and differing concentrations of sodium butyrate. Incorporation of [3H] Glc NAc into mucin by normal epithelium at least 10 cm distant from colonic cancer was increased in the presence of sodium butyrate in a dose dependent manner, with maximum effect (476%) at a concentration of 0.1 mM (number of specimens = 24 from six patients, p < 0.001). The increase in response to butyrate was not seen when specimens were incubated in the presence of the beta oxidation inhibitor sodium bromo-octanoate 0.05 M. The striking increase in mucin synthesis that results when butyrate is added to standard nutrient medium suggests that this may be an important mechanism affecting the rate of mucin synthesis in vivo and may also explain the therapeutic effect of butyrate in colitis.

The role of volume-sensitive Cl- channels in the stimulation of chloride absorption by short-chain fatty acids in the rat colon.

The short-chain fatty acids acetate, propionate and butyrate induced a concentration-dependent decrease in short-circuit current (Isc) of the rat colon in vitro. The decrease in Isc, being more pronounced in the distal than in the proximal colon, was dependent on the presence of Cl- ions and partly on the presence of HCO3-. In the distal colon, the fall in Isc could be inhibited by amiloride, indicating that the activity of the Na+/H+ exchanger is necessary for the induction of this response. The decrease in Isc was diminished by the Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate, and the lipoxygenase inhibitor, nordihydroguaiaretic acid. In contrast, inhibitors of the leukotriene pathway or a Cl- channel blocker did not affect the Isc response in the proximal colon. Measurements of unidirectional fluxes revealed that butyrate caused a stimulation of the mucosa to serosa fluxes (Fms) of Na+ and Cl- in the distal, but only of FNams in the proximal colon. Unidirectional Rb+ fluxes were not altered. The stimulation of Fclms correlated with the degree of metabolism of the short-chain fatty acid. The increase in FClms was most pronounced for butyrate, smaller for acetate and not observed with the poorly metabolizable short-chain fatty acid, isobutyrate. Consequently, two factors seem to be responsible for the stimulation of Cl- absorption by short-chain fatty acids in the distal colon: (1) the intracellular production of HCO3- during the oxidation of short-chain fatty acids as substrate for the apical Cl-/HCO3- exchanger, and (2) the activation of volume-sensitive basolateral Cl- channels.

The effect of short-chain fatty acids on Cl- and K+ conductance in rat colonic crypts.

The effect of butyrate on membrane potential and membrane currents of colonic enterocytes was studied with the whole-cell patch-clamp method. Superfusion of crypts from the rat distal colon with butyrate-containing solutions induced a membrane depolarization of 16.5 +/- 2.3 mV. This response was only observed in the upper third of the crypt. The depolarization was dependent on the presence of Cl- and was accompanied by an increase in membrane inward current, indicating that it is caused by an increase in Cl- conductance. Membrane outward current, however, behaved inconsistently. Whereas in most cells an increase was observed, about 25% of the cells responded with a decrease. This unexpected inhibition of the outward current probably represents a decrease of K+ conductance caused by the cellular acidification in the presence of butyrate. Manoeuvres carried out to acidify the cell interior, like perfusion with acid buffer solutions or inhibition of the Na+/H+ exchanger by amiloride, mimicked this inhibition of the K+ conductance. Orientating cell-attached patch-clamp recordings performed in parallel revealed an activation of previously silent basolateral Cl- channels by butyrate. They had a linear current/voltage relationship and a single-channel conductance of 20-30 pS. The butyrate-induced depolarization was not dependent on intracellular adenosine 5'-triphosphate (ATP) and was also observed when the buffer capacity of the pipette for Ca2+ was increased. It was also not inhibited by guanosine-5'-O(2-thiodiphosphate) (GDP[beta S]).(ABSTRACT TRUNCATED AT 250 WORDS)

Activity of the AIB factor observed in prokaryotic and eukaryotic microorganisms.

Streptomyces cinnamonensis produces a new substance named AIB (for anti-isobutyrate) factor which, on a solid medium, efficiently counteracts toxic concentrations not only of isobutyrate but also of other salts of short-chain monocarboxylic acids. In the present study we demonstrate that the AIB factor activity is widely spread because this effect was positively detected in 25 of 31 randomly chosen microorganisms (streptomycetes, ascomycetes, zygomycetes and basidiomycetes). The AIB factor produced by the tested microorganisms on an agar media allows for germination, growth, and sporulation of the testing Streptomyces coelicolor on an agar medium containing 20 mmol/L acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, and 2-methylbutyrate. The activity of the AIB factor from different sources towards these substances differs.