Effect of sodium butyrate on lymphocyte activation.
(1/1990)
Butyrate, in relatively low concentrations, has been shown to induce synthesis of enzymes, cause changes in cell morphology, and inhibit growth of a variety of mammalian cells in tissue culture (reviewed in [1]). In this communication, we report our observations on the effect of butyrate on lymphocyte activation. Butyrate completely and reversibly inhibits mitogen-induced blast formation. We present evidence that it does not interfere with the binding of mitogens, that it does not inhibit a number of the "early" reactions involved in activation, and that it does not affect ongoing DNA synthesis for an extended period of time. However, butyrate rapidly inhibits any increase in the rate of DNA synthesis. (+info)
Antisense RNA strategies for metabolic engineering of Clostridium acetobutylicum.
(2/1990)
We examined the effectiveness of antisense RNA (as RNA) strategies for metabolic engineering of Clostridium acetobutylicum. Strain ATCC 824(pRD4) was developed to produce a 102-nucleotide asRNA with 87% complementarity to the butyrate kinase (BK) gene. Strain ATCC 824(pRD4) exhibited 85 to 90% lower BK and acetate kinase specific activities than the control strain. Strain ATCC 824(pRD4) also exhibited 45 to 50% lower phosphotransbutyrylase (PTB) and phosphotransacetylase specific activities than the control strain. This strain exhibited earlier induction of solventogenesis, which resulted in 50 and 35% higher final concentrations of acetone and butanol, respectively, than the concentrations in the control. Strain ATCC 824(pRD1) was developed to putatively produce a 698-nucleotide asRNA with 96% complementarity to the PTB gene. Strain ATCC 824(pRD1) exhibited 70 and 80% lower PTB and BK activities, respectively, than the control exhibited. It also exhibited 300% higher levels of a lactate dehydrogenase activity than the control exhibited. The growth yields of ATCC 824(pRD1) were 28% less than the growth yields of the control. While the levels of acids were not affected in ATCC 824(pRD1) fermentations, the acetone and butanol concentrations were 96 and 75% lower, respectively, than the concentrations in the control fermentations. The lower level of solvent production by ATCC 824(pRD1) was compensated for by approximately 100-fold higher levels of lactate production. The lack of any significant impact on butyrate formation fluxes by the lower PTB and BK levels suggests that butyrate formation fluxes are not controlled by the levels of the butyrate formation enzymes. (+info)
Sustained induction of fetal hemoglobin by pulse butyrate therapy in sickle cell disease.
(3/1990)
High levels of fetal hemoglobin (Hb F) protect from many of the complications of sickle cell disease and lead to improved survival. Butyrate and other short chain fatty acids were previously shown to increase Hb F production in erythroid cells in vitro and in animal models in vivo. However, butyrates are also known to inhibit the proliferation of many cell types, including erythroid cells. Experience with the use of butyrate in animal models and in early clinical trials demonstrated that the Hb F response may be lost after prolonged administration of high doses of butyrate. We hypothesized that this loss of response may be a result of the antiproliferative effects of butyrate. We designed a regimen consisting of intermittent or pulse therapy in which butyrate was administered for 4 days followed by 10 to 24 days with no drug exposure. This pulse regimen induced fetal globin gene expression in 9 of 11 patients. The mean Hb F in this group increased from 7.2% to 21.0% (P <.002) after intermittent butyrate therapy for a mean duration of 29.9 weeks. This was associated with a parallel increase in the number of F cells and F reticulocytes. The total hemoglobin levels also increased from a mean of 7.8 g/dL to a mean of 8.8 g/dL (P <.006). The increased levels of Hb F were sustained in all responders, including 1 patient who has been on pulse butyrate therapy for more than 28 months. This regimen, which resulted in a marked and sustained increase in Hb F levels in more than two thirds of the adult sickle cell patients enrolled in this study, was well tolerated without adverse side effects. These encouraging results require confirmation along with an appropriate evaluation of clinical outcomes in a larger number of patients with sickle cell disease. (+info)
Effects of praeruptorine C on the intracellular free calcium in normal and hypertrophied rat ventricular myocytes.
(4/1990)
AIM: To study the intracellular free calcium ([Ca2+]i) in normal and hypertrophic left ventricular myocytes isolated from adult rat hearts and the effects of praeruptorine C (Pra-C) on them. METHODS: [Ca2+]i of single myocyte was measured with Fura 2-AM. RESULTS: The resting [Ca2+]i was 87 +/- 4 nmol.L-1 in normal left ventricular myocytes, 123 +/- 7 nmol.L-1 in hypertrophied myocytes. After exposure to KCl (20, 40, and 60 mmol.L-1), the [Ca2+]i were increased by 66%, 141%, and 268% in normal myocytes, and 77%, 185%, and 243% in hypertrophic myocytes, respectively. Pra-C (1, 10, and 100 mumol.L-1) concentration-dependently inhibited the [Ca2+]i elevation caused by KCl (35 mmol.L-1) or norepinephrine (20 mumol.L-1) in both normal and hypertrophied myocytes. All of the effects of Pra-C were similar to that of nifedipine. CONCLUSION: [Ca2+]i of hypertrophied myocytes was higher than that of normal ones and Pra-C decrease the [Ca2+]i elevation in left ventricular myocytes resulted from its calcium channel blockade. (+info)
Effect of ornithine and lactate on urea synthesis in isolated hepatocytes.
(5/1990)
1. In hepatocytes isolated from 24 h-starved rats, urea production from ammonia was stimulated by addition of lactate, in both the presence and the absence of ornithine. The relationship of lactate concentration to the rate of urea synthesis was hyperbolic. 2. Other glucose precursors also stimulated urea production to varying degrees, but none more than lactate. Added oleate and butyrate did not stimulate urea synthesis. 3. Citrulline accumulation was largely dependent on ornithine concentration. As ornithine was increased from 0 to 40 mM, the rate of citrulline accumulation increased hyperbolically, and was half-maximal when ornithine was 8-12 mM. 4. The rate of citrulline accumulation was independent of the presence of lactate, but with pyruvate the rate increased. 5. The rate of urea production continued to increase as ornithine was varied from 0 to 40 mM. 6. It was concluded that intermediates provided by both ornithine and lactate are limiting for urea production from ammonia in isolated liver cells. It was suggested that the stimulatory effect of lactate lies in increased availability of cytosolic aspartate for condensation with citrulline. (+info)
Butyrate augments interferon-alpha-induced S phase accumulation and persistent tyrosine phosphorylation of cdc2 in K562 cells.
(6/1990)
Interferon-alpha (IFN-alpha) is a clinically useful cytokine for treatment of a variety of cancers, including chronic myelocytic leukaemia (CML). Most CML cells are sensitive to IFN-alpha; however, its biological effects on leukaemic cells are incompletely characterized. Here, we provide evidence that IFN-alpha induces a significant increase in the S phase population in human CML leukaemic cell line, K562, and that the S phase accumulation was augmented by sodium butyrate. In contrast, neither sodium butyrate alone, nor sodium butyrate plus IFN-gamma, affected the cell cycle in K562 cells. These data suggest that the effect of sodium butyrate depended upon IFN-alpha-mediated signalling. The ability of leukaemic cells to exhibit the S phase accumulation after stimulation by IFN-alpha plus sodium butyrate correlated well with persistent tyrosine phosphorylation of cdc2, whereas treatment with IFN-gamma plus sodium butyrate did not affect its phosphorylation levels. Considering that dephosphorylation of cdc2 leads to entry to the M phase, the persistent tyrosine phosphorylation of cdc2 may be associated with the S phase accumulation induced by IFN-alpha and sodium butyrate. In addition, another human CML leukaemic cell line, MEG-01, also showed the S phase accumulation after stimulation with IFN-alpha plus sodium butyrate. Taken together, our studies reveal a novel effect of sodium butyrate on the S phase accumulation and suggest its clinical application for a combination therapy with IFN-alpha, leading to a great improvement of clinical effects of IFN-alpha against CML cells. (+info)
Demonstration of a new mammalian isoleucine catabolic pathway yielding an Rseries of metabolites.
(7/1990)
1. Normal human urine contains small amounts (less than 4 mg/g of creatinine) of 2-ethylhydracrylic acid, formed, we believe, by a previously undisclosed endogenous catabolic pathway for the oxidation of a newly described series of R metabolites of isoleucine. 2. Urinary excretion of 2-ethylhydracrylic acid is variably increased in defects of isoleucine oxidation at distal steps in the catabolic pathway (3-oxoacyl-CoA thiolase deficiency and methylmalonyl-CoA mutase deficiency) and is diminished when proximal steps of the oxidative pathway are blocked as in branched-chain oxo acid decarboxylase deficiency ('maple-syrup-urine' disease). 3. Precursors of R-pathway metabolites [R(-)-2-methylbutyrate and 2-ethylacrylate ] lead to increased 2-ethylhydracrylate excretion in the mammal(rat, rabbit and dog); the corresponding S metabolites [S(+)-2-methylbutyric acid and tiglic acid ], when given in equimolar amounts, have little effect on its excretion, suggesting that little or no interconversion between S and R metabolites occurs in vivo. 4. Studies with 2H-labelled precursors indicate that conversion of R 2-methylbutyrate into 2-ethylhydracrylic acid occurs by a direct pathway (apparently via 2-ethylacrylic acid). 5. The further oxidation of 2-ethylhydracrylic acid to ethylmalonic acid was demonstrated, and may be analogous to S-metabolite oxidation via methyl malonate. 6. Valine metabolites do not interact with the R=isoleucine pathway under the conditions of these experiments in vivo. (+info)
Molecular recognition of fatty acids by peroxisome proliferator-activated receptors.
(8/1990)
The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors for fatty acids (FAs) that regulate glucose and lipid homeostasis. We report the crystal structure of the PPAR delta ligand-binding domain (LBD) bound to either the FA eicosapentaenoic acid (EPA) or the synthetic fibrate GW2433. The carboxylic acids of EPA and GW2433 interact directly with the activation function 2 (AF-2) helix. The hydrophobic tail of EPA adopts two distinct conformations within the large hydrophobic cavity. GW2433 occupies essentially the same space as EPA bound in both conformations. These structures provide molecular insight into the propensity for PPARs to interact with a variety of synthetic and natural compounds, including FAs that vary in both chain length and degree of saturation. (+info)