Transformation of natural products into more potent compounds: chemical modification of monensin

Monensin (1) is a representative compound of polyether ionophore antibiotics, which selectively transport Na+ ions. In order to obtain potent Na+ ionophores, the modification of the carboxyl group of monensin was carried out to yield monensylamino acids (2) and monensylamino acid-1,29-lactones (3). The Na+ permeability of ion through the erythrocyte membrane of 2 and 3 was evaluated by the 23Na-NMR method. Compound 2 showed less Na+ ion transport activity than monensin, probably due to the lower lipophilicity caused by the conformational change of the chain moiety of the molecules. Although 3 showed higher lipophilisity than 1, 3 had no Na+ ion permeability, probably due to loss of the carboxyl group. As more lipophilic compounds possessing a carboxyl group was supposed to have more ion transport activity, 7-O-acylmonensins (8) and 7-O-alkylmonensins (11) were synthesized. Among these compounds, the value of Na+ ion permeability of 7-O-benzylmonensin (11c) was 1.4 time that of 1. Further investigation was carried out by preparing various 7-O-(substituted benzyl)monensins (13), and 7-O-(p-ethylbenzyl)monensin (13b) exhibited the largest Na+ ion permeability, about twice the value of 1. In order to convert monensin (1) to Ca2+ ionophore, 7-carboxylmethylmonensin (18) via protected 7-oxomonensin (15), and 25-carboxylmonensin (26) were prepared. In the course of the synthesis, 15 was clarified as a useful intermediate to give 7-amino and 7-alkyl derivatives. Ca2+ ion transport activities of 18 and 26 were determined by a CHCl3 liquid membrane system. 25-carboxylmonensin (26) showed 70% of the activity of Ca2+ ionophore, lasalocid A, and compound 26 could be the lead compound for the preparation of a new Ca2+ ionophore.     

Lactate transport in macrophages

Macrophages perform phagocytic and effector activities in a number of different tissues. The environment of the inflammatory foci in which they function is often acidic and contains an abundance of lactate. We characterized the ability of thioglycollate-elicited mouse peritoneal macrophages to accumulate lactate from the medium and to use this lactate to maintain intracellular energy stores. Lactate uptake was stereospecific for L-lactate and was inhibited by the organic anion transport blocker probenecid but not by concentrations of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid that block anion exchangers. L-[14C]Lactate uptake was not affected by variation of the extracellular Na+ concentration but was enhanced by acidification of the extracellular medium, suggesting that lactate uptake was mediated by a proton cotransport system. The enhanced accumulation of [14C]-lactate seen in medium at pH 6.0 to 6.5 was inhibited by probenecid or by an excess of unlabeled L-lactate. When macrophages were incubated in PBS without glucose for 6 h, intracellular stores of phosphocreatine were 13 nmol/mg of protein, compared with 44 nmol/mg of protein in cells incubated in medium containing glucose. When lactate was substituted for glucose, phosphocreatine stores were 32 nmol/mg of protein. These studies reveal that macrophages take up L-lactate in a pH-dependent manner and that lactate uptake occurs via a probenecid-inhibitable monocarboxylate transporter; they suggest that macrophages can utilize this lactate as an energy source.     

Glass fragility and the stability of pharmaceutical preparations--excipient selection

L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles L-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K(m) of 39.2 +/- 4.8 mM and a Jmax of 8.9 +/- 0.7 nmoles mg protein-1 sec-1. A very small conductive pathway for L-lactate is present in basolateral membranes. In brush border membrane vesicles both Na+ and H+ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H(+)-lactate cotransporter, whereas in the apical membrane both H(+)-lactate and Na(+)-lactate cotransporters are present, even if they exhibit a low transport rate.     

Distinct regions of frequent loss of heterozygosity of chromosome 5p and 5q in human esophageal cancer

Twenty strains of Streptococcus bovis grew more slowly on lactose (1.21 +/- 0.12 h-1) then than on glucose (1.67 +/- 0.12 h-1), and repeated transfers or prolonged growth in continuous culture (more than 200 generations each) did not enhance the growth rate on lactose. Lactose transport activity was poorly correlated with growth rate, and slow growth could not be explained by the ATP production rate (catabolic rate). Batch cultures growing on lactose always had less intracellular fructose 1,6-bisphosphate (Frul,6P2) than cells growing on glucose (6.6 mM compared to 16.7 mM), and this difference could be explained by the pathway of carbon metabolism. Glucose and the glucose moiety of lactose were metabolized by the Embden-Meyerhoff-Parnas (EMP) pathway, but the galactose moiety of lactose was catabolized by the tagatose pathway, a scheme that by-passed Frul,6P2. A mutant capable of co-metabolizing lactose and glucose grew more rapidly when glucose was added, even though the total rate of hexose fermentation did not change. Wild-type S. bovis grew rapidly with galactose and melibiose, but these galactose-containing sugars were activated by galactokinase and catabolized via EMP. On the basis of these results, rapid glycolytic flux through the EMP pathway is needed for the rapid growth (more than 1.2 h-1) of S. bovis.     

Energy-dependent uptake of calcium by the yeast Schizosaccharomyces pombe

1. In resting cells of the fission yeast Schizosaccharomyces pombe, the uptake of calcium is stimulated by the addition of 90 mM glucose in the presence as in the absence of respiration and inhibited by Antimycin A in the absence of exogenous carbon source. This uptake therefore requires fermentative or respiratory metabolic energy. 2. The calcium uptake by S. pombe exhibits saturation kinetics and high affinity for calcium. At external pH 4.5, the apparent Km is 45 muM ca2+ 400 muM of other divalent cations exert competitive inhibitions of calcium uptake in the following order of affinities: Sr2+ greater than Mn2+ greater than Co2+ greater than Mg2+. Inhibition by KCl is also observed but is of non-competitive type and requires high concentrations of the order of 40 mM. 3. At 30 degrees C, the uptake rate of calcium is about 10-times higher at pH 8925 than at pH 4.0. An extrusion of 45Ca2+, the rate of which is estimated to be lower than one-fifth of the uptake, is observed in the presence of glucose when the external pH is acid. 4. At external pH 4.5, low concentrations of lanthanum chloride, ruthenium red and hexamine cobaltichloride are inhibitory for the uptake of calcium by the yeast cells. 5. In presence of Antimycin A, the uncouplers: NaN3, dinitrophenol, and concentrations of crobonylcyanide m-chlorophenylhydrazone higher than 80 muM inhibit the calcium uptake by glycolysing cells. In the presence of glucose, the K+ ionophore Dio-9 dnhances severalfold the uptake of calcium even at 2 degrees C. 6. It is concluded that S. pombe possess an active transport system for low concentrations of calcium. This transport seems to be dependent on an electric potential (negative inside) across the cellular membrane.     

Role of sodium and potassium ions in regulation of glucose metabolism in cultured astroglia

Effects of increasing extracellular K+ or intracellular Na+ concentrations on glucose metabolism in cultures of rat astroglia and neurons were examined. Cells were incubated in bicarbonate buffer, pH 7.2, containing 2 mM glucose, tracer amounts of [14C]deoxyglucose ([14C]dGlc), and 5.4, 28, or 56 mM KCl for 10, 15, or 30 min, and then for 5 min in [14C]dGlc-free buffer to allow efflux of unmetabolized [14C]dGlc. Cells were then digested and assayed for labeled products, which were shown to consist of 96-98% [14C]deoxyglucose 6-phosphate. Increased K+ concentrations significantly raised [14C]deoxyglucose 6-phosphate accumulation in both neuronal and mixed neuronal-astroglial cultures at 15 and 30 min but did not raise it in astroglial cultures. Veratridine (75 microM), which opens voltage-dependent Na+ channels, significantly raised rates of [14C]dGlc phosphorylation in astroglial cultures (+20%), and these elevations were blocked by either 1 mM ouabain, a specific inhibitor of Na+,K(+)-ATPase (EC 3.6.1.37), or 10 microM tetrodotoxin, which blocks Na+ channels. The carboxylic sodium ionophore, monensin (10 microM), more than doubled [14C]dGlc phosphorylation; this effect was only partially blocked by ouabain and unaffected by tetrodotoxin. L-Glutamate (500 microM) also stimulated [14C]dGlc phosphorylation in astroglia--not through N-methyl-D-aspartate or non-N-methyl-D-aspartate receptor mechanisms but via a Na(+)-dependent glutamate-uptake system. These results indicate that increased uptake of Na+ can stimulate energy metabolism in astroglial cells.     

Impact of Initial pH and Linoleic Acid (C18:2) on Hydrogen Production by a Mesophilic Anaerobic Mixed Culture

Hydrogen production from glucose using a mixed anaerobic culture was assessed under batch conditions by adjusting the initial pH and adding linoleic acid (LA). At an initial pH of 5.0, hydrogen (1.9?mol?H2?mol?1 glucose) was detected after the first glucose injection in the controls (no LA added). At the latter initial pH and in cultures fed 2,000?mg?l?1 LA, the yield reached a maximum of 2.4?mol?H2?mol?1 glucose when glucose was injected again. Hydrogen was detected after the second glucose injection in cultures with the initial pH adjusted to 5.0 and 6.0. In cultures receiving LA and adjusted to pH 7.6, the hydrogen yield reached 1.4?mol?H2?mol?1 glucose when glucose was injected again. Acetate, propionate, and butyrate were detected under all conditions; however, the quantities were variable and dependent on the conditions examined. In comparison to the amount of volatile fatty acids produced, relatively low quantities of alcohols (ethanol, i-propanol, n-butanol, and i-butanol) were detected during the initial lag phase of 96?to?120?h.     

Effects of organic acid and monensin treatment on in vitro mixed ruminal microorganism fermentation of cracked corn

The objective of this study was to determine the effects of organic acids and monensin on the in vitro fermentation of cracked corn by mixed ruminal microorganisms. Ruminal fluid was collected from a steer fed 36.3 kg of wheat silage and 4.5 kg of concentrate supplement once daily. Mixed ruminal microorganisms were incubated in anaerobic media that contained 20% (vol/vol) ruminal fluid and .4 g of cracked corn. Incubations were carried out in batch culture for 24 h at 39 degrees C. Organic acids (L-aspartate, fumarate, and DL-malate) were added to serum bottles (n = 4) to achieve final concentrations of 0, 4, 8, or 12 mM. Monensin, dissolved in ethanol, was included in serum bottles at a final concentration of 0 or 5 ppm of culture fluid. The addition of 8 and 12 mM organic acids to cracked corn fermentations increased final pH (P < .05), tended to increase total gas production and CO2 concentration, and decreased the acetate:propionate ratio (P < .05). Organic acids tended to decrease methane concentrations and hydrogen concentration was not altered. DL-Malate addition at all levels reduced (P < .05) lactate accumulation. Additive effects of monensin and organic acids were observed in some fermentations. In conclusion, organic acid addition to in vitro mixed ruminal microorganism fermentations yielded beneficial results independent of monensin treatment by decreasing the acetate: propionate ratio and increasing final pH.     

Cell proliferation and ploidy of human solid tumours: methodological experience with in vivo bromodeoxyuridine and DNA flow cytometry

The divalent cation selective ionophores A23187 and ionomycin were compared for their effects on the Ca2+ contents, nucleotide contents, and protein synthetic rates of several types of cultured cells. Both ionophores reduced amino acid incorporation by approximately 85% at low concentrations (50-300 nmol/L) in cultured mammalian cells without reducing ATP or GTP contents. At these concentrations A23187 and ionomycin each promoted substantial Ca2+ efflux, whereas at higher concentrations a large influx of the cation was observed. Ca2+ influx occurred at lower ionophore concentrations and to greater extents in C6 glioma and P3X63Ag8 myeloma than in GH3 pituitary cells. The ATP and GTP contents of the cells and their ability to adhere to growth surfaces declined sharply at ionophore concentrations producing increased Ca2+ influx. Prominent reductions of nucleotide contents occurred in EGTA-containing media that were further accentuated by extracellular Ca2+. Ionomycin produced more Ca2+ influx and nucleotide decline than comparable concentrations of A23187. The inhibition of amino acid incorporation and mobilization of cell-associated Ca2+ by ionomycin were readily reversed in GH3 cells by fatty acid-free bovine serum albumin, whereas the effects of A23187 were only partially reversed. Amino acid incorporation was further suppressed by ionophore concentrations depleting nucleotide contents. Mitochondrial uncouplers potentiated Ca2+ accumulation in response to both ionophores. At cytotoxic concentrations Lubrol PX abolished protein synthesis but did not cause Ca2+ influx. Nucleotide depletion at high ionophore concentrations is proposed to result from increased plasmalemmal Ca2+-ATPase activity and dissipation of mitochondrial proton gradients and to cause intracellular Ca2+ accumulation. Increased Ca2+ contents in response to Ca2+ ionophores are proposed as an indicator of ionophore-induced cytotoxicity.     

Effects of glucose deprivation, chemical hypoxia, and simulated ischemia on Na+ homeostasis in rat spinal cord astrocytes

A steep inwardly directed Na+ gradient is essential for glial functions such as glutamate reuptake and regulation of intracellular ion concentrations. We investigated the effects of glucose deprivation, chemical hypoxia, and simulated ischemia on intracellular Na+ concentration ([Na+]i) in cultured spinal cord astrocytes using fluorescence ratio imaging with sodium-binding benzofuran isophthalate (SBFI) AM. Glucose removal or chemical hypoxia (induced by 10 mM NaN3) for 60 min increased [Na+]i from a baseline of 8.3 to 11 mM. Combined glycolytic and respiratory blockage by NaN3 and 0 glucose saline caused [Na+]i to increase by 20 mM, similar to the [Na+]i increases elicited by blocking the Na+/K+-ATPase with ouabain. Recovery from large [Na+]i increases (>15 mM) induced by the glutamatergic agonist kainate was attenuated during glucose deprivation or NaN3 application and was blocked in NaN3 and 0 glucose. To mimic in vivo ischemia, we exposed astrocytes to NaN3 and 0 glucose saline containing L-lactate and glutamate with increased [K+] and decreased [Na+], [Ca2+], and pH. This induced an [Na+]i decrease followed by an [Na+]i rise and a further [Na+]i increase after reperfusion with standard saline. Similar multiphasic [Na+]i changes were observed after NaN3 and 0 glucose saline with only reduced [Na+]e. Our results suggest that the ability to maintain a low [Na+]i enables spinal cord astrocytes to continue uptake of K+ and/or glutamate at the onset of energy failure. With prolonged energy failure, however, astrocytic [Na+]i rises; with loss of their steep transmembrane Na+ gradient, astrocytes may aggravate metabolic insults by carrier reversal and release of acid, K+, and/or glutamate into the extracellular space.     

Ionophore-mediated uptake of ciprofloxacin and vincristine into large unilamellar vesicles exhibiting transmembrane ion gradients

A new method, based on the ion-translocating properties of the ionophores nigericin and A23187, is described for loading large unilamellar vesicles (LUVs) with the drugs vincristine and ciprofloxacin. LUVs composed of distearoylphosphatidylcholine/cholesterol (DSPC/Chol) (55:45 mol/mol) or sphingomyelin (SPM)/Chol (55:45 mol/mol) exhibiting a transmembrane salt gradient (for example, internal solution 300 mM MnSO4 or K2SO4; external solution 300 mM sucrose) are incubated in the presence of drug and, for experiments involving divalent cations, the chelator EDTA. The addition of ionophore couples the outward movement of the entrapped cation to the inward movement of protons, thus acidifying the vesicle interior. External drugs that are weak bases can be taken up in response to this induced transmembrane pH gradient. It is shown that both nigericin and A23187 facilitate the rapid uptake of vincristine and ciprofloxacin, with entrapment levels approaching 100% and excellent retention in vitro. Following drug loading, the ionophores can be removed by gel exclusion chromatography, dialysis, or treatment with biobeads. In vitro leakage assays (addition of 50% mouse serum) and in vivo pharmacokinetic studies (in mice) reveal that the A23187/Mn2+ system exhibits superior drug retention over the nigericin/K+ system, and compares favorably with vesicles loaded by the standard DeltapH or amine methods. The unique features of this methodology and possible benefits are discussed.     

Sex specific action of insulin on the glucose metabolism of lymphatic tissue of rats and humans (author's transl)

Glucose metabolism of lymphoid cells isolated from thymus and spleen of Wistar rats, weighing 140-150 g, was found to be sensitive to insulin. The influence of insulin on glucose uptake by isolated cells from both lymphoid tissues displayed a significant sex specificity. Insulin at 0.1-10 nmol/1 stimulated glucose uptake of cells from male rats, whereas in cells from females, inhibition of glucose uptake was observed. However, lactate production was enhanced in lymphoid cells of both sexes. Cortisol (100 nmol/1) displayed a significant anti-insulin action on glucose uptake and lactate release by thymocytes of male rats. In contrast, in cells from females, cortisol and insulin both exhibited an inhibitory action on glucose uptake, whereas the hormones were found to antagonise lactate production. The sex specific stimualtory influence of insulin on glucose uptake by thymocytes of male rats was reversed and the hormone became inhibitory when animals were castrated. The action of insulin on glucose uptake was also shown to be age-dependent. In experiments with rats weighing either about 10 g or 40 g, sex-specific effects on glucose influx were found, that were similar to those of rats weighing 140-150 g. However, stimulation of glucose uptake was found with thymocytes from rats of both sexes weighing about 80 g. Comparable results were obtained with isolated thymocytes from immature humans (2 months-10 years old). Incubation of thymocytes from males with insulin (10 nmol/1) stimulated glucose uptake and lactate release, whereas insulin caused an inhibition of glucose uptake and an enhancement of lactate production in thymocytes from female. Age dependence and sex specificity of insulin action on glucose metabolism in lymphoid cells and tissues may explain the contradictory results reported by other authors.     

Non-insulin- and insulin-mediated glucose uptake in dairy cows

Four mid-lactation Holstein dairy cows (mean milk yield on day of experiments 26.1 kg/d) were used in a series of experiments to establish the contribution of non-insulin-mediated glucose uptake to total glucose uptake at basal insulin concentrations. A secondary objective was to determine whether somatostatin affects the action of infused insulin. In part I of the experiment a primed continuous infusion [6,6-2H]glucose (45.2 micrograms/kg per min) was begun at time 0 and continued for 5 h. After 3 h of [6,6-2H]glucose infusion (basal period) a primed continuous infusion of insulin (0.001 i.u./kg per min) was administered for 2 h. Coincidental with the insulin infusion, normal glucose was also infused in order to maintain the plasma glucose concentration at euglycaemia. Part II of the experiment was the same as part I except that somatostatin was infused for 2 h (0.333 micrograms/kg per min) instead of insulin. In part III of the experiment both insulin and somatostatin were infused for the final 2 h. Plasma insulin levels were increased by insulin infusion (to 0.1476 to 0.1290 i.u./l for parts I and III respectively) and were reduced by somatostatin infusion in part II (to 0.006 i.u./l) relative to the basal periods (mean 0.021 i.u./l). Glucose uptake during somatostatin infusion (2.50 mg/kg per min; part II) was 92.0% of that observed in the respective basal period (2.72 mg/kg per min). Circulating insulin levels were much lower than the dose of insulin that causes a half maximal effect on glucose uptake (0.06-0.10 i.u./l for ruminants); consequently insulin-mediated glucose uptake was probably absent in part II. Secondly, glucose uptake following insulin only infusion (4.05 mg/kg per min) was significantly lower than that observed when insulin plus somatostatin was infused (4.69 mg/kg per min), indicating that somatostatin either directly or indirectly enhanced the action of insulin on glucose uptake.     

Inhibition of membrane lipid peroxidation by a radical scavenging mechanism: a novel function for hydroxyl-containing ionophores

In the present study we show that K+/H+ hydroxyl-containing ionophores lasalocid-A (LAS) and nigericin (NIG) in the nanomolar concentration range, inhibit Fe2+-citrate and 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP)-induced lipid peroxidation in intact rat liver mitochondria and in egg phosphatidylcholine (PC) liposomes containing negatively charged lipids--dicetyl phosphate (DCP) or cardiolipin (CL)--and KCl as the osmotic support. In addition, monensin (MON), a hydroxyl-containing ionophore with higher affinity for Na+ than for K+, promotes a similar effect when NaCl is the osmotic support. The protective effect of the ionophores is not observed when the osmolyte is sucrose. Lipid peroxidation was evidenced by mitochondrial swelling, antimycin A-insensitive O2 consumption, formation of thiobarbituric acid-reactive substances (TBARS), conjugated dienes, and electron paramagnetic resonance (EPR) spectra of an incorporated lipid spin probe. A time-dependent decay of spin label EPR signal is observed as a consequence of lipid peroxidation induced by both inductor systems in liposomes. Nitroxide destruction is inhibited by butylated hydroxytoluene, a known antioxidant, and by the hydroxyl-containing ionophores. In contrast, valinomycin (VAL), which does not possess alcoholic groups, does not display this protective effect. Effective order parameters (Seff), determined from the spectra of an incorporated spin label are larger in the presence of salt and display a small increase upon addition of the ionophores, as a result of the increase of counter ion concentration at the negatively charged bilayer surface. This condition leads to increased formation of the ion-ionophore complex, the membrane binding (uncharged) species. The membrane-incorporated complex is the active species in the lipid peroxidation inhibiting process. Studies in aqueous solution (in the absence of membranes) showed that NIG and LAS, but not VAL, decrease the Fe2+-citrate-induced production of radicals derived from piperazine-based buffers, demonstrating their property as radical scavengers. Both Fe2+-citrate and ABAP promote a much more pronounced decrease of LAS fluorescence in PC/CL liposomes than in dimyristoyl phosphatidylcholine (DMPC, saturated phospholipid)-DCP liposomes, indicating that the ionophore also scavenges lipid peroxyl radicals. A slow decrease of fluorescence is observed in the latter system, for all lipid compositions in sucrose medium, and in the absence of membranes, indicating that the primary radicals stemming from both inductors also attack the ionophore. Altogether, the data lead to the conclusion that the membrane-incorporated cation complexes of NIG, LAS and MON inhibit lipid peroxidation by blocking initiation and propagation reactions in the lipid phase via a free radical scavenging mechanism, very likely due to the presence of alcoholic hydroxyl groups in all three molecules and to the attack of the aromatic moiety of LAS.     

Effect of monensin on milk production and efficiency of dairy cows fed two diets differing in forage to concentrate ratios

Four primiparous Holstein cows were gradually introduced, according to a Latin square design, to four diets obtained from the factorial combination of two forage to concentrate ratios (70:30 and 50:50) and two concentrations of monensin sodium (0 and 300 mg/d per cow). Addition of monensin tended to depress feed intake and milk fat content without affecting milk production and without interactions with forage to concentrate ratios. Ruminal propionate percentage was increased more by the addition of monensin to the low forage diet than by the addition of monensin to the high forage diet. Serum urea and concentrations of nonesterified fatty acids tended to decrease when monensin was added to the high forage diet but did not change when monensin was added to the low forage diet. The results suggested that monensin had moderate positive effects on efficiency of milk production and might have an antiketogenic effect with high forage diets.     

Comparison of three DNA extraction methods on bone and blood stains up to 43 years old and amplification of three different gene sequences

The objective of this study was to determine the effects of cellobiose and monensin on the in vitro fermentation of organic acids (L-aspartate, fumarate, and DL-malate) by mixed ruminal bacteria. Ruminal fluid was collected from a steer fed 36.7 kg of forage and 4.5 kg of concentrate supplement once per day. Ruminal fluid was centrifuged to sediment feed particles and protozoa, and the resulting supernatant, which contained bacteria, was added (33%, vol/vol) to anaerobic media (500 ml). Incubations (n = 2) were performed in batch culture at 39 degrees C and sampled at 0, 2, 4, 6, 8, 12, and 24 h. Organic acids were added to achieve a final concentration of 7.5 mM. Cellobiose was added to obtain a final concentration of 5 mM, and monensin dissolved in ethanol was included at concentrations of 0 or 5 ppm. Addition of cellobiose to organic acid fermentations increased the rate of organic acid utilization by the mixed bacterial population. Total concentrations of volatile fatty acids were increased by the addition of cellobiose to all fermentations. A lag period (< or = 8 h) occurred in fermentations that were treated with monensin before organic acids were utilized. Total concentrations of volatile fatty acids were increased, and the acetate to propionate ratio was decreased, by monensin treatment. When cellobiose and monensin were added together, propionate production and organic acid utilization were increased. Both cellobiose and monensin affected the in vitro fermentation of organic acids by mixed ruminal bacteria by providing a carbon and energy source and by influencing electron disposal.     

The importance of pH in the regulation of ruminal acetate to propionate ratio and methane production in vitro

Grain feeding often causes a decrease in ruminal pH, and experiments were conducted to define the role of pH in regulating the acetate to propionate ratio and production of CH4. Cows that were fed 90% concentrate had lower ruminal pH values (6.22 vs. 6.86), higher VFA concentrations (85 vs. 68 mM), and lower acetate to propionate ratios (2.24 vs. 4.12) than did cows that were fed forage only. When mixed ruminal bacteria from cows that were fed 90% concentrate or 100% forage were incubated (48 h) with hay (10 g/L) or cracked corn (5 g/L) in a medium containing bicarbonate (38 mM) and tricarballylate (50 mM), the final pH values were less than 0.3 units lower than the initial pH. At final pH values less than 5.7, hay fermentation was inhibited, the acetate to propionate ratio and CH4 production declined more than twofold, and the inoculum source was without effect. Small amounts of H2 were detected at pH values less than 5.5. Total VFA production from cracked corn decreased when pH declined, but only if the inoculum was obtained from cows that were fed 90% concentrate. The acetate to propionate ratio of cracked corn incubations declined from 1.2 to 0.6 when final pH was decreased from 6.5 to 5.3, and CH4, as a percentage of total VFA production, also decreased. At pH values less than 5.3, the acetate to propionate ratio of cracked corn increased more than fourfold, and large amounts of H2 could be detected. Over the final pH range of 6.5 to 5.3, CH4 production was highly correlated with acetate to propionate ratio, which was dependent on pH and substrate (CH4 = 0.02 + 0.05 pH; r2 = 0.80). Calculations based on the differences between pH 6.5 and 5.8 indicated that as much as 25% of the decrease in acetate to propionate ratio could be explained by the effect of pH alone.     

Energy transduction in the thermophilic anaerobic bacterium Clostridium fervidus is exclusively coupled to sodium ions

The thermophilic, peptidolytic, anaerobic bacterium Clostridium fervidus is unable to generate a pH gradient in the range of 5.5-8.0, which limits growth of the organism to a narrow pH range (6.3-7.7). A significant membrane potential (delta psi approximately -60 mV) and chemical gradient of Na+ (-Z delta pNa approximately -60 mV) are formed in the presence of metabolizable substrates. Energy-dependent Na+ efflux is inhibited by the Na+/H+ ionophore monensin but is stimulated by uncouplers, suggesting that the Na+ gradient is formed by a primary pumping mechanism rather than by secondary Na+/H+ antiport. This primary sodium pump was found to be an ATPase that has been characterized in inside-out membrane vesicles and in proteoliposomes in which solubilized ATPase was reconstituted. The enzyme is stimulated by Na+, resistant to vanadate, and sensitive to nitrate, which is indicative of an F/V-type Na(+)-ATPase. In the proteoliposomes Na+ accumulation depends on the presence of ATP, is inhibited by the ATPase inhibitor nitrate, and is completely prevented by the ionophore monensin but is stimulated by protonophores and valinomycin. These and previous observations, which indicated that secondary amino acid transport uses solely Na+ as coupling ion, demonstrate that energy transduction at the membrane in C. fervidus is exclusively dependent on a Na+ cycle.     

Distinct regulation of glucose transport by interleukin-3 and oncogenes in a murine bone marrow-derived cell line

Growth factors and oncogenes promote glucose uptake, but the extent to which increased uptake is regulated at the level of glucose transporter function has not been clearly established. In this paper, we show that interleukin-3 (IL-3), a cytokine growth factor, and the transforming oncogenes ras and abl alter the activation state of glucose transporters by distinct mechanisms. Using bone marrow-derived IL-3-dependent 32Dc13 (32D clone 3) cells and 32D cells transformed with ras and abl oncogenes, we demonstrated that IL-3 enhanced [3H]-2-deoxyglucose (2-DOG) uptake in parental 32Dc13 cells by 40-50% at 0.2 mM 2-DOG, and this was associated with a 2.5-fold increase in transporter affinity for glucose (reduced Km). In comparison, ras and abl oncogenes enhanced 2-DOG uptake by 72-112%, associated with a 2-fold greater transporter affinity for glucose. The tyrosine kinase inhibitor genistein reversed the effects of both IL-3 and oncogenes on glucose uptake and reduced transporter affinity for glucose. Likewise, with exponentially growing 32D cells in the presence of IL-3, a protein kinase C inhibitor, staurosporine, and a phosphatidylinositol 3-kinase (PI-3) kinase inhibitor, wortmannin, inhibited 2-DOG uptake and decreased transporter affinity for glucose. In contrast, in oncogene-transformed cells, staurosporine inhibited 2-DOG uptake but failed to decrease transporter affinity for glucose, whereas wortmannin did not affect 2-DOG uptake. Inhibition of protein tyrosine phosphatases with vanadate enhanced 2-DOG uptake and transporter affinity for glucose in parental cells and in ras-transformed cells but had little effect on abl-transformed cells. Consistently, the serine/threonine phosphatase type 2A inhibitor okadaic acid enhanced 2-DOG uptake and transporter affinity for glucose in parental cells but had little effect on ras- or abl-transformed cells. These results demonstrate differences in the regulation of glucose transport in parental and oncogene-transformed 32D cells. Thus, IL-3 responses are dependent upon tyrosine, serine/threonine, and PI-3 kinases, whereas ras and abl effects on glucose transport depend upon tyrosine phosphorylation but are compromised in their dependence upon serine/threonine and PI-3 kinases.     

The effectiveness of intravenous administration of large quantities of glucose in the treatment of bovine ketosis

Glucose in a 40% (w/v) solution (200 g, 400 g or 600 g) was infused intravenously in clinically healthy cattle. On urinalysis, 13%, 19% and 26% respectively of the infused glucose was excreted. In cattle with elevated urinary ketone bodies, only the infusion of 400 or 600 g glucose led to a significant rise in the serum glucose concentration compared to initial values. Only the infusion of 600 g of glucose was followed by a significant decrease in serum beta-hydroxybutyrate 24 hours later compared to initial values. The present results indicate a possibility of improving the results of treatment of ketosis by infusion of high amounts of glucose.