Metabolic flux in cellulose batch and cellulose-fed continuous cultures of Clostridium cellulolyticum in response to acidic environment.
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Clostridium cellulolyticum, a nonruminal cellulolytic mesophilic bacterium, was grown in batch and continuous cultures on cellulose using a chemically defined medium. In batch culture with unregulated pH, less cellulose degradation and higher accumulation of soluble glucides were obtained compared to a culture with the pH controlled at 7.2. The gain in cellulose degradation achieved with pH control was offset by catabolite production rather than soluble sugar accumulation. The pH-controlled condition improved biomass, ethanol and acetate production, whereas maximum lactate and extracellular pyruvate concentrations were lower than in the non-pH-controlled condition. In a cellulose-fed chemostat at constant dilution rate and pH values ranging from 7.4 to 6.2, maximum cell density was obtained at pH 7.0. Environmental acidification chiefly influenced biomass formation, since at pH 6.4 the dry weight of cells was more than fourfold lower compared to that at pH 7.0, whereas the specific rate of cellulose assimilation decreased only from 11.74 to 10.13 milliequivalents of carbon (g cells)(-1) h(-1). The molar growth yield and the energetic growth yield did not decline as pH was lowered, and an abrupt transition to washout was observed. Decreasing the pH induced a shift from an acetate-ethanol fermentation to a lactate-ethanol fermentation. The acetate/ethanol ratio decreased as the pH declined, reaching close to 1 at pH 6.4. Whatever the pH conditions, lactate dehydrogenase was always greatly in excess. As pH decreased, both the biosynthesis and the catabolic efficiency of the pyruvate-ferredoxin oxidoreductase declined, as indicated by the ratio of the specific enzyme activity to the specific metabolic rate, which fell from 9.8 to 1.8. Thus a change of only 1 pH unit induced considerable metabolic change and ended by washout at around pH 6.2. C. cellulolyticum appeared to be similar to rumen cellulolytic bacteria in its sensitivity to acidic conditions. Apparently, the cellulolytic anaerobes studied thus far do not thrive when the pH drops below 6.0, suggesting that they evolved in environments where acid tolerance was not required for successful competition with other microbes. (+info)
Issues in cDNA microarray analysis: quality filtering, channel normalization, models of variations and assessment of gene effects.
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We consider the problem of comparing the gene expression levels of cells grown under two different conditions using cDNA microarray data. We use a quality index, computed from duplicate spots on the same slide, to filter out outlying spots, poor quality genes and problematical slides. We also perform calibration experiments to show that normalization between fluorescent labels is needed and that the normalization is slide dependent and non-linear. A rank invariant method is suggested to select non-differentially expressed genes and to construct normalization curves in comparative experiments. After normalization the residuals from the calibration data are used to provide prior information on variance components in the analysis of comparative experiments. Based on a hierarchical model that incorporates several levels of variations, a method for assessing the significance of gene effects in comparative experiments is presented. The analysis is demonstrated via two groups of experiments with 125 and 4129 genes, respectively, in Escherichia coli grown in glucose and acetate. (+info)
Acetic acid feeding enhances glycogen repletion in liver and skeletal muscle of rats.
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To investigate the efficacy of the ingestion of vinegar in aiding recovery from fatigue, we examined the effect of dietary acetic acid, the main component of vinegar, on glycogen repletion in rats. Rats were allowed access to a commercial diet twice daily for 6 d. After 15 h of food deprivation, they were either killed immediately or given 2 g of a diet containing 0 (control), 0.1, 0.2 or 0.4 g acetic acid/100 g diet for 2 h. The 0.2 g acetic acid group had significantly greater liver and gastrocnemius muscle glycogen concentration than the control group (P < 0.05). The concentrations of citrate in this group in both the liver and skeletal muscles were >1.3-fold greater than in the control group (P > 0.1). In liver, the concentration of xylulose-5-phosphate in the control group was significantly higher than in the 0.2 and 0.4 g acetic acid groups (P < 0.01). In gastrocnemius muscle, the concentration of glucose-6-phosphate in the control group was significantly lower and the ratio of fructose-1,6-bisphosphate/fructose-6-phosphate was significantly higher than in the 0.2 g acetic acid group (P < 0.05). This ratio in the soleus muscle of the acetic acid fed groups was <0.8-fold that of the control group (P > 0.1). In liver, acetic acid may activate gluconeogenesis and inactivate glycolysis through inactivation of fructose-2,6-bisphosphate synthesis due to suppression of xylulose-5-phosphate accumulation. In skeletal muscle, acetic acid may inhibit glycolysis by suppression of phosphofructokinase-1 activity. We conclude that a diet containing acetic acid may enhance glycogen repletion in liver and skeletal muscle. (+info)
Agonist-induced internalization and mitogen-activated protein kinase activation of the human prostaglandin EP4 receptor.
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We examined the pathway of prostaglandin E(2) (PGE(2))-induced internalization of the prostaglandin EP4 receptor in HEK 293 cells. Co-expression of dominant negative beta-arrestin (319-418) or dynamin I (K44A) with the EP4 receptor reduced internalization. The activated receptor co-localized with GFP-arrestin 2 and GFP-arrestin 3, confirming the requirement for beta-arrestins in internalization. Inhibition of clathrin-coated vesicle-mediated internalization resulted in inhibition of sequestration, whereas inhibition of caveola-mediated internalization had no effect. PGE(2) stimulation of the EP4 receptor resulted in rapid mitogen-activated protein (MAP) kinase activation. Examination of an internalization-resistant mutant and co-expression of mutant accessory proteins with EP4 revealed that MAP kinase activation proceeds independently of internalization. (+info)
Perception of trigeminal chemosensory qualities in the elderly.
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One hundred healthy elderly subjects (65-88 years) were tested for their ability to: (i) assign verbal labels from a list of trigeminal type descriptors to six odorants known to have a strong trigeminal component; (ii) discriminate between intensity-matched pairs of these odorants in an odd-ball paradigm. Their performance was compared with that of 100 young controls (23--36 years). Young controls judged menthol and cineole as distinctly cool and fresh, acetic cid as pungent and sour and acetone as pungent, but showed no clear descriptive profile for ethanol and propanol. The descriptive profiles given by the elderly subjects correlated significantly with those given by the young controls for all six odorants and thus indicate a high degree of conformity in trigeminal perception of chemosensory qualities between the two age groups. In the odd-ball test the young controls correctly discriminated an average of 8.0 of 9 stimulus pairs presented, with most mistakes occurring in response to pairs with a similar trigeminal profile. With an average of 6.4 of 9 items correct, the discrimination performance of the elderly subjects was significantly poorer than that of the young controls but nevertheless significantly above chance at the group level with all 9 stimulus pairs. These results suggest that the nasal trigeminal system may experience some degree of age-related impairment but still contributes considerably to the perception and discrimination of chemosensory qualities in the elderly. (+info)
[1-(11)C]Acetate as a quantitative perfusion tracer in myocardial PET.
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Our objective was to investigate the properties of [1-(11)C]acetate as a quantitative perfusion tracer for myocardial PET studies. METHODS: We determined the flow dependence of the effective acetate extraction by a comparison with [(13)N]ammonia in 24 patients at rest (n = 8) and under pharmacologic vasodilation (n = 16). Furthermore, we compared the statistical quality of the perfusion values derived with both tracers. Quantification was based on an irreversible 2-compartment model for [(13)N]ammonia and a reversible 1-compartment model for [1-(11)C]acetate. Area-conserving polar maps were used to determine the correlation between the unidirectional uptake parameters of both tracers on a pixel-by-pixel basis for the whole left ventricular myocardium. RESULTS: A fit of a generalized Renkin-Crone formula to the data yielded the unidirectional acetate extraction fraction E(f) = 1 - 0.64e(-1.20/f). An extraction correction based on this formula led to good quantitative agreement of perfusion values derived with [(13)N]ammonia and [1-(11)C]acetate over the whole observed flow range (average difference of flow values, 3%; correlation coefficient, 0.96). This agreement proved the applicability of acetate as a quantitative perfusion tracer even under stress conditions. An analysis of the statistical properties of the parameter estimates showed, moreover, that statistical errors were reduced by a factor of nearly 2 in comparison with ammonia. CONCLUSION: [1-(11)C]acetate allows accurate quantification of myocardial perfusion with PET at rest as well as under stress conditions. The use of acetate leads to distinctly improved statistical accuracy for the perfusion estimates in comparison with ammonia. This accuracy facilitates the generation of reliable parametric polar maps, which are especially useful for clinical application of myocardial perfusion quantification. (+info)
Preferential labeling of glial and meningial brain tumors with [2-(14)C]acetate.
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Acetate is preferentially transported into and metabolized by astrocytes, rather than synaptosomes or neurons, and labeled acetate is used as a glial reporter molecule to assess glial metabolism and glial-neuronal interactions. Because monocarboxylic acid transporter specificity might confer a phenotype to help localize, detect, and characterize brain tumors of glial origin, use of [2-(14)C]acetate and [(14)C]deoxyglucose (a glucose analog metabolized by all brain cells) was compared in rat and human brain tumors. METHODS: Cultured C6 glioma or U-373 glioblastoma/astrocytoma tumor cells were injected into the caudate nucleus of anesthetized CDF Fisher rats; 2--3 wk later, an intravenous pulse of [2-(14)C]acetate or [(14)C]deoxyglucose was given, and timed blood samples were drawn during the 5- or 45-min experiment, respectively. Local (14)C levels in the brain were assayed by quantitative autoradiography, and acetate uptake or glucose use was calculated. Uptake and metabolism of the [(14)C]acetate was also assayed in C6 glioma and human surgical tumor samples in vitro. RESULTS: [(14)C]Acetate uptake into rat brain C6 tumors was 9.9 +/- 2.1 mL/100 g/min, compared with 3.9 +/- 1.0 mL/100 g/min in contralateral tissue (n = 6; P < 0.001), and was much higher than that into other brain structures (e.g., 5:1 for white matter and 2:1 for cortical gray matter). Glucose use in C6 tumors was 111 +/- 34 micromol/100 g/min, versus 81 +/- 5 micromol/100 g/min in contralateral tissue (n = 6; P = 0.08); no left-right differences in glucose use or acetate uptake were seen in other brain structures. The tumor-to-contralateral-tissue ratio for acetate (2.3 +/- 0.3) exceeded that for deoxyglucose (1.4 +/- 0.5) (P < 0.05), indicating that acetate is a sensitive C6 glioma marker. [(14)C]Acetate uptake also demarcated a few 3-wk-old C6 tumors that had unlabeled necrotic cores. U-373 tumors were smaller than C6 tumors in rat brain and were detected equally well with [(14)C]acetate and [(14)C]deoxyglucose. In vitro uptake of [(14)C]acetate into human glioblastoma or meningioma tumors was higher than uptake into pituitary adenoma. Rat C6 and human tumors with high uptake metabolized acetate to acidic compounds and amino acids. CONCLUSION: Tumor imaging with radiolabeled acetate can help to localize and classify brain tumors. Transporter and metabolic substrate specificity are traits that can be exploited further for in vivo imaging of brain glial tumors. (+info)
Tachykinin NK(2) receptors and enhancement of cholinergic transmission in the inflamed rat colon: an in vivo motility study.
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In the gastrointestinal tract, tachykinin NK(2) receptors are localized both on smooth muscle and nerve fibres. NK(2) receptor antagonists reduce exaggerated intestinal motility in various diarrhoea models but the site of action contributing to this effect is unknown. In this study we investigated the effects of atropine (1.4 micromol kg(-1), i.v.), hexamethonium (13.5 micromol kg(-1), i.v.), and nepadutant (0.1 micromol kg(-1), i.v.), a selective tachykinin NK(2) receptor antagonist, on distension (0.5 and 1 ml)-, or irritation (acetic acid, 0.5 ml of 7.5% v v(-1))-induced motility in the rat distal colon in vivo. The effects of atropine, hexamethonium or N(omega)-nitro-L-argininemethylester (L-NAME, 1.85 micromol kg(-1), i.v.) on [betaAla(8)]NKA(4-10) (10 nmol kg(-1), i.v.)-induced colonic contractions were also investigated. When the colonic balloon was filled with a subthreshold volume (0.5 ml), the intraluminal instillation of acetic acid triggered a high-amplitude phasic colonic motility which was partially reduced by nepadutant and suppressed by either hexamethonium or atropine. Filling of the balloon with 1 ml evoked reflex (hexamethonium-sensitive), atropine-sensitive phasic colonic motility: nepadutant had no significant effect on the distension-evoked motility. Neither hexamethonium nor atropine significantly reduced [betaAla(8)]NKA(4-10)-induced colonic contractions, whereas nepadutant suppressed them. Following L-NAME pretreatment, [betaAla(8)]NKA(4-10)-induced colonic contractions were inhibited by both atropine and hexamethonium. In hexamethonium-pretreated animals, an atropine-sensitive component of [betaAla(8)]NKA(4-10)-induced colonic contractions was also evident. These results indicate that the application of irritants onto the colonic mucosa induces the release of endogenous tachykinins which enhance excitatory cholinergic mechanisms through the stimulation of NK(2) receptors. (+info)