Effect of diet composition on coenzyme A and its thioester pools in various rat tissues

Three coenzyme A (CoA) molecular species, i.e., acetyl-CoA, malonyl-CoA, and nonesterified CoA (CoASH), in 13 types of fasted rat tissue were analyzed. A relatively larger pool size of total CoA, consisting of acetyl-CoA, malonyl-CoA, and CoASH, was observed in the medulla oblongata, liver, heart, and brown adipose tissue. Focusing on changes in the CoA pool size in response to the nutrient composition of the diet given, total CoA pools in rats continuously fed a high-fat diet for 4 weeks were significantly higher in the hypothalamus, cerebellum, and kidney, and significantly lower in the liver and skeletal muscle than those of rats fed a high-carbohydrate or high-protein diet. In particular, reductions in the liver were remarkable and were caused by decreased CoASH levels. Consequently, the total CoA pool size was reduced by approximately one-fifth of the hepatic contents of rats fed the other diets. In the hypothalamus, which monitors energy balance, all three CoA molecular species measured were at higher levels when rats were fed the high-fat diet. Thus, it was of interest that feeding rats a high-fat diet affected the behaviors of CoA pools in the hypothalamus, liver, and skeletal muscle, suggesting a significant relationship between CoA pools, especially malonyl-CoA and/or CoASH pools, and lipid metabolism in vivo.     

Steady-state concentrations of coenzyme A, acetyl-coenzyme A and long-chain fatty acyl-coenzyme A in rat-liver mitochondria oxidizing palmitate

1. Fluorimetric assays are described for CoASH, acetyl-CoA and long-chain fatty acyl-CoA, and are sensitive to at least 50mumumoles of each. 2. Application of these assays to rat-liver mitochondria oxidizing palmitate in the absence and presence of carnitine indicated two pools of intramitochondrial CoA. One pool could be acylated by palmitate and ATP, and the other pool acylated by palmitate with ATP and carnitine, or by palmitoylcarnitine alone. 3. The intramitochondrial content of acetyl-CoA is increased by the oxidation of palmitate both in the absence and presence of l-malate. 4. The conversion of palmitoyl-CoA into acetyl-CoA by beta-oxidation takes place without detectable accumulation of acyl-CoA intermediates.     

Regulation of pantothenate kinase by coenzyme A and its thioesters

Pantothenate kinase catalyzes the rate-controlling step in the coenzyme A (CoA) biosynthetic pathway, and its activity is modulated by the size of the CoA pool. The effect of nonesterified CoA (CoASH) and CoA thioesters on the activity of pantothenate kinase was examined to determine which component of the CoA pool is the most effective regulator of the enzyme from Escherichia coli. CoASH was five times more potent than acetyl-CoA or other CoA thioesters as an inhibitor of pantothenate kinase activity in vitro. Inhibition by CoA thioesters was not due to their hydrolysis to CoASH. CoASH inhibition was competitive with respect to ATP, thus providing a mechanism to coordinate CoA production with the energy state of the cell. There were considerable differences in the size and composition of the CoA pool in cells grown on different carbon sources, and a carbon source shift experiment was used to test the inhibitory effect of the different CoA species in vivo. A shift from glucose to acetate as the carbon source resulted in an increase in the CoASH:acetyl-CoA ratio from 0.7 to 4.3. The alteration in the CoA pool composition was associated with the selective inhibition of pantothenate phosphorylation, consistent with CoASH being a more potent regulator of pantothenate kinase activity in vivo. These results demonstrate that CoA biosynthesis is regulated through feedback inhibition of pantothenate kinase primarily by the concentration of CoASH and secondarily by the size of the CoA thioester pool.     

A role for malonyl-CoA in glucose-stimulated insulin secretion from clonal pancreatic beta-cells

To gain insight into the relationship between acyl coenzyme A (CoA) esters and glucose-induced insulin release, acyl-CoA profiles were determined in clonal pancreatic beta-cells (HIT). A high sensitivity high performance liquid chromatography method was used to measure malonyl, succinyl, beta-hydroxy beta-methylglutaryl and acetyl-CoA esters and free CoASH. Malonyl-CoA content increased more than 3-fold following exposure of HIT cells to 10 mM glucose. The rise in malonyl-CoA, which preceded insulin secretion, was evident 2 min after exposure to glucose and was sustained for at least 30 min. The increase in malonyl-CoA was associated with inhibition of fatty acid oxidation, increased de novo lipid synthesis and a rise in diacylglycerol content. Succinyl-CoA levels, which may reflect anaplerotic influx into the citric acid cycle, were elevated in the presence of glucose. The concentration of acetyl-CoA and the ratio of free CoASH to acetyl-CoA was unchanged. The data are consistent with a metabolic model in which malonyl-CoA mediates the switch from fatty acid catabolism to lipid synthesis during glucose stimulation of beta-cells. We suggest that these changes in lipid metabolism, by leading to increased diacylglycerol synthesis or protein acylation could play a pivotal role in the regulation of the sustained phase of insulin secretion.     

Putative anaerobic activity in aerated composts

It has been suggested that anaerobic microenvironments develop in aerobic composts, regardless of the aeration system used, and that anaerobic activity is responsible for odor generation and nitrogen losses. This study was designed to measure levels of microorganisms capable of anaerobic growth in two aerated composts: municipal solid waste, a relatively nutrient-rich compost, and pulp and paper-mill solid waste, which is relatively nutrient-poor. Anaerobic microorganisms were isolated from both composts at mesophilic and thermophilic temperatures. The majority of the anaerobic mesophiles were facultative anaerobes, whereas facultative, anaerobic thermophiles varied from 0 to 100%. Serially-diluted samples were spot-plated onto various media to preserve microbial consortia. Levels of aerobic and anaerobic exoenzyme production on spot-plates were similar on cell-wall, starch, and casein media. Although microbial levels on spread plates indicate that aerobes are present in much higher numbers than anaerobes (in 47 of 56 subsamples, 90% of the population were aerobes), microbial growth levels and exoenzyme production on spot-plates indicate that anaerobes may be responsible for a large portion (greater than or equal to 72%) of the metabolic activity in anaerobic microenvironments of aerobic composts.     

The aerobic and anaerobic microbiology of pustular acne lesions

Specimens from 32 pustular acne lesions that were inoculated on media supportive for the growth of aerobic and anaerobic bacteria showed bacterial growth. Only aerobic or facultative bacteria were recovered in 15 (47%) specimens, only anaerobic bacteria in 11 (34%) specimens, and mixed aerobic and anaerobic bacteria in 6 (18%) specimens. A total of 57 isolates, 31 anaerobes (1.0 per specimen) and 26 aerobes (0.8 per specimen) were recovered. The predominant isolates were Staphylococcus sp. (19 isolates), Peptostreptococcus sp. (15), and Propionibacterium sp. (10). Twelve (37.5%) of the comedones yielded only one organism. This retrospective study highlighted the polymicrobial nature of over two-thirds of culture positive pustular acne lesions and suggests the potential for pathogenic role of aerobic and anaerobic organisms other than P. acnes and Staphylococcus sp. in acne vulgaris.     

Growth of a facultative anaerobe under oxygen-limiting conditions in pure culture and in co-culture with a sulfate-reducing bacterium

Abstract The occurrence and properties were studied of glucose-metabolizing bacteria present in the anaerobic sediment 5–10 cm below the surface of an estuarine tidal mud-flat. Of all these bacteria (10⁴– 10⁵ per g wet sediment) 80–90% were facultatively anaerobic species. Chemostat enrichments on glucose under aerobic, oxygen-limited and alternately aerobic-anaerobic conditions also yielded cultures dominated by facultative anaerobes. One of the dominant species, tentatively identified as a Vibrio sp., was studied in more detail under oxygen-limiting conditions. Fermentative and respiratory metabolisms were found to operate simultaneously, and the ratio between the two was regulated by the extent of oxygen limitation. A small fraction of the acetate formed under such growth conditions was shown to be subsequently respired. A co-culture was established of the Vibrio sp. and a sulfate-reducing bacterium ( Desulfovibrio HL21 ) in an aerated chemostat. The importance of these observations is discussed in relation to the role of facultative anaerobes in anaerobic habitats.     

Relationship between acid-soluble carnitine and coenzyme A pools in vivo

The relationship between the acid-soluble carnitine and coenzyme A pools was studied in fed and 24-h-starved rats after carnitine administration. Carnitine given by intravenous injection at a dose of 60μmol/100g body wt. was integrated into the animal's endogenous carnitine pool. Large amounts of acylcarnitines appeared in the plasma and liver within 5min of carnitine injection. Differences in acid-soluble acylcarnitine concentrations were observed between fed and starved rats after injection and reflected the acylcarnitine/carnitine relationship seen in the endogenous carnitine pool of the two metabolic states. Thus, a larger acylcarnitine production was seen in starved animals and indicated a greater source of accessible acyl-CoA molecules. In addition to changes in the amount of acylcarnitines present, the specific acyl groups present also varied between groups of animals. Acetylcarnitine made up 37 and 53% of liver acid-soluble acylcarnitines in uninjected fed and starved animals respectively. At 5min after carnitine injection hepatic acid-soluble acylcarnitines were 41 and 73% in the form of acetylcarnitine in fed and starved rats respectively. Despite these large changes in carnitine and acylcarnitines, no changes were observed in plasma non-esterified fatty acid or β-hydroxybutyrate concentrations in either fed or starved rats. Additionally, measurement of acetyl-CoA, coenzyme A, total acid-soluble CoA and acid-insoluble CoA demonstrated that the hepatic CoA pool was resistant to carnitine-induced changes. This lack of change in the hepatic CoA pool or ketone-body production while acyl groups are shunted from acyl-CoA molecules to acylcarnitines suggests a low flux through the carnitine pool compared with the CoA pool. These results support the concept that the carnitine/acid-soluble acylcarnitine pool reflects changes in, rather than inducing changes in, the hepatic CoA/acyl-CoA pool.     

Separation and measurement of short-chain coenzyme-A compounds in rat liver by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed to measure short-chain CoA compounds in freeze-clamped liver. Seventeen CoA compounds can be quantitated in 37 min using a 3-micron octadecylsilica column (4.6 mm X 7.5 cm). The chromatographic separation of CoA compounds is conducted with a gradient system of sodium phosphate and acetonitrile. The large amount of uv-absorbing, non-CoA material present in liver extracts is eluted earlier than the CoA compounds when the phosphate concentration is 0.2 M. The CoA compounds that can be resolved by this method include acetoacetyl-CoA, acetyl-CoA, butyryl-CoA, CoASH, crotonyl-CoA, dephospho-CoA, glutathione-CoA, 3-hydroxy-3-methylglutaryl-CoA, isobutyryl-CoA, isovaleryl-CoA, malonyl-CoA, 3-methylcrotonyl-CoA, methylmalonyl-CoA, oxidized-CoA, propionyl CoA, succinyl-CoA, and valeryl-CoA. Comparisons at pH 3 and 6 showed that the stability of the CoA compounds is much greater when perchloric acid extracts of rat liver are adjusted to pH 3. Recovery of CoA standards added in tissue extracts ranged from 83 to 107%. The method is linear over the range of 12 to 700 pmol, and this sensitivity allows acetyl-CoA content to be determined in extracts of as little as 0.1 mg of liver. The values for CoA compounds obtained for freeze-clamped liver from starved rats include (units are nmol/g wet weight +/- SE) malonyl-CoA, 1.50 +/- 0.14; glutathione-CoA, 6.57 +/- 1.72; CoASH, 56.06 +/- 2.90; methylmalonyl-CoA, 4.60 +/- 1.27; succinyl-CoA, 13.52 +/- 0.76; 3-hydroxy-3-methylglutaryl-CoA, 7.06 +/- 0.89; and acetyl-CoA, 100.5 +/- 6.4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of plant Fatty Acid biosynthesis : analysis of acyl-coenzyme a and acyl-acyl carrier protein substrate pools in spinach and pea chloroplasts

In previous work (D. Post-Beittenmiller, J.G. Jaworski, J.B. Ohlrogge [1991] J Biol Chem 266: 1858-1865), the in vivo acyl-acyl carrier protein (ACP) pools were measured in spinach (Spinacia oleracea) leaves and changes in their levels were compared to changes in the rates of fatty acid biosynthesis. To further examine the pools of substrates and cofactors for fatty acid biosynthesis and to evaluate metabolic regulation of this pathway, we have now examined the coenzyme A (CoA) and short chain acyl-CoA pools, including acetyl- and malonyl-CoA, in isolated spinach and pea (Pisum sativum) chloroplasts. In addition, the relationships of the acetyl- and malonyl-CoA pools to the acetyl- and malonyl-ACP pools have been evaluated. These studies have led to the following conclusions: (a) Essentially all of the CoA (31-54 μm) in chloroplasts freshly isolated from light-grown spinach leaves or pea seedling was in the form of acetyl-CoA. (b) Chloroplasts contain at least 77% of the total leaf acetyl-CoA, based on comparison of acetyl-CoA levels in chloroplasts and total leaf. (c) CoA-SH was not detected either in freshly isolated chloroplasts or in incubated chloroplasts and is, therefore, less than 2 μm in the stroma. (d) The malonyl-CoA:ACP transacylase reaction is near equilibrium in both light- and dark-incubated chloroplasts, whereas the acetyl-CoA:ACP transacylase reaction is far from equilibrium in light-incubated chloroplasts. However, the acetyl-CoA:ACP transacylase reaction comes nearer to equilibrium when chloroplasts are incubated in the dark. (e) Malonyl-CoA and -ACP could be detected in isolated chloroplasts only during light incubations, and increased with increased rates of fatty acid biosynthesis. In contrast, both acetyl-CoA and acetyl-ACP were detectable in the absence of fatty acid biosynthesis, and acetyl-ACP decreased with increased rates of fatty acid biosynthesis. Together these data have provided direct in situ evidence that acetyl-CoA carboxylase plays a regulatory role in chloroplast fatty acid biosynthesis.     

感染根管的厌氧菌培养结果分析

从64只感染根管中的58只根管分离到144株无芽胞厌氧菌,其中类杆菌54株,厌氧性链球菌23株,韦荣氏球菌17株,真杆菌11株,梭杆菌10株,放线菌8株,双岐杆菌2株,消化链球菌和消化球菌19株。40只根管为厌氧菌和兼性厌氧菌或需氧菌混合感染,18只根管和6只根管分别为单独厌氧菌和兼性厌氧菌感染。33只根尖周炎根管分别采集牙髓和根尖渗出物样本进行培养,实验结果表明牙髓样本中革兰氏阳性厌氧杆菌检出率较高,根尖渗出物中以产黑素类杆菌属的细菌检出率较高。根尖周炎和牙槽脓肿患者的感染根管中产黑素类杆菌属的细菌检出率明显高于蜂窝组织炎患者。     

Anaerobic degradation of malonatevia malonyl-CoA bySporomusa malonica,Klebsiella oxytoca,andRhodobacter capsulatus

Anaerobic decarboxylation of malonate to acetate was studied withSporomusa malonica, Klebsiella oxytoca, andRhodobacter capsulatus. WhereasS. malonica could grow with malonate as sole substrate (Y=2.0 g·mol^–1), malonate decarboxylation byK. oxytoca was coupled with anaerobic growth only in the presence of a cosubstrate, e.g. sucrose or yeast extract (Y _s =1.1–1.8 g·mol malonate^–1).R. capsulatus used malonate anaerobically only in the light, and growth yields with acetate and malonate were identical. Malonate decarboxylation in cell-free extracts of all three bacteria was stimulated by catalytic amounts of malonyl-CoA, acetyl-CoA, or Coenzyme A plus ATP, indicating that actually malonyl-CoA was the substrate of decarboxylation. Less than 5% of malonyl-CoA decarboxylase activity was found associated with the cytoplasmic membrane. Avidin (except forK. oxytoca) and hydroxylamine inhibited the enzyme completely, EDTA inhibited partially. InS. malonica andK. oxytoca, malonyl-CoA decarboxylase was active only after growth with malonate; malonyl-CoA: acetate CoA transferase was found as well. These results indicate that malonate fermentation by these bacteria proceedsvia malonyl-CoA mediated by a CoA transferase and that subsequent decarboxylation to acetyl-CoA is catalyzed, at least withS. malonica andR. capsulatus, by a biotin enzyme.Abbreviations CoASH Coenzyme A - EDTA ethylenediamine tetraacetate     

Kinetic studies of the fatty acid synthetase multienzyme complex from Euglena gracilis variety bacillaris.

A fatty acid synthetase multienzyme complex was purified from Euglena gracilis variety bacillaris. The fatty acid synthetase activity is specifically inhibited by antibodies against Escherichia coli acyl-carrier protein. The Euglena enzyme system requires both NADPH and NADH for maximal activity. An analysis was done of the steady-state kinetics of the reaction catalysed by the fatty acid synthetase multienzyme complex. Initial-velocity studies were done in which the concentrations of the following pairs of substrates were varied: malonyl-CoA and acetyl-CoA, NADPH and acetyl-CoA, malonyl-CoA and NADPH. In all three cases patterns of the Ping Pong type were obtained. Product-inhibition studies were done with NADP+ and CoA. NADP+ is a competitive inhibitor with respect to NADPH, and uncompetitive with respect to malonyl-CoA and acetyl-CoA. CoA is uncompetitive with respect to NADPH and competitive with respect to malonyl-CoA and acetyl-CoA. When the concentrations of acetyl-CoA and malonyl-CoA were varied over a wide range, mutual competitive substrate inhibition was observed. When the fatty acid synthetase was incubated with radiolabelled acetyl-CoA or malonyl-CoA, labelled acyl-enzyme was isolated. The results are consistent with the idea that fatty acid synthesis proceeds by a multisite substituted-enzyme mechanism involving Ping Pong reactions at the following enzyme sites: acetyl transacylase, malonyl transacylase, beta-oxo acyl-enzyme synthetase and fatty acyl transacylase.     

Acyl coenzyme A carboxylase of Propionibacterium shermanii: detection and properties.

An acyl coenzyme A (CoA) carboxylase, which catalyzes the adenosine triphosphate-dependent fixation of CO2 into acetyl-, propionyl-, and butyryl-CoA, was detected in fractionated cell extracts of Propionibacterium shermanii. Catalytic activity was inhibited by avidin but was unaffected by avidin pretreated with excess biotin. The carboxylase levels detected were relatively small and were related to cellular growth. Maximal carboxylase activity was detected in cells grown for about 96 h. Thereafter, the activity declined rapidly. Optimal CO2 fixation occurred at pH 7.5. Other parameters of the assay system were optimized, and the apparent Km values for substrates were determined. The end product of the reaction (with acetyl-CoA as the substrate) was identified as malonyl-CoA. The stoichiometry of the reaction was such that, for every mole of acetyl-CoA and adenosine triphosphate consumed, 1 mol each of malonyl-CoA, adenosine diphosphate, and orthophosphate was formed. These data provide the first evidence for the presence of another biotin-containing enzyme, an acyl-CoA carboxylase, in these bacteria in addition to the well-characterized methylmalonyl-CoA carboxyltransferase.     

Radioisotopic assay of picomolar amounts of coenzyme A

A two-step method of determining reduced coenzyme A (CoASH) concentrations in tissue or cell extracts is described. In the first step, CoASH is reacted with acetylphosphate in a reaction catalyzed by phosphotransacetylase to yield acetyl-CoA. Acetyl-CoA is then condensed with [14C]oxaloacetate by citrate synthase to give [14C]citrate. This method allows the measurement of 10-200 pmol of CoASH. By omitting the phosphotransacetylase step, measurement of the same amount of acetyl-CoA is possible.     

The use of fatty acid methyl esters as biomarkers to determine aerobic, facultatively aerobic and anaerobic communities in wastewater treatment systems

The use of fatty acid methyl esters (FAME) as biomarkers to identify groups of microorganisms was studied. A database was constructed using previously published results that identify FAME biomarkers for aerobic, anaerobic and facultatively aerobic bacteria. FAME profiles obtained from pure cultures were utilized to confirm the predicted presence of biomarkers. Principal component analysis demonstrated that the FAME profiles can be used to determine the incidence of these bacterial groups. The presence of aerobic, anaerobic and facultatively aerobic bacteria in the communities, in four bioreactors being used to treat different wastewaters, was investigated by applying FAME biomarkers.     

Comparison of the Effects of Orally Administered Linoleic Acid,and Its Hydroperoxides and Secondary Autoxidation Products on Hepatic Lipid Metabolism in Rats

Linoleic acid, and its hydroperoxides and secondary autoxidation products were orally administered to rats (400 mg/rat). Their effects on hepatic lipid metabolism were examined. Linoleic acid reduced the activities of de novo synthesis of fatty acids and acetyl-CoA carboxylase. It decreased the CoASH level and caused the accumulation of long-chain acyl-CoA. Hydroperoxides changed the compositions of unsaturated fatty acids in the hepatic lipids and lowered the content of neutral lipids. Secondary products stimulated carnitine palmitoyltransferase and decreased the content of neutral lipids. They reduced the activities of de novo synthesis of fatty acids and acetyl-CoA carboxylase, and the levels of CoASH and acetyl-CoA. Thus, the effect of secondary products was apparently different from those of linoleic acid and its hydroperoxides.     

Changes in sensitivity of clinical strains of bacteria to dioxidine from 1984 to 1988

Dioxidine sensitivity of 7291 strains of aerobic bacteria and 163 strains of anaerobic bacteria was assayed with the disk diffusion method. The sensitivity of the aerobes was studied in the time course from 1984 to 1988. It was shown that during the 5-year period, the sensitivity of gram-positive bacteria to dioxidine gradually decreased. At the same time no increase in resistance of gram-negative organisms to dioxidine was observed. A high dioxidine sensitivity of obligate anaerobes, i.e. Clostridium spp., Bacteroides spp., Fusobacterium spp., anaerobic cocci and others was demonstrated.     

儿童乳牙根管感染的细菌学分析

对18例3～8岁儿童乳牙的根管感染以无菌技术进行定量取样,按种于12种选择性培养基和2种非选择性培养基上,进行需氧、微需氧和厌氧培养,并对细菌菌落计数。对牙髓拟杆菌和牙龈拟杆菌作半定量免疫荧光染色计数;并对其中9例病牙进行了菌相分析。检出的所有细菌中,厌氧菌占绝对优势;其中检出率较高的菌为:产黑色素拟杆菌属,厌氧革兰氏阳性球菌,微需氧革兰氏阳性球菌等.本试验证明,儿童乳牙根管感染是以厌氧菌为主的混合感染,其中以产黑色素拟杆菌属等最常见.