Detection of luciferase gene sequences in nonluminescent bacteria from the Chesapeake Bay

Washed excised roots of rice (Oryza sativa) produced H(2), CH(4) and fatty acids (millimolar concentrations of acetate, propionate, butyrate; micromolar concentrations of isovalerate, valerate) when incubated under anoxic conditions. Surface sterilization of the root material resulted in the inactivation of the production of CH(4), a strong reduction of the production of fatty acids and a transient (75 h) but complete inhibition of the production of H(2). Radioactive bicarbonate was incorporated into CH(4), acetate, propionate and butyrate. About 20-40% of the fatty acid carbon originated from CO(2) reduction. In the presence of phosphate, CH(4) was exclusively produced from H(2)/CO(2), since phosphate selectively inhibited acetoclastic methanogenesis. Acetoclastic methanogenesis was also selectively inhibited by methyl fluoride, while chloroform or 2-bromoethane sulfonate inhibited CH(4) production completely. Production of CH(4), acetate, propionate and butyrate from H(2)/CO(2) was always exergonic with Gibbs free energies <-20 kJ mol(-1) product. Chloroform inhibited the production of acetate and the incorporation of radioactive CO(2) into acetate. Simultaneously, H(2) was no longer consumed and accumulated, indicating that acetate was produced from H(2)/CO(2). Chloroform also resulted in increased production of propionate and butyrate whose formation from CO(2) became more exergonic upon addition of chloroform. Nevertheless, the incorporation of radioactive CO(2) into propionate and butyrate was inhibited by chloroform. The accumulation of propionate and butyrate in the presence of chloroform probably occurred by fermentation of organic matter, rather than by reduction of acetate and CO(2). [U-(14)C]Glucose was indeed converted to acetate, propionate, butyrate, CO(2) and CH(4). Radioactive acetate, CO(2) and CH(4) were also products of the degradation of [U-(14)C]cellulose and [U-(14)C]xylose. Addition of chloroform and methyl fluoride did not affect the product spectrum of [U-(14)C]glucose degradation. The application of combinations of selective inhibitors may be useful to elucidate anaerobic metabolic pathways in mixed microbial cultures and natural microbial communities.     

The lipogenic role of leucine in animal skeletal muscles

After incubation of rat, pig and cattle skeletal muscle homogenates with [U-14C]leucine, 80.4%, 37.0% and 57.0% of radioactivity was found in the proteins, 9.4%, 58.7% and 40.9% in the lipids, and 10.2%, 4.3% and 2.1% in 14CO2. This suggests that along-side with utilization in protein synthesis, leucine plays an essential role in lipid synthesis in muscle tissues of agricultural animals. The contribution of [U-14C]leucine to lipogenesis with substrates is greater than that of [U-14C]acetate and [U-14C]glucose in cattle skeletal muscles in vitro and greater than that of [U-14C]acetate in pig muscle. The CO2 production during oxidation of the [U-14C]leucine carbohydrate chain is higher than that during [U-14C]glucose and [U-14C]palmitate oxidation in skeletal muscles of rat and pig. In skeletal muscles of all animal species under study [U-14C]acetate is oxidized far more intensively than the other substrates tested.     

Pattern of non-methanogenic and methanogenic degradation of cellulose in anoxic rice field soil

Rice field soils turn anoxic upon flooding. The complete mineralization of organic matter, e.g. cellulose, to gaseous products is then accomplished by the sequential reduction of nitrate, ferric iron, sulfate and finally by methanogenesis. Therefore, the anaerobic turnover of [U-(14)C]cellulose was investigated in fresh, non-methanogenic and in preincubated, methanogenic slurries of Italian rice field soil. In anoxic soil slurries freshly prepared from air-dried soil [U-(14)C]cellulose was converted to (14)CO(2) and (14)CH(4) in a ratio of 3:1. In methanogenic soil slurries, on the other hand, which had been preincubated for 45 days under anaerobic conditions, [U-(14)C]cellulose was converted to (14)CO(2) and (14)CH(4) in the ratio of 1:1. The turnover times (7-14 days) of cellulose degradation were not significantly different (P0.05) in fresh and methanogenic soil. Chloroform addition abolished CH(4) production, but only slightly (30%) inhibited cellulose degradation in both fresh and methanogenic soil. Under both soil conditions, [(14)C]acetate was the only labeled intermediate detected. A maximum of 24% of the applied radioactivity was transiently accumulated as [(14)C]acetate in both fresh and methanogenic soil slurries. However, when methanogenesis was inhibited by chloroform, 46% and 66% of the applied radioactivity were recovered as [(14)C]acetate in fresh and methanogenic soil, respectively. Only non-radioactive propionate accumulated during the incubation with [U-(14)C]cellulose, especially in the presence of chloroform, indicating that propionate was produced from substrates other than cellulose.     

Anaerobic metabolism of immediate methane precursors in Lake Mendota.

Lake Mendota sediments and the immediate overlying water column were studied to better understand the metabolism of the methanogenic precursors H2/CO2 and acetate in nature. The pool size of acetate (3.5 microns M) was very small, and the acetate turnover time (0.22h) was very rapid. The dissolved inorganic carbon pool was shown to be large (6.4 to 8.3 mM), and the turnover time was slow (111 H.). CO2 was shown to account for 41 +/- 5.5% of the methane produced in sediment. Acetate and H2/CO2 were simultaneously converted to CH4. The addition of H2 to sediments resulted in an increase specific activity of CH4 from H(14)CO3- and a decrease in specific activity of CH4 from [2-14C]acetate. Acetate addition resulted in a decrease in specific activity of CH4 from H(14)CO3-. The metabolism of H(14)CO3- or [2-14C]acetate to 14CH4 was not inhibited by addition of acetate or H2. After greater than 99% of added [2-14C]acetate had been turned over, 42% of the label was recovered as 14CH4 20% was recovered as 14CO2 and 38% was incorporated into sediment. Inhibitor studies of [2-14C]acetate metabolism in sediments demonstrated that CHCl3 completely inhibited CH4 formation, but not CO2 production. Air and nitrate addition inhibited CH4 formation and stimulated CO2 production, whereas fluoroacetate addition totally inhibited acetate metabolism. The oxidation of [2-14C]acetate to 14CO2 was shown to decrease with time when sediment was incubated before the addition of label, suggesting depletion of low levels of an endogenous sediment electron acceptor. Acetate metabolism varied seasonally and was related to the concentration of sulfate in the lake and interstitial water. Methanogenesis occurred in the sediment and in the water immediately overlying the sediment during period of lake stratification and several centimeters below the sediment-water interface during lake turnovers. These data indicate that methanogenesis in Lake Mendota sediments was limited by "immediate" methane precursor availability (i.e., acetate and H2), by competition for these substrates by nonmethanogens, and by seasonal variations which altered sediment and water chemistry.     

Anaerobic metabolism of immediate methane precursors in Lake Mendota.

Lake Mendota sediments and the immediate overlying water column were studied to better understand the metabolism of the methanogenic precursors H2/CO2 and acetate in nature. The pool size of acetate (3.5 microns M) was very small, and the acetate turnover time (0.22h) was very rapid. The dissolved inorganic carbon pool was shown to be large (6.4 to 8.3 mM), and the turnover time was slow (111 H.). CO2 was shown to account for 41 +/- 5.5% of the methane produced in sediment. Acetate and H2/CO2 were simultaneously converted to CH4. The addition of H2 to sediments resulted in an increase specific activity of CH4 from H(14)CO3- and a decrease in specific activity of CH4 from [2-14C]acetate. Acetate addition resulted in a decrease in specific activity of CH4 from H(14)CO3-. The metabolism of H(14)CO3- or [2-14C]acetate to 14CH4 was not inhibited by addition of acetate or H2. After greater than 99% of added [2-14C]acetate had been turned over, 42% of the label was recovered as 14CH4 20% was recovered as 14CO2 and 38% was incorporated into sediment. Inhibitor studies of [2-14C]acetate metabolism in sediments demonstrated that CHCl3 completely inhibited CH4 formation, but not CO2 production. Air and nitrate addition inhibited CH4 formation and stimulated CO2 production, whereas fluoroacetate addition totally inhibited acetate metabolism. The oxidation of [2-14C]acetate to 14CO2 was shown to decrease with time when sediment was incubated before the addition of label, suggesting depletion of low levels of an endogenous sediment electron acceptor. Acetate metabolism varied seasonally and was related to the concentration of sulfate in the lake and interstitial water. Methanogenesis occurred in the sediment and in the water immediately overlying the sediment during period of lake stratification and several centimeters below the sediment-water interface during lake turnovers. These data indicate that methanogenesis in Lake Mendota sediments was limited by "immediate" methane precursor availability (i.e., acetate and H2), by competition for these substrates by nonmethanogens, and by seasonal variations which altered sediment and water chemistry.     

Increased heat production proportional to oxygen consumption in human neutrophils activated with phorbol-12-myristate-13-acetate

The heat produced by neutrophils was measured with a flow microcalorimeter. 02 consumption, ATP concentration, lactic acid production and 14CO2 production from oxidation of [1-(14)C]-glucose [6-(14)C]-glucose and [U-14C]-glucose were evaluated. Experiments were also carried out in the presence of the metabolic inhibitors, N-ethylmaleimide and NaF. Heat effects were correlated to the enthalpy change of aerobic and anaerobic glucose catabolism. Two different heat contributions related to two different nonmitochondrial 02 reduction pathways are present during the metabolic burst. Theoretical and experimental data indicate that the reducing power is derived from the catabolism of glucose both through the hexose monophosphate shunt and glycolysis.     

Microbial mineralization of VC and DCE under different terminal electron accepting conditions

Production of 14CO2 from [1,2-14C] dichloroethene (DCE) or [1,2-14C] vinyl chloride (VC) was quantified in aquifer and stream-bed sediment microcosms to evaluate the potential for microbial mineralization as a pathway for DCE and VC biodegradation under aerobic, Fe(III)-reducing, SO4-reducing, and methanogenic conditions. Mineralization of [1,2-14C] DCE and [1,2-14C] VC to 14CO2 decreased under increasingly reducing conditions, but significant mineralization was observed for both sediments even under anaerobic conditions. VC mineralization decreased in the order of aerobic > Fe(III)-reducing > SO4-reducing > methanogenic conditions. For both sediments, VC mineralization was greater than DCE mineralization under all electron-accepting conditions examined. For both sediments, DCE mineralization was at least two times greater under aerobic conditions than under anaerobic conditions. Although significant microbial mineralization of DCE was observed under anaerobic conditions, recovery of 14CO2 did not differ substantially between anaerobic treatments.     

Turnover of glucose and acetate coupled to reduction of nitrate, ferric iron and sulfate and to methanogenesis in anoxic rice field soil

Turnover of glucose and acetate in the presence of active reduction of nitrate, ferric iron and sulfate was investigated in anoxic rice field soil by using [U-(14)C]glucose and [2-(14)C]acetate. The turnover of glucose was not much affected by addition of ferrihydrite or sulfate, but was partially inhibited (60%) by addition of nitrate. Nitrate addition also strongly reduced acetate production from glucose while ferrihydrite and sulfate addition did not. These results demonstrate that ferric iron and sulfate reducers did not outcompete fermenting bacteria for glucose at endogenous concentrations. Nitrate reducers may have done so, but glucose fermentation may also have been inhibited by accumulation of toxic denitrification intermediates (nitrite, NO, N(2)O). Addition of nitrate resulted in complete inhibition of CH(4) production from [U-(14)C]glucose and [2-(14)C]acetate. However, addition of ferrihydrite or sulfate decreased the production of (14)CH(4) from [U-(14)C]glucose by only 70 and 65%, respectively. None of the electron acceptors significantly increased the production of (14)CO(2) from [U-(14)C]glucose, but all increased the production of (14)CO(2) from [2-(14)C]acetate. Uptake of acetate was faster in the presence of either nitrate, ferrihydrite or sulfate than in the unamended control. Addition of ferrihydrite and sulfate reduced (14)CH(4) production from [2-(14)C]acetate by 83 and 92%, respectively. Chloroform completely inhibited the methanogenic consumption of acetate. It also inhibited the oxidation of acetate, completely in the presence of sulfate, but not in the presence of nitrate or ferrihydrite. Our results show that, besides the possible toxic effect of products of nitrate reduction (NO, NO(2)(-) and N(2)O) on methanogens, nitrate reducers, ferric iron reducers and sulfate reducers were active enough to outcompete methanogens for acetate and channeling the flow of electrons away from CH(4) towards CO(2) production.     

Interaction of acetogens and methanogens in anaerobic freshwater sediments

Anaerobic decomposition processes in the profundal sediments of Blelham Tarn (English Lake District) are often limited during late summer by the input of organic carbon. The concentration of acetate in the interstitial water fell from about 100 microM (immediately after sedimentation of the spring diatom bloom) to a relatively constant value of about 20 microM in late summer, during which acetate utilization appeared to be balanced by production. Addition of chloroform and molybdate caused an accumulation of cold acetate in large sediment cores and of [14C]acetate in small cores to which [14C]bicarbonate had been added. In both cases chloroform caused the greater accumulation, implying that acetoclastic methanogens were the more active consumers. The conversion of 14CO2 to [14C]acetate was inversely related, with depth, to its conversion to 14CH4. Methanogenesis from CO2 decreased during late summer, whereas acetogenesis and acetoclastic methanogenesis increased over the same time period. The production of acetate from CO2 was generally equivalent to less than 10% of the acetate carbon utilized but could be as high as 25% of that value. Hydrogen consumption by acetogens could be as high as 50% of that utilized in methanogenesis. The role of acetogenic bacteria in anaerobic processes may therefore be of greater significance in lakes such as Blelham Tarn than in more eutrophic systems.     

Interaction of acetogens and methanogens in anaerobic freshwater sediments.

Anaerobic decomposition processes in the profundal sediments of Blelham Tarn (English Lake District) are often limited during late summer by the input of organic carbon. The concentration of acetate in the interstitial water fell from about 100 microM (immediately after sedimentation of the spring diatom bloom) to a relatively constant value of about 20 microM in late summer, during which acetate utilization appeared to be balanced by production. Addition of chloroform and molybdate caused an accumulation of cold acetate in large sediment cores and of [14C]acetate in small cores to which [14C]bicarbonate had been added. In both cases chloroform caused the greater accumulation, implying that acetoclastic methanogens were the more active consumers. The conversion of 14CO2 to [14C]acetate was inversely related, with depth, to its conversion to 14CH4. Methanogenesis from CO2 decreased during late summer, whereas acetogenesis and acetoclastic methanogenesis increased over the same time period. The production of acetate from CO2 was generally equivalent to less than 10% of the acetate carbon utilized but could be as high as 25% of that value. Hydrogen consumption by acetogens could be as high as 50% of that utilized in methanogenesis. The role of acetogenic bacteria in anaerobic processes may therefore be of greater significance in lakes such as Blelham Tarn than in more eutrophic systems.     

Microbial methanogenesis and acetate metabolism in a meromictic lake.

Methanogenesis and the anaerobic metabolism of acetate were examined in the sediment and water column of Knaack Lake, a small biogenic meromictic lake located in central Wisconsin. The lake was sharply stratified during the summer and was anaerobic below a depth of 3 m. Large concentrations (4,000 mumol/liter) of dissolved methane were detected in the bottom waters. A methane concentration maximum occurred at 4 m above the sediment. The production of (14)CH(4) from (14)C-labeled HCOOH, HCO(3) (-), and CH(3)OH and [2-(14)C]acetate demonstrated microbial methanogenesis in the water column of the lake. The maximum rate of methanogenesis calculated from reduction of H(14)CO(3) (-) by endogenous electron donors in the surface sediment (depth, 22 m) was 7.6 nmol/h per 10 ml and in the water column (depth, 21 m) was 0.6 nmol/h per 10 ml. The methyl group of acetate was simultaneously metabolized to CH(4) and CO(2) in the anaerobic portions of the lake. Acetate oxidation was greatest in surface waters and decreased with water depth. Acetate was metabolized primarily to methane in the sediments and water immediately above the sediment. Sulfide inhibition studies and temperature activity profiles demonstrated that acetate metabolism was performed by several microbial populations. Sulfide additions (less than 5 mug/ml) to water from 21.5 m stimulated methanogenesis from acetate, but inhibited CO(2) production. Sulfate addition (1 mM) had no significant effect on acetate metabolism in water from 21.5 m, whereas nitrate additions (10 to 14,000 mug/liter) completely inhibited methanogenesis and stimulated CO(2) formation.     

Evidence for aceticlastic methanogenesis in the presence of sulfate in a gas condensate-contaminated aquifer

The anaerobic metabolism of acetate was studied in sediments and groundwater from a gas condensate-contaminated aquifer in an aquifer where geochemical evidence implicated sulfate reduction and methanogenesis as the predominant terminal electron-accepting processes. Most-probable-number tubes containing acetate and microcosms containing either [2-(14)C]acetate or [U-(14)C]acetate produced higher quantities of CH(4) compared to CO(2) in the presence or absence of sulfate.(14)CH(4) accounted for 70 to 100% of the total labeled gas in the [(14)C]acetate microcosms regardless of whether sulfate was present or not. Denaturing gradient gel electrophoresis of the acetate enrichments both with and without sulfate using Archaea-specific primers showed identical predominant bands that had 99% sequence similarity to members of Methanosaetaceae. Clone libraries containing archaeal 16S rRNA gene sequences amplified from sediment from the contaminated portion of the aquifer showed that 180 of the 190 clones sequenced belonged to the Methanosaetaceae. The production of methane and the high frequency of sequences from the Methanosaetaceae in acetate enrichments with and without sulfate indicate that aceticlastic methanogenesis was the predominant fate of acetate at this site even though sulfate-reducing bacteria would be expected to consume acetate in the presence of sulfate.     

Thermoanaerobacteriaceae oxidize acetate in methanogenic rice field soil at 50°C

Rice field soils contain a thermophilic microbial community. Incubation of Italian rice field soil at 50°C resulted in transient accumulation of acetate, but the microorganisms responsible for methane production from acetate are unknown. Without addition of exogenous acetate, the δ(13)C of CH(4) and CO(2) indicated that CH(4) was exclusively produced by hydrogenotrophic methanogenesis. When exogenous acetate was added, acetoclastic methanogenesis apparently also operated. Nevertheless, addition of [2-(13)C]acetate (99% (13)C) resulted in the production not only of (13)C-labelled CH(4) but also of CO(2), which contained up to 27% (13)C, demonstrating that the methyl group of acetate was also oxidized. Part of the (13)C-labelled acetate was also converted to propionate which contained up to 14% (13)C. The microorganisms capable of assimilating acetate at 50°C were targeted by stable isotope probing (SIP) of ribosomal RNA and rRNA genes using [U-(13)C] acetate. Using quantitative PCR, (13)C-labelled bacterial ribosomal RNA and DNA was detected after 21 and 32 days of incubation with [U-(13)C]acetate respectively. In the heavy fractions of the (13)C treatment, terminal restriction fragments (T-RFs) of 140, 120 and 171 bp length predominated. Cloning and sequencing of 16S rRNA showed that these T-RFs were affiliated with the bacterial genera Thermacetogenium and Symbiobacterium and with members of the Thermoanaerobacteriaceae. Similar experiments targeting archaeal RNA and DNA showed that Methanocellales were the dominant methanogens being consistent with the operation of syntrophic bacterial acetate oxidation coupled to hydrogenotrophic methanogenesis. After 17 days, however, Methanosarcinacea increasingly contributed to the synthesis of rRNA from [U-(13)C]acetate indicating that acetoclastic methanogens were also active in methanogenic Italian rice field soil under thermal conditions.     

Ketone body utilization for energy production and lipid synthesis in isolated rat brain capillaries

Isolated brain capillaries from 2-month-old rats were incubated for 2 h in the presence of [3-14C]acetoacetate, D-3-hydroxy[3-14C]butyrate, [U-14C]glucose, [1-14C]acetate or [1-14C]butyrate. Labelled CO2 was collected as an index of oxidative metabolism and incorporation of label precursors into lipids was determined. The rate of CO2 production from glucose was slightly higher than from the other substrates. Interestingly, acetoacetate was oxidized at nearly the same rate as glucose. This shows that ketone bodies could be used as a source of energy by brain capillaries. Radiolabelled substrates were also used for the synthesis of lipids, which was suppressed by the addition of albumin. The incorporation of [U-14C]glucose in total lipids was 10-times higher than that from other precursors. However, glucose labelled almost exclusively the glycerol backbone of phospholipids, especially of phosphatidylcholine. Ketone bodies as well as glucose were incorporated mainly into phospholipids, whereas acetate and butyrate were mainly incorporated into neutral lipids. The contribution to fatty acid synthesis of various substrates was in the following order: butyrate greater than or equal to acetate greater than ketone bodies greater than or equal to glucose. All precursors except glucose were used for sterol synthesis. Glucose produced almost exclusively the glycerol backbone of phospholipids.     

Gluconeogenesis from acetone in starved rats.

To non-anaesthetized rats starved for 3 days, [U-14C]acetone, NaH14CO3, L-[U-14C]lactate, [2-14C]acetate or D-[U-14C]- plus D-[3-3H]-glucose was injected intravenously. From the change in the plasma concentration of labelled acetone versus time after the injection, the metabolic clearance rate of acetone was calculated as 2.25 ml/min per kg body wt., and its rate of turnover as 0.74 mumol/min per kg. The extent and time course of the labelling of plasma glucose, lactate, urea and acetoacetate were followed and compared with those observed after the injection of labelled lactate, acetate and NaHCO3. The labelling of plasma lactate was rapid and extensive. Some 1.37% of the 14C atoms of circulating glucose originated from plasma acetone, compared with 44% originating from lactate. By deconvolution of the Unit Impulse Response Function of glucose, it was shown that the flux of C atoms from acetone to glucose reached a peak at about 100 min after injection of labelled acetone. In comparable experiments the transfer from lactate reached a peak at 14 min after the injection of labelled lactate. It was concluded that acetone is converted into lactate to a degree sufficient to account for the labelling of plasma glucose and is thus a true, albeit minor, substrate of glucose synthesis in starved rats.     

Anaerobic degradation of polycyclic aromatic hydrocarbons and alkanes in petroleum-contaminated marine harbor sediments.

Although polycyclic aromatic hydrocarbons (PAHs) have usually been found to persist under strict anaerobic conditions, in a previous study an unusual site was found in San Diego Bay in which two PAHs, naphthalene and phenanthrene, were oxidized to carbon dioxide under sulfate-reducing conditions. Further investigations with these sediments revealed that methylnaphthalene, fluorene, and fluoranthene were also anaerobically oxidized to carbon dioxide in these sediments, while pyrene and benzo[a]pyrene were not. Studies with naphthalene indicated that PAH oxidation was sulfate dependent. Incubating the sediments with additional naphthalene for 1 month resulted in a significant increase in the oxidation of [14C]naphthalene. In sediments from a less heavily contaminated site in San diego Bay where PAHs were not readily degraded, naphthalene degradation could be stimulated through inoculation with active PAH-degrading sediments from the most heavily contaminated site. Sediments from the less heavily contaminated site that had been adapted for rapid anaerobic degradation of high concentrations of benzene did not oxidize naphthalene, suggesting that the benzene- and naphthalene-degrading populations were different. When fuels containing complex mixtures of alkanes were added to sediments from the two sites, there was significant degradation in the alkanes. [14C]hexadecane was also anaerobically oxidized to 14CO2 in these sediments. Molybdate, a specific inhibitor of sulfate reduction, inhibited hexadecane oxidation. These results demonstrate that a wide variety of hydrocarbon contaminants can be degraded under sulfate-reducing conditions in hydrocarbon-contaminated sediments, and they suggest that it may be possible to use sulfate reduction rather than aerobic respiration as a treatment strategy for hydrocarbon-contaminated dredged sediments.     

The metabolism of acetylcarnitine and acetate by bovine and hamster epididymal spermatozoa

Since acetylcarnitine has been identified in the epididymal plasma of many mammalian species, we investigated whether acetylcarnitine could serve as an energy substrate for epididymal bull and hamster spermatozoa. Intact caudal cells from both species oxidized [I-14C]acetyl-l-carnitine to 14CO2, in vitro, and the amount oxidized was dependent on time, substrate concentration, and cell number. Within each species, the rate of oxidation was the same as the rate at which free [1-14C]acetate was oxidized. Spermatozoa incubated with [3H]acetyl-L-carnitine hydrolyzed the compound and [3H]acetate accumulated in the medium. Unlabeled acetate added to the incubation medium competed with cellular uptake of [3H]acetate and resulted in further increase in [3H]acetate accumulation in the medium. Furthermore, the acetyl group of acetylcarnitine was oxidized by spermatozoa without concomitant uptake of the carnitine group. Purified plasma membrane vesicles contained an acetylcarnitine hydrolase activity that was solubilized from whole cells by detergents and that could be distinguished from acetylcholinesterase also present in the cells. The solubilized acetylcarnitine hydrolase activity was inhibited by p-hydroxymercuriphenylsulfonate, but not by the specific acetylcholinesterase inhibitors, eserine or BW63C47. The sulfhydryl blocker also inhibited the production of 14CO2 from [1-14C]acetylcarnitine by intact cells; acetylcholinesterase inhibitors did not. From estimates of sperm energy requirements, our results indicate that extracellular acetylcarnitine serves as a physiologically important energy substrate for maturing sperm cells.     

Effect of CO 2 concentration on phospholipid metabolism in the isolated perfused rat lung

Studies have been carried out on the incorporation of [U-(14)C]glucose, [2-(14)C]pyruvate, [2-(14)C]acetate, and [1-(14)C]-palmitate into the phospholipids of the isolated perfused rat lung in the presence of either 6 or 45 mm total CO(2) concentration in the perfusion medium. Incorporation of [U-(14)C]glucose into total phospholipid and into the phosphatidylcholine fraction was increased 19-53% over the 2-hr perfusion period in lungs perfused with medium containing 45 as compared with 6 mm CO(2). The incorporation of [2-(14)C]acetate, [2-(14)C]-pyruvate, and [1-(14)C]palmitate was not affected by the change in medium CO(2) concentration. Increased incorporation of [U-(14)C]glucose combined with a shift toward greater incorporation into the fatty acids of the phosphatidylcholine fraction produced a maximum increase of 90% in [U-(14)C]glucose incorporation into the fatty acids of phosphatidylcholine after 2 hr of perfusion in the presence of medium containing 45 mm CO(2) as compared with 6 mm CO(2). The increase in medium CO(2) concentration produced as much as a 150% increase in [U-(14)C]glucose incorporation into palmitate derived from the phosphatidylcholine fraction. The results provide evidence that glucose functions as an important precursor of palmitate in the phosphatidylcholine fraction of lung phospholipids and that the CO(2) concentration of the perfusion medium affects the incorporation of glucose into palmitate.     

Alteration in Neuronal-Glial Metabolism of Glutamate by the Neurotoxin Kainic Acid

The effect of the excitotoxin kainic acid on glutamate and glutamine metabolism was studied in cerebellar slices incubated with D-[2-14C]glucose, [U-14C]gamma-aminobutyric acid, [3H]acetate, [U-14C]glutamate, and [U-14C]glutamine as precursors. Kainic acid (1 mM) strongly inhibited the labeling of glutamine relative to that of glutamate from all precursors except [2-14C]glucose and [U-14C]glutamine. Kainic acid did not inhibit glutamine synthetase directly. The data indicate that in the cerebellum kainic acid inhibits the synthesis of glutamine from the small pool of glutamate that is thought to be associated with glial cells. Kainic acid also markedly stimulated the efflux of glutamate from cerebellar slices and this release was not sensitive to tetrodotoxin. Kainic acid stimulated efflux of both glucose- and acetate-labeled glutamate. In contrast, veratridine released glucose-labeled glutamate preferentially via a tetrodotoxin-sensitive mechanism. Kainic acid did not release [U-14C]glutamate from synaptosomal fractions. These results suggest that the bulk of the glutamate released from cerebellar slices by kainic acid comes from nonsynaptic pools.     

Oxidation of leucine by Leishmania donovani.

The metabolism of leucine by Leishmania donovani was investigated. Washed promastigotes were incubated with [1-14C]- or [U-14C]leucine or [1-14C]alpha-ketoisocaproate (KIC) and 14CO2 release was measured. The amount of KIC-derived acetyl-CoA oxidized in the citric acid cycle was computed. Promastigotes from mid-stationary phase cultures oxidized each of these labeled substrates less rapidly than cells from late log phase cultures, and significantly less acetyl-CoA derived from KIC oxidation was oxidized in the citric acid cycle. Glucose was a stronger inhibitor than was acetate of CO2 formation in the citric acid cycle in log phase promastigotes, but the reverse was observed in cells from mid-stationary phase. Alanine also inhibited leucine catabolism, but glutamate had little effect. Acute hypo-osmotic stress did not affect leucine catabolism, but hyper-osmotic stress caused appreciable inhibition of leucine oxidation. Cells grown under hypo- or hyper-osmotic conditions showed no changes in the effects of hypo- or hyper-osmotic stress on leucine catabolism, i.e. L. donovani is not an osmoconformer with respect to leucine metabolism. Leucine utilization in L. donovani was insensitive to a number of drugs that affect leucine metabolism in mammalian cells, indicating that the leucine pathway in L. donovani is not regulated in the same manner as in mammalian cells.