Synthesis of cyclopropane fatty acid and its effect on freeze-drying survival of Lactobacillus bulgaricus L2 at different growth conditions

In order to correlate cyclopropane fatty acid of the membrane of Lactobacillus bulgaricus L2 with freeze-drying survival at different growth conditions, fatty acid methyl esters (FAME) from extracts grown at difference fermentation pH (5.0, 5.5, 6.0, 6.5) and temperature (30, 35, 37, 39°C) were obtained and analyzed. Results showed that cultures grown at 30°C and pH 5.0, 35°C and pH 5.0, 39°C and pH 6.0 exhibited more resistance to the freeze-drying process than cultures grown in other conditions, cells cultured at 30°C and pH 5.0 had a highest survival rate. On the other hand, cells grown at 37°C displayed poor resistance to adverse conditions possible because of the lower cycC19:0 content. It was concluded that the improved cryotolerance observed during freeze-drying would be associated with an increase in cycC19:0 content and cycC19:0/SFA ratio and vice versa.     

Leishmania mexicana: Energy metabolism of amastigotes and promastigotes

The utilisation of substrates by Leishmania mexicana amastigotes and promastigotes differed significantly. The rates of uptake and catabolism of nonesterified fatty acids were up to 10-fold higher with amastigotes. Almost all the available exogenous fatty acids were consumed during amastigote transformation and by stationary phase of promastigote growth. The results suggest that fatty acids are important energy substrates for amastigotes, whereas promastigote utilisation may reflect the requirement for these substrates in anabolism. Glucose was utilised by amastigotes and promastigotes but the rate of catabolism was up to 10-fold higher in promastigotes. Uptake of glucose occurred throughout amastigote transformation and growth in vitro of promastigotes. High-subpassage promastigotes exhibited markedly lower glucose but higher amino acid utilisation than low-subpassage promastigotes. Asparagine, glutamine, glutamate, leucine, lysine, methionine, and threonine were consumed in large quantities by amastigotes and promastigotes, whereas alanine and glycine were excreted. Proline was catabolised to CO₂ by amastigotes and promastigotes but only at a low rate, and it was excreted in large amounts throughout promastigote growth. The major end products of energy metabolism were found to be CO₂ and succinate with both forms of the parasite and there was a secretion of up to 12 and 16% of the total protein synthesised by transforming amastigotes and growing promastigotes, respectively. Catabolism in amastigotes and promastigotes was found to be sensitive to cyanide and amytal, whereas 2-mercaptoacetate and 4-pentenoate primarily affected β-oxidation in the amastigote.     

Enzymes of Carbohydrate Metabolism in Leishmania donovani Amastigotes1

A method for the isolation of Leishmania donovani amastigotes from infected hamster spleen and liver tissues is described. Over 85% of the isolated amastigotes were viable as judged by acridine orange-ethidium bromide staining and in vitro transformation to the promastigote form. A comprehensive survey of the enzymes of carbohydrate metabolism in L. donovani amastigotes and promastigotes was conducted. Amastigotes and promastigotes possess all of the enzymes of the Embden-Meyerhof pathway, hexose monophosphate shunt, and tricarboxylic acid cycle. Cell-free extracts of both forms show pyruvate dehydrogenase activity which permits entry of pyruvate into the tricarboxylic acid cycle. Both forms demonstrate an active glutamate dehydrogenase, thus linking amino acid metabolism with carbohydrate metabolism. Pyruvate carboxylase, the enzyme responsible for replenishment of C₄ acids by heterotrophic CO₂ fixation into pyruvate, was also demonstrable in the tissue and insect forms. In general, activities of promastigote enzymes are higher than the amastigote enzymes. Differences between the vertebrate (amastigote) and invertebrate (promastigote) forms in their potential to utilize carbohydrates as substrates would appear to be quantitative rather than qualitative.     

The effect of volatile fatty acids on the inactivation of <Emphasis Type="Italic">Clostridium perfringens</Emphasis> in anaerobic digestion

The application of sludge digestion systems to remove pathogens has been employed to generate biosolids suitable for reuse in agriculture. Traditionally, temperature is considered the principal agent responsible for pathogen reduction in anaerobic digestion. However, other substances such as volatile fatty acids may also have an antimicrobial effect. The objective of this study was to assess the impact of fatty acid mixtures on the inactivation of C. perfringens over a range of digestion temperatures. An equimolar mixture of acetic acid, propionic acid and butyric acid was applied to digester effluent for a period of 24 h at temperatures of 35 °C, 42 °C, 49 °C and 55 °C. C. perfringens inactivation in digester effluents, when dosed with volatile organic acids, was found to depend on pH, acid concentration and temperature. Temperatures above 55 °C appeared to increase the inhibitory effects of the organic acids at higher concentrations. An interaction between temperature and pH on survival of C. perfringens was observed. The results suggest that high concentrations of organic acids at a pH value of 4.5–5.5 during thermophilic digestion substantially reduce concentrations of C. perfringens in municipal sludge.     

Deletion of mitochondrial associated ubiquitin fold modifier protein Ufm1 in Leishmania donovani results in loss of β‐oxidation of fatty acids and blocks cell division in the amastigote stage

Recently, we described the existence of the ubiquitin fold modifier 1 (Ufm1) and its conjugation pathway in Leishmania donovani. We demonstrated the conjugation of Ufm1 to proteins such as mitochondrial trifunctional protein (MTP) that catalyses β‐oxidation of fatty acids in L. donovani. To elucidate the biological roles of the Ufm1‐mediated modifications, we made an L. donovani Ufm1 null mutant (Ufm1^?/?). Loss of Ufm1 and consequently absence of Ufm1 conjugation with MTP resulted in diminished acetyl‐CoA, the end‐product of the β‐oxidation in the Ufm1^?/? amastigote stage. The Ufm1^?/? mutants showed reduced survival in the amastigote stage in vitro and ex vivo in human macrophages. This survival was restored by re‐expression of wild‐type Ufm1 with concomitant induction of acetyl‐CoA but not by re‐expressing the non‐conjugatable Ufm1, indicating the essential nature of Ufm1 conjugation and β‐oxidation. Both cell cycle analysis and ultrastructural studies of Ufm1^?/? parasites confirmed the role of Ufm1 in amastigote growth. The defect in vitro growth of amastigotes in human macrophages was further substantiated by reduced survival. Therefore, these studies suggest the importance of Ufm1 in Leishmania pathogenesis with larger impact on other organisms and further provide an opportunity to test Ufm1^?/? parasites as drug and vaccine targets.     

Induction of a Stringent Metabolic Response in Intracellular Stages of Leishmania mexicana Leads to Increased Dependence on Mitochondrial Metabolism

Leishmania parasites alternate between extracellular promastigote stages in the insect vector and an obligate intracellular amastigote stage that proliferates within the phagolysosomal compartment of macrophages in the mammalian host. Most enzymes involved in Leishmania central carbon metabolism are constitutively expressed and stage-specific changes in energy metabolism remain poorly defined. Using ¹³C-stable isotope resolved metabolomics and ²H₂O labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state. This state is characterized by a global decrease in the uptake and utilization of glucose and amino acids, a reduced secretion of organic acids and increased fatty acid β-oxidation. Isotopomer analysis showed that catabolism of hexose and fatty acids provide C4 dicarboxylic acids (succinate/malate) and acetyl-CoA for the synthesis of glutamate via a compartmentalized mitochondrial tricarboxylic acid (TCA) cycle. In vitro cultivated and intracellular amastigotes are acutely sensitive to inhibitors of mitochondrial aconitase and glutamine synthetase, indicating that these anabolic pathways are essential for intracellular growth and virulence. Lesion-derived amastigotes exhibit a similar metabolism to in vitro differentiated amastigotes, indicating that this stringent response is coupled to differentiation signals rather than exogenous nutrient levels. Induction of a stringent metabolic response may facilitate amastigote survival in a nutrient-poor intracellular niche and underlie the increased dependence of this stage on hexose and mitochondrial metabolism.     

A soluble phosphodiesterase in Leishmania donovani negatively regulates cAMP signaling by inhibiting protein kinase A through a two way process involving catalytic as well as non-catalytic sites

Intracellular cAMP level and cAMP mediated responses are elevated when Leishmania are exposed to macrophage phagolysosome conditions (37 °C and pH 5.5). Phosphodiesterases play major role in cAMP regulation and in the present study we have cloned and characterized a 2.1 kb cytosolic isoform of phosphodiesterase from Leishmania donovani (LdPDED) which plays important role in cAMP homeostasis when the promastigotes are exposed to macrophage phagolysome conditions for converting to axenic amastigotes. Domain characterization suggested the presence of two pseudo-substrate sites similar to the ones present in the regulatory subunit of cAMP-dependent protein kinase A (PKA) and a putative PKA phosphorylation site at T⁷⁰⁸ of C-terminus of LdPDED. Deletion constructs and site directed mutagenesis revealed the ability of LdPDED to interact with L. donovani PKA catalytic subunits (LdPKAC1 and LdPKAC2) resulting in inhibition of kinase activity in one hand and increase of phosphodiesterase activity through PKA mediated phosphorylation at putative phosphorylation site on the other hand. This study therefore identifies a unique phosphodiesterase in L. donovani which appears to regulate cAMP-dependent PKA signaling through a two way process.     

Chronic heat-shock treatment driven differentiation induces apoptosis in Leishmania donovani

The present study investigates the role of apoptosis in the regulation of cell numbers of Leishmania donovani during the in vitro differentiation of promastigote stage to amastigote stage in axenic conditions. We report that apoptosis is induced in Leishmania donovani due to chronic heat-shock treatment of 37 ^°C that also mediates the differentiation of promastigotes to amastigotes. This is characterized by the fragmentation of DNA, blebbing in the parasite cell membrane, nuclear condensation, formation of preapoptotic bodies and involvement of Ca⁺⁺ in the apoptotic process. The flowcytometric analysis shows an early and steep rise in percentage apoptotic nuclei till 48-hour stage of differentiation and then a gradual decline, suggesting synergistic action of Ca⁺⁺ ATPase and probably Hsp70. Hsp70 might be rescuing cells from apoptosis in the death signaling pathway. Incubation of the culture with Ca⁺⁺ chelator EGTA (1 mM) brings down the percentage of apoptotic nuclei considerably showing thereby that calcium is needed for the process of cell death here that occurs by apoptosis. The survival of the infective individuals appears to be decided by the parasite in the early stages of its differentiation. Our studies show the potential of the physiological temperature of 37 ^°C in inducing apoptosis in Leishmania donovani and the therapeutic use it can be put to.     

Membrane bound pyrophosphatase and P-Type adenosine triphosphatase of <Emphasis Type="Italic">Leishmania donovani</Emphasis> as possible chemotherapeutic targets: Similarities and differences in inhibitor sensitivities

The activities of inorganic pyrophosphatase (PPase) and adenosine triphosphatase (ATPase) were studied in the plasma membrane of Leishmania donovani promastigotes and amastigotes. It was shown that the specific activity of PPase was greater than that of ATPase in the promastigote plasma membrane. We characterized H⁺-PPase present in the plasma membrane of L. donovani and investigated its possible role in the survival of promastigote and amastigote. PPase activity was stimulated by K⁺ and sodium orthovanadate and inhibited by pyrophosphate analogs (imidodiphosphate and alendronate), KF, N,N′-dicyclohexylcarbodiimide (DCCD), thiol reagents (p-chloromercuribenzenesulfonate (PCMBS), N-ethylmaleimide (NEM), and phenylarsine oxide (PAO)), the ABC superfamily transport modulator verapamil, and also by the F₁F_o-ATPase inhibitor quercetin. ATPase activity was stimulated by K⁺ and verapamil, inhibited by DCCD, PCMBS, NEM, sodium azide, sodium orthovanadate, and quercetin, and was unaffected by PAO. We conclude that there are significant differences within promastigote, amastigote, and mammalian host in cytosolic pH homeostasis to merit the inclusion of PPase transporter as a putative target for rational drug design.     

Leishmania donovani: correlation among assays of amastigote viability

The viability of Leishmania donovani amastigotes was evaluated using two in vitro assays: the initiation of differentiation of viable cells toward the promastigote stage in tissue culture Medium 199 and the staining of nonviable amastigotes with erythrosin B. The results of these assays correlated with those of a previously described in vivo assay in which viability was related to the minimum number of amastigotes in mouse liver following intravenous injection. Results indicate that erythrosin B staining should be routinely used to evaluate viability of amastigote populations, but that the initiation of differentiation of amastigotes is a more sensitive assay. For best results with the latter method, it is suggested that no more than 1.2 × 10⁷ amastigotes in 1 ml of Medium 199 be incubated at 25 C in 5% CO₂ in air. The data presented indicate that routine manipulations may affect amastigote viability.     

Impact of fermentation pH and temperature on freeze-drying survival and membrane lipid composition of <Emphasis Type="Italic">Lactobacillus coryniformis</Emphasis> Si3

During the industrial stabilization process, lactic acid bacteria are subjected to several stressful conditions. Tolerance to dehydration differs among lactic acid bacteria and the determining factors remain largely unknown. Lactobacillus coryniformis Si3 prevents spoilage by mold due to production of acids and specific antifungal compounds. This strain could be added as a biopreservative in feed systems, e.g. silage. We studied the survival of Lb. coryniformis Si3 after freeze-drying in a 10% skim milk and 5% sucrose formulation following different fermentation pH values and temperatures. Initially, a response surface methodology was employed to optimize final cell density and growth rate. At optimal pH and temperature (pH 5.5 and 34 °C), the freeze-drying survival of Lb. coryniformis Si3 was 67% (±6%). The influence of temperature or pH stress in late logarithmic phase was dependent upon the nature of the stress applied. Heat stress (42 °C) did not influence freeze-drying survival, whereas mild cold- (26 °C), base- (pH 6.5), and acid- (pH 4.5) stress significantly reduced survival. Freeze-drying survival rates varied fourfold, with the lowest survival following mild cold stress (26 °C) prior to freeze-drying and the highest survival after optimal growth or after mild heat (42 °C) stress. Levels of different membrane fatty acids were analyzed to determine the adaptive response in this strain. Fatty acids changed with altered fermentation conditions and the degree of membrane lipid saturation decreased when the cells were subjected to stress. This study shows the importance of selecting appropriate fermentation conditions to maximize freeze-drying viability of Lb. coryniformis as well as the effects of various unfavorable conditions during growth on freeze-drying survival.     

Leishmania in the chick embryo: IV. Effects of embryo age and hatching,and behavior of L. donovani in cultures of chick fibroblasts

On incubation Days 9, 11, 12, 14, or 15, chick embryos were injected intravenously with 4.0 × 10⁶L. donovani amastigotes. Embryos were incubated at 33 C immediately after infection. Numbers of amastigotes found in the liver 1 hr after injection increased as the age of embryo recipients increased. Most 14- or 15-day infected embryos hatched when allowed to do so, but many younger embryos were unable to survive at 33 C. Numbers of amastigotes in the liver of chicks, hatched after infection as embryos, decreased as the cloacal temperature of the chicks increased. Despite a 31 C incubation temperature, chicks exhibited a mean 38.3 C cloacal temperature 1 day after hatching.Chick fibroblast cultures were initiated as explants of embryo brain and infected with amastigotes from hamster spleen. Only amastigotes were seen in cultures kept at 37 C, but extracellular promastigotes and intracellular amastigotes were present in cultures at 33 C. Although promastigotes increased in number in the medium overlay at 33 C, amastigotes decreased in number at 33 C and 37 C. One intracellular amastigote was seen in a culture which had been incubated at 25 C after inoculation with promastigotes.     

Effect of environmental factors and precursors on oxalic acid production,mycelial biomass and virulence of a potential bioherbicide isolate of Sclerotium rolfsii SC64 produced in liquid culture

The fungus Sclerotium rolfsii is presently under development as a bioherbicide for broadleaf weed species using fungus-infested substrates as application material in this laboratory. The effect of environmental factors and three precursors (citric acid, ascorbic acid, and sodium succinate) on mycelial growth, oxalic acid production, and virulence by SC64 in liquid culture were investigated. The results showed that for mycelia growth the optimum liquid medium was Modified Richard's solution (MRS) among the five tested media, but potato dextrose broth (PDB) produced the maximum oxalic acid production and virulence on detached Solidago canadensis leaves. When PDB was used as the basic medium, the oxalic acid/mycelial dry weight (mg g^–1) ratio reached the peak 4 days after inoculation. The optimum temperature for oxalic acid production was at 27°C, but increased mycelial dry weight and virulence were observed at 30°C. The optimum range of initial pH value for oxalic acid accumulation was 4.0–6.0, with the optimal pH 5.0; highest mycelial growth was with an initial pH 3.5–6.0 (optimum pH 5.0) and subsequently pH 3.5–5.5 (maximum at pH 3.5). Both mycelial dry weight and oxalic acid production showed a decreasing trend as a result of the precursor of oxalic acid being added to PDB. Among the three precursors, the greatest decrease in mycelial dry weight, and oxalic acid production was caused by sodium succinate. This clarification of optimal conditions for production of mycelial biomass while insuring high concentrations of oxalic acid and high virulence should be useful for further development of this fungus as biocontrol agent.     

<Emphasis Type="Italic">Ancylobacter abiegnus</Emphasis> sp. nov., an oligotrophic member of the xylotrophic mycobacterial community

A new species of the genus Ancylobacter, exemplified by strain Z-0056, was isolated from dystrophic humified waters formed by xylotrophic fungi grown on decaying spruce wood. The cells of strain Z-0056 (0.65–0.9 μm) are coccoid, gram-negative, with fimbriae, and nonmotile. The strain is pleomorphic and reproduces by nonuniform division. Strain Z-0056 is an aerobic organoheterotroph and a mesophilic and moderately acidophilic oligotrophic microorganism. As an inhabitant of dystrophic ultrafresh waters, strain Z-0056 is sensitive to NaCl. The bacterium utilizes organic acids (acetate, pyruvate, oxalate, gluconate, malate, succinate, and citrate), as well as xylose and xylan. The microorganism grows in a pH range of 4.0–8.0, with an optimum at pH 5.5. The temperature range for growth is 15–25°C, with an optimum at 20°C. According to its ecophysiological properties, strain Z-0056 belongs to the group of ombrophilic dissipotrophs. The DNA G+C base content is 66.8 mol %. According to phylogenetic analysis, strain Z-0056 belongs to the genus Ancylobacter. Strain Z-0056 showed the highest similarity (98.3%) with the type strain of the species A. oerskovii. The phenotypic and phylogenetic properties of strain Z-0056 support classification of this microorganism within the genus Ancylobacter as the novel species Ancylobacter abiegnus sp. nov.     

Characterization of mitochondrial bioenergetic functions between two forms of Leishmania donovani – a comparative analysis

Leishmaniasis is a growing health problem in many parts of the world partly due to drug resistance of the parasite. This study reports on the fisibility of studying mitochondrial properties of two forms of wild-type L. donovani through the use of selective inhibitors. Amastigote forms of L. donovani exhibited a wide range of sensitivities to these inhibitors. Mitochondrial complex II inhibitor thenoyltrifluoroacetone and F_oF₁-ATP synthase inhibitors oligomycin and dicyclohexylcarbodiimide were refractory to growth inhibition of amastigote forms, whereas they strongly inhibited the growth of promastigote forms. This result indicated that complex II and F_oF₁-ATP synthase were not functional in amastigote forms suggesting the presence of attenuated oxidative phosphorylation in the mitochondria of amastigote forms. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited cellular multiplication and substrate level phosphorylation in amastigote forms, suggesting the role of complex I and complex III for the survival of amastigote forms. Further we studied the mitochondrial activities of both forms by measuring oxygen consumption and ATP production. In amastigote form, substantial ATP formation by substrate level phosphorylation was observed in NADPH-fumarate, NADH-fumarate, NADPH-pyruvate and NADH-pyruvate redox couples. None of the redox couple generated ATP formation was inhibited by F_oF₁-ATP synthase inhibitor oligomycin. Therefore, we may conclude that there are significant differences between these two forms of L. donovani in respect of mitochondrial bioenergetics. Our results demonstrated bioenergetic disfunction of amastigote mitochondria. Therefore, these alterations of metabolic functions might be a potential chemotherapeutic target.     

Thryoid control over biomembranes. Rat liver mitochondrial inner membranes.

The effects of hypothyroidism and one injection of l-thyroxine on oxidative phosphorylation and the composition of proteins and phospholipids were examined in vesicles prepared from rat liver mitochondria by digitonin extractions. At 30 °C, the rates of ADP phosphorylation in sites I and II were below normal, and Mg²⁺-ATPase activity was greater than normal in vesicles from hypothyroid rats. At temperatures below 20 °C and above 30 °C, the Mg²⁺-ATPase was not accelerated above normal rates, a feature of temperature dependence shared by ADP phosphorylation (Chen, Y.-D. I., and Hoch, F. L., 1976, Arch. Biochem. Biophys.172, 741–744). Respiration at 30 °C was undiminished in hypothyroid vesicles, as were the flavin and cytochrome contents, and thyroxine administration corrected the phosphorylation rate at 30 °C in 3 days without changing either respiration or electron-carrier contents. The 30 °C phosphorylation defect comprised a decreased V and K_m for ADP and a decrease in the number of phosphorylating sites (measured with oligomycin) that accounted for most of the decreased phosphorylation rates, either dependent on or independent of the adenine nucleotide carrier. Vesicles from hypothyroid rats were not detectably depleted in major protein subunits, but were abnormal in phospholipid fatty acid contents. Thyroxine injection corrected the low unsaturation index of the fatty acids and the membrane contents of linoleic acid and its fatty acyl metabolites. Hypothyroidism appears to affect oxidative phosphorylation through the altered inner membrane lipid environment, which implies that previously reported direct, reversible effects of thyroxine may mimic repletion of the membranes with unsaturated fatty acyl groups.     

Trypanosoma cruzi: A Specific Surface Marker for the Amastigote Form1

ABSTRACT. Among the known life cycle stages of Trypanosoma cruzi only the amastigote form bound lactoferrin (LF), a glycoprotein produced by neutrophils. This capacity was readily demonstrable by indirect immunofluorescence in amastigotes derived from mice, a mammalian cell culture, or grown in an axenic medium. No LF binding was detectable on trypomastigotes from blood or mammalian cells, insect-derived metacyclics or epimastigotes, or on epimastigotes grown in Warren's medium. Serum levels of LF were increased in mice acutely infected with T. cruzi, and amastigotes from the spleens of these animals were found to have the glycoprotein on their surface. The amastigote LF receptor may have biological significance in parasite-host interaction since mononuclear phagocytes also express a LF receptor, and treatment of these cells with LF has been shown to increase their capacities to take up and kill T. cruzi amastigotes in vitro. The LF receptor is the first marker for T. cruzi amastigotes for which a naturally occurring ligand has been described.     

The Mechanism of Onset of the Stationary Phase in Euglena gracilis Grown with 10 mM Succinate: Intracellular pH Values*

SYNOPSIS. When Euglena gracilis were grown with 10mM succinate at pH 3.5 the extracellular pH averaged 3.62 and the cultures had produced 6 × 10⁵ cells/ml when the stationary phase began. Oxygen consumption values reached a maximum of 30 μliters/10⁶ cells/hr. Total protein and dry weights per cell remained constant during the logarithmic phase and began to decline when the late logarithmic phase was reached. Added succinate caused the cultures in stationary phase to commence logarithmic growth once more. Onset of the stationary phase in cultures grown at pH 3.5 was due to depletion of succinate. When cultures were grown at pH 6.9 the extracellular pH averaged 7.62 and the cultures produced 3 × 10⁵ cells/ml when the stationary phase began. Oxygen consumption values reached a maximum of 20 μliters/10⁶ cells/hr during the logarithmic phase. The decline in total protein and dry weights per cell began at the beginning of the logarithmic phase and continued into the stationary phase of growth. Cultures grown at pH 3.5 should produce a larger number of cells/ml than cultures grown at pH 6.9 if the cells are responding to the unionized moiety of succinate and not the ionized moiety. At pH 3.5 83% of the succinate is unionized, whereas at pH 6.9 0.20% of the succinate is unionized. The onset of the stationary phase in cultures grown at pH 3.5 and pH 6.9 is due to lack of an adequate amount of extracellular unionized succinate. Intracellular pH values were determined in cultures grown at pH 6.9 using the weak acid DMO (5.5-dimethyl-2,4-oxazolidinedione). As the extracellular pH increased from 6.90 to 7.62, the intracellular pH increased from 5.89 to 6.89. As the extracellular pH increased from 7.62 to 8.44, the intracellular pH increased from 6.89 to 7.50.     

Adenylate energy charge of rat and human cultured hepatocytes

Summary A simple and rapid method for the assay of adenine nucleotides (ATP, ADP, and AMP) was established to evaluate the adenylate energy charge (ATP+ADP/2)/(ATP+ADP+AMP) of cultured hepatocytes. The effects of inhibitors of glycolysis, fatty acid oxidation, or oxidative phosphorylation on the energy charge were examined. The energy charges of cultured hepatocytes in rats and human were almost identical and were maintained at a high level between 6 and 24 h after changing the media (rat: 0.908±0.008n=9, human: 0.918±0.014n=6, mean ± SD). Inhibition of glycolysis with sodium fluoride or oxidative phosphorylation with antimycin A irreversibly reduced both the adenine nucleotide contents and the energy charge. However, the inhibition of fatty acid oxidation with 2-tetradecylglycidic acid did not affect the nucleotide contents, and the energy charge only decreased transiently to recover within 8 h. When the inhibitor of oxidative phosphorylation was removed, the recovery in the energy charge preceded the recovery in the adenine nucleotide contents. These findings suggest that the adenylate energy charge is a more sensitive measure of the changes in energy metabolism than the adenine nucleotide contents. Furthermore, energy charge regulates adenine nucleotide contents in cultured hepatocytes. It is important to confirm that the high energy charge of the cultured hepatocytes is maintained when these cells are used for metabolic studies.     

Regulation of the synthesis of unsaturated fatty acids by growth temperature in Bacillus subtilis

Bacillus subtilis synthesizes, almost exclusively, saturated fatty acids, when grown at 37° C. When cultures were transferred from 37° C to 20° C, a chloramphenicol- and rifampicin-sensitive synthesis of a C-16 unsaturated fatty acid was observed. Synthesis of this compound reached a plateau after 5 h at 20° C, reaching levels of 20% of the total fatty acid content. [¹⁴C]-labelled fatty acids attached as thioesters to acyl-carriers compounds, such as coenzyme A (CoA) or acyl-carrier protein (ACP) synthesized de novo by glycerol-requiring auxotrophs deprived of glycerol to arrest phospholipid synthesis, could not be desaturated at 20° C. Desaturation of these fatty acids was readily observed when glycerol was restored to the cultures allowing resumption of transfer of acyl-moieties from acyl-thioesters to phospholipid. It was also observed that depletion of the pools of CoA and ACP by starvation of pantothenate auxotrophs had no effect on the observed synthesis of unsaturated fatty acid at 20° C. The overall results indicate that synthesis of unsaturated fatty acids in B. subtilis is a cold-inducible process and that phospholipids are obligate intermediates in this fatty acid desaturation pathway.