Synthesis of β-hydroxy fatty acids and β-amino fatty acids by the strains of Bacillus subtilis producing iturinic antibiotics

The iturinic antibiotics, which contain long chain β-amino acids, are produced by Bacillus subtilis. Screening these strains for the presence of a possible precursor of the iturinic antibiotics, we isolated a lipopeptide containing β-hydroxy fatty acids. The structure of this compound was studied and it appears to be identical or structurally very similar to surfactin. The carbon chain of its β-hydroxy fatty acids was n C₁₆, iso C₁₆, iso C₁₅ or anteiso C₁₅. The percentages of each β-hydroxy fatty acids varied according to the strain producing iturinic antibiotics and were influenced by addition of branched-chain α-amino acids to the culture medium. These results demonstrate for the first time that iso C₁₄ β-hydroxy fatty acid is a constituent present in such a surfactin like lipopeptide. Besides, the presence of radioactive β-hydroxy fatty acids in the phospholipids when the strains were grown in the presence of sodium [¹⁴C]acetate seems also characterize the different strains producing iturinic antibiotics.     

Production of farnesoic acid and methyl farnesoate by mandibular organs of the crayfish,Procambarus clarkii

The mandibular organs (MO) of crustaceans secrete methyl farnesoate (MF) and farnesoic acid (FA). To better understand the secretory activity of MO, the kinetics of production and release of both compounds were determined in vitro by following incorporation of [2-¹⁴C]acetate and l-[³H-methyl]methionine into MF and [2-¹⁴C]acetate into FA by MO of Procambarus clarkii. MO released more FA than MF but contained more MF. In medium lacking unlabeled acetate, the percentage incorporation of [¹⁴C]acetate into MF, relative to [³H]methionine, was between 21 and 40%, suggesting that there may be an alternative source of C₂ units.MO produce similar amounts of MF at concentrations of acetate from 0.08 to 10 mM. However, the addition of exogenous unlabelled FA to incubation media did not stimulate the biosynthesis of MF, raising the possibility that unlike JH biosynthesis in insects, the last step in MF production may be rate-limiting. Nonetheless, exogenous FA significantly reduced the incorporation of [¹⁴C]acetate into MF, suggesting that the glands use exogenous FA to synthesize MF. The absence of stimulation of FA production by exogenous FA indicates that there is no feedback effect of this product on the early steps in the biosynthetic pathway.     

Biosynthesis and characterization of a phosphatidic acid analog containing beta-hydroxy fatty acid

A new phospholipid was shown to be biosynthesized in liver mitochondria from labeled 1-alkyl-$\text{sn}$-glycerol-3-phosphate and labeled fatty acid in the presence of ATP and CoA and its structure was shown to be 1-alkyl-2-(3-hydroxy)acyl-$\text{sn}$-glycerol-3-phosphate. Fatty acids in mitochondria were oxidized to the β-hydroxy derivatives which were utilized for the acylation of alkyl glycerophosphate. Free long chain β-hydroxy acids were also utilized by mitochondria and microsomes in the presence of ATP and CoA for the acylation of glycerophosphate derivatives to form the phosphatidate analogs.     

Simple enzymatic detection method for urinary sulfated 7α-hydroxy bile acids in normal subjects and in patients with acute hepatitis

Urinary sulfated primary bile acids, 7α-hydroxy bile acids, are detected by an enzymatic method using 7α-hydroxysteroid dehydrogenase (EC 1.1.1.-, 7α-HSD) after chromatographic fractionation on Sephadex G-25. Urinary sulfated or glucuronated bile acids are hydrolyzed by β-glucuronidase/sulfatase (EC 3.2.1.31/EC 3.1.6.1) from Helix pomatia and then released 7α-hydroxy bile acids are detected with 7α-HSD in the presence of β-NAD⁺, diaphorase (EC 1.6.99.2, from Clostridium kluyveri) and 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride. The absorbance of formazan formed during the enzymic reaction is measured at 500 nm. Excretion values of 7α-hydroxy bile acids in normal subjects and in patients with acute hepatitis were compared. This enzymatic detection method for the excretion pattern of urinary 7α-hydroxy bile acids may be useful for clinical diagnosis.     

Stable Carbon Isotope Ratios of Lipid Biomarkers of Sulfate-Reducing Bacteria

We examined the potential use of natural-abundance stable carbon isotope ratios of lipids for determining substrate usage by sulfate-reducing bacteria (SRB). Four SRB were grown under autotrophic, mixotrophic, or heterotrophic growth conditions, and the δ¹³C values of their individual fatty acids (FA) were determined. The FA were usually ¹³C depleted in relation to biomass, with Δδ¹³C_{(FA − biomass)} of −4 to −17‰; the greatest depletion occurred during heterotrophic growth. The exception was Desulfotomaculum acetoxidans, for which substrate limitation resulted in biomass and FA becoming isotopically heavier than the acetate substrate. The δ¹³C values of FA in Desulfotomaculum acetoxidans varied with the position of the double bond in the monounsaturated C₁₆ and C₁₈ FA, with FA becoming progressively more ¹³C depleted as the double bond approached the methyl end. Mixotrophic growth of Desulfovibrio desulfuricans resulted in little depletion of the i17:1 biomarker relative to biomass or acetate, whereas growth with lactate resulted in a higher proportion of i17:1 with a greater depletion in ¹³C. The relative abundances of 10Me16:0 in Desulfobacter hydrogenophilus and Desulfobacterium autotrophicum were not affected by growth conditions, yet the Δδ¹³C_{(FA − substrate)} values of 10Me16:0 were considerably greater during autotrophic growth. These experiments indicate that FA δ¹³C values can be useful for interpreting carbon utilization by SRB in natural environments.     

Epicuticular wax of Poa ampla leaves

Leaf wax of a glaucous variety of Poa ampla contains hydrocarbons (5%, C₂₃–C₃₅), esters (9%, C₃₆–C₅₆), free acids (3%, C₁₆–C₃₄), free alcohols (6%, mainly C₂₆); hentriacontane-14,16-dione (14%), 5-oxohentriacontane-14,16-dione (1%); hydroxy β-diketones (56%) and unidentified material (6%). The hydroxy β-diketones, which are more abundant in this wax than in others, were shown by ¹³C NMR to consist of 4-hydroxy (15%), 5-hydroxy (70%) and 6-hydroxy (15%) hentriacontane-14,16-diones.     

Relative importance of acetate, acetoacetate and D-beta-OH-butyrate in the transport of acetyl CoA from the mitochondria to the cytoplasm for fatty acid synthesis in mice.

Mice were injected intravenously with either 3-¹⁴C acetoacetate, 3-¹⁴C D-β-OH-butyrate or 1-¹⁴C acetate and the radioactivity of the fatty acids was measured. In liver, the values obtained for acetoacetate and β-OH-butyrate were identical and slightly higher than those for acetate. In carcass and adipose tissue, the values obtained for the β-OH-butyrate were lower than for the other two. In particle-free supernatant of liver and adipose tissue, almost no radioactivity was obtained from β-OH-butyrate, and only the acetoacetate and the acetate were used efficiently (in vitro studies). The incorporation of acetate and acetoacetate by adipose tissue supernatant is higher than that of citrate by liver supernatant.The cytoplasmic acetoacetyl CoA synthetase and acetyl CoA synthetase activity was found to be higher in adipose tissue than in the liver. β-OH-butyryl CoA synthetase was found to be much less active than the other two synthetases. Acetoacetyl CoA thiolase is very active in the mitochondria and supernatant of adipose tissue.Our results show that, in mice adipose tissue in particular, where the citrate-cleavage enzyme is not very active, acetyl CoA is probably transformed into acetoacetate so that it can leave the mitochondria to participate in cytoplasmic fatty acid synthesis.     

Effects of doxorubicin and its aglycone metabolite on calcium sequestration by rabbit heart, liver, and kidney mitochondria

Previous investigators have shown that following doxorubicin treatment heart mitochondria appear swollen and contain intramitochondrial dense inclusion bodies identified as calcium phosphate. In vitro studies have shown that similar morphological changes occur in mitochondria previously loaded with excess calcium. The present studies were performed to determine the effects of doxorubicin and its aglycone metabolite on ⁴⁵Ca²⁺ uptake by mitochondria isolated from the heart, liver, and kidney of the rabbit. Doxorubicin (100 μM) significantly inhibited the initial rate of ⁴⁵Ca²⁺ accumulated by mitochondria isolated from the three tissues. In contrast, the aglycone metabolite (100 μM) induced the reverse effect. In preloaded mitochondria the aglycone stimulated the release of calcium while doxorubicin was without effect. Mitochondria from the heart were significantly more sensitive to the effects of these anthracyclines than were mitochondria from the other two tissues. If these in vitro effects also occur in vitro, then the aglycone metabolite would be a more likely candidate in explaining the morphological changes in heart mitochondria previously described.     

Effect of yttrium on calcium-dependent processes in vertebrate myocardium

Inoptopic effect of yttrium acetate (Y³⁺) on myocardium of the marsh frog Rana ridibunda and its effect on ion transport across the inner mitochondrial membrane (IMM) of rat heart was studied. Y³⁺ was found to decrease the rate of heart contractions and to stimulate ion transport in the rat heart mitochondria in media with 10 mM glutamate and 2 mM malate. Presence of Y³⁺ induced inhibition of energy-dependent Ca²⁺ transport into mitochondria, which was expressed as a marked decrease of their swelling in the media containing 125 mM NH₄NO₃ and Ca²⁺ or 25 mM potassium acetate, 100 mM sucrose and Ca²⁺. It is suggested that the Y³⁺-induced decrease in rat muscle contractions is determined not only by direct suppressing effect of Y³⁺ on potential-modulated Ca²⁺-channels of pacemaker and contractile cardiomyocytes (CM), but also by its indirect effect on Ca²⁺-carrier in IMM. The data confirming that Y³⁺ activates energy-dependent K⁺ transport catalyzed by mitochondrial uniporter and blocks Ca²⁺-channels in the mitochondrial membrane are important for more complete understanding of mechanisms of the Y³⁺ action on vertebrates and human CM.     

Comparative study of Y<Superscript>3+</Superscript> effect on calcium-dependent processes in frog cardiac muscle and mitochondria of rat cardiomyocytes

Inotropic effects of yttrium acetate (Y³⁺) on contractions of myocardium preparations of the frog Rana ridibunda, as well as on respiration and the inner membrane potential (ΔΨ_mito) of isolated rat heart mitochondria were studied. 2 mM yttrium in Ringer solution was found to significantly reduce the amplitude of myocardium contractions, evoked by electric stimulation, and increase the half-relaxation time (n = 5). In experiments with Ca²⁺, Y³⁺ decreased the Ca²⁺-dependent basal respiration rate in rat heart mitochondria, energized by glutamate and malate, impeded the reduction in respiration of these mitochondria operating in state 3 after Chance or uncoupled by 2,4-dinitrophenol, and inhibited a Ca²⁺-induced reduction in their inner membrane potential. The data obtained are important for better understanding the mechanism underlying Y³⁺ effects on the myocardial Ca²⁺-dependent processes. Possible mechanisms of the negative inotropic effect of Y³⁺ on myocardium and its influence on the Ca²⁺-dependent processes in rat mitochondria are discussed.     

Mitochondrial activity,hemocyte parameters and lipid composition modulation by dietary conditioning in the Pacific oyster Crassostrea gigas

Several parameters can affect membrane lipid composition in bivalves, including diet. Although two fatty acids (FA) 22:6n-3 and 20:5n-3 are essential membrane components, they are sparingly synthesized by bivalves and must be obtained from their diet. Here, effects of dietary modifications of membrane lipid composition were studied at both cellular and subcellular levels in the oyster Crassostrea gigas. To this end, we compared oysters fed two monoalgal diets that differed markedly in their FA composition and a mix of both. As expected, algae impacted phospholipids, in particular 22:6n-3 and 20:5n-3, reflecting differences of dietary microalgae FA composition. Meantime, total saturated FA, total monounsaturated FA, total polyunsaturated FA and total non-methylene-interrupted FA varied little and phospholipid class composition was only slightly affected by diets. Measures made in hemocytes indicated that only mitochondrial membrane potential was affected by diets. Total ROS production as well as mitochondrial superoxide production did not differ with diet. There was no difference in phosphorylating (state 3) and non-phosphorylating (state 4) rates of oxygen consumption rates or in cytochrome c oxidase activity of mitochondria isolated from gills between the three diets. Similarly, neither cytochromes a, b, c or c ₁ content nor citrate synthase activities were changed, suggesting that number and morphology of mitochondria were not affected by dietary treatment. These results suggest that oysters could possess high homeostatic capabilities, at both cellular and subcellular levels, to minimize the effect of dietary FA and related membrane lipid FA modifications on mitochondrial functions. These capabilities could be a means to face variations in diet composition in their natural environment and to preserve important oyster physiological functions such as growth and reproduction.     

Calcium uptake by two preparations of mitochondria from heart

Ca²⁺ transport and respiratory characteristics of two preparations of cardiac mitochondria (Palmer, J.W., Tandler, B. and Hoppel, C.L. (1977) J. Biol. Chem. 252, 8731–8739) isolated using polytron homogenization (subsarcolemmal mitochondria) and limited Nagarse exposure (intermyofibrillar mitochondria) are described.The Nagarse procedure yields mitochondria with 50% higher rates of oxidative phosphorylation than the polytron-prepared mitochondria in both rat and dog. Rat hear intermyofibrillar mitochondria contain 50% more cytochrome aa₃ than the polytron preparation, whereas in the dog, cytochrome aa₃ content is not significantly different. Cytochrome oxidase activities and cytochrome c, c₁ and b contents were comparable in both populations of rat and dog heart mitochondria.The V of succinate-supported Ca²⁺ accumulation for Nagarse-prepared mitochondria from rat heart was 1.8-fold higher than the polytron-prepared mitochondria. In dog heart, the Nagarse preparation showed a 3.0-fold higher V for Ca²⁺ uptake compared to the polytron preparation. A lower apparent affinity for Ca²⁺ was demonstrated in the intermyofibrillar mitochondria for both species (K_m is 2–2.5-fold higher). The Hill coefficient was 1 both mitochondrial types. Subsarcolemmal mitochondria from both species were treated with Nagarse to determine the role of this treatment on the observed differences. Nagarse did not alter any kinetic parameter of Ca²⁺ uptake.The properties of these mitochondria with reference to their presumed intracellular location may pertain to the role of mitochondria as an intracellular Ca²⁺ buffering mechanism in contractile tissue.     

Origin of acetate in spinach leaf cell

Mitochondria were isolated from spinach (Spinacia oleracea L.) leaves using a Percoll gradient step. The high purity of the organelle fraction is demonstrated by electron microscopy and biochemical parameters. In the matrix space of these mitochondria, a short-chain acyl-coenzyme A hydrolase is present that converts acetyl-coenzyme A to acetate and coenzyme A with reasonable rates (K_m, 150 micromolar; V_max, 140 nanomoles acetate formed milligram¹ protein hour⁻¹). The enzyme is product inhibited by coenzyme A-sulfhydryl, other thiols are ineffective; however, the disulfides 5,5′-dithio-bis-(2-nitrobenzoate) and cystamine stimulate the hydrolysis. The possible role of this mitochondrial enzyme as a means of generating free acetate from pyruvate via acetyl-coenzyme A in the mitochondria of mature spinach leaves is discussed. It is suggested that free acetate moves rapidly from the mitochondrion to the chloroplast where acetyl-coenzyme A synthetase, solely localized in this organelle, converts the metabolically inert free acetate to the highly active acetyl-coenzyme A.     

Ruthenium red-sensitive and Ruthenium Red-insensitive release of calcium by mitochondria isolated from rat liver and from rat heart

EGTA (ethanedioxybis(ethylamine)tetra-acetic acid) induced a release of Ca²⁺ from mitochondria isolated from both rat liver and rat heart that was inhibited by Ruthenium Red. The concentration of Ruthenium Red giving half-maximal inhibition was about 350 pmol/mg of protein, a value approximately 7 times greater than that giving half-maximal inhibition of the initial rate of Ca²⁺ transport. The EGTA-induced release of Ca²⁺ was temperature-dependent and was inhibited by the local anaesthetic, nupercaine.Pi, acetate, and tributyltin in the presence of Cl^?, inhibited the Ruthenium Red-sensitive Ca²⁺ release induced by EGTA, whereas these agents enhanced the Ruthenium Red-insensitive release of Ca²⁺ induced by acetoacetate in liver and heart mitochondria and by Na⁺ in heart mitochondria.     

The biosynthesis of ubiquinone in isolated rat heart cells

Beating heart cells were isolated from the adult rat and the biosynthesis of ubiquinone was studied. These cells were able to incorporate p-hydroxy[U-¹⁴C]benzoate into ubiquinone and some unidentified compounds, presumably intermediates in the biosynthesis of ubiquinone. The unidentified compounds were labile to alkali and were also labeled by [5-³H]-mevalonate and [methyl-³H]methionine, but not by p-hydroxy[carboxy-¹⁴C]benzoate. They appear to be chromatographically different from 5-demethoxy ubiquinone and 5-desmethyl ubiquinone. Addition of unlabeled mevalonate stimulated the incorporation of p-hydroxy [U-¹⁴C]benzoate into ubiquinone and the other compounds. The addition of dimethylsulfoxide to the isolated cells or the isolation medium caused inhibition of ubiquinone biosynthesis. Adriamycin was not inhibitory to the biosynthesis of ubiquinone in the cells. The advantages of these cells are the rapidity and ease in studying the biosynthesis of ubiquinone from various precursors and its regulation.     

Osmotic swelling of heart mitochondria in acetate and chloride salts. Evidence for two pathways for cation uptake.

Swelling of nonenergized heart mitochondria suspended in acetate salts appears to depend on the activity of an endogenous cation/H⁺ exchanger. Passive swelling in acetate shows a characteristic cation selectivity sequence of Na⁺ >Li⁺ >K⁺, Rb⁺, Cs⁺, or tetramethylammonium, a sharp optimum at pH 7.2–7.3, activation by Ca²⁺, and loss of activity on aging which can be related to loss of endogenous K⁺. The reaction is nearly insensitive to either addition of exogenous Mg²⁺ or removal of membrane Mg²⁺ with EDTA. Each of these characteristics of passive swelling in acetate salts is duplicated in chloride media when tripropyltin is added to induce Cl^?/OH^? exchange. In contrast to nonenergized mitochondria, swelling of respiring mitochondria has been postulated to depend on electrophoretic uptake of cations in response to an interior negative membrane potential. Respiration-dependent swelling in acetate shows an indistinct cation selectivity sequence with Li⁺ and Na⁺ supporting higher rates of swelling at higher efficiency than K⁺, Rb⁺, and Cs⁺. The high rates of respiration-dependent swelling in Li⁺ and Na⁺ are inhibited by low levels of exogenous Mg²⁺ (K_i of 5–10 μm), but a significant swelling with almost no cation selectivity persists in the presences of 2 mm Mg²⁺. Removal of membrane Mg²⁺ by addition of EDTA strongly activates the rate of respiration-dependent swelling and converts a sigmoid dependency of swelling rate on Li⁺ concentration to a hyperbolic one with a Km of about 14 mm Li⁺. The cation selectivity and Mg²⁺ dependence of the reaction induced in chloride salts by tripropyltin are identical to these properties in acetate. Energy-dependent swelling in acetate shows optimum activity at pH 6.5 which appears related to the availability of free acetic acid, since the corresponding reaction induced in chloride shows a broad optimum at about pH 7.5. These studies support the concept that monovalent cations enter nonenergized mitochondria by electroneutral exchange with protons but penetrate respiring mitochondria by electrophoretic movement through one or more uniport pathways. They further suggest that both a Mg²⁺-sensitive uniport with high activity for Na⁺ and Li⁺ and a Mg²⁺-insensitive pathway with little cation discrimination are available in the membrane.     

Fatty acid composition of membrane lipids and energy metabolism in mitochondria of pea seedlings under water deficit

The impacts of water deficit and melamine salt of bis(oximethyl)phosphonic acid (melaphen) on the fatty acid (FA) composition of membrane lipids and energy metabolism in mitochondria of 5-day-old pea (Pisum sativum L., cv. Flora-2) seedlings were studied. Insufficient watering resulted in the accumulation of saturated and a decrease in the content of unsaturated FAs with 18 and 20 carbon atoms. Seed treatment with 3 × 10^?10 M melaphen prevented these changes in the FA composition in the mitochondrial membrane lipids. Changes in the FA compositions of membrane lipids were correlated with changes in energy metabolism in mitochondria: the efficiency of oxidative phosphorylation and the rate of NAD-dependent substrate oxidation in the presence of ADP and FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) were reduced. A close correlation was observed between changes in the highest rates of NAD-dependent substrate oxidation and the relative content of FAs with 18 (r = 0.76489) and 20 (r = 0.9637) carbon atoms. The regulatory role of C₁₈ and C₂₀ unsaturated FAs in the mitochondrial energy metabolism of pea seedlings is discussed.     

Long chain esters of Virola species

The esters of n-fatty acids and ω-hydroxy n-fatty acids of β-sitosterol, D-glucose and ferulic acid (trans and cis) as well as β-sitosterol, fatty acids and β-sitosteryl-β-D-glucoside were isolated from three Virola species and identified by optical data and chemical reactions. A novel series of acidic esters derived from C₂₂C₂₉ ω-hydroxy fatty acids and cis- and trans-ferulic acid is reported for the first time. These compounds also occurred as the corresponding diester 1-monoglycerides whereas the ω-hydroxy acids themselves were also present as the corresponding glucosyl esters.     

Fatty acid composition of <Emphasis Type="Italic">Wautersia eutropha</Emphasis> lipids under conditions of active polyhydroxyalkanoates synthesis

The fatty acid composition of the lipids of a Wautersia eutropha polyhydroxyalkanoate-producing strain was studied by chromato-mass spectrometry. A total of 27 fatty acids were identified; their distribution in the cell fractions was determined. In the cytoplasmic membrane, palmitic, palmitoleic, and cis-vaccenic acids were the major components. Long-chain β-hydroxy acids and myristic acids (components of the lipopolysaccharides of the cell envelope) predominated in the fraction of strongly bound lipids. When the polymer was actively synthesized, the content of cyclopropane acids in the easily extracted lipids increased and the content of the corresponding monoenoic acids decreased. The strongly bound lipids had a high content of long-chain β-hydroxy acids (more than 50% of the total fatty acids). These results made it possible to determine the source of polyhydroxyalkanoate (PHA) contamination and to choose the strategy for their purification.     

Synthesis of [2H]gibberellins from steviol using the fungus Gibberella fujikuroi

[²H]Steviol (ent-13-hydroxykaur-16-en-19-oic acid) was synthesized from steviol acetate norketone (ent-13-acetoxy-16-oxo-17-norkauran-19-oic acid) by the Wittig reaction using (methyl-d₃)triphenylphosphonium bromide. A mixture of steviol analogs was produced containing from one to four ²H/molecule. [²H]Steviol was fed to strain LM-45-399 of the fungus Gibberella fujikuroi which was grown on synthetic medium (ICI, 0% N) in the presence of the growth retardant CCC. [²H]GA₁, [²H]GA₁₈, [²H]GA₂₃ and [²H]GA₅₃ were isolated from the fungal medium after 4 days. This strain converted steviol to 13-hydroxy GAs in the highest yields of the four Gibberella strains tested, and in amounts suitable for metabolic studies with higher plants.