The Effect of Temperature on the Fatty Acid Composition of Tetrahymena pyriformis WH-14

SYNOPSIS. A reduction in the growth temperature of Tetrahymena pyriformis strain WH-14 from 35 C to 15 C resulted in distinct alterations in the fatty acid composition of the glycerophospholipids. The proportion of normal saturated acids declined from 26 to 19%; palmitoleic acid increased by 6%, and the composition of the polyunsaturated fatty acids increased in 18:2 Δ^6,11(n) and decreased in 18:2 Δ^9,12(n) and 18:3 Δ^6,9,12(n). The unsaturation index (the average number of double bonds/100 molecules) did not change with a shift in temperature.
Two biosynthetic pathways exist in Tetrahymena for the formation of unsaturated fatty acids. The observed changes in fatty acid composition that accompany a lowering of the environmental temperature can be accounted for by a reduction in the accumulation of products of the fatty acid pathway leading to the formation of γ-linolenic acid [16:0(n) → 18:0(n) → 18:1 Δ⁹(n) → 18:2 Δ^9,12(n) → 18:3 Δ^6,9,12(n)] and an increase in the components of the pathway leading to the formation of 18:2 Δ^6,11(n) [16:0(n) → 16:1 Δ⁹(n) → 18:1 Δ¹¹(n) → 18:2 Δ^6,11(n)]. The data suggest that the regulatory mechanism in Tetrahymena differs from that found in some bacteria where a simple substitution of unsaturated fatty acids for saturated fatty acids occurs at low culture temperatures.     

Biosurfactant production by Pseudomonas aeruginosaGS3 from molasses

Patterns of long-chain faecal fatty acids were studied by gas–liquid chromatography in 55 newborn infants in a neonatal intensive care unit. Decreased fractions of fatty acid C16 : 1⁹ and increased fractions of C16 : 0 and C17 : 0 were associated with the occurrence of abdominal distension. Decreased fractions of C16 : 1⁹ and C18 : 2^9,1 were associated with diarrhoea. Flatulence was found in infants who had relatively smaller amounts of fatty acids C17 : 0D and C15 : 0 in their faecal samples. The differences in the patterns of faecal fatty acids are due to differences in bacterial flora. The results support the hypothesis that the initial intestinal colonization plays a role in the later gastrointestinal signs of newborn infants.     

Evidence for the synthesis of the multi-positional isomers of monounsaturated fatty acid in Methylococcus capsusatus by the anaerobic pathway

Abstract The biosynthesis of the positional isomers of the monounsaturated fatty acids of Methylococcus capsulatus (Bath) has been investigated by studying the incorporation of [2-¹⁴C]malonyl CoA into long-chain fatty acids in vitro. The major unsaturated products were Δ ⁹ 16:1 and Δ ¹¹ 18:1; however, Δ ⁸, Δ ¹⁰ and Δ ¹¹ 16:1, as well as, Δ ¹⁰, Δ ¹² and Δ ¹³ 18:1 were also synthesized. The exclusion of O₂ from the reaction vessel did not affect the synthesis of unsaturated fatty acids or the double bonds positions. Cerulenin inhibited the synthesis of unsaturated fatty acid more than saturated fatty acid. The use of both [1-¹⁴C] octanoate and [1-¹⁴C] decanoate as substrate resulted in the synthesis of long-chain fatty acids, however, unsaturates were only synthesized from octanoate. These results imply that the unique positional isomers of M. capsulatus are not synthesized by an aerobic mechanism.     

Nisin induces changes in membrane fatty acid composition of Listeria monocytogenes nisin-resistant strains at 10°C and 30°C

Listeria monocytogenes isolates resistant to 10⁵ IU ml^-1 nisin were obtained at 30°C (NR30) and at 10°C (NR10). Nisin prolonged the lag phase of isolate NR30 at 10°C. Isolates NR30 and NR10 did not produce a nisinase. Protoplasts of isolate NR30 were unaffected by exposure to nisin. The fatty acid composition from the wild-type strain and NR isolates was determined. As expected, temperature-induced differences in the C15/C17 fatty acid ratios were found. Growth of the NR strains in the presence of nisin resulted in significantly different C15/C17 ratios and a significant increase in the percentage of C16:0, C16: 1, C18:0 and C18: 1 fatty acids at 10°C and 30°C. Both the NR10 and NR30 isolates had similar growth rates at low temperatures, but these were slower than the wild-type strain. These results indicate that 'nisin resistance'is an environmentally defined phenotype and that nisin induces changes in the fatty acid composition of the membrane in L. monocytogenes nisin-resistant isolates regardless of the growth temperature.     

Poikilothermic traits and thermoregulation in the Afrotropical social subterranean Mashona mole-rat (Cryptomys hottentotus darlingi) (Rodentia: Bathyergidae)

When acclaimated for two months at 26 C the social Mashona mole-rat Cryptomys hottentotus darlingi (±S.D.) resting metabolic rate (RMR) of 0·98±0.·14cm²O₂g _-1 h_-1 ( n =21), within a thermal neutral zone (TNZ) of 28 31·5 C ambient temperature (Ta). The body temperature (Tb) of the mole-rat is very low. 33·3±0·5 C, and remained stable between 25 31·5 C ( n =28). Above 33 C. Tb increased to a mean of 34·±0· C (n=28) (Ta range 33 39 C). Below Ta 25 C. Tb showed strong poikilothermic tendencies, with Tb dropping to a mean of 26·8±1·16 C. whereas above Ta25 C. Tb varied in a typically endothermic pattern. The conductance is high 0·19±0·03 cm² O_2g¹ C ¹ (n=28) at the lower limit of thermoneutrality. The mean RMR at 18 C (the lowest Ta tested) was 2·63 ± 0·55 cm³ O₂g ¹ h ¹ (n=7) which is 2·6 times that of the resting metabolic rate in the TNZ.     

Utilization of Uniformly Labeled 13C-Polyunsaturated Fatty Acids in the Synthesis of Long-Chain Fatty Acids and Cholesterol Accumulating in the Neonatal Rat Brain

Abstract: Polyunsaturated fatty acids are needed for normal neonatal brain development, but the degree of conversion of the 18-carbon polyunsaturated fatty acid precursors consumed in the diet to their respective 20-and 22-carbon polyunsaturates accumulating in the brain is not well known. In the present study, in vivo ¹³C nuclear magnetic resonance spectroscopy was used to monitor noninvasively the brain uptake and metabolism of a mixture of uniformly ¹³C-enriched 16-and 18-carbon polyunsaturated fatty acid methyl esters injected intragastrically into neonatal rats. In vivo NMR spectra of the rat brain at postnatal days 10 and 17 had larger fatty acid signals than in uninjected controls, but changes in levels of individual fatty acids could not be distinguished. One day after injection of the U-¹³C-polyunsaturated fatty acid mixture, ¹³C enrichment (measured by isotope ratio mass spectrometry) was similar in brain phospholipids, free fatty acids, free cholesterol, and brain aqueous extract; ¹³C enrichment remained high in the phospholipids and cholesterol for 15 days. ¹³C enrichment was similar in the main fatty acids of the brain within 1 day of injection but 15 days later had declined in all except arachidonic acid while continuing to increase in docosahexaenoic acid. These changes in ¹³C enrichment in brain fatty acids paralleled the developmental changes in brain fatty acid composition. We conclude that, in the neonatal rat brain, dietary 16-and 18-carbon polyunsaturates are not only elongated and desaturated but are also utilized for de novo synthesis of long-chain saturated and monounsaturated fatty acids and cholesterol.     

Release of Neurotransmitter Amino Acids from Synaptosomes: Enhancement of Calcium-Independent Efflux by Oleic and Arachidonic Acids

Abstract: The release of preloaded [¹⁴C]neuroactive amino acids (glutamic acid, proline, γ-aminobutyric acid) from rat brain synaptosomes can occur via a time-dependent, Ca²⁺ -independent process. This Ca²⁺-independent efflux is increased by compounds that activate Na⁺ channels (veratridine, scorpion venoms), by the ionophore gramicidin D, and by low concentrations of unsaturated fatty acids (oleic acid and arachidonic acid). Saturated fatty acids have no effect on the efflux process. Neither saturated nor unsaturated fatty acids have an effect on the release of [¹⁴C]leucine, an amino acid not known to possess neurotransmitter properties. The increase in the efflux of neuroactive amino acids by oleic and arachidonic acids can also be demonstrated using synaptosomal membrane vesicles. Under conditions in which unsaturated free fatty acids enhance amino acid efflux, no effect on ²²Na⁺ permeability is observed. Since Na⁺ permeability is not altered by fatty acids, the synaptosomes are not depolarized in their presence and, thus, the Na⁺ gradient can be assumed to be undisturbed. We conclude that unsaturated fatty acids represent a potentially important class of endogenous modulators of neuroactive amino acid transport in nerve endings and further postulate that their action is the result of an uncoupling of amino acid transport from the synaptosomal Na⁺ gradient.     

Identification of the Palmitoylation Site in Rat Myelin P0 Glycoprotein

Abstract: P₀ glycoprotein, the major protein of PNS myelin, contains approximately 1 mol of covalently bound long-chain fatty acids. To determine the chemical nature of the fatty acid-protein linkage, P₀ was labeled in rat sciatic nerve slices with [³H]palmitic acid and subsequently treated with various reagents. The protein-bound palmi-tate was released by incubation with the reducing agents dithiothreitol and 2-mercaptoethanol, and with 1 M hydrox-ylamine at pH 7.5. In addition, P₀ was deacylated by treatment with 10 m M NaBH₄ with the concomitant production of [³H]hexadecanol, indicating that the fatty acid is bound in a thioester linkage. This conclusion was supported further by the fact that deacylation with hydroxylamine generated free thiol groups, which were titrated with [¹⁴C]-iodoacetamide. To identify the cysteine residue involved in the thioester linkage, [¹⁴C]carboxyamidomethylated P₀was digested with trypsin and the resulting peptides analyzed by reversed-phase HPLC. Identification of the radioactive protein fragments by amino acid analysis and amino-terminal peptide sequencing revealed that Cys¹⁵³ in rat P₀ glycoprotein is the acylation site. The acylated cysteine is located at the junction of the putative transmem-brane and cytoplasmic domains. This residue is also present in the P₀ glycoprotein of other species, including human, bovine, mice, and chicken.     

Nocardioides halotolerans sp. nov., isolated from soil on Bigeum Island, Korea

A strictly aerobic, Gram-positive, motile, coccoid-shaped, halotolerant actinobacterium (10% NaCl, w/v), designated MSL-23^T, was isolated from a soil sample on Bigeum Island, Korea. Results of 16S rRNA gene sequence analysis indicated that the isolate belonged to the genus Nocardioides, with the highest sequence similarity (95.63%) being to Nocardioides kribbensis KCTC 19038^T. The major menaquinone was MK-8(H₄), and the predominant cellular fatty acids were i-C_16:0, ai-C_17:0, C_18:1 ω9c and 10-methyl-C_16:0. The DNA G+C content was 69.7 mol%. The 16S rRNA gene sequence of strain MSL 23^T and its chemotaxonomic properties showed it to be unique in the genus Nocardioides. Phenotypic characteristics distinguished strain MSL-23^T from other Nocardioides species. On the basis of the phenotypic, chemotaxonomic and phylogenetic data strain MSL-23^T represents a novel species, for which the name Nocardioides halotolerans sp. nov. is proposed, with MSL-23^T (=KCTC 19274^T=DSM 19273^T) as the type strain.     

Fatty acid and lipid compositions of Conidiobolus

The fatty acid composition of the total lipids from two Conidiobolus species was studied by gas—liquid chromatography. The major fatty acids of C. lamprauges were palmitic acid (C_16:0), oleic acid (C_18:1), linolenic acid (C_18:3), and arachidonic acid (C_20:4). For C. eurymitus , myristic acid (C_14:0), C_16:0, and linoleic acid (C_18:2) were the most abundant acids. The fatty acid composition of C. eurymitus was quite different from that of the Conidiobolus species as mentioned in other reports. The lipid composition of the total lipids of C. lamprauges and C. eurymitus was also studied by thinchrography on quartz rods. Triglycerides and phospholipids were the major components in the two Conidiobolus species.     

Phosphatidylcholine molecular species involved in Γ-linolenic acid biosynthesis in microsomes from borage seeds

Boraginaceae seeds are particularly rich in Γ -linolenic acid (6,9,12-octadecatrienoic acid, Γ -18:3). In microsomes, the analysis of phosphatidylcholine (PC) molecular species by HPLC led to identification of 15 different molecular species; among them 4 contained Γ -18:3, mostly at position 2 of sn -glycerol. Time courses of acylation and desaturation in PC molecular species were examined when [¹⁴C]oleoyl-CoA or [¹⁴C]linoleoyl-CoA was provided as substrates to isolated microsomes. With [¹⁴C]oleoyl-CoA or [¹⁴C]linoleoyl-CoA and in the absence of NADH, 3 main labelled PC molecular species were found: 18:2/[¹⁴C]18:1, 16:0/[¹⁴C]18:1 and 18:1/[¹⁴C]18:1. When NADH was present in the incubation medium, the fatty acids were progressively desaturated by the Δ12- and Δ6-desaturases successively (with [¹⁴C]oleoyl-CoA as precursor) or by the Δ6-desaturase alone (with [¹⁴C]linoleoyl-CoA as precursor). In both types of experiments, 7 final desaturation products in microsomes were evidenced; among them, 3 contained radioactive Γ -18:3, i.e . 18:2/[¹⁴C] Γ -18:3, 18:1/[¹⁴C] Γ -18:3 and 16:0/[¹⁴C] Γ -18:3. While the Δ12-desaturase had no specificity for position on the glycerol backbone, labelled Γ -linolenic acid was recovered exclusively in the sn -2 position.     

Cations Affect [3H]Mazindol and [3H]WIN 35,428 Binding to the Human Dopamine Transporter in a Similar Fashion

Abstract: The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn²⁺, Mg²⁺, Hg²⁺, Li⁺, K⁺, and Na⁺ were assessed on the binding of [³H]mazindol and [³H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn²⁺ (30–100 µ M ) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [³H]mazindol; Mg²⁺ (0.1–100 µ M ) had no effect; Hg²⁺ at ∼3 µ M stimulated binding to membranes, with a relatively smaller effect for [³H]mazindol than [³H]WIN 35,428 at 0°C, and at 30–100 µ M inhibited both intact cell and membrane binding; Li⁺ and K⁺ substitution (30–100 m M ) inhibited binding to membranes more severely than to intact cells; and Na⁺ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn²⁺ at 0 and 21°C and Hg²⁺ at 0°C.     

Carbon isotope-labelling experiments indicate that ladderane lipids of anammox bacteria are synthesized by a previously undescribed, novel pathway

Ladderane lipids are unusual membrane lipids of bacteria that anaerobically oxidize ammonium to dinitrogen gas (anammox). Ladderane lipids contain linearly concatenated cyclobutane rings for which the pathway of biosynthesis is currently unknown. To investigate the possible biosynthetic routes of these lipids, 2-¹³C-labelled acetate was added to a culture of the anammox bacterium Candidatus Brocadia fulgida. Labelling patterns obtained by high-field ¹³C nuclear magnetic resonance spectroscopy of isolated lipids indicated that C . Brocadia fulgida synthesizes C_16:0 and iso C_16:0 fatty acids according to the known pathway of type II fatty acid biosynthesis. The ¹³C-labelling pattern of the C₈ alkyl chain of the C₂₀ [3] ladderane monoether also indicated the use of this route. However, carbon atoms in the cyclobutane rings and the cyclohexane ring were nonspecifically labelled and did not correspond to known patterns of fatty acid synthesis. Taken together, our results indicate that it is unlikely that ladderane lipids are formed from the cyclization of polyunsaturated fatty acids as hypothesized previously and suggest an alternative, although as yet unknown, pathway of biosynthesis.     

Measurement of the Membrane Potential of Isolated Nerve Terminals by the Lipophilic Cation [3H]Triphenylmethylphosphonium Bromide

Abstract: The lipophilic cation [³H]triphenylrnethylphosphonium bromide ([³H]TPMP⁺) was investigated as a measure of the membrane potential of synaptosomes. Conditions under which [³H]TPMP⁺ achieved an equilibrium distribution were tested. The toxicity of TPMP has been studied relative to its inhibitory effects on [³H]y-aminobutyric acid ([³H]GABA) transport. In some experiments the distribution of ⁸⁶RbZ⁺ and [³H]TPMP⁺ was changed upon incubation in the presence of elevated levels of K⁺, ouabain, or KCN, or at 0°C in a way that would be expected from the membrane potential. In normal incubation conditions a membrane potential of ∼−60 mv was calculated.     

Occurrence of esterified triterpenoid alcohols in the leaves of Pilosella officinarum

The mature winter leaves of Pilosella officinarum coll. Schultz and Schultz contained 29 mg esterified triterpenoid alcohols (g dry weight)⁻¹, of which over 80% was esterified with long-chain (C16-C18) fatty acids. The major fatty acids were 16:0, 18:2ω6 and 18:3ω3 and their amounts varied according to the season and stage of leaf development. In mature leaves the amount of triterpenoid alcohols esterified with long-chain fatty acids varied only slightly with the season. The changes in the fatty acid proportions in late winter-early spring, however, indicated a turnover in the esterified triterpenoid alcohol pool. In late winter after the snow had melted a 50% decrease occurred in the amount of triterpenoid alcohols esterified with short-chain fatty acids. The amount of esterified triterpenoid alcohols in immature leaves was relatively low [4 mg (g dry weight)⁻¹].
In the mature leaves esterified triterpenoid alcohols were found in globules whose appearance varied greatly according to the season. The globules were partly dissolved in late winter, and this together with the activation of respiration, suggests that the globules function as short-term energy reservoirs. Relationships between the appearance of lipid globules and the amount of esterified triterpenoid alcohols are discussed.     

Detection and quantification of Desulforhabdus amnigenus in anaerobic granular sludge by dot blot hybridization and PCR amplification

A species-specific 16S rRNA oligonucleotide probe (ASRB1) was developed for the detection of Desulforhabdus amnigenus in anaerobic granular sludge. The presence of nucleic acids from cells of D. amnigenus in granular sludge was determined using ASRB1 as a specific primer for polymerase chain reaction (PCR) amplification or as a probe for dot blot hybridizations. The detection threshold and the reproducibility of these two methods were determined with sludge amended with 10⁴–10¹⁰ D. amnigenus cells per gram of volatile suspended solids (VSS). For D. amnigenus cells with a ribosomal RNA content of 15 fg cell⁻¹, the lowest number of target cells detected by hybridization was 1 × 10⁸ cells g⁻¹ VSS. With the PCR amplification method the lowest number of target cells which could be detected was 1 × 10⁷ g⁻¹ VSS. This corresponds to a threshold level for hybridization of 0·1–0·001‰ of the total bacterial sludge population, while the threshold level obtained with the PCR approach amounted to 0·01–0·0001‰. The rRNA content of D. amnigenus was found to be affected by the growth rate and the growth phase, and it ranged from 19 fg cell⁻¹ in slow-growing cultures to 90 fg cell⁻¹ in fast-growing cultures. Therefore, the detection threshold of the dot blot hybridization method for fast-growing cells is lower than for slow-growing cells.     

Effect of chill and freezing temperatures on survival of Vibrio parahaemolyticus inoculated in homogenates of oyster meat

Survival of Vibrio parahaemolyticus was determined in oyster meat homogenates at various temperatures. (4°C, 0°C, -18°C and -24°C) and bacterial levels (10², 10⁴, 10⁵ and 10⁷ ml^-1). In all cases, the numbers of V. parahaemolyticus were a logarithmic function of log time. This study indicates that high numbers of V. parahaemolyticus can be inactivated at low temperatures. The time of total inactivation depends on the initial number of micro-organisms and incubation temperature. It is possible to use this information to determine the storage time necessary to reduce V. parahaemolyticus hazards in fish.     

Temperature-dependent changes in phospholipid and fatty acid composition and membrane lipid fluidity of Yersinia enterocolitica

Temperature-dependent compositional changes of phospholipids and their fatty acids were analysed in Yersinia enterocolitica grown at 5°, 25° and 37°C. The relative amounts of the four phospholipids, phosphatidylethanolamine (75–78%), phosphatidylglycerol (10–11%), cardiolipin (<7%) and lysophosphatidylethanolamine (<5%), were essentially the same at all growth temperatures. The degree of fatty acid unsaturation of the four phospholipids increased with decrease in growth temperature, mainly due to an increase of C_16:1 and C_18:1 and a corresponding decrease of C_16;0, C_18:0 and cyclo C_17:0. An electron spin resonance spectroscopic study of the membrane lipids showed that membrane lipid fluidity was enhanced by decreasing the growth temperatures. The changes in fatty acid composition of phospholipids in response to varied temperatures were consistent with the temperature-dependent changes in the membrane lipid fluidity of Y. enterocolitica , and were similar to those reported for other bacteria.     

Physiological mechanisms of buoyancy in eggs from brackish water cod

Newly fertilized eggs of brackish water (Gotland, Baltic Sea) and marine (Lofoten, Norway) cod were investigated with regard to specific gravity, wet and dry weight, water content, chorion weight, and content of protein, free amino acids (FAA), and ions. The eggs had neutral buoyancies equivalent to a salinity of 14^.3% (range 11^.5–16^.2%) in brackish water, and 33^.0% (range 31^.8–34^.5%) in the marine environment. A buoyancy model was developed and showed that this difference was mainly caused by differences in egg water content which was 96.6 ± 0^.47% and 92.7 ± 0.45% in the brackish and marine eggs, respectively. The higher water content of the brackish eggs resulted from increased water uptake during final oocyte maturation due to higher intracellular contents of FAA, Cl ^– and NH₄⁺. SDS polyacrylamide gel electrophoresis of eggs and oocytes, and measurements of egg protein content suggested that the FAA pool of both egg types originated from hydrolysis of specific yolk proteins. The main contributor seemed to be a protein with a molecular weight of 100 kDa.     

Description of Oxalicibacterium horti sp. nov. and Oxalicibacterium faecigallinarum sp. nov., new aerobic, yellow-pigmented, oxalotrophic bacteria

Three strains of aerobic, Gram-negative, rod-shaped, non-spore-forming, yellow-pigmented bacteria (OD1^T, YOx^T and NS13), which were isolated in previous studies by enrichment in a mineral medium with potassium oxalate as the sole carbon source, were characterized. On the basis of 16S rRNA gene sequence similarity, strains OD1^T, YOx^T and NS13 belong to the Betaproteobacteria , most closely related to Oxalicibacterium flavum TA17^T (97.2–99.7% sequence similarity). The major whole-cell fatty acids were C_16:0, C_16:1ω7c and C_17:0 cyclo. The results of DNA–DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strains OD1^T and YOx^T from O. flavum TA17^T and from each other. Therefore, it is concluded that the strains OD1^T and YOx^T represent novel species within the genus Oxalicibacterium , for which the names Oxalicibacterium horti sp. nov. (type strain OD1^T=DSM 21640^T=NBRC 13594^T) and Oxalicibacterium faecigallinarum sp. nov. (type strain YOx^T=DSM 21641^T=CCM 2767^T) are proposed.