Metabolic responses to intracerebroventricular leptin and restricted feeding.

Leptin is a hormone secreted by adipocytes, which plays an important role in the control of food intake and metabolic processes. In the current study, a dose-dependent relationship was shown between a bolus intracerebroventricular rat recombinant leptin administration and reductions in food intake and body weight in Sprague-Dawley rats. During the 24 h postinjection period, food intake was decreased by 24, 26, and 52% with 0.625, 2.5, and 10 microg of leptin, respectively. Body weight was reduced by 2, 3, and 5% at 24 h after leptin administration at the doses of 0.156, 2.5, and 10 microg, respectively. Furthermore, indirect calorimetry demonstrated that five daily i.c.v. injections of leptin resulted in an increase in heat production per unit of metabolic body size and fat oxidation by approximately 10 and 48%, respectively. In contrast, food-restricted rats that consumed the equivalent amount of food as leptin-treated rats for 5 days decreased their energy expenditure by 10%. Food restriction was found to decrease respiratory quotient in a similar pattern as the leptin administration. When ad lib feeding was resumed, food-restricted rats quickly recovered their normal food intakes, body weights, and metabolism. Conversely, leptin treatment has prolonged effects on body weight resulting from different metabolic responses than food restriction. Leptin not only suppresses food intake, but also enhances energy expenditure to reduce fat depots.

Virulence plasmid pJM1 prevents the conjugal entry of plasmid DNA into the marine fish pathogen Vibrio anguillarum 775.

Studies involving the introduction of cloned homologous genes into Vibrio anguillarum revealed that several plasmids could not be conjugally introduced into V. anguillarum 775(pJM1), but were transmissible to the pJM1-cured derivative H775-3. Recombinant pBR322 plasmids containing V. anguillarum genomic DNA inserts were mobilized from Escherichia coli donors, using pRK2013, into V. anguillarum H775-3 recipients at frequencies of 10(-6) to 10(-5) per recipient. When identical matings were performed with V. anguillarum 775(pJM1) recipients, the infrequent exconjugants recovered carried the pBR322-based plasmid but had lost the large virulence plasmid pJM1. Similar studies were carried out with plasmid RP4 and with recombinant derivatives of the closely related broad-host-range plasmid pRK290. While RP4 was transmissible from E. coli to V. anguillarum H775-3 at frequencies of 6.7 x 10(-2) per recipient, transmission to V. anguillarum 775(pJM1) recipients occurred at frequencies of only 2.5 x 10(-7). When pRK290 contained V. anguillarum DNA inserts, the only exconjugants recovered had lost pJM1, or contained pJM1 and a deletion derivative of the recombinant pRK290 plasmid where all of the DNA insert had been deleted. The use of Dam-, Dcm-, or EcoK- methylation-deficient E. coli donor strains failed to result in appreciable numbers of V. anguillarum 775(pJM1) exconjugants that contained the desired transferred plasmids. Following UV mutagenesis, a derivative of V. anguillarum 775(pJM1) was isolated that would accept conjugally transferred plasmid DNAs at frequencies similar to those observed when using V. anguillarum H775-3 recipients. These data suggest that virulence plasmid pJM1 mediates a restriction system that prevents conjugal transmission of plasmid DNA from E. coli donors into V. anguillarum 775(pJM1). This putative restriction system appears not to be directed towards Dam-, Dcm-, or EcoK-methylated DNA, and appears not to involve a Type II restriction endonuclease.

A new restriction endonuclease from the anaerobic bacterium, Desulfovibrio desulfuricans, Norway.

The purification and characterization of a new restriction endonuclease, Dde 1, from a sulfate-reducing, anaerobic bacterium, Desulfovibrio desulfuricans, Norway, is reported. The enzyme recognizes the sequence (see formula index) and cleaves at the position indicated by the arrows. The enzyme preparation obtained is suitable for restriction mapping an ligation.

Cellular lipids of the Legionnaires' disease bacterium.

We qualitatively and quantitatively analyzed the simple and complex lipids of 10 Legionnaires' disease bacteria. The phospholipids in decreasing order of concentration were phosphatidylcholine, phosphatidylethanolamine, cardiolipin, phosphatidylmonomethylethanolamine, phosphatidylglycerol, and phosphatidyldimethylethanolamine. The total phospholipids averaged 96 micromoles per gram dry cell weight. Phospholipid fatty acids were solely branched-chain fatty acids and were, in decreasing order of concentration, iso-C16:0, anteiso-C15:0, anteiso-C17:0, iso-C14:0, iso-C16:1, and an unidentified fatty acid. Neutral lipids identified were free fatty acid, ubiquinone, triglyceride, diglyceride, monoglyceride, and wax ester. Neutral lipid fatty acids consisted predominately of branched-chain fatty acids, normal fatty acids, and a minor unidentified fatty acid. Analysis of the cellular lipids of 10 Legionnaires' disease bacteria revealed an unusual and novel microorganism.

Hydrolysis of phosphatidylethanolamine and phosphatidylglycerol by outer membrane phospholipase A from Acinetobacter sp. HO1-N.

An outer membrane phospholipase A active against phosphatidylglycerol and phosphatidylethanolamine was characterized from Acinetobacter sp. HO1-N.

Phospholipid composition of methane-utilizing bacteria.

The phospholipids of Methylococcus capsulatus, Methylosinus trichosporium, La Paz, and OBT were examined in relation to their qualitative and quantitative composition. M. capsulatus exhibited a phospholipid composition consisting of phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and phosphatidyl-choline. The esterified fatty acids were predominantly C16:0 and C16:1. M. trichosporium, La Paz, and OBT exhibited an essentially identical phospholipid composition consisting of phosphatidylmonomethylethanolamine, phosphatidyl-dimethylethanolamine, phosphatidylcholine, and phosphatidylglycerol. Only trace amounts (less than 1%) of cardiolipin were found in these organisms. The major esterified fatty acid in these organisms was C18:1 (87 to 90%). The monounsaturated fatty acids from all four organisms consisted of both cis and trans isomers, each of which contained delta8, delta9, delta10, and delta11 double-bond positional isomers.

Identification and synthesis of acyl-phosphatidylglycerol in Acinetobacter sp. HO1-N.

A minor phospholipid from Acinetobacter sp. HO1-N was identified as acyl-phosphatidylglycerol. Acyl-phosphatidylglycerol synthesis by outer-membrane preparations appeared to be a result of phospholipase A activity.

Characterization of lysocardiolipin from Acinetobacter sp. HO1-N.

Triacyl-lysocardiolipin (triacyl-LCL) and diacyl-LCL were isolated from Acinetobacter sp. HO1-N, and their structures were determined by chemical, physical, and enzymatic procedures. Deacylation of triacyl-LCL and diacyl-LCL yielded bis-glycerylphosphorylglycerol. Periodate oxidation of both lysolipids was negative. Diglyceride and 2-monoglyceride resulted from the acetic acid hydrolysis of triacyl-LCL, whereas 2-monoglyceride was the sole product obtained from diacyl-LCL. Cardiolipin (CL)-specific phospholipase D treatment of triacyl-LCL yielded lysophosphatidylglycerol and phosphatidic acid. Pancreatic lipase treatment of CL yielded triacyl-LCL and diacyl-LCL. 31P nuclear magnetic resonance spectrometry showed two resonance peaks separated by 40 HZ for CL, two overlapping peaks separated by 14 HZ for triacyl-LCL, and one peak for diacyl-LCL. The proportion of lysocardiolipin increased as a function of cell age, representing 2 to 3% of the total phospholipids in early- and mid-exponential growth, 5 to 7% in late-exponential growth, and 12% in the stationary growth phase.

Outer membrane phospholipase A from Acinetobacter sp. HO1-N.

A phospholipase A1 activity that hydrolyzed cardiolipin to triacyl- and diacyl-lysocardiolipin was localized in outer membrane preparations derived from Acinetobacter sp. HO1-N. The specific activity of the enzyme derived from hexadecane-grown cells was 2.5 to 3 times higher than that derived from NBYE-grown cells. An apparent Km of 2.22 mM was determined, although inhibition kinetics resulted at the higher cardiolipin substrate concentrations. Optimal reaction conditions established on metal requirements. Enzyme activity was obligately dependent on Triton X-100 (0.5%) and was inhibited by cationic and anionic detergents. Cardiolipin-specific phospholipase D converted triacyl-lysocardiolipin to lysophosphatidylglycerol and phosphatidic acid. The specific activity of this enzyme was approximately 100 times greater than that reported for other membrane preparations derived from microorganisms.

Metabolism of the alkane analogue n-dioctyl ether by Acinetobacter species.

Metabolism of n-dioctyl ether by Acinetobacter species HO1-N resulted in formation of 8-n-octoxy-1-octanoic acid and 2-n-octoxy-1-acetic acid. The 16-carbon ether acid was incorporated into the cellular lipids, whereas the 10-carbon ether acid accumulated in the growth medium. Qualitative and quantitative characteristics of the cellular phospholipids were similar to hexadecane-grown cells. The growth of Acinetobacter on dioctyl ether occurred at the expense of six-carbon atoms of dioctyl ether.

Phospholipid composition and phospholipase A activity of Neisseria gonorrhoeae.

Exponential-phase cells of Neisseria gonorrhaeae 2686 were examined for phospholipid composition and for membrane-associated phospholipase A activity. When cells were harvested by centrifugation, washed, and lyophilized before extraction, approximately 74% of the total phospholipid was phosphatidylethanolamine, 18% was phosphatidylglycerol, 2% was cardiolipin, and 10% was lysophosphatidylethanolamine. However, when cells still suspended in growth medium were extracted, the amount of lysophosphatidylethanolamine decreased to approximately 1% of the phospholipid composition. This suggests that a gonococcal phospholipase A may be activated by conditions encountered during centrifugation and/or lyophilization of cells preceding extraction. Phospholipase A activity associated with cell membranes was assayed by measuring the conversion of tritiated phosphatidylethanolamine to lysophosphatidylethanolamine. Optimal activity was demonstrated in 10% methanol at pH 8.0 to 8.5, in the presence of calcium ions. The activity was both detergent sensitive and thermolabile. Comparisons of gonococcal colony types 1 and 4 showed no significant differences between the two types with respect to either phospholipid content or phospholipase A activity.

Isolation and characterization of an ornithine-containing lipid from Desulfovibrio gigas.

The isolation and characterization of an ornithine-containing lipid obtained from Desulfovibrio gigas are reported. The general structure for this aminolipid is represented by NH2-CH2-(CH)2-CHNH(CO-CH2CH(O-COR2)-R1)-COOH, where R1 represents 3-hydroxy palmitate linked through an amide bond to the alpha-amino group of ornithine, and R2 represents a complex variety of fatty acids esterified to the hydroxyl group of 3-hydroxy palmitate. Fatty acids characterized were n-C14:0 (21%), iso-C14:0 (14%) anteiso-C15:0 (43%), n-C16:0 (2%), n-C18:0 (8%), and n-C 18:1 (11%). The quantitative relationships between aminolipid and phospholipids showed the aminolipid to represent the major polar lipid. Isolation of the cytoplasmic and outer membranes of D. gigas showed the aminolipid to be evenly distributed between both membrane fractions, suggesting a compensatory role in phospholipid-deficient membranes.

Comparative analysis of the lipids of Acinetobacter species grown on hexadecane.

A comparative analysis of the cellular and extracellular lipids of Acinetobacter species HO1-N indicated basic physiological differences in hexadecane-grown cells. The cellular lipids obtained from hexadecane-grown cells were characterized by 3- and 18-fold increases in the phospholipid fraction and the mono- and diglyceride fraction, respectively, over that obtained from nutrient broth-yeast extract-grown cells. The cellular-associated pools of hexadecane were shown to comprise approximately 8% of the dry cell weight of hexadecane-grown cells. The extracellular lipids obtained from the culture broths of hexadecane-grown cells were comprised of triglyceride, mono- and diglyceride, free fatty acid, and wax ester. These lipids were either absent or present in minor concentrations in the culture broths of nutrient broth-yeast extract-grown cells. The exponential growth of Acinetobacter sp. on hexadecane was characterized by the significant accumulation of free fatty acid, monoglyceride, and diglyceride in the culture medium. Wax ester was shown to represent a minor portion of the extracellular lipids during the exponential growth phase, appearing in significant proportion only after the culture had entered the stationary phase of growth.

Phospholipid composition of Desulfovibrio species.

The phospholipids of Desulfovibrio desulfuricans, Norway strain, D. vulgaris, and D. gigas were examined in relationship to their qualitative and quantitative composition. D. desulfuricans and D. vulgaris exhibited an essentially identical phospholipid composition consisting of phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and lysophosphatidylserine. Phosphatidylserine (10.9%) was present in D. desulfuricans but was not detected in D. vulgaris. D. gigas was found to contain only two phospholipids, phosphatidylethanolamine (30%) and phosphatidylglycerol (70%). An ornithine-containing lipid was detected in D. gigas which was not present in the other two Desulfovibrio species.

Microbial assimilation of hydrocarbons: cellular distribution of fatty acids.

The distribution of cellular fatty acids in defined lipid classes was analyzed in Micrococcus cerificans after growth on specified hydrocarbons. Neutral lipid, phospholipid, and cell residue fatty acids were qualitatively and quantitatively determined for M. cerificans grown on nutrient broth, tetradecane (C(14)), pentadecane (C(15)), hexadecane (C(16)), and heptadecane (C(17)), respectively. Percentage of total cellular fatty acid localized in defined lipid classes from cells grown on the above growth substrates was (i) neutral lipid-11.8, 1.81, 7.74, 23.1, and 2%; (ii) phospholipid-74.5, 65, 66.43, 62.1, and 86%; (iii) cell residue lipid-13.5, 33.29, 25.82, 14.78, and 11.9%. Phospholipid fatty acid chain length directly reflected the carbon number of the alkane substrate, with 40, 84, 98, and 77% of the fatty acids being 14, 15, 16, and 17 carbons when cells were grown on C(14), C(15), C(16), and C(17)n-alkanes, respectively. The bound lipids of the cell residue after chloroform-methanol extraction were characterized by 2-hydroxydodecanoic and 2-hydroxytetradecanoic acids plus a broad spectrum of fatty acids ranging from C(10) to C(17) chain length. An increase in total unsaturated fatty acid localized in the phospholipids was noted from cells grown on alkanes greater than 15 carbons long. An extracellular accumulation of free fatty acid (FFA) was demonstrated in hexadecane-grown cultures that was not apparent in non-hydrocarbon-grown cultures. Identification of extracellular FFA demonstrated direct derivation from hexadecane oxidation. Studies supporting inhibition of de novo fatty acid biosynthesis in relationship to extracellular FFA and hexadecane oxidation are described. The ability to alter the fatty acid composition of membrane polar lipids in a predictable manner by the alkane carbon source provides an excellent model system for the investigation of membrane structure-function relationships in M. cerificans.