A.M. Municio

In vitro fatty acid and lipid biosynthesis during development of insects

1. 1. In vitro biosynthesis of fatty acids from [1-14C]acetate, desaturation of [1-14C] palmitic and [1-14C]stearic acids and their incorporation into different classes of lipids have been investigated with homogenates of larvae and pharate adults of Ceratitis capitata. 2. 2. The time-course of [1-14C]acetate incorporation shows clear differences in the behaviour of larval and pharate adult homogenates. Specific radioactivities of fatty acids synthesised by larval homogenates exhibit a net distinction between mediumchain and long-chain fatty acids. This distinction is not exhibited in the [14C]acetate incorporation by pharate adult homogenates. 3. 3. Both developmental stages also show differences in the incorporation of radioactivity into different classes of lipids. Triglycerides or phospholipids become rapidly labelled by either larval or pharate adult homogenates, respectively. This different behaviour of both developmental stages also applies to the specific radioactivity of the individual fatty acids present in the lipid classes. Decanoic acid has the highest specific radioactivity of the fatty acids incorporated into triglycerides by larval homogenates. The fatty acids of triglycerides from pharate adults, on the other hand, do not exhibit any incorporation of radioactivity. 4. 4. Desaturation of [1-14C]palmitic and [1-14C]stearic acids has been investigated in larval and pharate adult homogenates. Pharate adult homogenates do not desaturate these fatty acids, whereas larval homogenates exhibit a certain degree of desaturation. [14C]Palmitic and [14C]stearic acids are effectively incorporated by larval homogenates while they remain in a free form during incubation with pharate adult homogenate. However, stearic acid shows a lower metabolic efficiency than palmitic acid. Desaturation of palmitic acid is favoured by aerobic conditions and palmitoleic acid is preferentially incorporated into phospholipids.

Biochemistry of the development of the insect Ceratitis capitata. Changes of the positional distribution of fatty acids in diacylethanolamine phosphoglycerides.

Abstract The positional distribution of fatty acids in diacylethanolamine phosphoglycerides from the Dipterous Ceratitis capitata has been investigated in the egg, larval, pharate adult and adult stages of development. The patterns of distribution confirm the general rule that in animal phospholipids unsaturated acids are mainly located at the 2-position while saturated ones are situated at the 1-position. In spite of that some differences in the distribution have been found during metamorphosis. The unsaturated fatty acid 18: 1ω7 is only present in eggs and acylates exclusively at the 1-position whereas the isomer 18: 1ω9 is incorporated into both positions. A net tendency to increase the unsaturation degree during metamorphosis in ethanolamine phosphoglycerides has been shown.

In vitro elongation and desaturation of fatty acids during development of insects

Abstract 1. 1. In vitro elongation and desaturation reactions of fatty acids using labelled 10:0, 12:0, 14:0 and 16:0 have been investigated with homogenates of larvae and pharate adults of Ceratitis capitata. The obtained results show clear differences in the behaviour of larval and pharate adult stages of development. 2. 2. Larval homogenates desaturate and elongate the labelled substrates according to their chain length. The longer the length of the fatty acids used as substrates the more pronounced the abundance of the corresponding monounsaturated fatty acids, as well as the smaller activity of the elongation pathway. Direct unsaturation and elongation of fatty acids by pharate adult homogenates are insignificant. 3. 3. Labelled acetate incorporation into fatty acids by the larval homogenates is notably stimulated in the presence of malonate, whereas the fatty acid synthesis by pharate adult homogenates is practically unaffected. The fatty acids 10: 0 and 12: 0 are elongated by larval homogenates more effectively in the presence of acetate than that of malonate. 4. 4. The larval homogenate incorporates considerably more labelled fatty acids into triglycerides than into phospholipids, whereas the radioactivity remains mainly as free fatty acids in the presence of pharate adult homogenates. 5. 5. Pharate adult mitochondria preparations incorporate more acetate than larval itochondria preparations. Incorporation by larval mitochondria was not influenced by the presence of malonate and the resulting fatty acids are present mainly as phospholipids, in a clear contrast with the incorporation achieved by the whole larval homogenates.

Variation of the levels of cyclic AMP and cyclic GMP during development of the insect Ceratitis capitata.

Abstract Changes in the levels of adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) during development were studied in the Dipterous Ceratitis capitata. The developmental patterns were different to each other. Cyclic AMP showed a sharp maximum in the larval stage to decrease afterwards during adult development. Changes of cyclic GMP exhibited an opposite pattern, although its levels were always higher than those of cyclic AMP.

Comparison between intra- and extracellular surfactant in respiratory distress induced by oleic acid

The present study compares the phospholipid distribution and protein content in bronchoalveolar lavage, purified extracellular surfactant and lamellar bodies isolated from rabbits killed at intervals of 2.5, 12 and 24 h after oleic acid administration. The data suggest that the alteration of pulmonary surfactant could be partially due to the type II cell response to the injury.

The binding of Escherichia coli endotoxin to isolated rat hepatocytes

The mechanisms through which bacterial endotoxins exert their biological activity is not well understood. A characteristic property of lipopolysaccharides (LPS) is their marked heterogeneity in terms of physical state, molecular size and charge. Also, LPS contain varying amounts of impurities according to the different methods of extraction and this might explain the various results reported with different preparations. Investigations in the field of bacterial endotoxins suggested the major responsibility of the lipid component for the endotoxic activities of LPS [1-4]. Some types of interactions between LPS and cell membranes have been reported for red cells [5], lymphocytes [6], granulocytes [7] and platelets [8]. The cell membrane of eukaryotic cells contains significant amounts of phospholipids that probably play a role in the binding of LPS to cell membranes [9]; thus, the variation in the phospholipid composition among cell types might account for the specificity in the biological reactions elicited by LPS [10-12] and it has been reported that there is a direct relation between the susceptibility of different strains of mice to the lethal effects of Salmonella endotoxin and the affinity of their red cells for endotoxin [13]. However, it is generally accepted that liver exerts a LPS clearance function with the participation of both Kupffer cells [14-17] and parenchymal cells [18]. Also, there is now general agreement that, in the liver, endotoxemia causes hepatocyte mitochondrial alterations that can be characterized by both morphologic and functional alterations [ 19,20]. This paper is concerned with the binding properties of 14C-labelled lipopolysaccharide (14C-LPS) to isolated hepatocytes from rats. Endotoxin labelled with 14C was obtained from Escherichia eoli 011 la,011 lb: K58:H21 (ATCC) grown in a medium with [14C]glucose as the only carbon source. The extraction of 14C-LPS was carried out according to Romanowskas modification [21 ] of the general method of Westphal. Specific activity was 2.23/ICi/mg; A26o/A24o was determined for purity verification. Hepatocytes were isolated from male rats (SpragueDawley, 250-300 g, fasted over 20 h with water ad libitum), by the perfusion technique in [22] using collagenase (Sigma, St Louis MO; type IV, 170 U/mg) (30 mg) in 131 ml Krebs-Ringer-bicarbonate solution, without Mg 2+. The cell suspension was purified by repeated centrifugation at 165 g for 20 s. Cell viability was monitored by trypan blue exclusion and cell preparations had 85-90% viability. Gluconeogenesis from lactate was measured in isolated cells incubated with 10 mM substrate in KrebsRinger-bicarbonate solution (10 6 cells/500 gd). Glucose levels were determined in the supernatants enzymically using glucoseoxidase and peroxidase (Kit Carlo Erba). Control of structure was carried out by electron microscopy of thin sections of Epon-embedded cell suspensions, cut on an LKB Ultrotome III ultramicrotome, and examined in a Jeol 100 electron microscope. Stimulation of gluconeogenesis by glucagon (Novo, Spain) was determined by incubating the hormone with hepatocytes in various experimental conditions. The standard binding assay was based on the method in [23] and consisted of incubating hepatocytes at 25°C in complete Krebs-Ringer-bicarbonate solution containing 14C-LPS. Hepatocyte-LPS

Fatty acid synthetase complex from the insect Ceratitis capitata.

Fatty acid synthesis capacity of the insect Ceratitis capitata has been investigated in vitro from [1-14C]acetyl-CoA using homogenates at different stages of development. A maximum activity was observed after 5--6 days of larval development. But homogenates of the pharate adult insect did not show synthetic capacity of fatty acids. Fatty acid synthetase complex has been isolated from the particle-free supernatant fraction of homogenates from the 6-day C. capitata larvae. The enzyme complex was purified 182-fold with respect to the protein contained in the crude extract. The complex was homogeneous when analysed by gel filtration and by polyacrylamide-gel electrophoresis. The molecular weight was 5.2X10(5). The enzyme was dissociated into half-molecular subunits. Amino acid analysis, general properties, stability and kinetic constants (V and Km) for the substrates are reported. The fatty acid synthetase complex from the insect contains 42+/-1-SH residues and one phosphopatetheine moiety per 5.2X10(5). Activity was dependent on the presence of NADPH; FMN strongly inhibited the enzyme activity promoted by NADPH. The enzyme complex synthesized a range of fatty acid (10:0--18:0), palmitate being the predominant end product. The proportions of fatty acids synthesized varied with substrate concentrations. Fatty acids released from the complex were almost completely in the free form.

Phospholipase C-diglyceride lipase is a major pathway for arachidonic acid release in macrophages.

Macrophages are a rich source of arachidonic acid oxygenated metabolites and play a remarkable role in a number of physiopathological situations. The synthesis and secretion of arachidonic acid metabolites are triggered at the cytoplasmic membrane level. The present study was outlined to further investigate the cellular mechanisms controlling arachidonic acid release in macrophages. The results presented here strongly suggest that the amount of arachidonic acid released in macrophages in response to phagocytic challenge could be accounted for by a phospholipase C-diglyceride lipase system being unnecessary the presence of phospholipase A2 whose activity, on the other hand, was found vanishingly small in macrophage homogenates.

The fluidity of plasma membranes from ethanol-treated rat liver

SummaryMale Wistar rats were maintained for 35–40 days on a liquid diet containing 36% of calories as ethanol. Ethanol was replaced by carbohydrates in the isocaloric diet fed to control animals. The effect of ethanol consumption has been studied on the fluorescence polarization of rat liver plasma membranes and artificial lipid vesicles and on the lipid composition of the membranes. Fluorescence polarization in both membranes and vesicles was determined using DPH and TMA-DPH as fluorescence markers; from these data, the polarization term (ro/r−1)−1 and flow activation energy (ΔE) were calculated. The ethanol consumption induces a more fluid environment within the membrane core of liver plasma membranes; the ethanol-fed rat membranes are more resistant to the in vitro effect of ethanol disordering the membrane structure. Vesicles obtained with lipids from either control membranes or ethanol-fed rat membranes were treated with ethanol and the changes in polarization paralleled to those exhibited by the membranes. The absence of phase transitions and of ΔE changes was also shown in temperature-dependence studies. The lower cholesterol content found in ethanol-fed rat plasma membranes might be responsible for observed variations in the microviscosity.

Intracellular calcium and pH alterations induced by Escherichia coli endotoxin in rat hepatocytes

In this study, the fluorescent Ca2+ probe fura-2 and the fluorescent pH indicator BCECF have been used to monitor cytosolic free Ca2+ and intracellular pH (pHi), respectively, in isolated and cultured hepatocytes treated with Escherichia coli O111:B4 endotoxin. Uptake of 45Ca2+ was also measured to study the effect of endotoxin on the extracellular calcium influx. Endotoxin treatment produced a progressive increase of cytosolic Ca2+ in a dose-dependent manner caused by both induction of a significant release of Ca2+ from intracellular stores and stimulation of the extracellular calcium influx. The perturbation of Ca2+ homeostasis by endotoxin may cause an abnormal stimulation of physiological processes, developing lethal cell injury. Endotoxin also produced a significant decrease in the pHi of hepatocytes which can justify important metabolic alterations during endotoxicosis.