Differences in the fatty acid profile and beta-oxidation by heart homogenates of rats fed cis and trans octadecenoic acids.

Female Wistar rats were fed a fat-free diet containing either 5% partially hydrogenated corn oil (52.2% elaidate) or 5% oleic acid (67% oleate) with 8.6% linoleate providing 1% of calories 2 weeks before mating and were maintained on this diet throughout pregnancy and lactation. Fatty acid analysis of the developing organs as well as beta-oxidation by heart homogenates with [1-14C]palmitate, [1-14C]elaidate and [1-14C]oleate of the developing male and female progeny were determined and compared with age-matched controls on a stock diet. Results show that irrespective of the cis and trans 18:1 in the diet, the maternal plasma at term contained mostly cis 18:1, with 5% trans for the rats on the trans diet. The placenta and fetal liver contained 40 and 60% less trans, respectively, than did the maternal plasma. trans 18:1 was not detected in fetal brain or heart. Regardless of diet or sex, the order of preference for the heart was palmitate greater than elaidate greater than oleate. There was an increase in the rate of beta-oxidation of all the substrates, especially in the females on the trans diet, suggesting a stimulation of one or more of the enzymes involved. Above all, the myocardium showed a unique capacity to retain n-6 and n-3 fatty acids when the levels of these decreased in the serum.

Nicotine and amyloid formation.

The major protein constituents of amyloid deposits in Alzheimer's disease (AD) are the 40-residue beta-amyloid (Abeta) (1-40) peptide and the 42-residue Abeta(1-42) peptide. The Abeta(1-42) is more pathogenic and produced in greater quantities in familial forms of AD. A major goal of research is to uncover a suitable inhibitor that either slows down or inhibits Abeta formation (beta-amyloidosis). During beta-amyloidosis, structural changes associated with the conversion of monomeric Abeta peptide building blocks into the aggregated fibrillar beta-sheet structures occur (alpha-helix-->beta-sheet or random, extended chain-->beta-sheet). In previous work, we and others established that nicotine, a major component of cigarette smoke, inhibits beta-amyloidosis of the Abeta(1-42), which may result from nicotine binding to the alpha-helical structure. These conclusions were based on solution nuclear magnetic resonance (NMR) spectroscopic studies with the nonnative 28-residue Abeta(1-28). This information suggests that, when administered therapeutically to AD patients, nicotine may not only affect cholinergic activation, but could also conceivably alter amyloid deposition. In this report, NMR studies were augmented with the naturally occurring Abeta(1-42), under conditions where the peptide folds into a predominantly alpha-helical or random, extended chain structure. The major result is that nicotine shows only modest binding to these conformations, indicating that the nicotine inhibition to beta-amyloidosis probably results from binding to a small, soluble beta-sheet aggregate that is NMR invisible.

Cloning, sequencing and overexpression of the Desulfovibrio gigas ferredoxin gene in E. coli.

We have cloned the gene encoding Desulfovibrio gigas ferredoxin using a photodigoxigenin-labelled probe synthesized with the polymerase chain reaction. The DNA sequence of the gene predicts a polypeptide of 58 residues after removal of the initial formyl methionine (polypeptide M(r) = 6,276). The ferredoxin gene was expressed in aerobically grown E. coli behind the lac promoter of pUC18 resulting in a high level of ferredoxin expression which comprises about 10% of the total cell protein. EPR analysis of recombinant ferredoxin revealed the presence of a [3Fe-4S] cluster which is characteristic of native D. gigas ferredoxin II.

Carboxy-terminal processing of the large subunit of [NiFe] hydrogenases.

Two electrophoretic forms of the large subunit of the soluble periplasmic [NiFe] hydrogenase from Desulfovibrio gigas have been detected by Western analysis. The faster moving form co-migrates with the large subunit from purified, active enzyme. Amino acid sequence and composition of the C-terminal tryptic peptide of the large subunit from purified hydrogenase revealed that it is 15 amino acids shorter than that predicted by the nucleotide sequence. Processing of the nascent large subunit occurs by C-terminal cleavage between His536 and Val537, residues which are highly conserved among [NiFe] hydrogenases. Mutagenesis of the analogous residues, His582 and Val583, in the E. coli hydrogenase-1 (HYD1) large subunit resulted in significant decrease in processing and HYD1 activity.

Effect of lysophosphatidylcholine on atherosclerotic rabbit arteries.

We report here on the effect of an endothelium-dependent vascular smooth muscle relaxant, lysophosphatidylcholine (LPC) on rabbit aortic strips and on hemodynamic changes by LPC in atherosclerotic animals. Cyclic GMP changes induced by LPC in atherosclerotic vessels were also determined. Atherosclerosis was produced by feeding a high cholesterol and saturated fatty acid diet. LPC was injected into the left atrium and coronary flow was measured by radioactive microspheres; in vitro, relaxation of precontracted aortic strips by lysophosphatidylcholine was also recorded. LPC failed to increase coronary flow in the presence of atherosclerosis. In isolated aortic strips, dose-response curves with acetylcholine and LPC showed diminished relaxation in atherosclerotic preparations, and cyclic GMP production following LPC was reduced. The results demonstrate that vascular relaxation by LPC, together with its ability to activate guanylate cyclase is dependent on the functional and morphological integrity of the vascular wall.

Calcium, calmodulin and 3',5'-cyclic nucleotide phosphodiesterase activity in human muscular disorders.

3',5'-Cyclic nucleotide phosphodiesterase (PDE) is known to play an important role in the regulation of cyclic nucleotide levels in various tissues including the muscle. Previous studies have estimated the level of this enzyme in several neuromuscular disorders but the results have been variable. Moreover, there was no attempt made to correlate the enzyme levels with the levels of calcium and calmodulin, both of which regulate diverse biological processes including muscle contraction. In the present study we have estimated phosphodiesterase in the muscle of normal controls as well as patients with myotonic (MyD) and Duchenne muscular dystrophy (DMD) and amyotrophic lateral sclerosis (ALS). PDE was found to be increased significantly in all of the diseased muscles as compared to controls (P less than 0.01). But the increase could be coupled with an increase in calcium and calmodulin only in Duchenne dystrophic muscle.

The effects of mild congenital methylmercury intoxication on the metabolism of 3-hydroxybutyrate and glucose in the brains of suckling rats.

We studied the effects of mild congenital methylmercury toxicity upon intermediary metabolism in the developing brain. Female rats were injected intravenously with 10 mg/kg methylmercury chloride on the 4th day of impregnation. Controls received saline. At postnatal days 1, 7, 14 and 21, pup brain slices were incubated with either 3-hydroxy[3-14C]butyrate or [U-14C] glucose. The rate of oxidation of 3-hydroxybutyrate was significantly reduced at days 14 and 21 in the methylmercury-treated pups. There was a marked reduction in the incorporation of label from both substrates into total brain lipids during the most rapid phase of myelination. Incorporation of 14C from [U-14C] glucose into proteins was decreased at all ages. Since there was no decrease in the incorporation of [1-14C]leucine into proteins in the methylmercury-treated pups, this decrease could have resulted from changes in pool sizes of certain amino acids in the brain.

Correlation of lysophosphatidylcholine-induced vs spontaneous relaxation to cyclic GMP levels in rabbit thoracic aorta.

We demonstrated previously that 10(-5)M micellar solution of lysophosphatidylcholine (LPC) produced relaxation of rabbit thoracic aorta in a dose-dependent manner. It was also noticed that histamine (HA)-precontracted strips relaxed spontaneously in the absence of LPC. We now measured the cyclic GMP changes during these two types of relaxation. Results indicate that while LPC-induced relaxation was mediated through cyclic GMP, spontaneous relaxation was not. The vasodilating effect of LPC was not due to its interference with cellular viability by solubilizing the membrane, because repeated additions of LPC produced identical degrees of relaxation and after three consecutive additions of LPC, the strips continued to relax to the same degree with acetylcholine (Ach) demonstrating the functional integrity of the endothelium. LPC/Cholesterol dispersions (1:0.5 mole percent) relaxed the strips to the same extent as the micellar solution of LPC, while 1:1 mole percent did not. The results stress the role of cyclic GMP in LPC-induced relaxation. They further suggest that LPC/Cholesterol ratio may determine the availability of LPC and regulate LPC-mediated processes.

A model to study physiological activation of phospholipase A2 and vasorelaxation by lysophosphatidylcholine.

Earlier we demonstrated that micellar solutions of LPC caused endothelium-dependent relaxation of rabbit thoracic aorta and bovine intrapulmonary artery and vein through a cyclic GMP-dependent mechanism. The availability of LPC for vasorelaxation depends on its production by deacylation of PC by PLA2. We assessed the possible activation of PLA2 by commonly used vasorelaxants such as acetylcholine, bradykinin, calcium ionophore A23187 and thrombin and vasoconstrictors like histamine and phenylephrine in the presence of indomethacin in a model system where 14C PC was incorporated into bovine intrapulmonary arterial segments. Taking the ratio of 14C PC:LPC formed by exogenous PLA2 as an index of deacylation, we found that while all the agents relaxed the strips in an endothelium-dependent manner, only thrombin caused relaxation followed by an increase in 14C LPC and a concomittant decrease in 14C PC indicating activation of PLA2. Our data show that PC/PLA2 system can be activated to generate LPC for vascular relaxation under specific physiological conditions. This model system can be used to monitor PLA2 activity and LPC production to compensate flow and pressure induced changes in arteries.

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.

Restriction of maternal food intake inhibits fatty acid activation in developing rat hearts.

We studied the effect of restricting the diet of pregnant and lactating rats on the beta-oxidation of fatty acids by the developing heart in suckling pups. Control pregnant rats were fed a stock diet ad libitum. For the experimental group, food was restricted to half of the control intake on the seventh day of pregnancy and continued through lactation. The pups on the restricted diet were significantly smaller than the controls. At postnatal days 5, 14 and 21, the beta-oxidation of [1-14C] palmitate by heart homogenates was determined in the presence of ATP, carnitine and CoA. At day 21, the production of 14CO2 was 60% lower in the group on the restricted diet. Consequently, the possibility of inhibiting activation or intramitochondrial transport of fatty acids by heart mitochondria was studied in vitro using [1-14C] palmitate, [1-14C] palmitoyl CoA and [1-14C] palmitoyl carnitine. With [1-14C] palmitate, the rate of 14CO2 produced was 2464 +/- 317 cpm/mg protein/min for the control and 1682 +/- 91 for the restricted diet group. With [1-14C] palmitoyl CoA and [1-14C] palmitoyl carnitine, the oxidation rate of the experimental group was similar to control values, showing clearly that the inhibition of oxidation was from a problem with activation. A significant decrease in palmitoyl CoA synthetase activity in the heart homogenates and mitochondria of the diet-restricted pups took place.

Accumulation of lipid peroxidation products in human myotonic dystrophic muscle.

The possibility of oxygen radical-induced injury contributing to the pathogenesis of muscle disorders was studied. Significant increases in fluorescent lipid peroxidation products was found in the muscle samples of myotonic dystrophy (MyD) patients as compared to controls as well as patients with Duchenne Muscular dystrophy (DMD), Amyotrophic Lateral Sclerosis (ALS), Polymyositis and Limb Girdle Dystrophy (LGD). The results demonstrate the possibility that in MyD the primary genetic disorder leads to the rapid generation of oxygen radicals, tissue depletion of antioxidants followed by peroxidation of membrane lipids, impaired calcium homeostasis and finally atrophy of the muscle.

Elevated levels of prostaglandins E2 and F2( in human neuromuscular disorders.

Prostaglandins (Pgs), especially PGE2 and F2( ( have been implicated in variety of pathological processes including proteolysis of normal and dystrophic muscle. Using a modified radioimmunoassay which did not involve extraction in organic solvents and purification, thus eliminating acid artifacts, we measured PGE2 and F2( levels in patients with myotonic (MyD), Duchenne (DMD), and limb girdle (LGD) dystrophies and amyotrophic lateral sclerosis (ALS). There was a significant increase in PGE2 and PGF2 in the muscle samples of all the disorders studied as compared to normal controls. It is proposed that increased influx of calcium activated phospholipase A2 (PLA2) leading to the accumulation of arachidonic acid and hence prostaglandins. It appears that the relative increases in PGE2 and PGF2( which are implicated in protein degradation and synthesis respectively in vitro, may reflect the extent of degeneration and regeneration occurring in the diseased muscles.