Characterization of highly purified, inactivated HIV-1 particles isolated by anion exchange chromatography.

This report characterizes inactivated, gp120 depleted, HIV-1 particles purified by an anion exchange chromatography production process. This antigen formulated with incomplete Freund's adjuvant constitutes Remune, which is being evaluated in a phase III clinical endpoint trial to determine the effect of this immune-based therapy on clinical progression of HIV-1 seropositive patients. Multiple production lots of the inactivated HIV-1 antigen strain HZ321, isolated by anion exchange chromatography, exhibit purity of > 95% by gel filtration. These findings are corroborated by thin section electron microscopy showing a homogenous field of intact particles. Analyses of the purified virus particles for protein, lipid, carbohydrate and RNA show structural retention of the envelope proteins, lipid bilayer and core components after large scale processing. The qualitative identification of at least 85% of total HIV-1 protein is determined by ELISA, Western blot, HPLC and amino acid sequencing analyses. Quantitative values are assigned to 50% of these proteins. The data confirm the presence of virally encoded proteins p6, p7, pI15, p17, p24, p32, pI39Gag, gp41, pp55Gag, p66/51, Vpr, Vif and Nef. Excellent consistency between production lots and equivalency to HIV-1 preparations purified by sucrose density gradient sedimentation has been established for protein and lipid composition, and overall purity. These findings further establish that non-viral encoded proteins and lipids are integral structural components of the intact virion and are not contaminants unique to a particular isolation method. The data confirm the presence of multicomponent antigens in the viral particles for stimulating a broad HIV-1 specific immune response. Finally, the work demonstrates that the two inactivation procedures (beta-propiolactone and gamma irradiation), which achieve efficient viral inactivation meeting US FDA guidelines, do not damage the protein antigens of the viral particles.

Modulation of the excitability of avian peripheral nerves by vitamin D: relation to calbindin-D28k, calcium status and lipid composition.

Calbindin-D28k (CaBP), previously localized in some of the cell bodies of ganglia of the avian intestinal (Remark's) nerve, was shown to be vitamin D-dependent. In the present studies, the effect of vitamin D3 on electrophysiological properties of this nerve was examined in vitro. Electrical stimulation of the nerve yielded a compound action potential with two primary components, Peaks I and II. Peak II, suppressed by hexamethonium bromide or Ca(2+)-free buffer, is synaptically mediated. The transit time between the two peaks was unaffected by vitamin D3. The apparent conduction velocity, defined as [(activation time + transit time)/nerve length], was increased by vitamin D-deficiency and decreased by vitamin D3 repletion, the latter decrease due entirely to an increase in activation time. Activation time after vitamin D-repletion was correlated with an increase in CaBP and plasma Ca2+ levels. However, normalization of plasma Ca2+ by supplementation of vitamin D-deficient diets with excess calcium (2.5 and 4.0%) also resulted in an increase in activation time, without affecting neuronal CaBP levels. Vitamin D3 also decreased the conduction velocity and increased CaBP of the vagus nerve and, by lipid analysis, was shown to increase and decrease its phosphatidylcholine and phosphatidylethanolamine content, respectively, and to decrease its phospholipid/cholesterol ratio. Modulation of peripheral nerve activity by vitamin D3 is related to calcium status and perhaps to changes in lipid composition. The functional role of CaBP in the behaviour of this complex nerve remains unknown.

Effect of a high fat diet on phospholipid class distribution and fatty acid composition in rat liver.

1. Long term consumption (20 weeks) of a high fat diet (65% of the energy content as fat) rich in either saturated [30% (w/w) coconut oil] or unsaturated [30% (w/w) sunflower oil] fatty acids resulted in strikingly similar alterations in the phospholipid class distribution and fatty acid composition in the liver of male Wistar rats. 2. The effect of these two diets was compared to a control group maintained on a 2% fat diet (w/w) for the same time interval. 3. In spite of the difference in the PUFA/SFA (polyunsaturated fatty acid/saturated fatty acid) ratio between the two high fat diets (0.1, saturated fatty acid diet; 5.4, unsaturated fatty acid diet), both diets resulted in a similar PUFA/SFA ratio in liver phospholipids, a similar reduction in palmitic acid (16:0), oleic acid (18:1, n-9) and arachidonic acid (20:4, n-6) and an elevation in stearic acid (18:0), linoleic acid (18:2, n-6) and docosahexaenoic acid (22:6, n-3). 4. Further, changes in the phospholipid classes were also similarly affected by both high fat diets.

Lipid composition and fluidity of the human immunodeficiency virus envelope and host cell plasma membranes.

Previous studies have indicated that human immunodeficiency virus (HIV) is enclosed with a lipid envelope similar in composition to cell plasma membranes and to other viruses. Further, the fluidity, as measured by spin resonance spectroscopy, is low and the viral envelope is among the most highly ordered membranes analyzed. However, the relationship between viral envelope lipids and those of the host cell is not known. Here we demonstrate that the phospholipids within the envelopes of HIV-1RF and HIV-2-L are similar to each other but significantly different from their respective host cell surface membranes. Further, we demonstrate that the cholesterol-to-phospholipid molar ratio of the viral envelope is approximately 2.5 times that of the host cell surface membranes. Consistent with the elevated cholesterol-to-phospholipid molar ratio, the viral envelopes of HIV-1RF and HIV-2-L were shown to be 7.5% and 10.5% more ordered than the plasma membranes of their respective host cells. These data demonstrate that HIV-1 and HIV-2-L select specific lipid domains within the surface membrane of their host cells through which to emerge during viral maturation.

Effects of the interaction between ferric iron and L-dopa melanin on T1 and T2 relaxation times determined by magnetic resonance imaging.

T1 and T2 relaxation times of agar phantoms containing L-dopa melanin and Fe3+ were measured under MRI conditions. Fe3+ shortened T1 and T2 relaxation times. Melanin influenced relaxation times only in the presence of Fe3+; thus, contrast in MR images of the basal ganglia may depend upon levels of both paramagnetic iron and neuromelanin.

Membrane function in mammalian hibernation.

For homeotherms the maintenance of a high, uniform body temperature requires a constant energy supply and food intake. For many small mammals, the loss of heat in winter exceeds energy supply, particularly when food is scarce. To survive, some animals have developed a capacity for adaptive hypothermia in which they lower their body temperature to a new regulatory set-point, usually a few degrees above the ambient. This process, generally known as hibernation, reduces the temperature differential, metabolic activity, as well as the energy demand, and thus facilitates survival during winter. Successful hibernation in mammals requires that the enzymatic processes are regulated in such a manner that metabolic balance is maintained at both the high body temperature of the summer-active animal (37 degrees C) and the low body temperature of the winter-torpid animal (approx. 5 degrees C). This means that the cellular membranes have thermal properties capable of maintaining a balanced metabolism at these extreme physiological temperatures. The available evidence indicates that, for some tissues, preparation for hibernation involves an alteration in the lipid composition and thermal properties of cellular membranes. Marked differences in the thermal response of cellular membranes have been observed on a seasonal basis and, in some membranes, differences in lipid composition have been associated with the torpid state. However, to date, no consistent changes in lipid composition which would account for, or explain, the changes in membrane thermal response, have been detected. An important point to emphasize is that the process of 'homeoviscous adaptation', which occurs in procaryotes and some poikilotherms during acclimation to low temperatures, is not a characteristic feature of most membranes of mammalian hibernators.

Thermotropic lipid phase separation in the human immunodeficiency virus.

The presence of thermodependent lipid domains in the envelope of the human immunodeficiency virus (HIV) was studied. HIV was propagated in Hut-78 cells and purified by differential-gradient centrifugation. Since the virus was highly infectious in cell culture and Western blots of detergent-inactivated HIV showed envelope proteins when exposed to sera containing anti-HIV antibodies, this viral preparation was not deficient in 'spike' or 'knob' particles. Electron spin resonance (ESR) studies of intact HIV labeled with 5-nitroxide stearate (5-NS) indicated that a temperature-dependent lipid phase separation occurs with a high onset at approx. 42 degrees C and a low onset at approx. 15 degrees C. Cooling below 42 degrees C induces 5-NS clustering. Similar phase separations with high onsets at approx. 37-38 degrees C were previously identified in 5-NS labeled human erythrocytes (cholesterol/phospholipid (C/P) molar ratio = 0.90) and cholesterol-loaded (C/P = 0.85-0.98) rat liver plasma membranes. These were attributed to a temperature-sensitive redistribution of endogenous lipid components such that 5-NS is excluded from cholesterol-rich domains and tends to reside in cholesterol-poor domains at low temperatures. Since HIV has a lipid envelope with a similarly high C/P of 0.88 (Aloia et al. (1988) Proc. Natl. Acad. Sci. USA 85, 900-904), cholesterol-rich and cholesterol-poor domains also probably exist in HIV at physiologic temperatures. The reduced stability and infectivity of HIV noted on heating above 42 degrees C may be due, in part, to the abolition of these thermodependent domains.

Effect of skim milk and whey diets on plasma lipid levels of rabbits in a cross-over study.

The influence of a skim milk and a whey-based diet on plasma lipids and lipoproteins in rabbits was investigated in a crossover design, in which the diets were switched after 12 weeks. The whey diet increased plasma lipid levels regardless of when it was consumed in the feeding protocol. The milk diet, on the other hand, modestly elevated the lipid levels if provided in the first half of the feeding protocol, but reduced lipid levels if given in the second half of the feeding regime. Both diets also induced similar changes in lipoprotein patterns when given in the first half of the feeding protocol, but opposite effects during the second half. Atherosclerotic changes were also observed in the aorta of animals receiving the whey diet during the second half of the experimental protocol.

The effect of a saturated fat diet on pentobarbital induced sleeping time and phospholipid composition of mouse brain and liver.

Mice maintained for 48 d on a diet containing 10% partially hydrogenated coconut oil (PHCO) slept more than twice as long as mice maintained on a standard, laboratory mouse chow (CHOW) diet when injected i.p. with pentobarbital. At this time, the liver of mice maintained on the PHCO diet exhibited a decrease in the mol% of phosphatidylethanolamine and an increase in phosphatidylcholine and phosphatidylinositol compared to the liver of control animals. In the brain, no changes in phospholipid composition were detected. However, in both brain and liver, alterations in the fatty acid composition of the phospholipid classes were observed, with eicosatrienoic acid (20:3, n-9) increasing and the polyunsaturates, particularly docosahexaenoic acid (22:6, n-3), decreasing significantly. These changes in lipids of the brain and liver may be related to the prolonged sleeping time of the animals on the PHCO diet by altering the metabolism of pentobarbital in the liver and/or altering the sensitivity of the brain membranes to the presence of the drug.

Mitochondrial membrane transitions in heart and other organs of a hibernator.

Critical temperatures (T) for transitions in both lipid structure and enzyme function of mitochondrial membranes from liver, kidney, brown fat, and heart tissues were determined for the hibernator Spermophilus lateralis at two weekly intervals from early summer to late autumn and during hibernation. For all tissues T fell into one of three groups: those below 4 degrees C (the minimal level of accurate determination), those centered about a mean of 11.9 +/- 1.4 degrees C, and those centered about a mean of 20.9 +/- 1.8 degrees C. The T for tissues from torpid animals and from heart, at all sampling periods, was below 4 degrees C. For liver, kidney, and brown fat the mean T was approximately 21 degrees C in early summer but was lowered later in the season in a two-step process, falling to below 4 degrees C before the animals were exposed to cold and entering torpor. It is concluded that for mitochondria the thermal response of the membrane lipids is altered such that the transition in structure and function is always below the minimum body temperature likely to be experienced by this animal. Heart tissue is exceptional in that the transition is at a temperature consistent with a body temperature of torpor even in summer-active animals.

Chronic alcohol treatment results in disturbed vitamin D metabolism and skeletal abnormalities in rats.

The effect of chronic alcohol consumption on the skeleton was investigated in rats. The treated group received ethanol administered as 38% of caloric intake in a liquid diet (Sustacal) for 10 months. The control rats were pair weighted to the ethanol-treated animals throughout the study; the growth curves of the two groups were the same. The controls were given the same liquid diet except that dextrin:maltose (3:1) was substituted isocalorically for ethanol. Ethanol-treated rats did not differ from the pair-weighted controls in mean serum calcium, phosphorous, or creatinine. In contrast, serum magnesium was reduced (p less than 0.02) in alcohol-treated rats. Ethanol treatment also resulted in changes in the serum concentrations of vitamin D metabolites; serum 25-hydroxyvitamin D3 was increased (p less than 0.001), while serum 1,25-dihydroxyvitamin D3 was decreased (p less than 0.01). Tibial length was reduced in ethanol-treated rats (p less than 0.05) but there was no change in femoral length. Medullary area was increased in tibial diaphyses from alcohol-treated rats compared to weight matched control animals (p less than 0.01), indicating a net increase in resorption. The cross-sectional area of the tibial diaphysis of ethanol-treated rats was the same as the matched controls. Trabecular bone was decreased in the tibial metaphysis of ethanol-treated rats compared to the matched controls (p less than 0.05) indicating a net loss of trabecular bone. Ethanol treatment did not have an effect on the organic weight of the femur but the ash weight was reduced (p less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid composition and fluidity of the human immunodeficiency virus.

Lipid analyses of the human immunodeficiency virus (HIV) propagated in Hut 78 cells indicated a low total lipid/protein ratio, a high cholesterol/phospholipid molar ratio, and major phospholipids consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and phosphatidylserine; comparable lipid profiles were noted for human erythrocytes and other RNA viruses. Electron spin resonance (ESR) studies of HIV labeled with 5-nitroxide stearate (N-oxy-4',4'-dimethyloxazolidine derivative of ketostearate) showed a low "fluidity" at 37 degrees C, similar to other enveloped RNA viruses and erythrocytes and probably due to the high cholesterol/phospholipid ratio. Ethanol (50%) completely disrupts the envelope, contributing to the rapid inactivation of HIV by ethanol. Contrarily, heating to 57 degrees C causes much less fluidization, and this heating may play a role in the slower viral inactivation at high temperatures. Should a critical minimum ordering in the HIV envelope be necessary for viral stability and infectivity, manipulating the lipid composition or fluidizing the HIV membrane, or both, may provide an untried therapeutic approach.

Effect of chronic alcohol consumption on rat brain microsome lipid composition, membrane fluidity and Na+-K+-ATPase activity.

The present study reports differences in phospholipid classes, fatty acids of individual phospholipids, and changes in membrane fluidity and Na+-K+-ATPase activity in brain microsomes of rats maintained on an alcohol diet for 35 days compared to sex, age and weight-matched control rats maintained on a calorically-equivalent, non-alcohol diet. Although no difference in Na+-K+-ATPase activity was found in microsomes from alcohol vs control rats when measured in the absence of added alcohol, the presence of low concentrations of ethanol (less than 100 mM) stimulated, while high concentrations (greater than 100 mM) inhibited enzyme activity. The stimulation was differentially expressed in that the microsomal enzyme from alcohol rats was stimulated to a lesser extent than the enzyme from control rats. However, the inhibiting effect of high concentrations of alcohol was similar in microsomes from both alcohol and control rats. Also in membranes from alcohol rats, there was a lower quantity of phosphatidylethanolamine (PE) and higher quantities of phosphatidylserine (PS) and phosphatidylinositol (PI) compared to membranes from control rats. The major change in fatty acid composition was a reduction in the level of polyunsaturated fatty acids, which was particularly evident in PI and PS. The linoleic acid: arachidonic acid ratio (18:2/20:4) and the saturation:unsaturation ratio were also increased in PI and PS in membranes from alcohol animals. However, the ratio of n-6/n-3 fatty acids remained the same or was reduced in membranes from alcoholic animals. Although no difference in the inherent "fluidity" of membranes from alcohol vs control rats could be demonstrated by electron paramagnetic resonance, molecular tolerance to ethanol was demonstrated in the membranes from alcohol rats by the resistance to the disordering effects of added ethanol.

The role of membrane fatty acids in mammalian hibernation.

During mammalian hibernation, cellular membranes continue to function at temperatures approaching 0 C. The molecular mechanisms that confer this capacity to the membranes are unknown but may be related to the fluidity of the membrane and to the level of unsaturated fatty acids. The basic tenets of membrane fluidity and the contribution of cholesterol, polar head groups, and fatty acids toward maintaining a fluid membrane in a liquid-crystalline state are examined in this review. It is shown that although unsaturated fatty acids can enhance membrane fluidity at low temperatures, there does not appear to be a consistent trend toward increased levels of unsatruated fatty acids during hibernation in all tissues of hibernators. Consequently, there may be some other role for the alterations in the composition of membrane fatty acids found during the hibernating cycle other than increasing membrane fluidity to permit continued activity at reduced temperatures.

Phospholipid composition of hibernating ground squirrel (Citellus lateralis) kidney and low-temperature membrane function.

1. Phospholipid analysis of kidney lipids from active and hibernating ground squirrels (Citellus lateralis) indicate that molar quantities of phosphatidyl choline increase, while sphingomyelin decreases in hibernating animals. 2. Both of these changes are in such a direction as to enhance membrane fluidity and possibly contribute to low-temperature membrane function in these organisms.

Spin label evidence for the role of lysoglycerophosphatides in cellular membranes of hibernating mammals.

The phospholipid composition of ground squirrel heart muscle changes during hibernation: more lysoglycerophosphatides are found in the hibernating state than in the active state. Phase transitions inferred from spin label motion occur in the usual manner typical of mammalian mitochondria for the mitochondria and mitochondrial lipids from active squirrels. However, a conspicuous absence of a spin label-detectable phase transition is observed in equivalent preparations from hibernating animals. The addition of lysolecithin to preparations from active squirrels removes the break and induces a straight line in the Arrhenius plot. The lack of a spin label-detectable phase transition in hibernating animals, therefore, is attributed to an increased content of lysoglycerophosphatides present in the phospholipids during hibernation.