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Cholesteryl-phosphoryl-choline (CPC), a hybrid between cholesterol and lecithin, is incorporated into sonicated liposomes and erythrocyte membranes similarly to cholesterol. The effect of CPC on lipid microviscosity and degree of order is smaller, but not significantly than that of cholesterol. It is proposed that CPC may be employed as an efficient modulator of lipid dynamics.
Precipitation of cholesterol crystals from vesicles is an important step in the pathogenesis of cholesterol gallstones. Little is known, however, about the kinetics and the mechanisms involved in cholesterol crystallization. Therefore, the time course of cholesterol crystal precipitation and lipid exchange between vesicles and micelles were monitored in a model bile system. Vesicles obtained from supersaturated model bile (cholesterol saturation index (CSI) 1.4; 10 g/dl) by KBr density gradient ultracentrifugation, were incubated with various bile salts: deoxycholate (DC), chenodeoxycholate (CDC), cholate (C), ursodeoxycholate (UDC), and their respective taurine and glycine conjugates. Vesicle integrity was assessed in a leakage-assay of carboxyfluorescein-loaded vesicles (0-15 min) and by the change in optical absorbance at 340 nm of a vesicle solution (0-50 min). Fluorescence increased within 1 min after addition of bile salt, and was stable within 5-10 min. After addition of bile salt, absorbance fell immediately and stabilized within 30 min. Fluorescence and absorbance were dependent on bile salt hydrophobicity and concentration. At several time points after addition of bile salt to vesicles (from 1 to 72 h), the extent of cholesterol nucleation was determined semiquantitatively and incubation mixtures were again subjected to ultracentrifugation to assess the lipid distribution among residual vesicles, de novo formed mixed micelles, and cholesterol crystals. Nucleation occurred within 0.5 h after exposure of vesicles to the hydrophobic bile salts DC or CDC, and the cholesterol/phospholipid (c/p) ratio of the vesicles showed a transient rise from 1.45 to 3-4 (at t = 0.5 h) that coincided with the appearance of mixed micelles. Then the vesicular c/p ratio decreased to 0.6-0.8 (at t = 24 h) concomitantly with increasing precipitation of cholesterol crystals. In the case of UDC, the most hydrophilic bile salt used, < 5% micellization, no nucleation, and a constant vesicular c/p ratio were observed. We conclude that under the conditions used in the present model study, the kinetics of cholesterol crystallization are governed by the hydrophobicity of the added bile salts and their capacity to form mixed micelles. The results emphasize the pivotal role of time, and the dynamic aspects of the processes involved in cholesterol crystal formation.
An assay detecting and quantifying cholesterol nucleation from low-density lipoproteins has been established. Förster resonance energy transfer between dehydroergosterol and dansylated lecithin becomes significantly alleviated as a consequence of conucleation of dehydroergosterol and cholesterol. The assay, in combination with dynamic light scattering, absorbance spectroscopy, and fluorescence microscopy, can be used to study aggregation and nucleation in model blood systems. Human plasma LDL was labeled with dehydroergosterol and dansylated lecithin by incubation with donor multilamellar liposomes and isolated by centrifugation. Exposure of labeled LDL (0.5 mg/mL of total lipids) to sphingomyelinase (0.0-0.2 unit/mL) led to modest particle aggregation but produced no changes in energy transfer and no crystallization. However, addition of sphingomyelinase produced significant particle aggregation, nucleation, and crystallization, in a dose-dependent fashion, in samples that were previously treated with the enzyme, cholesterol esterase (0.2 unit/mL). The combination of cholesterol esterase and sphingomyelinase led to a significant alleviation of energy transfer, which preceded by 24 h the appearance of fluorescent, microscopic sterol crystals. These results point to a synergistic effect between cholesterol esterase and sphingomyelinase, suggesting that mere aggregation of LDL is insufficient to promote nucleation, and crystal formation likely proceeds in the intracellular space after LDL uptake by macrophages.
The substituted triphenylethylene antiestrogen clomiphene (CLO) prevents cancellous bone loss in ovariectomized (OVX'd) rats. However, CLO is a mixture of two stereoisomers, enclomiphene (ENC) and zuclomiphene (ZUC), which have distinctly different activities on reproductive tissues and tumor cells. The purpose of the present dose response study was to determine the effects of ENC and ZUC on nonreproductive estrogen target tissues. These studies were performed in 7-month-old female rats with moderate cancellous osteopenia that was established by ovariectomizing rats 1 month before initiating treatment. OVX resulted in increases in body weight, serum cholesterol, endocortical resorption, and indices of cancellous bone turnover, as well as decreases in uterine weight, uterine epithelial cell height, bone mineral density, bone strength, and cancellous bone area. Estrogen treatment for 3 months restored body weight, uterine histology, dynamic bone measurements, and osteoblast and osteoclast surfaces in OVX'd rats to the levels found in the age-matched sham-operated rats. In contrast, estrogen only partially restored cancellous bone volume and uterine weight, and it reduced serum cholesterol to subnormal values. CLO was a weak estrogen agonist on uterine measurements and a much more potent agonist on body weight, serum cholesterol, and dynamic bone measurements. CLO increased trabecular thickness in osteopenic rats and was the most effective treatment in improving cancellous bone volume and architecture. ZUC was a potent estrogen agonist on all tissues investigated and had dose-dependent effects. In contrast, ENC had dose-dependent effects on most measurements similar to CLO and decreased the uterotrophic effects of ZUC. It is concluded that ENC antagonizes the estrogenic effects of ZUC on the uterus but that the beneficial effects of CLO on nonreproductive tissues in OVX'd rats is conferred by both isomers. Furthermore, the combined actions of the two isomers on bone volume and architecture were more beneficial than either isomer given alone.
High-density lipoproteins (HDL) function as cholesterol transporters, facilitating the removal of excess cholesterol from the body. Due to the heterogeneity of native HDL particles (both in size and shape), the details on how these protein-lipid particles form and the structure they assume in their lipid-associated states are not well characterized. We report here a study of the self-assembly of discoidal HDL particles using coarse-grained (CG) molecular dynamics. The microsecond simulations reveal the self-assembly of HDL particles from disordered protein-lipid complexes to form structures containing many of the features of the generally accepted double-belt model for discoidal HDL particles. HDL assembly is found to proceed in two broad steps, aggregation of proteins and lipids driven by the hydrophobic effect which occurs on a approximately 1 micros time scale, followed by the optimization of the protein structure driven by increasingly specific protein-protein interactions.
The nature and distribution of neutral sterols has been determined in the digestive tract of adult Large White sows maintained in a defined biological steady state (constant weight: 70 +/- 5 kg). The semi-synthetic diet contained 0.08 p. 100 cholesterol. Cholesterol was the main sterol of the intestinal contents. From the caecum, it was partially transformed into coprostanol (less than 6 p. 100). Just before the morning meal, the intestinal contents of the sows were rich in cholesterol (532 mg in the small intestine). As in rats, this rapidly renewed cholesterol of endogenous origin was not distributed homogeneously with the exogenous dietary cholesterol. During intestinal cholesterol transit, hydrolysis exceeded esterification. Using the principle of occupancy of every dynamic system, two isotopes were employed to measure the absorption coefficient: cholesterol-4-14C (10 microCi), incorporated into the meal and ingested at one time, and cholesterol-3H (400 microCi), injected intravenously once in a suspension of autologous red blood cells previously labelled in vitro. The absorption coefficient of dietary cholesterol in our experimental conditions reached 57 +/- 4 p. 100. The cholesterol was mainly absorbed in the first-half of the small intestine as in man, whereas in rat it is absorbed in the second-half. This dietary tracer appeared in the general circulation later in swine than in rat or man. After intake of a meal containing the cholesterol tracer, maximal plasma cholesterol radioactivity was reached after 48 hrs in the sows while it is reached after 10-12 hrs in rat or man.
A method for simulating a two-component lipid bilayer membrane in the mesoscopic regime is presented. The membrane is modeled as an elastic network of bonded points; the spring constants of these bonds are parameterized by the microscopic bulk modulus estimated from earlier atomistic nonequilibrium molecular dynamics simulations for several bilayer mixtures of DMPC and cholesterol. The modulus depends on the composition of a point in the elastic membrane model. The dynamics of the composition field is governed by the Cahn-Hilliard equation where a free energy functional models the coupling between the composition and curvature fields. The strength of the bonds in the elastic network are then modulated noting local changes in the composition and using a fit to the nonequilibrium molecular dynamics simulation data. Estimates for the magnitude and sign of the coupling parameter in the free energy model are made treating the bending modulus as a function of composition. A procedure for assigning the remaining parameters in the free energy model is also outlined. It is found that the square of the mean curvature averaged over the entire simulation box is enhanced if the strength of the bonds in the elastic network are modulated in response to local changes in the composition field. We suggest that this simulation method could also be used to determine if phase coexistence affects the stress response of the membrane to uniform dilations in area. This response, measured in the mesoscopic regime, is already known to be conditioned or renormalized by thermal undulations.
Quenching of the fluorescence of pyrene-labeled phospholipids by dibromolipids was used to determine the chain length dependence of the bilayer depths of the pyrenyl moieties. Six 1-palmitoyl-2-(pyrenyl-n-acyl)-phosphatidylcholines (PyrnPC) were examined, with end-labeled pyrenyl chains varying in length, n, from 4 to 14 carbons. These lipids were incorporated, at a concentration of 0.3 mol%, into bilayers composed of various mixtures of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and of one of three 1-palmitoyl-2-(x,y-dibromostearoyl)phosphatidylcholine quencher lipids (Brx,yPC; x,y = 6,7; 9,10; or 11,12). Parallel experiments were carried out with bilayers containing 50 mol % cholesterol. Quenching in these systems is dynamic, as demonstrated by the identical dependence of steady-state fluorescence intensities and excited state lifetimes of Pyr8PC on the mole fraction of Br6,7PC. Stern--Volmer analysis of the Brx,PC mole fraction dependence of PyrnPC fluorescence yielded apparent quenching constants, KSV, which show a systematic relation with both the length of the pyrenyl acyl chain and the position of the bromine atoms. The quenching data were further analyzed by plotting KSV as a function of n (defined above), or b (the average of the two bromine positions for each PyrnPC), or n--b (the separation between pyrenes and bromines). In all cases, the data were fit by Gaussian functions yielding estimates of the centers and the apparent 1/e half-widths of the transversal distributions of the pyrenyl moieties in methylene units (mu). Both in the absence and in the presence of cholesterol, the position of each PyrnPC Gaussian center is equal to the sum of n plus a constant d approximately 2.5 mu, corresponding to the distance from the effective center of the pyrenyl moiety to its point of attachment to the acyl chain.(ABSTRACT TRUNCATED AT 250 WORDS)
Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors such as the G-protein coupled serotonin1A receptor. In this paper, we have explored the organization and dynamics of bovine hippocampal membranes using environment-sensitive and differentially localized fluorescent probes NBD-PE and NBD-cholesterol, utilizing wavelength-selective and time-resolved fluorescence measurements. The NBD group in NBD-PE is localized at the membrane interface while in NBD-cholesterol it is localized deeper in the membrane. Our results show that native hippocampal membranes offer considerable motional restriction as evidenced from red edge excitation shift of NBD probes. However, this effect progressively decreases with increasing cholesterol depletion in the case of NBD-cholesterol, possibly indicating a reduction in membrane heterogeneity. In contrast, REES of NBD-PE in hippocampal membranes does not show any significant change upon cholesterol depletion indicating relative lack of sensitivity of the membrane interface to cholesterol depletion. These observations are supported by changes in fluorescence polarization with cholesterol depletion. Taken together, these results imply that the deeper hydrocarbon region of the hippocampal membrane is more sensitive to changes in membrane organization and dynamics due to cholesterol depletion than the interfacial region. The motional restriction in native membranes is maintained even in the absence of proteins. The fluorescence lifetimes of both the NBD probes show slight reduction upon cholesterol depletion indicating a change in micro-environmental polarity possibly due to water penetration. These results are relevant in understanding the complex organization of hippocampal membranes and could have possible functional implications.
The composition of vesicles determines the physical state and elasticity of their bilayers. Fatty acid spin labels were incorporated into vesicles, composed of the single chain non-ionic surfactant octaoxyethylenelaurate-ester (PEG-8-L), the sucrose laurate-ester L-595 and cholesterol sulfate (CS) to monitor local dynamic properties of lipid molecules in vesicle bilayers and to study the elasticity of vesicle bilayers. Studies with the spin label probes 5-, 12- and 16-doxyl stearic acid (DSA) indicated that both the order parameter and the rotational correlation times increased when the doxyl group was positioned closer to the headgroup region. These findings indicate that the fluidity of membranes decreased near the headgroup region. Comparing 16-DSA incorporated in vesicle formulations with either 30 or 70 mol% showed no difference in alkyl chain mobility as was reflected by the order parameter. The rotational correlation times, however, showed a slowdown from 0.38 to 0.71 and 1.13 ns when the PEG-8-L molar content was decreased from 100 to 70 and 30 mol% for PEG-8-L:L-595:CS vesicles, respectively. Extrusion measurements indicated an increase in elasticity of vesicle bilayers as the molar content of PEG-8-L was increased from 10 to 90 mol%. Incorporation of cholesterol sulfate stabilizes vesicles and thereby, decreases the elasticity. The increased elasticity correlated excellent with a reduction in the rotational correlation times observed. In conclusion, these results demonstrate that when the molar content of the single chain non-ionic surfactant PEG-8-L in vesicles is increased the elasticity is enhanced and the rotational correlation time is reduced. The enhanced elasticity might contribute to an optimal design of vesicles as drug carriers for transdermal application.
Pure 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) or mixed DPPC:1,2-dipalmitoyl phosphatidyletanolamine (DPPE):1,2-dipalmitoyl diphosphatidylserine (DPPS) (17:5:3) liposomes were incorporated with 5 mol% dietary carotenoids (beta-carotene, lutein and zeaxanthin) or with cholesterol (16 and 48 mol%) in the absence or presence of 15 mol% carotenoids, respectively. The carotenoid incorporation yields ranged from 0.42 in pure to 0.72 in mixed phospholipid liposomes. They decreased significantly, from 3 to 14%, in the corresponding cholesterol-doped liposomes, respectively. Highest incorporation yields were achieved by zeaxanthin and lutein in phospholipid liposomes while in cholesterol-containing liposomes, lutein was highest incorporated. The effects on membrane structure and dynamics were determined by differential scanning calorimetry, steady-state fluorescence and anisotropy measurements. Polar carotenoids and cholesterol cause similar, dose-dependent effects: ordering and rigidification revealed by broadening of the transition peak, and increase of anisotropy. Membrane hydrophobicity is determined by cholesterol content and carotenoid polarity. In cholesterol-doped liposomes, beta-carotene is less incorporated than in cholesterol-free liposomes. Our observations suggest effects of carotenoids, even at much lower effective concentrations than cholesterol (8 to 80-fold), on membrane structure and dynamics. Although they are minor constituents of animal membranes, carotenoids may act as modulators of membrane phase transition, fluidity, polarity and permeability, and therefore, can influence the membrane physiology and pathology.
11-ketotestosterone (11-KT), a potent male-specific androgen in fish, has important roles on spermatogenesis, male behavior, and nuptial coloration. The site of 11-KT synthesis and its role on male germ cell development during protogynous sex change is not clearly understood. We examined the dynamics of steroidogenic enzymes immunolocalization, viz cholesterol side-chain cleavage (P450scc), biomarker of steroids and cytochrome P45011beta-hydroxylase (P45011beta), downstream to 11-KT production, throughout the process of sex change in honeycomb grouper (Epinephelus merra). In female, P450scc immunoreactivity (-ir) was observed in the theca layer and tunica near blood vessels (BV). During the onset of sex change, P450scc reactive cells were observed in the remaining follicle layer of degenerated oocyte of the ovo-testis in early transitional (ET) and late transitional (LT). In male, P450scc-ir was localized in the interstitial Legdig cells of testis. P45011beta reactive cells were observed in the tunica near BV in female but not in theca layer. In ET and LT phases gonads, P45011beta localized in remaining follicle layer of degenerated oocyte and tunica near BV. On the other hand, in male, both interstices and tunica near BV showed strong signals against P45011beta. Moreover, in vivo and in vitro levels of 11-KT related with the changes in the nuclei diameter of P45011beta-positive cells in both tunica near BV and remaining follicle layer of degenerated oocyte to interstices during the progress of sex change. The present results suggest that 11-KT produced in the tunica near BV may provide the stimulus for female to degenerate oocytes and initiate sex change. However, 11-KT produced both in tunica near BV and remaining follicle layer of degenerated oocyte possibly plays critical role during testicular differentiation as well as gonadal restructuring at mid to late phases (ET to LT) of sex change in honeycomb grouper.
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Interendothelial and subendothelial monocytes/macrophages are extremely rare in normal rabbit aortas compared with humans and swine, as has been shown by the present study and previous investigations of surface preparations. Light microscopy was used to study the development of immune complex-induced hematogenic macrophage infiltrations in 26 rabbits from the 4th to the 28th day after the last antigen administration. Of particular interest was the intensity, localization and duration of the hematogenic cell infiltrates with a view to establishing the role of immunological stimuli for subendothelial monocyte and lymphocyte accumulation. It was found that immune complex-induced hematogenic macrophage infiltrates are very short-lived and disappear after 20-28 days following immune complex deposition. They concentrate in the ostium regions of the thoracic and abdominal aorta and are subject to flow dynamics similar to those of cholesterol-induced sudanophile lipid deposition. Subendothelial macrophage infiltrates increase the permeability of the aorta for low-molecular but not for macromolecular plasma particles, do not affect the endothelial silver line picture and do not lead to lipid accumulation.
Throughout evolution, organisms have developed immune-surveillance networks to protect themselves from potential pathogens. At the cellular level, the signalling events that regulate these defensive responses take place in membrane rafts--dynamic microdomains that are enriched in cholesterol and glycosphingolipids--that facilitate many protein-protein and lipid-protein interactions at the cell surface. Pathogens have evolved many strategies to ensure their own survival and to evade the host immune system, in some cases by hijacking rafts. However, understanding the means by which pathogens exploit rafts might lead to new therapeutic strategies to prevent or alleviate certain infectious diseases, such as those caused by HIV-1 or Ebola virus.
Sixteen untreated black patients with mild-to-moderate hypertension and no evidence of target organ damage were matched for age, sex, casual blood pressure (BP), and socioeconomic status with 16 white hypertensives. All patients were studied under standardized conditions in the hospital where they underwent continuous intraarterial ambulatory monitoring of BP and assessment of BP control mechanisms. BP characteristics over prolonged periods of recording were similar for both groups, as were sinoaortic baroreflex activity and pressor response to isometric and dynamic exercise and to cold. Fasting cholesterol and triglyceride levels in both groups were similar. Resting plasma renin activity (PRA) was significantly lower in blacks, but no difference was observed in resting plasma norepinephrine levels. Urinary excretion of NA+ and K+ was also similar in both groups. Thus, results showed that casual BPs matched for black and whites, and recorded over a prolonged period, were similar in pattern, variability, and response to pressor stimuli. It appears that, if BP contributes to the different patterns of morbidity in blacks and whites, it is more likely to be the actual level of BP rather than differences in BP characteristics.
Male and female, normotensive, Sprague-Dawley (S-D) rats, and spontaneously hypertensive rats (SHR) were subjected to acute and massive myocardial infarction with isoproterenol. Some of the animals were pre-treated (7 days) with the prolactin-lowering drug, bromocryptine. SHR survived in greater numbers than S-D but developed massive congestive heart failure of late onset. The adrenal glands and hearts became greatly hypertrophied in parallel with severely involuted thymus glands. ECG tracings demonstrated intense tachycardia and myocardial ischaemia. Bromocryptine reduction of prolactin (PRL) showed no effect on ECG tracings but reduced triglyceride, free fatty acid, total cholesterol and glucose levels. Isoproterenol caused dynamic increase in glucose, free fatty acids and triglycerides. CPK levels demonstrated greater cardiac damage in S-D vs SHR; greatly elevated SGOT and SGPT levels confirmed the presence of fatty liver in S-D and SHR. Myocardial infarction caused marked increase in circulating PRL in females only and sustained increases in aldosterone and corticosterone. SHR survivors had a high incidence of atrial and ventricular thrombi, left ventricular aneurysms, and intense fibroplasia and cartilaginous metaplasia in areas adjacent to damaged myocardium. It is suggested that adrenal steroidogenesis during an acute myocardial infarct favours survival and more complete myocardial repair in females vs males, and preexisting hypertension in SHR is associated with hormonal and metabolic response patterns different from normotensive S-D rats.
The lipid composition and protein content in extremely low-density lipoproteids. lipoproteid-lypase activity and breakdown dynamics of the mentioned [3H]cholesterol-labelled lipoproteids are studied in blood of young and old rabbits in norm and under experimental atherosclerosis. It is shown that in normal animals the content of all lipid components and protein decreases with ageing in the fraction of extremely low-density lipoproteids isolated from blood serum. The activity of lipoproteid lypase and breakdown of intravenously injected labelled extremely low-density lipoproteids are lowered in old animals as well. Under experimental atherosclerosis in extremely low-density lipoproteids there occurs accumulation of cholesterol esters in considerable amounts, the breakdown of the class lipoproteids is slown down. The enzymic activity manifests no significant changes in comparison with the norm. Disturbances in the composition of lipoproteids and their metabolism with the studied pathology are more pronounced in old rabbits than in young ones.