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Results for “Cholesterol dynamics”

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PubMed1994

Mechanical properties of model atherosclerotic lesion lipid pools.

Structural analysis of atherosclerotic coronary arteries has suggested that stress concentrations are associated with plaque rupture and that these stress concentrations are critically dependent on the geometry and mechanical properties of the fibrous cap and lipid pool. Recent clinical trials of lipid-lowering therapy have shown a significant reduction in cardiac events associated with plaque rupture perhaps because of the changing composition of subintimal lipid pools. To test the hypothesis that changes in lipid composition can change the mechanical properties of lipid pools, we measured the dynamic shear moduli of combinations of cholesterol monohydrate crystals, phospholipids, and triglycerides similar to those found in atherosclerotic lesions. Increasing the cholesterol monohydrate concentration from 0% to 50% increased the real component of the dynamic shear modulus (storage modulus or stiffness) by 4.5 times at a frequency of 1 Hz (P < .001). All specimens demonstrated an increase in stiffness with increasing frequencies of stress ranging from 0.1 to 3 Hz. We conclude that the stiffness of model atherosclerotic plaque lipid pools is related to the concentration of cholesterol monohydrate crystals. Because the relative concentration of cholesterol monohydrate increases during early regression of experimental atherosclerosis, the resultant stiffening of the lipid pool may reduce stresses in plaque caps. However, the magnitude of the contribution of changing lipid stiffness to the reduction of cardiac events seen in clinical studies is unclear.

Arteriosclerosis↗

PubMed1991

Diet-induced type IV-like hyperlipidemia and increased body weight are associated with cholesterol gallstones in hamsters.

Male Syrian hamsters (60-70 g) were fed purified diets containing 5% fat (American Fat Blend) and 15% fiber with or without 0.3% cholesterol (0.86 mg/kcal), for 12 weeks. Hamsters fed the cholesterol-supplemented challenge diet revealed a major increase in plasma triglyceride between 9 and 12 weeks, whereas plasma cholesterol (which reflected body weight dynamics) increased three-fold up to nine weeks and plateaued (342 +/- 22 vs. 122 +/- 5 mg/dL). The greatest increases in cholesterol occurred in the very low density lipoprotein (VLDL) and high density lipoprotein (HDL2) fractions. Gallstone incidence was similar (69% vs. 78%) for cholesterol-supplemented vs. control hamsters, but the type of stones differed. Of the cholesterol-supplemented hamsters with gallstones, 45% had cholesterol stones and 55% had pigment stones. Only pigment stones were seen in control hamsters. Hamsters with cholesterol stones were 25% heavier and transported most cholesterol in VLDL (33 +/- 5%), approximately double that in VLDL of cholesterol-supplemented hamsters with no stones (19 +/- 3%) or cholesterol-supplemented hamsters with pigment stones (21 +/- 3%). Hamsters with pigment stones or no stones (regardless of diet fed) transported the majority of their cholesterol in HDL2 (44%), whereas this figure was only 27% in hamsters that developed cholesterol stones. Thus pigment stones develop routinely in hamsters fed casein-based purified diets. Adding dietary cholesterol resulted in cholesterol gallstones only in those hamsters that gained the most weight and whose terminal VLDL/HDL cholesterol ratio exceeded 1.0, not unlike the lipoprotein profile of obese humans who develop cholesterol gallstones.

Animals↗

PubMed2004

Polarization of plasma membrane microviscosity during endothelial cell migration.

Cell movement is characterized by anterior-posterior polarization of multiple cell structures. We show here that the plasma membrane is polarized in moving endothelial cells (EC); in particular, plasma membrane microviscosity (PMM) is increased at the cell leading edge. Our studies indicate that cholesterol has an important role in generation of this microviscosity gradient. In vitro studies using synthetic lipid vesicles show that membrane microviscosity has a substantial and biphasic influence on actin dynamics; a small amount of cholesterol increases actin-mediated vesicle deformation, whereas a large amount completely inhibits deformation. Experiments in migrating ECs confirm the important role of PMM on actin dynamics. Angiogenic growth factor-stimulated cells exhibit substantially increased membrane microviscosity at the cell front but, unexpectedly, show decreased rates of actin polymerization. Our results suggest that increased PMM in lamellipodia may permit more productive actin filament and meshwork formation, resulting in enhanced rates of cell movement.

Actin Cytoskeleton↗

PubMed1995

Exercise-induced vasomotion of angiographically normal and stenotic coronary arteries improves after cholesterol-lowering drug therapy with bezafibrate.

OBJECTIVES: We attempted to determine whether the coronary vasomotor response to exercise improves after cholesterol-lowering drug therapy with bezafibrate. BACKGROUND: Hypercholesterolemia and other coronary risk factors are associated with impaired endothelium-dependent coronary vasomotor response to physiologic or pharmacologic stimuli, even in the absence of overt coronary atherosclerosis. It is still unknown whether the coronary artery vasomotor response to dynamic exercise improves under cholesterol-lowering drug therapy. METHODS: Of 15 male patients (age 51 +/- 7 years [mean +/- SD]) included in the study, 7 had markedly elevated cholesterol levels (> or = 6.5 mmol/liter, therapy group), and 8 had normal or slightly elevated cholesterol levels (< 6.5 mmol/liter, control group). At baseline and after 7 months of cholesterol-lowering therapy with bezafibrate (400 mg/day) in the therapy group, coronary vasomotor response to dynamic exercise (percent change in cross-sectional vascular area at maximal exercise vs. rest [100%]) in normal and stenotic, previously dilated vessels was assessed by quantitative coronary angiography. RESULTS: During follow-up, total serum cholesterol levels in the therapy group decreased from 7.8 +/- 1.1 to 5.8 +/- 1.1 mmol/liter (p = 0.0001) and did not change significantly in the control group (from 5.4 +/- 0.9 to 6.0 +/- 1.2 mmol/liter, p = NS). Exercise-induced vasomotor response (at similar work loads in the therapy and control groups) in both normal and dilated stenotic coronary arteries improved significantly in the therapy group, from 100 +/- 9% to 109 +/- 7% (p = 0.0001, cross-sectional area at rest 100%) and from 80 +/- 11% to 106 +/- 7% (p = 0.0002), respectively, but did not improve during follow-up in the control group. CONCLUSIONS: The present study indicates that cholesterol-lowering drug therapy with bezafibrate for 7 months improves exercise-induced vasomotion of angiographically normal coronary arteries. Seven months after coronary angioplasty, the reduction in serum cholesterol levels is, at least in part, associated with a restoration of the initially disturbed vasomotor response of stenotic vessel segments to exercise.

Bezafibrate↗

PubMed2005

A comparison of the effect of medium- vs. long-chain triglycerides on the in vitro solubilization of cholesterol and/or phytosterol into mixed micelles.

Despite clinical evidence of the cholesterol-lowering effects of phytosterols, the exact mechanisms involved are still unclear. Displacement of cholesterol by phytosterols from mixed micelles, which is due to their greater hydrophobicity, is one of the hypotheses for the lumenal effects contributing to the reduction of intestinal cholesterol absorption. In this study a dynamic in vitro lipolysis method was used to examine the solubilization behavior of cholesterol and/or phytosterols during lipolysis to probe the efficacy of cholesterol displacement from mixed micelles by phytosterols. The effects of lipid chain length on sterol solubilization were studied by using microcrystalline suspensions containing 17% phytosterol or cholesterol, formulated in long-chain TG (LCT) and medium-chain TG (MCT). When digesting cholesterol-suspended in LCT, the entire cholesterol dose was incorporated into the micellar phase. For the cholesterol formulation suspended in MCT, 50.3% of the initial dose was recovered in the micelles. Under the respective conditions, we observed lower solubilization of phytosterols than of cholesterol (roughly fourfold). Only 25% of the initial phytosterol dose was solubilized from suspensions formulated with LCT, and 13% was solubilized from MCT formulations. Co-administration of phytosterol and cholesterol suspensions showed a significant reduction of cholesterol solubilization, particularly when dosed in MCT, with approximately 25% of the cholesterol dose solubilized. Insignificant amounts of cholesterol were displaced by phytosterols when cholesterol was presolubilized in the mixed micelles. The results show that, compared with LCT, mixed micelles containing MCT lipolysis products have a reduced solubilizing capacity for cholesterol, which adds to the effectiveness of the phytosterols in displacing cholesterol. This suggests potential benefits of using medium chain length lipids in cholesterol-lowering phytosterol products.

Cholesterol↗

PubMedDate not supplied

High resolution 2D 1H-13C correlation of cholesterol in model membrane.

High resolution 2D NMR MAS spectra of liposomes, in particular 1H-13C chemical shifts correlations have been obtained on fluid lipid bilayers made of pure phospholipids for several years. We have investigated herein the possibility to obtain high resolution 2D MAS spectra of cholesterol embedded in membranes, i.e. on a rigid molecule whose dynamics is characterized mainly by axial diffusion without internal segmental mobility. The efficiency of various pulse sequences for heteronuclear HETCOR has been compared in terms of resolution, sensitivity and selectivity, using either cross polarization or INEPT for coherence transfer, and with or without MREV-8 homonuclear decoupling during t1. At moderately high spinning speed (9 kHz), a similar resolution is obtained in all cases (0.2 ppm for 1H(3,4), 0.15 ppm for 13C(3,4) cholesterol resonances), while sensitivity increases in the order: INEPT < CP(x4) < CP + MREV. At reduced spinning speed (5 kHz), the homonuclear dipolar coupling between the two geminal protons attached to C(4) gives rise to spinning sidebands from which one can estimate a H-H dipolar coupling of 10 kHz which is in good agreement with the known dynamics of cholesterol in membranes.

Cholesterol↗

PubMed1989

Slow motions in oriented phospholipid bilayers and effects of cholesterol or gramicidin. A 19F-NMR T1 rho study.

In an extension of our earlier work (Peng, Z.-y., V. Simplaceanu, I. J. Lowe, and C. Ho. 1988. Biophys. J. 54:81-95), the rotating-frame nuclear spin-lattice relaxation (T1 rho) technique has been used to investigate the slow molecular motions (10(-4) - 10(-6) s) in lipid bilayers prepared from protonated or perdeuterated 19F-labeled phospholipids in the absence and presence of cholesterol or gramicidin as membrane-interacting molecules. Complications caused by the 19F-1H cross-polarization observed previously can be removed by the substitution of 2H for 1H in the acyl chains. Only a weak dependence of the T-1(1 rho) on the locking field strength is found for a phospholipid molecule with perdeuterated acyl chains, indicating that there are no slow motions with a single, well-defined correlation time between 5 x 10(-6) and 4 x 10(-5) s. However, the orientation dependences of the T-1(1 rho) can be well fitted by motional models with either one slow motion having an unspecified geometry or with a superposition of two specific types of slow motions. Cholesterol and gramicidin show distinct effects in altering either the geometry or the weighting of slow motions in phospholipid bilayers, as reflected by changes in the orientation dependence. These two additives also exhibit quite different label-position specificities. A qualitative understanding of the induced effects of cholesterol and gramicidin on the dynamics of phospholipid bilayers will be discussed.

Cholesterol↗

PubMed2002

Morphological manipulation of bolaamphiphilic polydiacetylene assemblies by controlled lipid doping.

Morphological transformations of bolaamphiphilic polydiacetylene (L-Glu-Bis-3) lipid assemblies from helical ribbons to vesicles and flat sheets through controlled doping are described, and the role of specific lipid dopants in these processes is discussed. Upon doping with cell surface receptor G(M1) ganglioside, fluid vesicular structures start to emerge, coexisting with the micro-crystalline helical ribbons. The vesicle formation is further facilitated and stabilized by the introduction of cholesterol into the system, presumably through surface curvature variation induced by inhomogeneous distribution and dynamic clustering of G(M1) and cholesterol within the doped assemblies. Extended helical ribbons are "truncated" into patches of flat sheets when a sufficient amount of Bis-1, a structurally compatible symmetric bolaamphiphilic diacetylene lipid, is doped. The results reaffirm the important roles of packing geometry and headgroup chirality in the formation of extended helical ribbon structures. The doped assemblies of bolaamphiphiles allow for capture of intermediate structures of morphological transformation using transmission electron microscopy (TEM). A vesicle-to-ribbon transformation mechanism via lateral reorganization within relatively fluid vesicular microstructures has been suggested. Understanding of the doping-induced transformation process provides useful information for the design of advanced materials where the microscopic morphology of material is crucial to its function.

Acetylene↗

PubMed2002

MLN64 mediates mobilization of lysosomal cholesterol to steroidogenic mitochondria.

This study demonstrates that the steroidogenic acute regulatory protein-related lipid transfer (START) domain-containing protein, MLN64, participates in intracellular cholesterol trafficking. Analysis of the intracellular itinerary of MLN64 and MLN64 mutants tagged with green fluorescent protein showed that the N-terminal transmembrane domains mediate endocytosis of MLN64 from the plasma membrane to late endocytic compartments. MLN64 constitutively traffics via dynamic NPC1-containing late endosomal tubules in normal cells; this dynamic movement was inhibited in cholesterol-loaded cells, and MLN64 is trapped at the periphery of cholesterol-laden lysosomes. The MLN64 START domain stimulated free cholesterol transfer from donor to acceptor mitochondrial membranes and enhanced steroidogenesis by placental mitochondria. Expression of a truncated form of MLN64 (DeltaSTART-MLN64), which contains N-terminal transmembrane domains but lacks the START domain, caused free cholesterol accumulation in lysosomes and inhibited late endocytic dynamics. The DeltaSTART-MLN64 dominant negative protein was located at the surface of the cholesterol-laden lysosomes. This dominant negative mutant suppressed steroidogenesis in COS cells expressing the mitochondrial cholesterol side chain cleavage system. We conclude that MLN64 participates in mobilization and utilization of lysosomal cholesterol by virtue of the START domain's role in cholesterol transport.

Animals↗

PubMed2003

In vivo and in vitro peripheral-type benzodiazepine receptor polymerization: functional significance in drug ligand and cholesterol binding.

Peripheral-type benzodiazepine receptor (PBR) is an 18 kDa high-affinity drug ligand and cholesterol binding protein involved in various cell functions. Antisera for distinct PBR areas identified immunoreactive proteins of 18, 40, and 56 kDa and occasionally 72, 90, and 110 kDa in testicular Leydig and breast cancer cells. These sizes may correspond to PBR polymers and correlated to the levels of reactive oxygen species. Treatment of Leydig cells with human chorionic gonadotropin rapidly induced free radical, PBR polymer, and steroid formation. UV photoirradiation generates ROS species, which increased the size of intramembraneous particles of recombinant PBR reconstituted into proteoliposomes consistent with polymer formation, determined both by SDS-PAGE and by freeze-fracture electron microscopy. Spectroscopic analysis revealed the formation of dityrosines as the covalent cross-linker between PBR monomers. Moreover, photoirradiation increased PK 11195 drug ligand binding and reduced cholesterol binding capacity of proteoliposomes. Further addition of PK 11195 drug ligand to polymers increased the rate of cholesterol binding. These data indicate that reactive oxygen species induce in vivo and in vitro the formation of covalent PBR polymers. We propose that the PBR polymer might be the functional unit responsible for ligand-activated cholesterol binding and that PBR polymerization is a dynamic process modulating the function of this receptor in cholesterol transport and other cell-specific PBR-mediated functions.

Animals↗

PubMed1995

Abrupt modifications of phospholipid bilayer properties at critical cholesterol concentrations.

The fluorescence generalized polarization (GP) of 2-dimethylamino-6-lauroylnaphthalene (Laurdan) reveals different effects of cholesterol on the phase behavior of phospholipid bilayers. Phospholipid vesicles composed of gel, liquid-crystalline, and coexisting domains of the two phases have been studied at temperatures from 1 to 65 degrees C, without cholesterol and with cholesterol concentrations of 3-50 mol %. Laurdan GP measurements show the general effect of cholesterol of increasing the molecular dynamics of the gel and of decreasing the molecular dynamics of the liquid-crystalline phase. In the liquid-crystalline phase, the increased order yields Laurdan GP values close to those obtained in the gel phase. At cholesterol concentrations > 15 mol % a phase transition cannot be detected. Using the wavelength dependence of the excitation and emission GP spectra we determine that differences between the two phospholipid phases cannot be detected. In particular, in vesicles composed of coexisting gel and liquid-crystalline phases the GP wavelength dependence characteristic of coexisting domains cannot be observed at cholesterol concentrations > or = 15 mol %. Cholesterol causes the decrease in both the polarity and the dipolar relaxation effects on the neighborhood of the fluorescent naphthalene moiety of Laurdan. Probably because of a cholesterol-induced increase in the bilayer packing, these effects do not occur continuously with the increase of cholesterol concentration in the bilayer. Cholesterol concentrations inducing higher Laurdan GP values have been determined at about 5, 10, 15, 30, and 45 mol % with respect to phospholipids. We propose that the formation of ordered molecular microdomains at critical cholesterol concentrations can explain the occurrence of the observed discontinuities.

2-Naphthylamine↗

PubMed1999

[Effect of hypoxic hypoxia on development of atherosclerosis in rabbits].

The study was aim a evaluating effects of intermittent hypoxic training on experimental atherosclerosis. In a three-month experiment rabbits (n = 12) were fed with cholesterol (200 mg/kg of the body mass) and "ascended" to the altitude of 6000 m for 6 hrs per a day. The controls (n = 10) were fed with cholesterol only. Investigated was the dynamics of a spectrum of plasmatic lipoproteids, lipid peroxidation, morphologic alterations in the aorta and mononuclear phagocytes. Intermittent hypoxia caused less evident than in the control increases in total cholesterol (20.6 +/- 2.3 vs. 33.1 +/- 1.9 mmol/l, p < 0.05) and LDL cholesterol (19.4 +/- 2.2 vs. 32.3 +/- 2.3 mmol/l, p < 0.05). Unlike the controls, there were no atherosclerotic plaques in the aorta of the experimental rabbits. Atherosclerosis was diagnosed just by tiny dot- and strip-like lipid patches smaller in size when compared with the control group in which massive diffuse lipid deposits were found. The affected aortic areas made up 13.5% in the experimental group and 65% in the control. Hypoxic training brought about activation of the antioxidant system and was propitious for functional activity and oxygen-dependent and -independent metabolism of phagocytes. Exposure to hypoxia reduced the number of lipid inclusions in monocytes (303.0 +/- 12.5% vs. 370.0 +/- 4.5% in the control, p < 0.05), prevented the dramatic inhibition of the cells observed in the animals fed with cholesterol and, eventually, blocked up formation of spumescent cells. These data infer that intermittent chamber hypoxia exerts the antiatherogenic effect on experimental atherosclerosis.

Animals↗

PubMed1987

Static and dynamic components of renal cortical brush border and basolateral membrane fluidity: role of cholesterol.

Static polarization and differential polarized phase fluorimetry studies on rat renal cortical brush border (BBM) and basolateral membranes (BLM) were undertaken to determine the membrane components responsible for differences in BBM and BLM fluidity, whether these differences were due to the order or dynamic components of membrane fluidity and if a fluidity gradient existed within the bilayer. Surface membrane proteins rigidified both BBM and BLM fluidity. Neutral lipid extraction, on the other hand, caused a larger decrease in BBM than BLM fluorescence polarization (0.104 vs. 0.60, P less than 0.01) using diphenyl hexatriene (DPH). Cholesterol addition to phospholipid fractions restored membrane fluidity to total lipid values in both BBM and BLM phospholipids. The response to cholesterol in the BBM was biphasic, while the BLM response was linear. Lateral mobility, quantitated using dipyrenylpropane, was similar in both BBM and BLM fractions at 35 degrees C. BBM and BLM differed primarily in the order component of membrane fluidity as DPH-limiting anisotropy (r infinity) (0.212 vs. 0.154, P less than 0.01) differed markedly between the two membrane fractions. The two membrane components also differed with respect to 2 and 12-anthroyloxy stearate (2-AS, 12-AS) probes, indicating a difference in the dynamic component of membrane fluidity may also be present. DPH and 12-AS probes were also used to quantitate inner core membrane fluidity and showed the BBM was less fluid than the BLM for intact membranes, total lipid extracts and phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2001

Surface properties of cholesterol-containing membranes detected by Prodan fluorescence.

The fluorescent membrane probe 6-propionyl-2-dimethylaminonaphthalene (Prodan) displays a high sensitivity to the polarity and packing properties of lipid membrane. Contrary to 6-lauroyl-2-dimethylaminonaphthalene (Laurdan), Prodan can also monitor the properties of the membrane surface, i.e., the polar-head pretransition. In bilayers composed of coexisting gel and liquid-crystalline phases, Prodan shows a preferential partitioning in the latter, so that the detected membrane properties mainly belong to fluid domains. In the presence of cholesterol, the packing properties of the gel phase phospholipids are modified in such a way that Prodan can penetrate and label the membrane. Although Prodan labeling of the gel phase is a function of cholesterol concentration, 3 mol percent cholesterol is sufficient for a 60% Prodan labeling with respect to the maximum labeling reached at 15 mol percent cholesterol. We present steady-state and dynamical fluorescence measurements of Prodan in bilayers in the presence of cholesterol. Our results fit the liquid-ordered/liquid-disordered phase model for cholesterol-containing membranes and show that the presence of cholesterol, in addition to modification to the phase state of the hydrophobic portion of the bilayer, strongly affects the packing and the polarity of the membrane hydrophobic-hydrophilic interface.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1984

Amphotericin and model membranes. The effect of amphotericin B on cholesterol-containing systems as viewed by 2H-NMR.

The interactions between amphotericin B and sterol-containing model membranes were monitored by 2H-NMR of deuterium-labelled dimyristoylphosphatidylcholine (DMPC), cholesterol and epicholesterol. The addition of amphotericin B to a cholesterol/DMPC (3:7) system was perceived differently by the lipid, depending upon the depth in the bilayer: no structural change was manifest in the acyl chain region associated with the plateau in molecular ordering (C4'), whereas the lipid clearly senses two environments near the center of the bilayer (C13', C14'). The amount as well as the ordering properties of the more ordered antibiotic-induced component, sensed at C14', increased with decreasing temperature. The structural parameters of deuterium-labelled cholesterol in cholesterol/DMPC mixtures were unchanged upon addition of amphotericin B, regardless of the bilayer depth. Upon addition of amphotericin B, the lipid T1 values are unchanged, whereas the T2 values are reduced by a factor of 2. The minimum in T1 observed for cholesterol in DMPC at 32-35 degrees C was shifted towards 38-40 degrees C in the presence of amphotericin B. Epicholesterol-containing dispersions of DMPC had properties similar to those of their cholesterol-containing analogs; a noticeable difference between the systems was an approx. 10% increase in the segmental order parameters on the addition of amphotericin B to the system containing the alpha-isomer of cholesterol. The concept of a dynamic complexation between amphotericin B and sterol is discussed.

Amphotericin B↗

PubMed1988

Influence of molecular packing and phospholipid type on rates of cholesterol exchange.

The rates of [14C]cholesterol transfer from small unilamellar vesicles containing cholesterol dissolved in bilayers of different phospholipids have been determined to examine the influence of phospholipid-cholesterol interactions on the rate of cholesterol desorption from the lipid-water interface. The phospholipids included unsaturated phosphatidylcholines (PC's) (egg PC, dioleoyl-PC, and soybean PC), saturated PC (dimyristoyl-PC and dipalmitoyl-PC), and sphingomyelins (SM's) (egg SM, bovine brain SM, and N-palmitoyl-SM). At 37 degrees C, for vesicles containing 10 mol% cholesterol, the half-times for exchange are about 1, 13, and 80 h, respectively, for unsaturated PC, saturated PC, and SM. In order to probe how differences in molecular packing in the bilayers cause the rate constants for cholesterol desorption to be in the order unsaturated PC greater than saturated PC greater than SM, nuclear magnetic resonance (NMR) and monolayer methods were used to evaluate the cholesterol physical state and interactions with phospholipid. The NMR relaxation parameters for [4-13C]cholesterol reveal no differences in molecular dynamics in the above bilayers. Surface pressure (pi)-molecular area isotherms for mixed monolayers of cholesterol and the above phospholipids reveal that SM lateral packing density is greater than that of the PC with the same acyl chain saturation and length (e.g., at pi = 5 mN/m, where both monolayers are in the same physical state, dipalmitoyl-PC and palmitoyl-SM occupy 87 and 81 A2/molecule, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Radioisotopes↗

PubMed2006

Dynamic and 2D NMR studies on hydrogen-bonding aggregates of cholesterol in low-polarity organic solvents.

Self-diffusion coefficients (D) are measured for normal (nondeuterated) and deuterated cholesterol-d(6) (C26 and C27 methyl groups deuterated) in 1-octanol, chloroform, and cyclohexane at concentrations of 1-700 mM by varying the impurity water concentration (>2 mM) and temperature (30-50 degrees C). The pulsed field gradient spin-echo (PGSE) (1)H and (2)H NMR were used, respectively, at 600 and 92 MHz. At 30 degrees C, the hydrodynamic radius (R) obtained at 20 mM from the D value and solvent viscosity is 5.09, 7.07, and 6.17 A, respectively, in 1-octanol, chloroform, and cyclohexane when the impurity water is negligible. The R value in 1-octanol is the smallest and comparable with the average length of the molecular axes for the cholesterol molecule. In 1-octanol, R is invariant against the concentration variation, whereas in chloroform, R is larger and increases almost linearly with cholesterol concentration. At the highest concentration, 700 mM, the R in chloroform is 13.5 and 16.7 A, respectively, when the impurity water is at negligible and saturated concentrations. The R value larger than that in hydrogen-bonding 1-octanol indicates that cholesterol forms an aggregate through hydrogen bonding. The aggregate structure is confirmed by comparing NOESY spectra in chloroform and 1-octanol. The NOESY analysis reveals the presence of one extra cross peak (C4-C19) in chloroform compared to 1-octanol. Because the carbon atoms related to the cross peak are close to the hydroxyl group (C3-OH), cholesterol molecules are considered to be not piled but are found to be OH-centered in the aggregate. This is supported also by larger rotational hydrodynamic radii measured on cholesterol deuterated at positions C2, C3, C4, and C6. This shows that the aggregate formation is driven by the hydrogen-bonding between cholesterol molecules.

1-Octanol↗

PubMed2002

Dynamic changes in the expression of relaxin-like factor (INSL3), cholesterol side-chain cleavage cytochrome p450, and 3beta-hydroxysteroid dehydrogenase in bovine ovarian follicles during growth and atresia.

Relaxin-like factor (RLF) is a new member of the insulin-relaxin gene family known to be expressed in the ovarian follicular thecal cells of ruminants. To investigate the pattern of RLF expression in development and atresia of bovine follicles, antisera were raised in rats and rabbits to recombinantly expressed bovine pro-RLF and to chemically synthesized ovine RLF B chain, respectively. On dot blotting analysis, the rat antiserum bound to pro-RLF and less strongly to a synthetic mature ovine RLF lacking the C-domain, whereas the rabbit antiserum bound the mature form of ovine RLF. These antisera were used to immunostain bovine ovarian follicles of differing sizes and stages of health and atresia. 3beta-Hydroxysteroid dehydrogenase was colocalized with pro-RLF (n = 86 follicles), and cholesterol side-chain cleavage cytochrome P450 was localized in another section of many of the same follicles (n = 66). Not all follicles expressed pro-RLF in the theca interna, so the results are presented as the proportion of follicles expressing pro-RLF. Both mature and pro-RLF were immunolocalized to steroidogenic thecal cells of healthy follicles. As follicles enlarged to >5 mm, the proportion expressing pro-RLF declined (19/19 for <5 mm and 18/26 for >6 mm). Atresia was divided into antral (antral granulosa cells dying first) or basal (basal cells dying first) and further divided into early, middle, and late. For antral atresia of small follicles (2-5 mm), no decline in the proportion expressing pro-RLF was observed (early 6/6, middle 2/2) until the late stages (1/4). For basal atresia, which only occurs in small follicles (2-5 mm), the proportion expressing pro-RLF declined in the middle (2/5) and late (0/8) stages. In larger follicles (>6 to <10 mm), the proportion expressing pro-RLF also declined with atresia (1/13). These declines in RLF expression with atresia or increasing size were not accompanied by a decline in the expression of steroidogenic enzymes in the theca interna. A significant (P < 0.001) inverse relationship in the expression of pro-RLF and 3beta-hydroxysteroid dehydrogenase in the membrana granulosa was observed. We conclude that the expression of pro-RLF in the theca interna is switched off as follicles enlarge or enter atresia, whereas the expression of steroidogenic enzymes is maintained in the theca interna.

3-Hydroxysteroid Dehydrogenases↗