Cholesterol gallstone dissolution in bile. Dissolution kinetics of crystalline cholesterol monohydrate by conjugated chenodeoxycholate-lecithin and conjugated ursodeoxycholate-lecithin mixtures: dissimilar phase equilibria and dissolution mechanisms

Using compressed discs and microcrystals of cholesterol monohydrate, we evaluated the mechanisms and kinetics of dissolution in conjugated bile salt-lecithin solutions. In stirred conjugated ursodeoxycholate-lecithin and cheno-deoxycholate-lecithin solutions, dissolution of 10,000-psi discs was micellar and linear with time for 10 hours. The dissolution rate constants (k) decreased in proportion to the lecithin content and dissolution rates and k values were appreciably smaller in conjugated ursodeoxycholate-lecithin solutions. After dissolution for 5 to 10 days the discs incubated with ursodeoxycholate-lecithin systems became progressively transformed into macroscopic liquid crystals. Unstirred dissolution of 3,000-psi discs in "simulated" human bile containing physiological lecithin concentrations gave apparent k values that decreased in the following order: ursodeoxycholate-rich >/= chenodeoxycholate-rich > normal. In most cases the discs incubated with ursodeoxycholate-rich bile became covered with a microscopic liquid-crystalline layer. With 20-25 moles % lecithin, these layers eventually dispersed into the bulk solution as microscopic vesicles. During dissolution of microcrystalline cholesterol in conjugated ursodeoxycholate-lecithin systems, a bulk liquid-crystalline phase formed rapidly (within 12 hours) and the final cholesterol solubilities were greater than those in conjugated chenodeoxycholate-lecithin micellar systems. Prolonged incubation of cholesterol microcrystals with pure lecithin or lecithin plus bile salt liposomes did not reproduce these effects. Condensed ternary phase diagrams of conjugated ursodeoxycholate-lecithin-cholesterol systems established that cholesterol-rich liquid crystals constituted an equilibrium precipitate phase that coexisted with cholesterol monohydrate crystals and saturated micelles under physiological conditions. Similar phase dissolution-relationships were observed at physiological lecithin-bile salt ratios for a number of other hydrophilic bile salts (e.g., conjugated ursocholate, hyocholate, and hyodeoxycholate). In contrast, liquid crystals were not observed in conjugated chenodeoxycholate-lecithin-cholesterol systems except at high (nonphysiological) lecithin contents. Based on these and other results we present a molecular hypothesis for cholesterol monohydrate dissolution by any bile salt-lecithin system and postulate that enrichment of bile with highly hydrophilic bile salts will induce crystalline cholesterol dissolution by a combination of micellar and liquid crystalline mechanisms. Since bile salt polarity can be measured and on this basis the ternary phase diagram deduced, we believe that the molecular mechanisms of cholesterol monohydrate dissolution as well as the in vivo cholelitholytic potential of uncommon bile salts can be predicted.-Salvioli, G., H. Igimi, and M. C. Carey. Cholesterol gallstone dissolution in bile. Dissolution kinetics of crystalline cholesterol monohydrate by conjugated chenodeoxycholate-lecithin and conjugated ursodeoxycholate-lecithin mixtures: dissimilar phase equilibria and dissolution mechanisms.     

Structure of cholesterol/ceramide monolayer mixtures: implications to the molecular organization of lipid rafts

The structure of monolayers of cholesterol/ceramide mixtures was investigated using grazing incidence x-ray diffraction, immunofluorescence, and atomic force microscopy techniques. Grazing incidence x-ray diffraction measurements showed the existence of a crystalline mixed phase of the two components within a range of compositions of cholesterol/ceramide between 100:0 and 67:33. The mixed phase coexists with the ceramide crystalline phase in the range of compositions between 50:50 and 30:70; between 30:70 and 0:100 only the highly crystalline phase of ceramide was detected. The latter was determined and modeled. Immunolabeling was performed with an antibody specific to the cholesterol monohydrate crystalline arrangement. The antibody recognizes crystalline cholesterol monolayers, but does not interact with crystalline ceramide. Immunofluorescence and atomic force microscopy data show that in uncompressed ceramide monolayers, the highly crystalline phase coexists with a disordered loosely packed phase. In contrast, no disordered phase coexists with the new crystalline mixed phase. We conclude that the new mixed phase represents a stable homogeneous arrangement of cholesterol with ceramide. As ceramide incorporates the lipid backbone common to all sphingolipids, this arrangement may be relevant to the understanding of the molecular organization of lipid rafts.     

The phase behavior of hydrated cholesterol.

The thermotropic phase behavior of cholesterol monohydrate in water was investigated by differential scanning calorimetry, polarizing light microscopy, and x-ray diffraction. In contrast to anhydrous cholesterol which undergoes a polymorphic crystalline transition at 39 degrees C and a crystalline to liquid transition at 151 degrees C, the closed system of cholesterol monohydrate and water exhibited three reversible endothermic transitions at 86, 123, and 157 degrees C. At 86 degrees C, cholesterol monohydrate loses its water of hydration, forming the high temperature polymorph of anhydrous cholesterol. At least 24 hours were required for re-hydration of cholesterol and the rate of hydration was dependent on the polymorphic crystalline form of anhydrous cholesterol. At 123 degrees C, anhydrous crystalline cholesterol in the presence of excess water undergoes a sharp transition to a birefringent liquid crystalline phase of smectic texture. The x-ray diffraction pattern obtained from this phase contained two sharp low-angle reflections at 37.4 and 18.7 A and a diffuse wide-angle reflection centered at 5.7 A, indicating a layered smectic type of liquid crystalline structure with each layer being two cholesterol molecules thick. The liquid crystalline phase is stable over the temperature range of 123 to 157 degrees C before melting to a liquid dispersed in water. The observation of a smectic liquid crystalline phase for hydrated cholesterol correlates with its high surface activity and helps to explain its ability to exist in high concentrations in biological membranes.     

Effects of amiridin and tacrine, drugs effective in Alzheimer's disease, on the activity of monoamine oxidase A and B]

In vitro comparative studies of effects of amiridin (9-amino-2, 3, 5, 6, 7, 8-hexahydro-1H-cyclopentane (b) choline monohydrate hydrochloride) and tacrine physostigmine and piracetam on monoamine oxidase A (MAO-A) and B (MAO-B) activity in the rat brain were carried out. Piracetam (1 x 10(-4)-1 x 10(-3) M) dose-dependently increased MAO-A and MAO-B activity. At all concentrations used (1 x 10(-7)-5 x 10(-4) M) physostigmine had no effect on MAO-A and MAO-B activity. Amiridin was found to inhibit MAO-B activity at 5 x 10(-4) M concentration only. Tacrine inhibited MAO-A activity at 5 x 10(-4) M concentration. The therapeutical effects of amiridin and tacrine in treatment of Alzheimer disease were not related to their action on MAO-A and -B activity.     

Properties of mixtures of cholesterol with phosphatidylcholine or with phosphatidylserine studied by (13)C magic angle spinning nuclear magnetic resonance

The behavior of cholesterol is different in mixtures with phosphatidylcholine as compared with phosphatidylserine. In (13)C cross polarization/magic angle spinning nuclear magnetic resonance spectra, resonance peaks of the vinylic carbons of cholesterol are a doublet in samples containing 0.3 or 0.5 mol fraction cholesterol with 1-palmitoyl-2-oleoyl phosphatidylserine (POPS) or in cholesterol monohydrate crystals, but a singlet with mixtures of cholesterol and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). At these molar fractions of cholesterol with POPS, resonances of the C-18 of cholesterol appear at the same chemical shifts as in pure cholesterol monohydrate crystals. These resonances do not appear in samples of POPS with 0.2 mol fraction cholesterol or with POPC up to 0.5 mol fraction cholesterol. In addition, there is another resonance from the cholesterol C18 that appears in all of the mixtures of phospholipid and cholesterol but not in pure cholesterol monohydrate crystals. Using direct polarization, the fraction of cholesterol present as crystallites in POPS with 0.5 mol fraction cholesterol is found to be 80%, whereas with the same mol fraction of cholesterol and POPC none of the cholesterol is crystalline. After many hours of incubation, cholesterol monohydrate crystals in POPS undergo a change that results in an increase in the intensity of certain resonances of cholesterol monohydrate in (13)C cross polarization/magic angle spinning nuclear magnetic resonance, indicating a rigidification of the C and D rings of cholesterol but not other regions of the molecule.     

Bile salt micelle can sustain more cholesterol in the intermicellar aqueous phase than the maximal aqueous solubility

The intermicellar aqueous phase in equilibrium with micelle plays an important role in the uptake of sterol. To test the hypothesis whether cholesterol concentration in the intermicellar aqueous phase of a micellar solution is similar to its maximal aqueous solubility, cholesterol concentration in the intermicellar aqueous phase of a bile salt-cholesterol solution and maximal aqueous cholesterol solubility were quantitatively determined by capillary gas-liquid chromatography after filtration. Cholesterol concentration in the intermicellar aqueous phase increased linearly with cholesterol concentration in the micellar solution and reached 1.3 microM at its micellar solubility limit, while the maximal aqueous solubility of cholesterol was (1.2-1.4) x 10(-8) M. The intermicellar monomer concentration of taurocholate was 5.8 mM in which 26 x 10(-8) M cholesterol was solubilized. The results indicate the presence of a cholesterol concentration in the intermicellar aqueous phase that is significantly higher than its maximal aqueous solubility, which can be ascribed primarily to the presence of an intermicellar concentration of bile salt.     

The validity of the cholesterol nucleation assay.

The validity of the cholesterol nucleation assay rests on the assumption that all cholesterol crystals are removed at the start of the assay so that de novo formation of crystals can be studied. In this paper we have tested the validity of this assumption. Cholesterol crystals were added to supersaturated model bile. Subsequently the mixtures were either filtered over a 0.22 micron filter or centrifuged at 37 degrees C for 2 h at 100,000 x g. After ultracentrifugation the isotropic interphase was collected. Using polarized light microscopy no crystals could be visualized in this fraction. However, the nucleation time of the isotropic interphase decreased from 6.8 +/- 1.1 days to 1.8 +/- 0.2 days (mean +/- S.E., P less than 0.01, n = 5) when 10-100 micrograms/ml crystals were added prior to centrifugation. Similar results were observed when instead of centrifugation the mixtures containing crystals were filtered. After filtration over a 0.22 micron filter no crystals could be detected in the filtrate. Yet the nucleation time of the filtrate decreased from 6.4 +/- 0.7 days to 3.1 +/- 0.5 days (mean +/- S.E.) when 10 micrograms/ml cholesterol crystals were added before filtration (n = 10, P less than 0.01). Since no cholesterol crystals could be detected at the start of the assay the reduction in nucleation time must have been brought about by cholesterol microcrystals that passed through the filter. Supplementation of cholesterol crystals to model bile did not accelerate the nucleation time when the samples were passed over a 0.02 micron filter, indicating that the size of the microcrystals was larger than 20 nm. The effect of addition of cholesterol crystals prior to filtration over a 0.22 micron filter was also tested in the crystal growth assay recently developed by Busch et al. ((1990) J. Lipid Res. 31, 1903-1909). Addition of crystals had only a minor effect on the assay. In conclusion, the reduced nucleation time of biles from gallstone patients is probably not only due to the presence of promoting or the absence of inhibiting proteins, but can be caused by the presence of small cholesterol crystals in these biles.     

Fluoroimmunometric method of determining digitoxin]

Digitoxin at concentrations up to 5 x 10(-10) M (therapeutic concentrations are 2 x 10(-8) M) can be reliable measured by a fluoroimmunometric method with coproporphyrin as a tracer. The use of nylon filters with immobilized digitoxin to remove an excess of labelled antibodies increases reliability and reduces the time of measurements, and thus simplifies the assay.     

Effects of tetrahydrolipstatin, a lipase inhibitor, on absorption of fat from the intestine of the rat

Tetrahydrolipstatin (THL) derived by hydrogenation from lipstatin, a lipase inhibitor produced by Streptomyces toxytricini, has been shown to inhibit in vitro the activity of all three lipases secreted to the gastro-intestinal tract; gastric lipase, pancreatic lipase and carboxylester lipase (cholesterol ester hydrolase). The effects of THL on intestinal absorption of fat (transport to the thoracic duct chyle) has now been investigated after intraduodenal infusion in a rat model. Absorption of label from oleic acid when administered with monoolein in micellar bile salt solution was not affected by THL in concentrations up to 10(-4) M calculated on the volume of the aqueous phase. Absorption of free cholesterol in micellar bile salt solution of the lipolytic products of triolein; oleic acid and monoolein, is not significantly affected at a concentration of THL of 10(-4) M. Absorption of cholesterol from cholesteryl oleate under the same conditions is almost completely inhibited. The results indicate that absorption of free cholesterol is not dependent on the activity of pancreatic cholesterol ester hydrolase. The absorption of emulsified triolein was not significantly affected by 10(-5) M THL but decreased to around 30% of the controls by a concentration 10-times higher. There was no significant decrease of cholesterol absorption when administered in emulsified triolein while absorption of cholesteryl oleate was reduced at both concentrations of THL and almost completely at 10(-4) M. Radioactivity from [2-14C]THL when administered emulsified in triolein was recovered in urine, bile and thoracic duct lymph to 10-14, 8-13 and 1-3%, respectively, largely independent on dose administered. Label from [1"-14C] THL was recovered in the same amounts in lymph but much less in bile and urine indicating that the amino acid moiety has been split off early in the absorption process.     

Cholesterol crystallization in model biles: effects of bile salt and phospholipid species composition

Cholesterol in human bile is solubilized in micelles by (relatively hydrophobic) bile salts and phosphatidylcholine (unsaturated acyl chains at sn-2 position). Hydrophilic tauroursodeoxycholate, dipalmitoyl phosphatidylcholine, and sphingomyelin all decrease cholesterol crystal-containing zones in the equilibrium ternary phase diagram (van Erpecum, K. J., and M. C. Carey. 1997. Biochim. Biophys. Acta. 1345: 269-282) and thus could be valuable in gallstone prevention. We have now compared crystallization in cholesterol-supersaturated model systems (3.6 g/dl, 37 degrees C) composed of various bile salts as well as egg yolk phosphatidylcholine (unsaturated acyl chains at sn-2 position), dipalmitoyl phosphatidylcholine, or sphingomyelin throughout the phase diagram. At low phospholipid contents [left two-phase (micelle plus crystal-containing) zone], tauroursodeoxycholate, dipalmitoyl phosphatidylcholine, and sphingomyelin all enhanced crystallization. At pathophysiologically relevant intermediate phospholipid contents [central three-phase (micelle plus vesicle plus crystal-containing) zone], tauroursodeoxycholate inhibited, but dipalmitoyl phosphatidylcholine and sphingomyelin enhanced, crystallization. Also, during 10 days of incubation, there was a strong decrease in vesicular cholesterol contents and vesicular cholesterol-to-phospholipid ratios (approximately 1 on day 10), coinciding with a strong increase in crystal mass. At high phospholipid contents [right two-phase (micelle plus vesicle-containing) zone], vesicles were always unsaturated and crystallization did not occur. Strategies aiming to increase amounts of hydrophilic bile salts may be preferable to increasing saturated phospholipids in bile, because the latter may enhance crystallization.     

Cholesterol crystal formation and growth in model bile solutions

Cholesterol monohydrate crystal formation was studied in supersaturated model bile solutions, containing unlabeled cholesterol, sodium cholate and soybean phosphatidylcholine, and tracer amounts of [3H]cholesterol. Solutions were either seeded with cholesterol crystals to initiate growth, or not seeded to allow self-nucleation and subsequent crystal growth to occur. Crystal growth at 37 degrees C was measured by two methods. First, radioactive cholesterol crystals were isolated by filtration, and the mass of cholesterol that had precipitated was calculated. In unseeded solutions, there was a long lag period before crystal growth was detected. This lag time was decreased by increases in the cholesterol concentration, temperature, and lipid concentration. In seeded solutions, crystal growth also was dependent on the cholesterol concentration, temperature, and lipid concentration. The second method used to measure crystal growth involved the Coulter Counter. At 37 degrees C, reproducible results were not obtained using unseeded solutions due to blocking of the counter aperture with large crystals. In seeded solutions, crystal growth could be measured as an increase in total particle volume. However, comparison of growth rate estimates from the Coulter Counter with those obtained radiochemically revealed poor agreement between the two methods. It is probable that the Coulter Counter is inaccurate in measuring the volume of cholesterol monohydrate crystals due to their anisometric shape.     

Subunit dissociation in fish hemoglobins.

The tetramer-dimer dissociation equilibria (K 4,2) of several fish hemoglobins have been examined by sedimentation velocity measurements with a scanner-computer system for the ultracentrifuge and by flash photolysis measurements using rapid kinetic methods. Samples studied in detail included hemoglobins from a marine teleost, Brevoortia tyrannus (common name, menhaden); a fresh water teleost, Cyprinus carpio, (common name, carp); and an elasmobranch Prionace glauca (common name, blue shark). For all three species in the CO form at pH 7, in 0.1 M phosphate buffer, sedimentation coefficients of 4.3 S (typical of tetrameric hemoglobin) are observed in the micromolar concentration range. In contrast, mammalian hemoglobins dissociate appreciably to dimers under these conditions. The inability to detect dissociation in three fish hemoglobins at the lowest concentrations examined indicates that K 4,2 must have a value of 10(-8) M or less. In flash photolysis experiments on very dilute solutions in long path length cells, two kinetic components were detected with their proportions varying as expected for an equilibrium between tetramers (the slower component) and dimers (the faster component); values of K 4,2 for the three fish hemoglobins in the range 10(-9) to 10(-8) M were calculated from these data. Thus, the values of K 4,2 for liganded forms of the fish hemoglobins appear to be midway between the value for liganded human hemoglobin (K 4,2 approximately 10(-6) M) and unliganded human hemoglobin (K 4,2 approximately 10(-12) M). This conclusion is supported by measurements on solutions containing guanidine hydrochloride to enhance the degree of dissociation. All three fish hemoglobins are appreciably dissociated at guanidine concentrations of about 0.8 M, which is roughly midway between the guanidine concentrations needed to cause comparable dissociation of liganded human hemoglobin (about 0.4 M) and unliganded human hemoglobin (about 1.6 M). Kinetic measurements on solutions containing guanidine hydrochloride indicated that there are changes in both the absolute rates and the proportions of the fast and slow components, which along with other factors complicated the analysis of the data in terms of dissociation constants. Measurements were also made in solutions containing urea to promote dissociation, but with this agent very high concentrations (about 6 M) were required to give measureable dissociation and the fish hemoglobins were unstable under these conditions, with appreciable loss of absorbance spectra in both the sedimentation and kinetic experiments.     

Rat liver microsomal cholesterol biosynthesis and drug oxidase activity are affected by chromium ions (Cr3+) in vitro

Chromium ions (Cr3+)evoked a biphasic curve of changes of rat liver microsomal cholesterol biosynthesis using [14C]acetate and/or [14C]mevalonate as precursors. While for the lower range of Cr3+ concentrations the rate of cholesterol biosynthesis rises, at concentrations above 8 X 10(-6) M they evoke a decrease in the cholesterol biosynthesis, up to 50% down on its control value at a concentration of 8 X 10(-4) M. Differences were more pronounced when using [14C]mevalonate instead of [14C]acetate as precursor. The activity of the microsomal enzyme biphenyl-4-hydroxylase showed an equally intense rise to that of cholesterol biosynthesis up to a 8 X 10(-6) M Cr3+ concentration. Above this concentration, however, the activity of the enzyme starts to drop. NADPH-cytochrome c reductase and NADPH-oxidase were decreased at all Cr3+ concentrations used, which cover a 100-fold range. Lineweaver-Burk plots of the cytoplasmic glucose-6-phosphate dehydrogenase demonstrated an uncompetitive mechanism of inhibition by Cr3+ ions. The results are discussed in terms of the possible significance of the Cr3+ concentration-dependent effects on cholesterol biosynthesis, with the observed atherosclerosis in Cr-deficient humans.     

Size and shape of charged micelles of ionic surfactants in aqueous salt solutions

Light-scattering has been measured on aqueous NaCl solutions of dodecyldimethylammonium chloride and sodium dodecyl sulfate. From molecular weight determination it is confirmed that spherical micelles are formed at low NaCl concentrations, but at high NaCl concentrations the small micelles formed at the critical micelle concentration further associate to form large rod-like micelles with increasing micelle concentration. The reduction of repulsion between charged groups induces the sphere-rod transition of micelle shape. The dependence of molecular weight on ionic strength can be expressed by double logarithmic relations, which are dependent on the micelle shape. While dodecyldimethylammonium chloride dissolves even in 4.00 M NaCl, sodium dodecyl sulfate solutions exhibit some XXX in angular dissymmetry at NaCl concentrations higher than 0.50 M at low temperatures.     

Solubilization of cholesterol and polycyclic aromatic compounds into sodium bile salt micelles (part 2)

The aqueous solubility of cholesterol was determined over the temperature range from 288.2 to 318.2 K with intervals of 5 K by the enzymatic method. The solubility was (3.7+/-0.3)x10(-8) mol dm(-3) (average +/- S.D.) at 308.2 K. The maximum additive concentrations of cholesterol into the aqueous micellar solutions of sodium deoxycholate (NaDC), sodium ursodeoxycholate (NaUDC), and sodium cholate (NaC) were spectrophotometrically determined at different temperatures. The cholesterol solubility increased in the order of NaUDC相似文献   

Effects of norepinephrine on lung liquid production by in vitro lungs from fetal guinea pigs

Lungs from near-term fetal guinea pigs (61 +/- 2 days of gestation) were supported in vitro for 3 h; lung liquid production was monitored by a dye dilution method. Untreated control preparations produced fluid at 1.38 +/- 0.30 mL x kg(-1) body weight x h(-1), with no significant change (ANOVA; regression analysis); those given 1.24 x 10(-9) or 1.24 x 10(-8) M norepinephrine during the middle hour showed no significant change, but those given concentrations between 5.24 x 10(-8) and 1.24 x 10(-5) M all showed significant reductions or fluid reabsorption (based on 42 fetuses). The responses showed a linear relationship with the log concentration (r = 0.97). They appeared to involve alpha-adrenoreceptors, since responses to 10(-7) M norepinephrine were unaffected by 10(-6) M propranolol, but those to 10(-7) and 1.24 x 10(-6) M norepinephrine were abolished by 10(-6) and 1.78 x 10(-5) M phentolamine, respectively (based on 48 fetuses). Activation was through alpha2-adrenoreceptors, since responses to 10(-7) and 10(-5) M norepinephrine were abolished by 10(-4) M yohimbine, but not by 10(-5) M prazosin (based on 60 fetuses). The results show that norepinephrine is able to reduce lung liquid production when at plasma levels present at birth, and that it can produce reabsorption; unlike epinephrine, there was no reduction in responses at high concentrations. This work reintroduces a neglected factor, norepinephrine, into possible controls of lung liquid reabsorption, and opens up the potential for neural controls.     

Diffusion in bile and its implications on detergency.

Diffusion coefficients of bile salts, lecithin, and cholesterol above the critical micelle concentration have been measured with the diaphragm cell at varying concentrations of bile salts, lecithin, and added electrolyte. The diffusion of the bile salt can be five times faster than that of the solubilized lipids. This is shown not to be an artifact of multicomponent diffusion, but a result of a different transport mechanism of the bile salt. As a consequence, the concentration of bile salt and lipids at the surface of a cholesterol gallstone can differ from those in the bile solution. The effects of this upon growth and dissolution in detergent solutions are discussed.     

Quasi-elastic light-scattering studies of aqueous biliary lipid systems. Cholesterol solubilization and precipitation in model bile solutions

We have employed quasi-elastic light-scattering methods to characterize micellar aggregates and microprecipitates formed in aqueous solutions containing sodium taurocholate (TC), egg lecithin (L), and cholesterol (Ch). Particle size and polydispersity were studied as functions of Ch mole fraction (XCh = 0-15%), L/TC molar ratio (0-1.6), temperature (5-85 degrees C), and total lipid concentration (3 and 10 g/dL in 0.15 M NaCl). For XCh values below the established solubilization limits (XChmax) [Carey, M. C., & Small, D. M. (1978) J. Clin. Invest. 61, 998], added Ch has little influence on the size of simple TC micelles (type 1 systems), on the coexistence of simple and mixed TC-L micelles (type 2 systems), or on the growth of "mixed disc" TC-L micelles (type 3 systems). For supersaturated systems (XCh/XChmax greater than 1), 10 g/dL type 1 systems (L/TC = 0) exist as metastable micellar solutions even at XCh/XChmax = 5.3. Metastability is decreased in type 2 systems (0 less than L/TC less than 0.6), and "labile" microprecipitation occurs when XCh/XChmax exceeds approximately 1.6. In 10 g/dL mixtures, the microprecipitates initially range in size from 500 to 20000 A and later coalesce to form a buoyant macroscopic precipitate phase. In 3 g/dL mixtures, the microprecipitates are smaller (200-400 A) and remain as a stable, noncoalesced microdispersion. Transmission electron microscopy of the microprecipitates formed at both concentrations indicates a globular noncrystalline structure, and lipid analysis reveals the presence of cholesterol and lecithin in a molar ratio (Ch/L) of approximately 2/1, suggesting that the microprecipitates represent a metastable cholesterol-rich liquid-crystalline phase. In supersaturated type 3 systems (0.6 less than L/TC less than 2.0), the precipitate phase is a lecithin-rich liquid-crystalline phase which likewise coalesces in a 10 g/dL system but forms stable vesicle (liposomal) structures (600-800 A radius) in 3 g/dL systems. In conjunction with these experimental data, we present a quantitative thermodynamic analysis of Ch solubilization in model bile systems from which rigorous deductions of the free energy and enthalpy change for solubilization of cholesterol monohydrate in type 1 and type 2 systems are obtained. In addition, we employ homogeneous nucleation theory to analyze the origin of the metastable/labile limit in supersaturated systems and to deduce the interfacial tension between microprecipitates and solution. On the basis of these experimental data and theoretical analyses, we offer new hypotheses on the structure and physiology of bile and the pathogenesis of Ch gallstones. In particular, it is suggested that the "stable" microprecipitates observed in 3 g/dL type 2 systems may provide a secondary vehicle (in addition to micelles) for cholesterol transport in supersaturated hepatic bile.     

Sandwich hybridization as a convenient method for the detection of nucleic acids in crude samples

A method based on three-DNA-component, sandwich hybridization has been designed for the detection and quantitation of nucleic acids in crude samples using adenovirus DNA as a model. Two non-overlapping restriction fragments of adenovirus type 2 (Ad2) DNA were cloned into two vectors, the pBR322 plasmid and M13 phage. The recombinant plasmid DNA was immobilized onto nitrocellulose filters and the single-stranded recombinant phage DNA was labeled with 125I and used as a probe. When these two reagents were incubated under annealing conditions no radioactivity became filter-bound; only if denatured adenovirus DNA was added as the third reagent, it mediated the attachment of the radioactive probe to the filters. Hybridization efficiency was shown to be dependent on both the filter and probe DNA concentrations and on the hybridization conditions. When standardized, the assay is quantitative, and under the conditions used 0.2 ng of adenovirus DNA (8 X 10(-6) pmol) could be detected by an overnight incubation. The test is suitable for crude samples, e.g., solubilized cell extracts, without any purification steps. Less than 100 cells infected with Ad2 can be detected, implying that the assay could be applicable to virus diagnostics.     

Interaction between membrane properties and protein synthesis in reticulocytes — A two step inhibition of protein synthesis by Valinomycin

In this work we consider the differential effect of Valinomycin used at different concentrations both on the protein synthesis of reticulocytes and on 42K exchange. We demonstrate that there is a two step action of this antibiotic. At 10(-6)M and below the drug has no effect on the 42K exchange, but it stops, however reversibly, protein synthesis. At 10(-5)M the drug has a very sharp action on the 42K exchange and stops protein synthesis in an irreversible way. Ribosomal population checked by two ways, sucrose gradient and direct counting on E.M. sections shows that at low concentrations of Valinomycin (10(-8)M to 10(-6)M) there is no breakdown of the polysomes which can be detected by either one of these methods. On the contrary, after short incubation with 10(-5)M of Valinomycin the breakdown of ribosomes is very clear, as evidenced by sucrose gradient analysis. By direct ribosomes clusters counting on E.M. sections this breakdown is seen only after long incubation.