Influence of bile salt molecular species on cholesterol crystallization from supersaturated model biles

Time-sequential enzymatic determination of cholesterol (CH) crystals harvested by ultrafiltration, and concomitant polarizing light microscopy observations corroborated the striking importance of the bile salts (BS) species in determining CH crystals formation rate from supersaturated model biles incubated in vitro. The more hydrophilic tauroursodeoxycholate, taurohyocholate, glycohyocholate, taurohyodeoxycholate, glycohyodeoxycholate and glyco-3α, hydroxy-6 oxo-5ß-cholanate inhibited CH precipitation through the formation of a stabilized liquid-crystalline phase. In contrast, in all hydrophobic systems (taurine (T) and glycine (G) conjugates of cholate (C), deoxycholate (DC) and chenodeoxycholate (CDC)), CH crystals precipitated with time. When crystallized CH concentrations were plotted vs. time, the figures showed a sigmoidal pattern, consistent with the transition from metastable systems to stable equilibrium states. Over the equilibration period, the nucleation kinetics (as inferred from enzymatic measurements) and all crystallization events (as microscopically observed) were both shifted in time, depending on the BS species: they were earliest in CDC systems, then in DC systems, and finally in C systems. In the latter, the delay was clearly due to the formation of a transient labile liquid-crystalline phase. G-conjugation also induced a significant delay in CH precipitation, compared to T-conjugation. At last, maximum crystallized CH concentrations at equilibrium were in the decreasing order: C > CDC > DC and T-conjugates > G-homologues. All data are discussed in connection with BS hydrophobicities, with predictions from the phase equilibria of aqueous biliary lipid systems and with new insights into CH crystal habits.     

Bovine gallbladder mucin promotes cholesterol crystal nucleation from cholesterol-transporting vesicles in supersaturated model bile

This study examined the ability of purified gallbladder mucin to accelerate the nucleation of cholesterol monohydrate crystals from the cholesterol-transporting particles in supersaturated model bile. Mixed lipid micelles and cholesterol-phosphatidylcholine vesicles in supersaturated model bile were separated by Sephadex G-200 column chromatography. Mixed lipid micelles prepared by column chromatography had a low cholesterol-phosphatidylcholine ratio (0.30) and did not spontaneously nucleate cholesterol monohydrate crystals. In contrast, vesicles prepared by column chromatography had a cholesterol-phosphatidylcholine ratio of 1.00 and nucleated cholesterol crystals rapidly (P less than 0.001). Nucleation of cholesterol crystals was significantly accelerated in a concentration- and time-dependent manner by purified bovine gallbladder mucin in cholesterol containing vesicles, but not in mixed lipid micelles (P less than 0.001). A rapid filtration binding assay demonstrated significant binding of cholesterol and phosphatidylcholine in vesicles to gallbladder mucin but only minimal binding of cholesterol and phosphatidylcholine in mixed micelles. These data indicate that gallbladder mucin binds cholesterol and phosphatidylcholine in vesicles and accelerates the nucleation of cholesterol monohydrate crystals from these cholesterol-transporting particles in supersaturated model bile.     

Quantitative analysis of cholesterol nucleation with time in supersaturated model bile

The amount of cholesterol (Ch) crystals formed in supersaturated taurochenodeoxycholate (TCDC) - lecithin (L) solutions of the same Ch saturation index (CSI) but at different Ch thermodynamic activities (Ch A_T) was quantified at different time intervals. The initial Ch nucleation rate (i.e., amount of Ch crystals formed with respect to time) in a Ch A_T = 1.73 and TCDC to L molar ratio (TCDC:L) = 5.1 system was faster than that in a Ch A_T = 1.42 and TCDC:L = 3.4 system. Shaking could enhance the early appearance of Ch crystals and cause the fast initial Ch nucleation rates for the TCDC:L = 5.1 and the TCDC:L = 3.4 systems. The final Ch nucleation rates were faster than the initial Ch nucleation rates for the TCDC:L = 5.1 and the TCDC:L = 3.4 systems. According to a light scattering analysis of vesicle concentration in supersaturated TCDC–L solutions, vesicles provide nucleation sites only in the Ch nucleation process and the vesicle concentration may not be an important factor for the Ch nucleation rate. A model of a mixed TCDC–L micelle releasing Ch molecules together with the surface area of Ch crystals formed was used in the interpretation of the Ch nucleation.     

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.     

Factors affecting cholesterol monohydrate crystal nucleation time in model systems of supersaturated bile

We explored the influence of several compositional factors considered capable of influencing the nucleation time of model biles supersaturated in cholesterol. In addition to the classical techniques, e.g., electron microscopy and quasielastic light scattering, employed for size measurement and structural assessment, we employed a novel technique, i.e., video-enhanced microscopy, for particle evaluation in these polydisperse systems which often may simultaneously contain isolated small vesicles, their complex aggregates, and small cholesterol monohydrate crystals. The factors we studied included dilution, degree of cholesterol supersaturation, bile salt/lecithin molar ratio, and Ca2+ concentration. Dilution markedly raised the degree of cholesterol saturation, prolonged nucleation time for cholesterol monohydrate crystals, and favored formation of metastable small unilamellar vesicles. Increasing the degree of cholesterol supersaturation as an independent variable in more concentrated systems both shortened the nucleation time and favored spontaneous formation of a relatively small number of isolated vesicles. A decrease in bile salt/lecithin molar ratio within the physiologically relevant range was accompanied by a prolonged nucleation time and favored spontaneous vesicle formation. Large numbers of small unilamellar vesicles were observed even in concentrated model bile solutions (total lipids: 20 g/dl) when the bile salt/lecithin molar ratio was 1.9 or less. At physiological concentrations, Ca2+ promoted nucleation of cholesterol monohydrate crystals only in vesicle-containing solutions. Taken together, the following conclusions can be drawn. First, spontaneous vesicle formation in dilute systems prolongs solid cholesterol crystal nucleation. It can thus provide a supplementary non-micellar mode of cholesterol transport in micellar systems of supersaturated human bile. Second, dilution, degree of cholesterol supersaturation, and a decrease in bile salt/lecithin ratio prolong cholesterol crystal nucleation time and favor spontaneous vesicle formation. With increasing calcium concentrations, opposite effects are observed. Third, the presence of vesicles may help to account for the frequently observed and otherwise unexplained remarkable degree of metastable supersaturation and prolonged metastability (delayed nucleation time) for cholesterol in human bile.     

Accurate separation of vesicles, micelles and cholesterol crystals in supersaturated model biles by ultracentrifugation, ultrafiltration and dialysis.

Gel filtration with bile salts at intermixed micellar/vesicular concentrations (IMC) in the eluant has been proposed to isolate vesicles and micelles from supersaturated model biles, but the presence of vesicular aggregates makes this method unreliable. We have now validated a new method for isolation of various phases. First, aggregated vesicles and - if present - cholesterol crystals are pelleted by short ultracentrifugation. Cholesterol contained in crystals and vesicular aggregates can be quantitated from the difference of cholesterol contents in the pellets before and after bile salt-induced solubilization of the vesicular aggregates. Micelles are then isolated by ultrafiltration of the supernatant through a highly selective 300 kDa filter and unilamellar vesicles by dialysis against buffer containing bile salts at IMC values. Lipids contained in unilamellar vesicles are also estimated by subtraction of lipid contents in filtered micelles from lipid contents in (unilamellar vesicle+micelle containing) supernatant ('subtraction method'). 'Ultrafiltration-dialysis' and 'subtraction' methods yielded identical lipid solubilization in unilamellar vesicles and identical vesicular cholesterol/phospholipid ratios. In contrast, gel filtration yielded much more lipids in micelles and less in unilamellar vesicles, with much higher vesicular cholesterol/phospholipid ratios. When vesicles obtained by dialysis were analyzed by gel filtration, vesicular cholesterol/phospholipid ratios increased strongly, despite correct IMC values for bile salts in the eluant. Subsequent extraction of column material showed significant amounts of lipids. In conclusion, gel filtration may underestimate vesicular lipids and overestimate vesicular cholesterol/phospholipid ratios, supposedly because of lipids remaining attached to the column. Combined ultracentrifugation-ultrafiltration-dialysis should be considered state-of-the-art methodology for quantification of cholesterol carriers in model biles.     

Pre-assembled clusters distort crystal nucleation kinetics in supersaturated lysozyme solutions

Efficient determination of three-dimensional protein structures is critical for unraveling structure-function relationships and for supporting targeted drug design. A major impediment to these efforts is our lack of control over the nucleation and growth of high-quality protein crystals for X-ray structure determinations. While basic research on protein crystal growth mechanisms has provided valuable new insights, studies of crystal nucleation have been plagued by inconsistent and outright contradictory results. Using dynamic light scattering and SDS gel electrophoresis, we have investigated possible causes of these inconsistencies. We find that commercial sources of lyophilized hen-egg white lysozyme (HEWL) used in nucleation studies contain significant populations of large (approximately 100 nm), pre-assembled lysozyme clusters that can readily evade standard assays of sample purity. In supersaturated solutions, these clusters act as heterogeneous nucleation centers that enhance the rate of crystal nucleation and significantly deteriorate the quality of macroscopic crystals.     

Liquid crystals and cholesterol nucleation during equilibration in supersaturated bile analogs

In recent work, apparent liquid crystal agglomeration to form typical solid cholesterol microcrystals was frequently observed photomicrographically in bile samples from prairie dogs fed a cholesterol-enriched diet, prior to solid crystal formation. We therefore have conducted a systematic study of time-course lipid compositional changes in the mesophase and micellar phase constituents of bile analog solutions while undergoing cholesterol nucleation during equilibration. On the basis of these studies, we conclude that the nucleation process for microcrystal formation most likely occurs within the mesophase component which is only the first of a two-step transition in a sequential series of physical ordering processes. We deduce that mesophase formation must have a lower kinetic energy requirement and that the second step (microcrystal formation) must be rate limiting. In keeping with theoretical considerations, structural evidence for increased hydration is demonstrable near the point of complete equilibration when the mesophase is dissolving.     

Quantitation of cholesterol crystallization from supersaturated model bile

Cholesterol crystallization is an essential step in gallstone formation. Although spectrophotometry and nephelometry have been used for quantitation of crystallization, potential effects of crystal size and shape have not been evaluated. We determined crystallization in model biles [total lipid concentration 7.3 g/dl, egg yolk Phosphatidylcholine (EYPC)/(EYPC+taurocholate) molar ratio = 0.05, 0.15, or 0.30; cholesterol saturation index (CSI) = 1.2, 1.7, or 2.0; 37 degrees C] plotting in the central three-phase (micelles, vesicles, and crystals containing) zone or in the left two-phase (micelles and crystals containing) zone of the equilibrium ternary phase diagram. Extent of crystallization estimated by spectrophotometry and nephelometry was related to chemical determination of crystal mass and to crystal size or shape (by microscopy). With all methods, crystallization was less extensive when vesicles were present (central three-phase zone) and at lower CSIs. In the left two-phase zone, particularly at EYPC/(EYPC+taurocholate), ratio of 0.15, there were strong increases in spectrophotometric and nephelometric readings during the first days of incubation, but decreases at later stages, despite progressive increases in crystal mass by chemical measurement. Initially, there were large numbers of very small crystals (<10 microm) in these biles, which were subsequently replaced by large cholesterol monohydrate crystals. Decreasing sizes of harvested cholesterol monohydrate crystals by sonication increased spectrophotometric and nephelometric values despite identical crystal mass.When cholesterol crystal mass is assayed by indirect methods such as spectrophotometry or nephelometry, results are strongly influenced by crystal size.     

Cholesterol monohydrate nucleation in ultrathin films on water

The growth of a cholesterol crystalline phase, three molecular layers thick at the air-water interface, was monitored by grazing incidence x-ray diffraction and x-ray reflectivity. Upon compression, a cholesterol film transforms from a monolayer of trigonal symmetry and low crystallinity to a trilayer, composed of a highly crystalline bilayer in a rectangular lattice and a disordered top cholesterol layer. This system undergoes a phase transition into a crystalline trilayer incorporating ordered water between the hydroxyl groups of the top and middle sterol layers in an arrangement akin to the triclinic 3-D crystal structure of cholesterol x H(2)O. By comparison, the cholesterol derivative stigmasterol transforms, upon compression, directly into a crystalline trilayer in the rectangular lattice. These results may contribute to an understanding of the onset of cholesterol crystallization in pathological lipid deposits.     

Vesicle aggregation in model systems of supersaturated bile: relation to crystal nucleation and lipid composition of the vesicular phase

The presence of small vesicles composed of phospholipid and cholesterol has recently been demonstrated in super-saturated model and in dilute native human biles by several groups using differing methods. Among compositional factors shown to favor spontaneous vesicle formation and prolong the cholesterol monohydrate nucleation time in model bile systems are dilution, a raised cholesterol saturation index (CSI), and a low bile salt/phospholipid ratio. Time-lapse video-enhanced microscopy of a series of model bile systems representing systematically designed variations in the above factors revealed strong evidence for an essential linkage between antecedent vesicle aggregation and subsequent crystal nucleation. Stability of vesicles was inversely related to their degree of cholesterol saturation, i.e., the greater the degree of vesicular cholesterol saturation, the less their stability (metastability). Instability of vesicles was reflected by their early aggregation followed by rapid cholesterol crystal nucleation. The lowest degree of vesicular cholesterol saturation was found in dilute systems which also exhibited the greatest metastability despite a high degree of cholesterol solubility (raised CSI). Conversely, the more concentrated and least metastable systems exhibited both rapid vesicle aggregation and rapid onset of crystal nucleation. These systems, while influenced by the other compositional factors, were found to have a high degree of vesicular cholesterol saturation, i.e., cholesterol/phospholipid molar ratio = 2.0. An additional finding was the extreme variability in the proportionate distribution of total solution cholesterol distributed to the vesicular phase, i.e., from zero to as high as 37%. Higher solute concentration, raised bile salt/lecithin ratio, and raised CSI were interactive and almost equally capable of increasing the proportionate amount of cholesterol in the vesicular phase. In conclusion, lipid compositional differences in model bile systems drastically affect the cholesterol saturation of spontaneously formed phospholipid-cholesterol vesicles. This effect, in turn, exerts a potent influence upon the metastability of vesicles, subsequently affecting the cholesterol crystal nucleation time.     

Differential patterns of lipid-protein association in fast and slow cholesterol nucleating human gallbladder biles: implications for cholesterol nucleation from biliary lipid carriers

We compared the protein/lipid structure and Ch-nucleating capacity of individual lipid carriers in two groups of human gallbladder biles: 11 with Fast cholesterol nucleation (2.2 +/- 1.3 days) and 10 with Slow cholesterol nucleation (19.2 +/- 4.4 days). The groups had comparable cholesterol-saturation (1.31 vs. 1.28), total lipids (9.9 vs. 8.5 g/dl) and proteins (8.5 vs. 7.6 mg/ml). Bile was ultracentrifuged (2 h at 150,000 x g) and the resulting isotropic phase was incubated with [3H]Ch and [14C]lecithin and gel-chromatographed on a Superose 6 column with a buffer containing 7.0 mM sodium-taurocholate. Seven protein peaks were identified (280 nm and biochemistry), with the following molecular mass ranges (kDa): 1 (Void volume), 2 (155-205), 3 (50-79), 4 (20-29), 5 (6-15), 6 (3.5-6), 7 (2-3.5). Peaks 2 and 3 were identified as vesicles and micelles, respectively. Fast vs. Slow Ch nucleating biles had: (a) more (P less than 0.02) cholesterol coeluting with vesicles, (b) more (P less than 0.01) lecithin coeluting with low m.w. peaks (Nos. 5-6), (c) less (P less than 0.01) cholesterol and lecithin coeluting with micelles. An inverse correlation (P less than 0.001) was observed between the amount of proteins coeluting with the micellar peak and the cholesterol nucleation of both whole bile and isolated micellar fractions. A marked shift of cholesterol and lecithin from micelles to vesicles was apparent, in the whole bile, after cholesterol nucleation had occurred. Incubation and sequential analysis of isolated and radiolabeled micelles showed a progressive transfer of lecithin and cholesterol molecules to low molecular weight fractions and to vesicles before cholesterol nucleation. We conclude that pro-nucleating biliary vesicles develop from micelles, due to the phasing out and redistribution of micellar cholesterol and lecithin, which are probably induced by biliary proteins.     

Looking at physiological anthropology from a historical standpoint

As one way of thinking about physiological anthropology, let us survey it from a historical viewpoint. At the beginning of the 19th century, Blumenbach, considered the father of Physical Anthropology, wrote his "Handbook of Comparative Anatomy and Physiology." The subsequent research conducted and papers written by researchers such as Broca and Martin pointed in the direction of physiological anthropology; furthermore, the research carried out by the American researchers Demon and Baker had a physiological anthropology "feel." The courses in Physiological Anthropology taught by Tokizane exerted a major influence on physiological anthropology in Japan. The precursor of the Japan Society of Physiological Anthropology, organized by Sato in 1978, was extremely significant in the effect that it had on the subsequent development of physiological anthropology. The holding of the biennial International Congress of Physiological Anthropology, along with the allocation of the Research sub-field of Physiological Anthropology in the Grant-in-Aid for Scientific Research, would seem to suggest that the field of physiological anthropology is set to increasingly grow and evolve.     

Effects of hydrophobic and hydrophilic bile salt mixtures on cholesterol crystallization in model biles

[Ru(2,2'-bipyridine)(2)(4,4'-dicarboxy-2,2'-bipyridine)](2+) (RuBDc) is a very photostable probe that possesses favorable photophysical properties including long lifetime, high quantum yield, large Stokes' shift, and highly polarized emission. In the present study, we demonstrated the usefulness of this probe for monitoring the rotational diffusion of high-molecular-weight (MW) proteins. Using frequency-domain fluorometry with a high-intensity, blue light-emitting diode (LED) as the modulated light source, we compared the intensity and anisotropy decays of RuBDc conjugated to immunoglobulin G (IgG) and immunoglobulin M (IgM), which show a six-fold difference in MW We obtained slightly longer lifetimes for IgM (=428 ns in buffer) than IgG (=422 ns in buffer) in the absence and presence of glycerol, suggesting somewhat more efficient shielding of RuBDc from water in IgM than in IgG. The anisotropy decay data showed longer rotational correlation times for IgM (1623 and 65.7 ns in buffer) as compared to IgG (264 and 42.5 ns in buffer). Importantly, the ratio of the long rotational correlation times of IgM to IgG in buffer was 6.2, which is very close to that of MW of IgM to IgG (6.0). The shorter correlation times are most likely to be associated with domain motions within the proteins. The anisotropy decays reflect both the molecular size and shape of the immunoglobulins, as well as the viscosity. These results show that RuBDc can have numerous applications in studies of high-MW protein hydrodynamics and in fluorescence polarization immunoassays (FPI) of high-MW analytes.     

Cholesterol crystal nucleation from enzymatically modified low-density lipoproteins: combined effect of sphingomyelinase and cholesterol esterase

An assay detecting and quantifying cholesterol nucleation from low-density lipoproteins has been established. F?rster 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.     

Factors affecting plasmid production in Escherichia coli from a resource allocation standpoint

Background  

Plasmids are being reconsidered as viable vector alternatives to viruses for gene therapies and vaccines because they are safer, non-toxic, and simpler to produce. Accordingly, there has been renewed interest in the production of plasmid DNA itself as the therapeutic end-product of a bioprocess. Improvement to the best current yields and productivities of such emerging processes would help ensure economic feasibility on the industrial scale. Our goal, therefore, was to develop a stoichiometric model of Escherichia coli metabolism in order to (1) determine its maximum theoretical plasmid-producing capacity, and to (2) identify factors that significantly impact plasmid production.