Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Influence of mevinolin on metabolism of low density lipoproteins in primary moderate hypercholesterolemia

Mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, was used for treatment of 12 patients with moderate hypercholesterolemia, but not classical familial hypercholesterolemia. For most patients, measurements of turnover of low density lipoprotein-apolipoprotein B (LDL-apoB) were made on placebo and during treatment with two doses of mevinolin. LDL turnover was determined after injection of autologous 125I-labeled radioiodinated LDL. Compared to placebo, a low dose of mevinolin (10 mg, twice daily (BID] caused reductions of plasma total cholesterol and LDL-cholesterol averaging 15% and 20%, respectively; corresponding reductions on high doses of mevinolin (20 mg BID) were 22% and 31%, respectively. Triglyceride levels were unchanged by the drug. High density lipoprotein cholesterol levels rose significantly on the high dose, but not on the low dose. Neither dose produced a stastistically significant change in fractional catabolic rate (FCR) for LDL-apoB for the whole group, although several patients had increases in FCR on both doses. In contrast, both doses of mevinolin caused decreases in production rates of LDL-apoB. Thus, the fall in LDL levels in patients with moderate hypercholesterolemia can be explained more by a reduction in the input rate of LDL-apoB than by enhanced fractional removal of LDL from the circulation.     

Isopropanol precipitation method for the determination of apolipoprotein B specific activity and plasma concentrations during metabolic studies of very low density lipoprotein and low density lipoprotein apolipoprotein B

A method has been described for the measurement of apoB concentration and specific activity in very low density lipoprotein (VLDL) and low density lipoprotein (LDL) during metabolic studies. For measurement of specific activity, apoB was separated from other apolipoproteins and lipids by precipitation in, and subsequent washing with, isopropanol. For determination of apoB concentration, equal volumes of lipoprotein and isopropanol were mixed, and the protein content of the apoB precipitate was measured by the difference between total lipoprotein protein and the protein measured in the supernatant. Evidence that there was no apoB solubilization in isopropanol and that precipitated apoB was virtually free of soluble apolipoproteins was obtained by electrophoresis. Lipid contamination of the apoB precipitate was less than 1%. The methods were applicable to VLDL, intermediate density lipoprotein (IDL), and LDL from normolipemic patients with protein concentrations between 187 micrograms/ml and 1898 micrograms/ml. The data obtained using isopropanol were highly correlated with those using tetramethylurea, and recoveries of apoB were similar. Furthermore, the isopropanol method has been demonstrated to yield consistent data for apoB specific activities in a study of VLDL, IDL, and LDL metabolism.     

An evaluation of four methods for measuring cholesterol absorption by the intestine in man

Critical comparisons have been made in 12 patients of four methods for measuring cholesterol absorption from the intestine. Methods I-III depend on the use of labeled cholesterol (intravenously or continuous labeling orally) in conjunction with sterol balance measurements; Method IV can be carried out with only a single test dose containing labeled cholesterol plus labeled beta-sitosterol. In the latter technique absorption is calculated as the loss of cholesterol relative to beta-sitosterol during intestinal transit. Method III (isotopic steady-state method) proved to be undependable because of uncertainties in determining the existence of an isotopic steady state. However, Method IV gave good agreement with Methods I and II, and it appears to have certain practical as well as theoretical advantages. Although Method IV requires collections of stools for up to 8 days, it is nevertheless the most rapid and the simplest of all the methods for estimating absorption. It can also be used in certain situations, such as in fur-licking animals, when Methods I and II are inadequate. Therefore, this method would seem to be a valuable addition to other isotopic techniques for estimating cholesterol absorption in man.     

Effects of dietary cholesterol on the regulation of total body cholesterol in man

Studies on the interaction of cholesterol absorption, synthesis, and excretion were carried out in eight patients using sterol balance techniques. Absorption of dietary cholesterol was found to increase with intake; up to 1 g of cholesterol was absorbed in patients fed as much as 3 g per day. In most patients, increased absorption of cholesterol evoked two compensatory mechanisms: (a) increased reexcretion of cholesterol (but not of bile acids), and (b) decrease in total body synthesis. However, the amount of suppression in synthesis was extremely variable from one patient to another; one patient had no decrease in synthesis despite a large increment in absorption of dietary cholesterol, and two patients showed a complete suppression of synthesis. In the majority of cases the accumulation of cholesterol in body pools was small because of adequate compensation by reexcretion plus reduced synthesis, but in a few patients large accumulations occurred on high cholesterol diets when absorption exceeded the compensatory mechanisms. These accumulations were not necessarily reflected in plasma cholesterol levels; these increased only slightly or not at all.     

Dietary beta-sitosterol as an internal standard to correct for cholesterol losses in sterol balance studies

In the course of carrying out sterol balance studies in 19 patients, we gathered the following evidence that, in some but not all patients, considerable amounts of neutral sterols are "lost" during their passage through the intestinal tract. (a) Since plant sterols are largely nonabsorbable in man, they should be totally recovered in the feces; yet in many patients significantly less plant sterol than expected was recovered, the loss amounting to as much as 56% of daily intake. (b) In two patients in whom cholesterol-(14)C and beta-sitosterol-(3)H were instilled into the terminal ileum, from which neither sterol is absorbed, the feces contained 25% less of each isotope than was instilled. (c) In four patients fed radioactive cholesterol daily until the isotopic steady state was closely approximated, 28-50% of the isotope could not be accounted for. On the other hand, in five patients fed radioactive bile acids until the isotopic steady state was approximated, input equalled output as predicted. Since the amount of -sitosterol absorbed in man is limited (5% or less), this sterol can be used as an internal standard for upward correction of the figure obtained for the amount of neutral steroids excreted. The use of beta-sitosterol for this purpose is based on three considerations: (a) it passes through the intestine in the same physicochemical state as cholesterol; (b) it accompanies cholesterol at every step of its isolation and chromatographic measurement; and (c) it is lost to the same extent as cholesterol. Excretion data for fecal neutral steroids can therefore be corrected for irregular fecal flow as well as for the "unexpected loss" referred to. This loss seems to be due not to errors in stool collection or to technical errors, but to intestinal bacterial degradation of neutral 3beta-OH,Delta(5)-sterols to products not recognized as steroids in the analytical methods used.     

Responses to mitogenic stimulation of lymphocytes taken during and after pregnancy

Serial blood samples were collected during pregnancy, after delivery and several months postnatally from 28 women. The blastogenic responses of lymphocytes to varying concentrations of phytohaemagglutinin (PHA) were tested using autologous plasma or fetal calf serum (FCS) to support the lymphocyte cultures. Using FCS, the blastogenic response decreased as pregnancy progressed and remained depressed months after delivery. In contrast, when autologous plasma was used a 10-fold higher concentration of PHA was required to give optimal stimulation. Blastogenic responses were still suppressed during pregnancy but had returned to initial values by the time of delivery and were greater still in the post-partum and postnatal periods. We conclude that the inherent ability of lymphocytes to undergo blastogenesis is suppressed during pregnancy but that this is over-shadowed by a humoral effect of pregnancy plasma. The significance of these results is discussed.