R. Gordon Gould

Mixed connective tissue disease-an apparently distinct rheumatic disease syndrome associated with a specific antibody to an extractable nuclear antigen (ENA)

Abstract We describe the clinical and serologic findings in twenty-five patients with an apparently distinct rheumatic disease syndrome which we have termed mixed connective tissue disease. All these patients had hemagglutinating antibody to an extractable nuclear antigen (ENA) which consists mainly of protein and ribonucleic acid (RNA). A marked sensitivity of the hemagglutination antigen to ribonuclease indicated that the specificity of the antibody to ENA circulating in these patients was different from that of antibody to ENA which occurred in about 50 per cent of the patients with systemic lupus erythematosus. Serum from patients with mixed connective tissue disease also contained high titers of speckled pattern fluorescent antinuclear antibody which showed the same response of tissue antigens to enzyme digestion as found with hemagglutinating antibody. There was no detectable Sm antibody. Antibody to native deoxyribonucleic acid (DNA) was infrequent and of low titer, and serum complement levels were normal or elevated. The clinical characteristics of the patients with mixed connective tissue disease included a combination of features similar to those of systemic lupus erythematosus, scleroderma and polymyositis. Most of these abnormalities were responsive to corticosteroid therapy. Thus, the detection of antibody to ENA with a well defined specificity allows recognition of an apparently distinct mixed connective tissue disease syndrome which is characterized by an excellent response to corticosteroid therapy and a favorable prognosis.

Absorbability of β-sitosterol in humans

Abstract Tritium labeled β-sitosterol was administered orally to 5 normal humans and to 5 terminal patients. Isolation of tritium-labeled sterols from plasma and red blood cells of normal male subjects showed some absorption had occurred, but it was much less than for comparable doses of labeled cholesterol. The highest level observed after a single 50-mg. dose of β-sitosterol was about 0.015 mg. per 100 ml. of blood in the total sterol fraction. These values are uncorrected for dilution by any unlabeled β-sitosterol present in the diet and are therefore minimum values. The disappearance rate from blood was slightly higher than for labeled cholesterol. Larger doses of tritium labeled β-sitosterol administered to a series of terminal patients gave similar results for blood levels and disappearance rates. Isolation of sterols from a variety of tissues showed β-sitosterol to be widely distributed. High specific activity values were observed in bile sterols, suggesting that β-sitosterol is selectively excreted by this route. Aorta and other blood vessels had lower amounts of tritium labeled sterols than most other tissues. The total amount of β-sitosterol present in blood and principal visceral organs (excluding intestine) was estimated in one patient as about 0.5 per cent of a single dose of 258 mg. given 5 days previously. Although the distribution pattern of absorbed β-sitosterol was similar to that of fed labeled cholesterol, it did not become esterified to as great an extent.

Solubilization and partial purification of hepatic 3-hydroxy-3-methylglutaryl coenzyme a reductase☆

Abstract This paper describes an effective method for the solubilization of microsomal HMG-CoA reductase from rat liver. Exposing the microsomes to a freeze-thaw treatment solubilized 80% of the microsomal reductase activity. Subsequently, a 25-fold purification has led to an enzyme preparation with a specific activity of 10–14 nmoles MVA per min per mg of protein and an increased stability.

Studies on the mechanisms of the rapid modulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase in intact liver by mevalonolactone and 25-hydroxycholesterol☆

A rapid, biphasic inhibition of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase (mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34) was induced by intragastric administration of R,S-mevalonolactone. The initial phase had a t1/2 of 5.3 min. 30 min after drug administration the inhibition could be reversed in vitro by cytosol or a partially purified cytosolic activator. The reactivation was prevented by 50mM NaF. Thus the initial inhibition appeared to be the result of reversible inactivation possibly by phosphorylation of the enzyme. Consistent with this was the finding that the net reductase activator (phosphatase) activity present in cytosol was decreased 64% in these animals. The rapid reversible inhibition could not be reproduced in vitro by incubating microsomes or postmitochondrial supernatants with mevalonate suggesting the intact cell was necessary for expression of the effect. The second phase of inhibition due to mevalonate administration had a t1/2 of 1.3 h and was not reversible. It was attributed to inhibition of synthesis of reductase probably as the result of sterol accumulation in the cell. Perfusion of 25-hydroxycholesterol through livers isolated from animals at the circadian peak of cholesterol biosynthesis resulted in a rapid, 75-80% inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase. This inhibition was not reversed by incubation with cytosol or partially purified activator. Further, there was no apparent change in net activator levels in cytosol from the livers perfused with 25-hydroxycholesterol. This suggests the effect of this sterol on reductase does not involve reversible phosphorylation-dephosphorylation. On the basis of this study it is postulated that there are at least two mechanisms by which 3-hydroxy-3-methylglutaryl coenzyme A reductase activity can be rapidly suppressed in the intact liver. One is reversible and appears to be the result of alteration in the reductase kinase-phosphatase system. The second is irreversible and may be due to acceleration of the normal degradation system.

Correlation of rat liver chromatin-bound free and esterified cholesterol with the circadian rhythm of cholesterol biosynthesis in the rat

Cholesterol has been shown to be present in rat liver chromatin isolated by methods designed to avoid contamination by membrane fragments. Evidence that the cholesterol was actually a component of chromatin includes (a) the constancy of the amount (1.30 +/- 0.14 mug per mg DNA), (b) the striking difference in the ratio of free (i.e. unesterified) to esterified cholesterol between that in chromatin and that in membrane, and (c) the rapid and marked changes which occurred in this ratio during the circadian cycle in chromatin but not in membranes. Although the total amount of chromatin-bound cholesterol did not change throughout the circadian cycle, the concentration of free cholesterol increased sharply a short time before the peak of cholesterol synthetic activity was reached at about midnight; it reached a basal level about 6 h later at approximately the same time the rate of synthesis returned to its basal level. When labelled cholesterol was administered by stomach tube, it was detectable within 2 h in whole nuclei and in chromatin, indicating that chromatin-bound cholesterol is rapidly exchangeable with that in liver cytoplasm and in blood plasma. Removal of basic proteins from chromatin did not result in the loss of any cholesterol, but removal of most of the acidic as well as the basic proteins resulted in loss of most of the chromatin-bound cholesterol. These results indicate that cholesterol is bound either to the acidic proteins or to both the acidic proteins and DNA. The data are compatible with the hypothesis that cholesterol biosynthesis controlled at the nuclear level and suggest that the relative amounts of free and esterified cholesterol associated with chromatin may play a role.

Prevention of cold inactivation of 3-hydroxy-3-methylglutaryl coenzyme a reductase by NADPH

Solubilized 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) from rat liver microsomes has been reported to be reversibly inactivated by temperatures below 19 degrees C. Cold inactivation has now been found to be completely prevented by NADPH and by NADP+ at a concentration of 3 mM. NADPH, however, was more active than NADP+ at lower concentrations and prevented 50% of the cold inactivation at 0.2 mM, whereas a 1.1 mM NADPH+ without effect and the substrate 3-hydroxy-3-methylglutaryl coenzyme A prevented only 30% of the cold inactivation at a concentration 50 times greater than the Km value.

Characterization of microvesicles produced by shearing of human erythrocyte membranes

Abstract Human erythrocyte membranes were sheared into microvesicles with substantial loss of activity of the ATPases but not the nitrophenyl phosphatases. This loss of ATPase activity was not accompanied by alteration in lipid content, sialic acid content or orientation, or sulfhydryl group reactivity. Centrifugation in a linear sucrose gradient, resolved the microsvesicles into distinct classes which differed with respect to their lipid content, the reactivity of their sulfhydryl groups, and the activity of their ATPases and nitrophenyl phosphatases. Vesicles with low sulfhydryl reactivity had low activity of all enzymes tested. Vesicles containing relatively high lipid to protein and lipid to sialic acid ratios had low ATPase and K + -dependent nitrophenyl phosphatase activities. We have found that membranes could be sheared into readily separable membrane classes containing varying amounts of lipid relative to sialic acid and protein, and this observation supports a segmental concept of membrane structure. The parallelism of (Na + , K + )-ATPase and K + -dependent nitrophenylphosphatase activities in the separated microvesicle classes stands in contrast to their divergent behavior following shearing where the K + -dependent nitrophenyl phosphatase is not inactivated. This pattern suggested that K + -dependent nitrophenyl phosphatase probably accurately reflected the K + -dependent dephosphorylation phase of (Na + , K + )-ATPase; however, the prior step required a higher degree of structural integrity and was very susceptible to inactivation by chemical or mechanical perturbation.

GAS-LIQUID CHROMATOGRAPHY OF THE TETRAHYDRO DERIVATIVES OF CORTISOL ISOLATED FROM URINE.

Abstract Tetrahydrocortisone, tetrahydrocortisol, and allotetrahydrocortisol can be separated by gas—liquid chromatography and quantitated with an argon ionization detector. The retention times of these cortisol metabolites are identical with those of their 17-ketosteroid analogs. This technique has been used to monitor the compostion of fractions from liquid-liquid chromatograms of urine extracts.

INTERACTION OF apoHDL WITH HDL AND WITH OTHER LIPOPROTEINS

Abstract Ultracentrifugation at a density of 1.21 of mixtures of apoHDL and HDL in which one component was labeled with 125 I showed that interchange of labeled protein took place. Since no evidence of intermediates was obtained it seems more probable that these results are explained by interchange of the relatively small protein subunits (mol.wt. approx. 15,000) between apoHDL and the large HDL complex (mol.wt. approx. 200,000) than that lipids move from HDL to apoHDL, as has been previously suggested. The top fraction obtained by centrifuging a mixture of [ 125 I]apoHDL and HDL at d 1.21 lost less than 20% of the labeled protein when it was recentrifuged at the same density. The fraction of [ 125 I]apoHDL which floated at d 1.21 in the presence of HDL was larger than the fraction of [ 125 I]HDL which sedimented to the bottom in the presence of apoHDL, indicating the occurrence of some complex formation between HDL and [ 125 I]apoHDL in addition to the interchange of labeled protein subunits. Similar results were obtained on electrophoresis; [ 125 I]HDL after incubation with apoHDL gave zones corresponding to HDL and to apoHDL both of which contained 125 I. Complex formation between [ 125 I]apoHDL and LDL was demonstrated by ultracentrifugation of a mixture. A small fraction of the labeled protein floated at d 1.063 but on recentrifugation half of the labeled protein sedimented. When trace amounts of [ 125 I]apoHDL were mixed with serum and the lipoprotein fractions separated by ultracentrifugation, 60% of the label was recovered in the HDL fraction and only 1.5% in the LDL fraction.