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.     

Regulation of hepatic cholesterol and lipoprotein metabolism in ethinyl estradiol-treated rats

The regulation of hepatic cholesterol and lipoprotein metabolism was studied in the ethinyl estradiol-treated rat in which low density lipoprotein (LDL) receptors are increased many fold. Cholesterol synthesis was reduced at both its diurnal peak and trough by ethinyl estradiol. The diurnal variation in 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was abolished, whereas that for acyl coenzyme A: cholesterol acyltransferase (ACAT) was retained. LDL receptor number did not vary diurnally. Feeding these animals a cholesterol-rich diet for 48 h suppressed cholesterol synthesis and reductase activities to levels similar to those found in cholesterol-fed control animals, but ACAT activity was unaffected. LDL receptors were reduced about 50%. Intravenously administered cholesterol-rich lipoproteins suppressed HMG-CoA reductase and LDL receptors in 2 h but had a variable effect on ACAT activity. Intragastric administration of mevalonolactone reduced reductase and increased acyltransferase activity but had little effect on LDL receptors when given 2 or 4 h before death. Although animals fed a cholesterol-rich diet before and during ethinyl estradiol treatment became hypocholesterolemic, free and esterified cholesterol concentrations in liver were high as was ACAT activity. HMG-CoA reductase was inhibited to levels found in control animals fed the cholesterol-rich diet. LDL receptors were increased to a level about 50% of that reached in animals receiving a control diet and ethinyl estradiol. These data demonstrate that key enzymes of hepatic cholesterol metabolism and hepatic LDL receptors respond rapidly to cholesterol in the ethinyl estradiol-treated rat. Furthermore, estradiol increases LDL receptor activity several fold in cholesterol-loaded livers.     

The use of hollow fiber cross-flow microfiltration in bioaccumulation and continuous removal of heavy metals from solution by Saccharomyces cerevisiae

Cross-flow microfiltration was shown to retain Saccharomyces cerevisiae biomass utilized for heavy metal bioaccumulation. The passage of metal-laden influent through a series of sequential bioaccumulation systems allowed for further reductions in the levels of copper, cadmium, and cobalt in the final effluent than that afforded by a single bioaccumulation process. Serial bioaccumulation systems also allowed for partial separation of metals from dual metal influents. More than one elemental metal cation could be accumulated simultaneously and in greater quantities than when a single metal was present in the effluent (Cu(2+) 0.43 mmol, Cu(2+) + Cd(2+) 0.67 mmol, and Cu(2+) + Co(2+) 0.83 mmol/g yeast dry mass when the initial concentration of each of the metal species was 0.2 mmol.L(-1)). Co-accumulation of two different metal cations allowed higher total levels of bioaccumulation than found with a single metal. The flux rate was 2.9 x 10(2) L.h(-2)mum(-2) using a polypropylene microfiltration membrane (0.1 mum pore size) at 25 degrees C. (c) 1994 John Wiley & Sons, Inc.     

Endo-1,4-[beta]-Glucanase,Xyloglucanase, and Xyloglucan Endo-Transglycosylase Activities Versus Potential Substrates in Ripening Tomatoes

In ripening fruits of tomato (Lycopersicon esculentum L. var 83-G-38), the amounts of cellulose and xyloglucan (XG) remained constant during tissue softening, but the relative molecular weight (Mr) of XG decreased markedly and the Mr of cellulose declined slightly. These changes could have been due to activities of non-specific endo-1,4-[beta]-glucanases and/or buffer-soluble XG endo-transglycosylase, both of which increased when tissue firmness declined most rapidly. Tomato extracts also reduced the viscosity of XG solutions, especially in the presence of added XG oligosac-charides. This depolymerizing (XGase) capacity differed from [beta]-glucanase and XG transglycosylase activity (a) by being almost entirely buffer insoluble, and (b) by declining precipitously during fruit softening. Although it disappeared from ripe fruit, XGase may have functioned in promoting wall loosening at earlier stages of fruit development when its activity was highest. By contrast, during aging of fruit in the ripening-inhibited mutant rin there was no change in Mr of XG or cellulose, and activities of [beta]-glucanases and XG transglycosylase were lower than in wild-type tomato. Nevertheless, some softening of the fruit did take place over time and XG amounts declined, possibly because high XGase activity was maintained in the mutant, unlike in wild-type fruit.     

Prevalence of nine mutations among Jewish and non-Jewish Gaucher disease patients.

The frequency of nine different mutated alleles known to occur in the glucocerebrosidase gene was determined in 247 Gaucher patients, of whom 176 were of Jewish extraction, 2 were Jewish with one converted parent, and 69 were of non-Jewish origin. DNA was prepared from peripheral blood, active glucocerebrosidase sequences were amplified by using the PCR technique, and the mutations were identified by using the allele-specific oligonucleotide hybridization method. The N37OS mutation appeared in 69.77% of the mutated alleles in Jewish patients and in 22.86% of the mutated alleles in non-Jews. The 84GG mutation, which has not been found so far among non-Jewish patients, existed in 10.17% of the disease alleles among Jewish patients. The IVS + 1 mutation constituted 2.26% of the disease alleles among Jewish patients and 1.43% among the non-Jewish patients. RecTL, a complex allele containing four single-base-pair changes, occurred in 2.26% of the alleles in Jewish patients and was found in two (1.43%) of the patients of non-Jewish extraction. Another complex allele, designated "RecNciI" and containing three single-point mutations, appeared in 7.8% of alleles of non-Jewish patients and in only two (0.56%) of the Jewish families. The prevalence of the L444P mutation among non-Jewish Gaucher patients was 31.43%, while its prevalence among Jewish patients was only 4.24%. The prevalence of two other point mutations--D409H and R463C--was 5.00% and 3.57%, respectively, among non-Jewish patients and was not found among the Jewish Gaucher patient population. The prevalence of the R496H mutation, found so far only among Jewish patients, was 1.13%. The results presented demonstrate that seven mutations identify 90.40% of the mutations among Jewish patients and that these seven mutations allow diagnosis of only 73.52% of the non-Jewish patients. Identification of additional mutant alleles will enhance the accuracy of carrier detection.