Characterization of human platelet surface and intracellular membranes isolated by free flow electrophoresis.

High voltage free flow electrophoresis has been applied to the separation of human platelet membranes. After short treatment with neuraminidase at the whole cell level, three membrane vesicle subpopulations have been isolated. Using a surface label (125I-labeled Lens culinaris lectin), the marker enzyme NADH-cytochrome c reductase, and lipid analysis, two of the fractions have been identified as of surface origin and the other consists of intracellular membrane elements. The distribution of adenylate cyclase, leucyl aminopeptidase, 5'-nucleotidase and Ca2+-ATPase has also been investigated, and their usefulness as markers for the different membrane fractions has been evaluated. All three fractions are vesicular but differ in size and character. Their phospholipid and cholesterol contents have been determined, and the cholesterol/phospholipid ratios of the two surface fractions are over twice that of the intracellular membrane, which also has a significantly lower microviscosity as determined by fluorescence polarization using diphenyl hexatriene. The polypeptide profiles from sodium dodecyl sulfate-polyacrylamide gel electrophoresis are particularly distinctive, with actin present in the two surface membrane fractions and absent from the intracellular membranes. Myosin, confirmed by its ATPase characteristics, is almost exclusively localized in one of the surface membrane fractions, and actin-binding protein is a prominent feature of the other.

Lectin histochemistry of brains from a murine mutant carrying a storage disorder.

A strain of Balb/c mice with neurovisceral storage disorder exhibits metabolic and phenotypic manifestations similar to those found in Niemann-Pick type C and D patients. The storage material in the brain reacted positively with periodate-Schiff reagent. To identify the chemical nature of the storage material we applied lectin histochemistry on paraffin-embedded and frozen sections, using biotinylated lectins and avidin-biotin-peroxidase complex. Major abnormalities were noted in the neurons and glia cells. Swollen neurons were stained heavily by Con A and S-WGA, whereas glia cells, mainly astrocytes, which were abundant both in the cerebrum and cerebellum, were positive to RCA-I, GS-I, PNA, S-WGA and WGA. The myelin tracts reacted with PNA, SBA and RCA-I but to a lesser extent in affected animals when compared to normals. Frozen brain sections stained positively only after extraction with chloroform methanol prior to the lectin treatment and revealed a lectin binding pattern similar to that of the paraffin-embedded preparations. The data presented here show that the stored glucoconjugates in the neurons are of a different chemical composition than those found in glia cells. Since only paraffin embedded sections or lipid extracted frozen sections reacted with the lectins, we suggest that the stored glucoconjugates are glycoproteins or oligosaccharides rather than glycolipids.

Impaired cholesterol esterification in primary brain cultures of the lysosomal cholesterol storage disorder (LCSD) mouse mutant.

Esterification of cholesterol was investigated in primary neuroglial cultures obtained from newborn lysosomal cholesterol storage disorder (LCSD) mouse mutants. An impairment in 3H-oleic acid incorporation into cholesteryl esters was demonstrated in cultures of homozygous LCSD brain. Primary cultures derived from other phenotypically normal pups of the carrier breeders esterified cholesterol at normal levels or at levels which were intermediary between normal and deficient indicating a phenotypic expression of the LCSD heterozygote genotype. These observations on LCSD mutant brain cells indicate that the defect in cholesterol esterification is closely related to the primary genetic defect and is expressed in neuroglial cells in culture.

The cholesterol storage disorder of the mutant BALB/c mouse. A primary genetic lesion closely linked to defective esterification of exogenously derived cholesterol and its relationship to human type C Niemann-Pick disease.

An inborn murine cholesterol storage disorder exists which is characterized by a lesion in intracellular cholesterol esterification not accounted for by any discernible abnormality in acyl-CoA: cholesterol acyltransferase (Pentchev, P.G., Boothe, A.D., Kruth, H.S., Weintroub, H., Stivers, J., and Brady, R.O. (1984) J. Biol. Chem. 259, 5784-5791). Current studies have shown that the level of esterification of nonlipoprotein-derived [3H]cholesterol in cultured fibroblasts from heterozygous mutant mice was intermediary between the level found in normal fibroblasts and the deficient level found in fibroblasts from homozygous mutant mice. Homozygous-affected fibroblasts took up and converted [3H]desmosterol to [3H]cholesterol at a normal rate indicating that the murine mutation does not compromise the transport of exogenous sterol to microsomes. In contrast to the defect in esterification of exogenously derived cholesterol, synthesis of cholesteryl ester from [3H]mevalonic acid and [3H]squalene was normal in affected fibroblasts as was the stimulation of cholesteryl ester synthesis from endogenous cholesterol induced by 25-hydroxycholesterol. In surveying a number of mutant cell lines from human metabolic disorders with phenotypic manifestations similar in part to the mutant cholesterol storage mouse, Niemann-Pick C fibroblasts displayed a similar defect in esterification of exogenously derived cholesterol.

A genetic storage disorder in BALB/C mice with a metabolic block in esterification of exogenous cholesterol.

Cholesterol metabolism has been investigated in a strain of BALB/C mice that carry an autosomal recessive mutation associated with decreased sphingomyelinase and glucocerebrosidase activity and storage of sphingomyelin and glucocerebroside as well as cholesterol in lysosomes (Pentchev, P. G., Gal, A. E., Boothe, A. D., Omodeo-Sale, F., Fouks, J., Neumeyer, B. A., Quirk, J. M., Dawson, G., and Brady, R. O. (1980) Biochim. Biophys. Acta 619, 669-679). When affected animals are placed on a diet high in cholesterol, they develop hepatomegaly associated with an extensive accumulation of unesterified cholesterol in the liver. Cultured skin fibroblasts derived from these mice also manifest a defect in cholesterol esterification although the uptake and intracellular location of exogenous cholesterol is comparable to that of controls. Microsomal fatty acyl-CoA:cholesterol acyltransferase activity was normal or elevated in extracts of tissues from the affected animals. Furthermore, the subcellular distribution and membrane orientation of acyl-CoA:cholesterol acyltransferase appeared normal in microsomal preparations isolated from affected mice. The blockage of esterification of exogenous cholesterol in the presence of normal transferase activity is suggestive of a defect in a component involved in the intracellular disposition of this sterol. The attenuation in tissue levels of sphingomyelinase and glucocerebrosidase and the accumulation of sphingolipids may reflect alterations in lysosomal function resulting from an imbalance of unesterified cholesterol in these organelles.

Niemann-pick variant disorders: comparison of errors of cellular cholesterol homeostasis in group D and group C fibroblasts.

Fluorescence microscopic examination of filipin-stained cultured skin fibroblasts derived from two brothers with group D Niemann-Pick disease revealed abnormal storage of low density lipoprotein (LDL)-derived cholesterol. LDL stimulation of intracellular cholesteryl ester synthesis was severely compromised in the Niemann-Pick D fibroblasts, as it also was in fibroblasts obtained from Niemann-Pick C patients. Cholesteryl ester synthesis was intermediately deficient in cells derived from an obligate group-D heterozygous carrier. Activity of acyl-CoA:cholesterol acyltransferase was within the normal range in cell-free extracts of both LDL-depleted and LDL-supplemented cultures of Niemann-Pick C and D fibroblasts. Incubation of Niemann-Pick D fibroblasts with LDL did not lead to as high a level of intracellular cholesterol accumulation as the excessive storage observed with Niemann-Pick C fibroblasts. These findings suggest that the Niemann-Pick variant disorders may represent a family of specific and possibly individual mutations that disrupt cellular cholesterol homeostasis.

A lysosomal storage disorder in the BALB/c mouse: bone marrow transplantation.

The morphological and biochemical consequences of transplanting affected bone marrow from donor BALB/c mice with a lysosomal storage disorder (BALB/c LSD) into normal recipient mice were studied. Bone marrow was removed from normal BALB/c and BALB/c LSD mice and transfused into normal BALB/c recipient mice four hours after the mice received 850 rads of irradiation. Tissues of the recipient mice were examined 240 days later. This study revealed that the defective cells that constituted the visceral lesions of BALB/c LSD could be transplanted to normal BALB/c mice by the use of bone marrow from affected BALB/c LSD homozygote; that the defective cells of BALB/c LSD proliferated and disseminated throughout the mononuclear phagocytic system of the recipient; that there were increases in cholesterol, sphingolipids, and cystine with decreases in sphingomyelinase and glucocerebrosidase activity in tissues of the recipients; and that the recipients survived substantially longer than BALB/c LSD homozygotes and their lifespan was compromised mainly by the secondary effects of irradiation. These lesions, although not as extensive as in homozygous BALB/c LSD, paralleled the lesions which develop in BALB/c LSD. Since the recipient mice were not compromised by the short life span (70 days) of the BALB/c LSD mice, they may be used to study the long-term chronic effects of these metabolic lesions.