Type C Niemann-Pick disease. Lysosomal accumulation and defective intracellular mobilization of low density lipoprotein cholesterol

The intracellular accumulation of unesterified cholesterol was examined during 24 h of low density lipoprotein (LDL) uptake in normal and Niemann-Pick C fibroblasts by fluorescence microscopy with filipin staining and immunocytochemistry. Perinuclear fluorescence derived from filipin-sterol complexes was observed in both normal and mutant cells by 2 h. This perinuclear cholesterol staining reached its peak in normal cells at 6 h. Subsequent development of fluorescence during the remaining 18 h of LDL incubation was primarily limited to the plasma membrane region of normal cells. In contrast, mutant cells developed a much more intense perinuclear fluorescence throughout the entire 24 h of LDL uptake with little enhancement of cholesterol fluorescence staining in the plasma membranes. Direct mass measurements confirmed that internalized LDL cholesterol more readily replenishes the plasma membrane cholesterol of normal than of mutant fibroblasts. Perinuclear filipin-cholesterol fluorescence of both normal and mutant cells was colocalized with lysosomes by indirect immunocytochemical staining of lysosomal membrane protein. Abnormal sequestration of LDL cholesterol in mutant cells within a metabolically latent pool is supported by the finding that in vitro esterification of cellular cholesterol could be stimulated in mutant but not in normal cell homogenates by extensive disruption of the intracellular membranous structures of cells previously cultured with LDL. Deficient translocation of exogenously derived cholesterol from lysosomes to other intracellular membrane sites may be responsible for the delayed homeostatic responses associated with LDL uptake by mutant Niemann-Pick Type C fibroblasts.     

Quantitation of the Myelin-Associated Glycoprotein in Human Nervous Tissue from Controls and Multiple Sclerosis Patients

Myelin-associated glycoprotein (MAG) was measured by radioimmunoassay in the human CNS and peripheral nervous system (PNS). The level of MAG, expressed as ng/microgram of total protein, was approximately 20-fold higher in whole homogenates of cerebral white matter (4.7 +/- 0.60) than of peripheral nerve (0.12-0.28). MAG concentrations were only slightly higher in the isolated myelin fractions from these tissues: CNS myelin, 5.6 ng/microgram; PNS myelin, 0.37 ng/microgram. The levels of MAG were measured in nine plaques, periplaque regions, and areas of macroscopically normal-appearing white matter (NAWM) from six separate multiple sclerosis brains and compared with the levels of other myelin proteins in the same samples. MAG and other myelin proteins were reduced to very low levels in plaques. The levels of MAG and basic protein (BP) and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in periplaque areas were significantly lower than those in control white matter, and MAG and BP levels were also significantly reduced in NAWM. In a periplaque region and NAWM from the most rapidly progressing case of multiple sclerosis examined, the MAG content was between 30 and 35% of the control level, whereas BP and PLP levels and CNP activity were between 50 and 85% of control values. The reduction of MAG content in periplaque regions from all nine multiple sclerosis plaques examined was significantly greater than the reductions of BP level and CNP activity. In NAWM samples, the mean reduction of MAG content was also greater than the reductions of BP level and CNP activity, but the difference was only statistically significant in comparison to CNP.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of the suspensor and gibberellic acid on Phaseolus vulgaris embryo protein content

The role of the suspensor in the early development of the dicot embryo has not yet been defined. It has been described as merely an anchor and also as the major route of nutrients into the embryo. In order to further elucidate the role of the suspensor, early 0.2-mm and late heart stage 0.5-mm Phaseolus vulgaris (var. Taylor's Horticultural) embryos have been examined in tissue culture. It is known that Phaseolus embryos in culture at low osmotic potential will germinate precociously and that embryos in culture at high osmotic potential will either fail to grow or form callus. Optimum sucrose concentrations for continued, normal embryonic development of 0.2 mm and 0.5 mm P. vulgaris in tissue culture with Gamborg B5 medium were determined to be 12 and 6%, respectively. Protein content was examined in embryos and suspensors. Results showed that both 0.2- and 0.5-mm embryos required an attached suspensor for maximum protein content. Protein levels were substantially decreased when the embryo was cultured detached from or without the suspensor. Gibberellic acid at 10(-6) to 10(-7) M restored the protein content to that of freshly excised embryos.     

Interactions between the benzodiazepine receptor antagonist Ro 15-1788 (flumazepil) and the inverse agonist beta-CCE: behavioral studies with squirrel monkeys

The effects of Ro 15-1788 and ethyl-beta-carboline-3-carboxylate (beta-CCE) were studied alone and in combination on the behavioral performances of squirrel monkeys. Under one procedure, performances maintained by food were suppressed by electric shock presentation (punishment or "conflict" procedure). Under a second procedure, responding was maintained either by food or electric shock delivery under a 5-min fixed-interval schedule. Doses of beta-CCE between 0.1 and 3.0 mg/kg, i.m., produced graded decreases in punished responding which were reversed by pretreatment with Ro 15-1788 (1.0 - 10.0 mg/kg, i.m.). Low doses of beta-CCE (0.03 - 0.3 mg/kg, i.m.) increased responding of monkeys maintained by shock presentation, but did not affect food-maintained responding; higher doses of beta-CCE decreased responding under both schedules. These effects of beta-CCE are opposite those produced by the benzodiazepines under this procedure. Ro 15-1788 (1.0 mg/kg i.m.) antagonized the effects of beta-CCE, producing a shift to the right in the dose-response curves. These findings provide further support for the view that beta-CCE and Ro 15-1788 produce effects mediated by the same benzodiazepine receptor recognition site.     

Effects of glucagon, Arg-vasopressin, and angiotensin II on rat hepatic zinc thionein levels

Rat hepatic zinc thionein levels can be modulated by a variety of external and internal stimuli. Metals, such as zinc or copper, induce levels 20 to 50 fold over controls. Catecholamines can increase levels 10 to 20 fold, while glucocorticoids, such as dexamethasone, can increase levels modestly by 2-6 fold. We have investigated the ability of additional hormones, which have receptors on hepatocytes, to modulate the levels of hepatic zinc thionein. Glucagon, angiotensin II, and Arg-vasopressin were administered intravenously and intraperitoneally, one time and three times, over an 11 hour period. Zinc thionein levels in rat liver were increased 1.7 to 5.6 fold by glucagon and 1.7 to 3.6 fold by angiotensin II, but not at all by Arg-vasopressin, as compared to appropriate controls. Glucagon and angiotensin II, when administered in vivo, can modulate zinc thionein levels in rat liver to an extent similar to glucocorticoids. Hepatic zinc thionein levels must now be recognized to be affected in vivo by metals, glucocorticoids, catecholamines, and polypeptide hormones.     

Monoclonal antibodies against human beta-glucocerebrosidase

Monoclonal antibodies were obtained against the membrane-bound lysosomal enzyme beta-glucocerebrosidase (acid beta-glucosidase), which is deficient in Gaucher's disease. BALB/c mice were immunized with homogeneous enzyme protein extracted from a sodium dodecyl sulphate/polyacrylamide gel. The mice were subsequently hyperimmunized with partially purified enzyme prior to fusion of spleen cells with myeloma cells. After fusion, 32 primary hybrid cell populations were obtained which continued to produce antibodies against beta-glucocerebrosidase after prolonged time of culture. All antibodies reacted with both native and denatured enzyme. Four primary cell populations were subcloned and the antibodies produced were characterized. The antibodies were all four of the IgG1 subclass. Three of these antibodies bind to protein A whereas one does not. The results of binding assays indicated that three of the antibodies react with the same antigenic domain (epitope 1), but the fourth with a different one (epitope 2). Probably two antigenic determinants are present in epitope 1 since one of the antibodies with specificity for epitope 1 is inactivated after iodination by the chloramine-T procedure whereas a second one is not.     

Metabolism of zinc and copper in the neonate: Zinc thionein in developing rat brain,heart, lung,spleen, and thymus

In a continuing study of the importance of metallothionein (MT) in the growth and development of neonates, zinc and copper metabolism in rat brain, heart, lung, spleen, and thymus has been analyzed in 5, 10, 15, 20 and 25 day old rats. Total, cytosol, and MT zinc and copper concentrations and organ contents were determined. Zinc, but very little, if any copper was associated with MT in these organs. Concentrations ranged from 0.03 to 3.3 μg Zn in MT/g; organ contents ranged from 0.003 to 2.2 μg Zn in MT/organ. Brain exhibited the highest concentrations and contents of zinc in MT, approaching the levels found in kidneys. Rank order of organ contents of zinc in MT was brain > lung > heart, spleen, thymus, during this neonatal growth period. When organ growth was rapid, a large percentage (20–95%) of the cytosolic zinc present in these organs was associated with MT, as has been previously observed with liver, kidneys, and testes. None of these organs undergoes the dramatic changes in zinc and copper metabolism previously observed in neonatal rat liver and gastrointestinal tract, and in maturing testes. They are more comparable to kidneys in their concentrations of zinc in MT. Like testes, little copper is found in these organs.     

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.