Isolation of Dane particles containing a DNA strand by metrizamide density gradient.
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Dane-particle cores labelled with [3H]TTP were subjected to ultracentrifugation in a metrizamide density gradient. Two populations of core particles with different densities were obtained, and radioactivity was found only in the cores that sedimented at the lower density (1.19-1.23 g/cm3). All the cores in this group, when spread in a monolayer, were found to expel a closed circular double-stranded DNA molecule. In contrast, the core particles that sedimented at the higher density (1.23-1.27 g/cm3) were not associated with radioactivity, nor was any DNA strand extruded from them. These results show that metrizamide density gradients allow the separation of complete hepatitis B virions for the study of viral DNA. (+info)
Complex of DNA with chromatin proteins investigated by isopycnic centrifugation in metrizamide.
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Complexes of mouse main band DNA with a fraction of non-histone proteins (NHP), having a high affinity for DNA, in the absence or presence of histones have been investigated by gradient centrifugation in metrizamide. Two types of complexes were formed at an input ratio of NHP to DNA between 1 and 2.5. In metrizamide gradients a majority of DNA was found in the light complex (at the density of 1.14-1.16 g/cm3) even at the very high NHP to DNA ratio. When histones were present in the reaction mixture, most of the DNA was found in the heavy complex (1.19-1.21 g/cm3). The electrophoretic profiles of the proteins recovered from the heavy and light complexes were different; some fractions of nonhistone proteins were present only in the heavy component. (+info)
Techniques of facial nerve block.
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The efficacy of different techniques of facial nerve block for cataract surgery was investigated. Forty four patients underwent either modified O'Brien, Atkinson, van Lint, or lid blocks. Intentional muscle activity of the orbicularis oculi muscle was recorded and the area under the EMG curve calculated for quantitative comparison of muscle activity between the groups before and after injection of lignocaine with the vasoconstrictor naphazoline nitrate. In addition, the force of lid closure was measured and lid motility determined on a subjective score scale. Whereas the modified O'Brien and lid blocks nearly abolished the muscle activity recorded in the EMG (p < 0.003), the Atkinson and van Lint blocks did not significantly affect these variables. The O'Brien and lid blocks decreased the force of lid closure and lid movements far more effectively than the Atkinson and van Lint blocks (p < 0.0001). The topographic distribution of a mixture of metrizamide and lignocaine solutions was evaluated radiographically in eight additional patients, to assess potential causes for differences in the efficacy of the block techniques. The radiological results showed involvement of the region of the facial nerve trunk and its temporal and cervical divisions by the modified O'Brien block. The lid block, on the other hand, affected terminal branches of the facial nerve's temporal division. In this study, complete lid akinesia was achieved by both the modified O'Brien block and the lid block. However, because the modified O'Brien block involves the risk of neural injury to the facial nerve or its main divisions, the lid block is recommended as the most effective and safe method to achieve akinesia of the orbicularis oculi muscle. (+info)
Survival of newly postmitotic motoneurons is transiently independent of exogenous trophic support.
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We compared the survival requirements of early- and late-born motoneurons from E5 chicken spinal cord. Density gradient centrifugation followed by immunopanning using SC1 antibody allowed us to purify two size classes of motoneuron. Large motoneurons retained by 6.8% metrizamide were shown by BrdU labeling in ovo to be born on average 1.5 d earlier than the small motoneurons recovered from the metrizamide pellet. Large motoneurons were both biochemically and functionally more mature: they expressed higher levels of choline acetyltransferase and low-affinity neurotrophin receptor, and had an acute requirement for trophic support from muscle-derived factors. After 24 hr in culture in basal medium, all early-born motoneurons died, whereas 60% of late-born motoneurons survived. Small motoneurons can develop into large motoneurons in ovo, suggesting that they represent a general transitional stage in motoneuron development. Our results suggest that a defined period elapses between birth of a motoneuron and its acquisition of trophic dependence, possibly corresponding to the time required for target innervation. This property may have important consequences for the timing and regulation of developmental motoneuron death. (+info)
Biliary lipid secretion: immunolocalization and identification of a protein associated with lamellar cholesterol carriers in supersaturated rat and human bile.
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Feeding a 0.5% diosgenin plus 0.02% simvastatin diet to rats increases biliary cholesterol concentration and saturation to levels generally found in human native supersaturated bile. By using preparative ultracentrifugation, gel filtration chromatography, and electron microscopy, we isolated, purified, and identified lamellar structures (unilamellar vesicles and multilamellae) as a major biliary cholesterol transport in supersaturated human and rat bile. It was estimated that more than 60% of biliary cholesterol is transported in these lamellar carriers, which were identified by transmission electron microscopy as unilamellar vesicles and multilamellar bodies within bile canaliculi of rats with cholesterol supersaturated bile. By SDS-PAGE, a characteristic and constant protein profile was found associated to the purified lamellar carriers. One of these proteins, a 130-kDa protein, was isolated from human biliary lamellae and used for preparation of a rabbit polyclonal antibody, which cross-reacted with the homologous rat protein. By Western blotting, it was established that the purified low density fraction of bile-Metrizamide gradients, containing lamellae, was enriched with the 130-kDa protein. The 130-kDa protein was characteristically detected at the canalicular membrane by Western blotting of hepatic subcellular fractions and by immunohistochemistry of rat and human liver biopsies. Amino acid sequencing of the amino terminus of the 130-kDa protein demonstrated a complete identity with aminopeptidase N, a canalicular transmembrane hydrophobic glycoprotein. These studies show that biliary lipids may acquire an ordered multilamellar structure that is present in the canaliculi of rats with supersaturated bile. These biliary lamellae are similar to lamellar bodies and surfactant-like material frequently found in other epithelia, suggesting common biogenetic, structural, and functional properties. The identification of aminopeptidase N associated with biliary lamellae is consistent with the involvement of the canalicular membrane in the secretory mechanism of biliary lipids. (+info)
Skeletal muscle-derived trophic factors prevent motoneurons from entering an active cell death program in vitro.
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The purpose of the experiments reported here is to provide evidence that motoneurons (MTNs) isolated from chick embryo spinal cords go through an active process of cell death when deprived of trophic support in vitro. In order to analyze and characterize this process, MTNs were isolated with a metrizamide gradient technique and cultured in the presence of saturating concentrations of soluble muscle extract. When muscle extract was washed off from the cultures, MTNs entered a process of cell death that could be blocked with inhibitors of mRNA and protein synthesis. Two other additional criteria were used to define this process as an active one. First, ultrastructural analysis of MTNs dying as a consequence of muscle extract deprivation showed that some, but not all, of the MTNs displayed clear signs of apoptotic cell death. Those included cytoplasm condensation, fragmentation of chromatin, and preservation of cytoplasmic organelles. Second, internucleosomal degradation of DNA was detected in MTNs deprived of muscle extract. When DNA was analyzed by Southern hybridization techniques using digoxigenin-labeled genomic probes, a clear ladder pattern could be identified on muscle extract-deprived MTNs. The degradation of DNA upon trophic deprivation could be prevented by cycloheximide (CHX). In an attempt to characterize further the process of active cell death in MTNs, we found a time point of commitment to cell death of approximately 10 hr by using three different approaches: muscle extract deprivation plus readdition of muscle extract, muscle extract deprivation plus addition of CHX, and muscle extract deprivation plus addition of actinomycin D. Moreover, we show that MTNs deprived of trophic support from muscle extract but maintained alive with CHX could not be rescued from cell death by reading muscle extract if CHX was washed off the cultures within the first 15 hr of muscle extract deprivation. However, muscle extract alone was able to rescue MTNs that had been kept alive with CHX for periods of time longer than 24 hr after muscle extract deprivation. From these results we postulate that the activation of the cell death program after trophic deprivation is transient. (+info)
Use of glycyl-L-phenylalanine 2-naphthylamide, a lysosome-disrupting cathepsin C substrate, to distinguish between lysosomes and prelysosomal endocytic vacuoles.
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Lysosome-disrupting enzyme substrates have been used to distinguish between lysosomal and prelysosomal compartments along the endocytic pathway in isolated rat hepatocytes. The cells were incubated for various periods of time with 125I-labelled tyramine cellobiose (125I-TC) covalently coupled to asialoorosomucoid (AOM) (125I-TC-AOM); this molecule is internalized by receptor-mediated endocytosis and degraded in lysosomes, where the degradation products (acid-soluble, radio-labelled short peptides) accumulate, Glycyl-L-phenylalanine 2-naphthylamide (GPN) and methionine O-methyl ester (MOM), which are hydrolysed by lysosomal cathepsin C and a lysosomal esterase respectively, both diffused into hepatocytic lysosomes after electrodisruption of the cells. Intralysosomal accumulation of the hydrolysis products (amino acids) of these substrates caused osmotic lysis of more than 90% of the lysosomes, as measured by the release of acid-soluble radioactivity derived from 125I-TC-AOM degradation. The acid-soluble radioactivity coincided in sucrose-density gradients with a major peak of the lysosomal marker enzyme acid phosphatase at 1.18 g/ml; in addition a minor, presumably endosomal, acid phosphatase peak was observed around 1.14 g/ml. The major peak of acid phosphatase was almost completely released by GPN (and by MOM), while the minor peak seemed unaffected by GPN. Acid-insoluble radioactivity, presumably in endosomes, banded (after 1 h of 125I-TC-AOM uptake) as a major peak at 1.14 and a minor peak at 1.18 g/ml in sucrose gradients, and was not significantly released by GPN. GPN thus appears to be an excellent tool by which to distinguish between endosomes and lysosomes. MOM, on the other hand, released some radioactivity and acid phosphatase from endosomes as well as from lysosomes. (+info)
Synthesis and processing of the precursor to the major core protein of adenovirus type 2.
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An isopycnic Metrizamide-detergent gradient system was developed in which the newly synthesized precursor (polypeptide P-VII) to the major core protein of adenovirus type 2 (polypeptide VII) was confined to a spectrum of complexes with densities equal to or higher than that of adenovirions. The majority of the newly synthesized P-VII was, at the beginning of the logarithmic period of virus production, present as an entity of protein density. This pool of P-VII was efficiently depleted. P-VII was also associated with high-molecular-weight structures of intermediate density, sharing some properties with empty capsids or incomplete particles. The transfer of P-VII from the intermediate-density region was not quantitative, and only particles of true virion density subsequently contained polypeptide VII. No structures equivalent to the core structure of disrupted virions or identical to incomplete particles were detected in this system. A temperature-dependent transition of radioactivity from polypeptide P-VII into polypeptide VII was also detectable after in vitro incubation of P-VII-containing complexes. Addition of Ad2-infected cell extracts was required for processing of complexes derived from regions of protein density, whereas P-VII was processed spontaneously upon incubation in complexes of virion density. (+info)