Use of the baculovirus system to assemble polyomavirus capsid-like particles with different polyomavirus structural proteins: analysis of the recombinant assembled capsid-like particles.
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The genes encoding the structural proteins (VP1, VP2 and VP3) of murine polyomavirus were cloned into the p2Bac dual multiple cloning site vector, individually or jointly, and the corresponding proteins were expressed in Spodoptera frugiperda (Sf9) insect cells by cotransfecting Sf9 cells with the constructed vector and the linear DNA of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Recombinant capsid-like particles could be purified 5 days post-infection from Sf9 cells infected with AcMNPV-VP1, with or without the involvement of minor protein (VP2 or VP3). Although VP2 and VP3 alone could not generate recombinant particles, they became incorporated into these particles when expressed with VP1 in Sf9 cells. Recombinant particles with different polyomavirus structural protein(s) were obtained by using different combined expression of these proteins in Sf9 cells. Cellular DNA of 5 kbp in size was packaged in all of the recombinant particles, which showed the same diameter as that of native virions. Agarose gel electrophoresis indicated that DNA packaged in these recombinant particles had a different pattern than that of native virions. Two-dimensional gel electrophoresis of the VP1 species of recombinant particles showed more VP1 species than those of the native virions from mouse cells, and an additional species of VP1 when VP2 was co-expressed with VP1. The recombinant particles were also compared for their ability to compete for polyomavirus infection. The competition assay indicated that the recombinant particles containing VP2 were the most efficient in inhibiting the native polyomavirus infection of 3T6 cells. (+info)
Properties of beta-glucosidase in cultured skin fibroblasts from controls and patients with Gaucher disease.
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Membrane-bound beta-glucosidase from cultured skin fibroblasts can be solubilized in an active form by treatment of membrane preparations with a mixture of Triton X-100 and sodium taurocholate. Several properties of the solubilized enzyme have been studied in fibroblasts from normal, healthy individuals and from 14 patients with different clinical forms of Gaucher disease. The patients studied were classified as follows: group 1 consisted of 10 chronic patients, all (with one exception) of Ashkenazi Jewish origin; group 2 consisted of three black American patients with severe visceral symptoms, manifest from early childhood, but with no apparent neurological involvement; and group 3 consisted of a single white patient with the classical infantile form of the disease. Specific beta-glucosidase activity ranged from 6.6% to 16.5% mean control value in group 1 patients and from 4.1% to 5.8% in groups 2 and 3. When compared with the enzyme from control fibroblasts, the enzyme from chronic Gaucher patients (group 1) was more rapidly inactivated at 50 degrees C, had an altered pH curve, was less effectively inhibited by deoxycorticosterone-beta-glucoside, and was more effectively inhibited by deoxycorticosterone. The enzyme from patients in groups 2 and 3 was qualitatively indistinguishable from the control enzyme in terms of these parameters. No differences in Km (4-methylumbelliferyl-beta-glucoside) or sedimentation coefficient were found between the beta-glucosidases from control and Gaucher cells. The results demonstrate that cells from Ashkenazi Jewish patients with the chronic form of Gaucher disease contain a structurally altered form of beta-glucosidase. This enzyme differs both from normal beta-glucosidase and from the residual enzyme in patients of different ethnic origin and with clinically more severe forms of the disease. (+info)
Immature germ cell separation using a modified discontinuous Percoll gradient technique in human semen.
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The difficulty of identifying immature germ cells in unstained, fresh semen has led most laboratories to use the broad definition 'round cells' to indicate cells other than spermatozoa, thus grouping together both leukocytes and immature germ cells. This is also the case in research andrology, where very little attention has been given to immature germ cells in the semen apart from some rare exceptions, such as the attempts to study meiosis. Here we report on the use of a discontinuous Percoll gradient method modified to enable the best separation possible of immature germ cells from the other cells found in the ejaculate, in order to obtain a cellular suspension free of spermatozoa. Our technique (intra-assay variation in duplicates < 10%) demonstrated a high immature germ cell concentration in gradient fractions with 30% to 45% Percoll with a small contamination (1.5-6%) of leukocytes, confirmed by May-Grunwald-Giemsa staining, immunofluorescence and cytofluorimetry. The concentrations of immature germ cells ranged from zero in obstructive azoospermia to 2.0 x 10(6)/ml in oligozoospermia and genital tract infection. The purified immature germ cell suspensions obtained can be useful for diagnostic and research purposes. (+info)
Comparative evaluation of two density gradient preparations for sperm separation for medically assisted conception.
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To evaluate and optimize the sperm separation efficiency of a novel silane-coated silica bead (Puresperm), serial studies were carried out to compare the various sperm parameters between: (i) three-layer (90%-70%-40%) Puresperm and three-layer (90%-70%-40%) conventional polyvinylpyrrolidone (PVP)-coated silica bead (Percoll) gradients; (ii) three-layer (90%-70%-40%) and two-layer (90%-45%) Puresperm gradients and separately the same for Percoll; and (iii) large (3.0 ml) and small (0.75 ml) semen loading volumes on three-layer Puresperm gradients. Normozoospermic semen samples were treated and analysed in 12 replicates for each experiment. Manual evaluation of concentration, percentage motility, percentage vitality, percentage normal morphology; computer-assisted semen analysis evaluation of concentration, percentage motility, grade of motility, motion characteristics (curvilinear velocity, linearity, amplitude of lateral head velocity, beat cross frequency, percentage hyperactivation); and yields from the initial semen samples were compared. Percoll was found to be superior to Puresperm in concentration, percentage motility, percentage vitality and yields after three-layer density gradient centrifugation. There were no significant differences in sperm parameters between two- and three-layer Percoll gradients, but three-layer Puresperm gradients behaved significantly better than two-layer gradients. Large semen volume loads on three-layer Puresperm gradients resulted in greater sperm concentrations, percentage motility, percentage vitality and percentage normal morphology, but small semen volume loads produced greater yields of good-quality spermatozoa. In the light of Percoll being withdrawn from the shelf for the use of assisted reproduction because of the presence of PVP, three-layer Puresperm gradients with large semen loading volumes appear to be an attractive alternative for sperm separation in medically assisted conception. (+info)
Preliminary characterization of a reovirus isolated from golden ide Leuciscus idus melanotus.
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Some characteristics of a reovirus recently isolated from golden ide Leuciscus idus melanotus and tentatively designated as golden ide reovirus (GIRV) were determined. Spherical non-enveloped particles with an outer capsid of about 70 nm and an inner capsid of about 50 nm were observed by electron microscopy. The density of the virus determined in CsCl gradients was 1.36 g ml-1. The genome contained 11 segments of dsRNA. GIRV differed from other aquareoviruses by a slight reduction of infectivity after treatment with chloroform and by the absence of forming syncytia in cell monolayers. (+info)
Size and transforming activity of deoxyribonucleic acid in Diplococcus pneumoniae during thymidine starvation.
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The transforming activity and the molecular structure of DNA from cells of Diplococcus pneumoniae during thymidine starvation have been analyzed and the effects of thymidine starvation have been compared with the effects of single-strand breaks produced by deoxyribonucleases in DNA of unstarved cells. The decrease in transforming activity of lysates from starved cells as a function of the size of DNA particles, measured by centrifugation in neutral and alkaline sucrose gradients, does not follow the kinetics observed after enzymatic degradation of DNA of unstarved cells. Moreover, a strain lacking exo- and endonuclease activities is not protected from thymineless death. These results suggest that the basic lethal mechanism of thymidine starvation might have an origin other than the activation of nucleases. (+info)
Subunits of the alkaline phosphatase of Bacillus licheniformis: chemical, physicochemical, and dissociation studies.
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The alkaline phosphatase (orthophosphoric monoester phosphydrolase, EC 3.1.3.1) of Bacillus licheniformis MC14 was studied in an attempt to determine the number of subunits contained in the 120,000-molecular-weight native enzyme. Two moles of arginine was liberated per mole of native enzyme by carboxypeptidases A and B in the presence of sodium dodecyl sulfate. The effect on the native enzyme of progressively lowering the solvent buffer pH was monitored by determining the molecular weight by sedimentation equilibrium analysis, the sedimentation coefficient, the frictional coefficient, and the percent alpha-helix content of the enzyme. The alkaline phosphatase dissociates into two subunits around pH 4. At pH 2.8 a further decrease in S value, but no change in molecular weight, is observed, indicating a change in conformation. The frictional coefficients and percent alpha-helix content agree with this interpretation. A subunit molecular weight of 59,000 was calculated from sodium dodecyl sulfate gels. (+info)
Evidence for RNA linked to nascent DNA in HeLa cells.
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Rapidly labeled, i.e., nascent, DNA from HeLa cells was separated from the bulk DNA by ultracentrifugation. Further characterization of the rapidly labeled component revealed that its sedimentation coefficient is in the range of 4S and that it exists in a single- and double-stranded conformation. Moreover, analysis by nitrocellulose chromatography and CsSO4 density sedimentation of the nascent DNA labeled with 3H-uridine revealed that it is covalently linked to short chains of RNA, indicating that in HeLa cells RNA primer is involved in DNA replication. (+info)