Biomedical subjects
Publications and source records attributed to D T Brown.
Sindbis virus nucleocapsids were isolated from intact virus particles and from the cytoplasms of infected cells. These two isolates of purified nucleocapsids differed from one another in ribonuclease sensitivity and in sedimentation velocity. These findings suggest that a conformational change takes place in the Sindbis virus nucleocapsid during envelopment.
The effects of the lysosomotropic weak bases chloroquine, ammonium chloride, and amantadine as well as dansylcadaverine (an inhibitor of receptor mediated endocytosis) on the replication of Sindbis virus in tissue-cultured cells was examined. Chloroquine had no effect on the expression of virus-induced homologous interference. None of these drugs significantly affected the ability of a complex of a cell and single virion to form an infectious center. Chloroquine and ammonium chloride were found to inhibit the synthesis of virus RNA in established infections when added early in infection. These drugs also inhibited the production of progeny virions when added any time after infection. These results suggest that the antiviral activity of these agents may not be due to an ability to prevent transport of the virus genome into the cell cytoplasm.
A 6.9 kilobase Eco R1 fragment containing genes for two U1 RNAs has been isolated from a library of mouse DNA. The two genes code for an RNA which is very similar, if not identical, to mouse U1b RNA as judged by S1 nuclease mapping. This RNA is one base longer than the mouse U1a RNA, human U1 RNA, and rat U1 RNA and differs in six nucleotide substitutions from rat U1 RNA. The two genes are five kilobases apart and the U1 RNAs are coded for on opposite strands of the DNA with the 5' ends juxtaposed. The sequences flanking the genes are identical for 700 bases 5' to the gene and at least 80 bases 3' to the gene.
A comparative ultrastructural study of the organization of intermediate filaments (tonofilaments) within normal colonic epithelium and colonic carcinoma cells was carried out. The pattern of development of the specialized intermediate filament (IF) network, occurring during the differentiation of normal colonic epithelial cells, was examined to allow a more complete comparison with tumour cells of different degrees of differentiation. Essentially the IF system becomes more organized as the cells differentiate, resulting in the formation of a three-dimensional network loosely surrounding the nucleus, extending through the sub-terminal web and basal cytoplasm, and inserting into lateral desmosomes. Another system of IFs also runs directly between lateral desmosomes, lying parallel to the plasma-membrane and desmosomal plaque, without inserting directly into the junction. Alterations in the organization of the IF system were seen in tumour cells with the type and extent being dependent upon their location and degree of differentiation. Cells within areas of the tumour where some degree of glandular structure was retained exhibited major alterations in their microfilament-containing structures. However, their IF system was essentially intact and lateral desmosomes were still present in similar numbers to those seen in normal, partially differentiated mid-crypt cells. Apolar tumour cells within areas of the tumour where a gross loss of cryptal architecture had occurred exhibited a striking disorganization of their IF system. No parallel bundles of interdesmosomal tonofilaments were present beneath the cell surface and the majority of the three-dimensional network appeared to have collapsed around the nucleus, although some extensions to randomly distributed surface desmosomes still occurred. It appears that this disorganization of the IF system is associated with a loss of cell polarity and may involve alterations in putative IF-associated proteins important in the interaction of IFs with surface desmosomes or other cytoskeletal elements. A study of this phenomenon may shed light on both the means of IF organization and assembly and their role in normal cells as well as the possible role of alterations in cytoskeletal elements in the expression or maintenance of the malignant phenotype.
An ultrastructural analysis of the organisation of 10 nm intermediate filaments during mitosis, in normal and malignant human colonic epithelial cells present in resected material, was carried out. Similar changes in the integrity of the 10 nm filaments were seen both in normal mitotic cells (which have a highly specialised filamentous network during interphase), and malignant cells (which show a disorganised network during interphase). The 10 nm filaments were seen to shorten and 'fray' in early mitosis. This was associated with the formation of spheroidal aggregates of thin filaments and small granules, both free in the cytoplasm and associated with surface desmosomes. At mid-mitosis cells were essentially devoid of obvious filamentous material, (including the spheroidal aggregates). The filaments reformed in daughter cells after cytokinesis was completed.
Dextran solutions of low viscosity (7.5 centipoise) and high viscosity (2.2 poise) were instilled in turn into the middle ears of guinea pigs, using an open bulla preparation. The degree of conductive hearing loss induced was assessed by measurement of the cochlear microphonic. Both solutions produced a comparable degree of hearing loss which was frequency dependent, ranging from 30 dB at 500 Hz to almost zero at 4000 Hz. The results indicate that the degree of conductive hearing loss produced by a serous effusion in otitis media is due solely to an effect on compliance in the middle ear and is independent of the viscosity of the effusion.
Midazolam hydrochloride and diazepam in emulsion were compared as i.v. sedatives for spinal anaesthesia. Mean doses of midazolam 12 mg and diazepam 27 mg were required to induce sleep and provide good or adequate sedation throughout surgery of approximately 1 h duration. Neither drug caused loss of the airway or significant cardiorespiratory changes. The frequency of drowsiness after operation was greater following sedation with diazepam, but objective testing of recovery with the Trieger test showed no significant difference between the two drugs. The frequency of amnesia was greater following midazolam.
The ability of Sindbis virus to grow in enucleated BHK-21 (vertebrate) and Aedes albopictus (invertebrate) cells was tested to determine the dependence of this virus upon nuclear function in these two phylogenetically unrelated hosts. Although both cell types could be demonstrated to produce viable cytoplasts (enucleated cells) which produced virus-specific antigen subsequent to infection. BHK cytoplasts produced a significant number of progeny virions, whereas mosquito cytoplasts did not. The production of vesicular stomatitis virus in mosquito cells was not significantly reduced by enucleation. That such a host function was not essential for vesicular stomatitis virus growth in insect cells is supported by the observation that the production of this virus by mosquito cells is not actinomycin D sensitive. This result agrees with a previously published report in which it was shown that Sindbis virus maturation in invertebrate cells is inhibited by actinomycin D, indicating a possible requirement for host cell nuclear function (Scheefers-Borchel et al., Virology, 110:292-301, 1981).
The process of cell fusion mediated by Sindbis virus membrane proteins synthesized after infection was examined. At the times after infection at which virus proteins were detectable on the cell surface, Sindbis virus-infected BHK-21 cells were found to express a fusion function after brief treatment at acid pH. In studies employing wild-type virus and temperature-sensitive mutants and testing drug or protease inhibition of virus production, we made the following observations on Sindbis virus-mediated fusion from within. (i) Fusion requires the synthesis of virus glycoproteins and their transport to the cell surface. (ii) Modification of the cell plasma membrane by polypeptides PE2 and E1 alone is not sufficient for expression of the fusion function. (iii) The proteolytic conversion of plasma membrane-associated PE2 to E2 is not essential for fusion. (iv) Glycosylation of virus plasma membrane proteins is essential for fusion. (v) The lesions of Sindbis virus temperature-sensitive mutants do not affect their ability to fuse cells.
The attachment of high multiplicities of Sindbis virus to tissue-cultured cells followed by brief treatment at low pH has been shown to produce cell fusion (fusion from without). In this report, experiments to determine the effects of low pH on the physical and biological properties of Sindbis virus are described. Exposure of purified Sindbis virions to mildly acidic conditions resulted in a rapid and irreversible alteration in particle density and sedimentation characteristics, followed by a slower loss of infectivity. Infectivity was not restored by a return to neutral pH; rather, the loss of virus infectivity seemed to be initiated by exposure to low pH but continued at neutral pH. The formation of a virus-cell complex in which virions were attached to the cell surface protected the particles from low-pH inactivation, although low pH could still expose virus functions responsible for cell fusion. Low pH was found to induce a conformational change in the E2 polypeptide of the intact virion. These results are discussed with respect to the process of Sindbis virus infection of tissue-cultured cells.
Human tracheal secretions were collected from patients with chronic tracheostomies in the intensive care unit and through an endotracheal tube from pediatric surgical patients under general anesthesia. These secretions were dialyzed, fractionated on an agarose column, and lyophilized to recover the tracheal mucin fraction. Analysis of pooled mucin samples included determination of amino acids, carbohydrates, and sulfates. Intrinsic viscosity, microrheometry, and mucociliary transport studies were also performed. Structural and functional differences between several pools of specimens are cited with an emphasis on possible clinical correlations.
The effects of a wide range of mucolytic agents on the viscoelastic nature of a purified mucus glycoprotein gel have been investigated. The gel was produced by ultrafiltration of a solution containing high molecular weight glycoproteins obtained by gel chromatography in potassium thiocyanate. The fact that compounds as diverse as dithiothreitol and protease reduced the viscoelastic properties indicates that the model gel is appropriate for screening such compounds. Since it is homogenous and highly reproducible then it would appear to be ideal for such purposes. Also, it has been shown that mucus gels can be formed containing nothing but glycoproteins.
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The genes coding for the two major small nuclear RNAs in the sea urchin are organized in independent tandem repeating units. The small nuclear RNAs, N1 and N2 were purified from gastrula embryos of Lytechinus variegatus. These RNAs are analogous to the U series of RNA in mammalian cells as judged by their identical 5' termini and the sequence homology of the N1 urchin RNA and U1 mouse RNA. These RNAs were polyadenylated with E. Coli adenylate transferase. A 32PO4 labeled copy of each RNA was made with RNA-dependent DNA polymerase. This copy was used to probe the gene organization of these RNAs by hybridizing to restriction enzyme digests of sperm DNA. Each of these RNAs is coded in a tandemly repeated cluster (at least 30 kb) with a repeat length of 1100-1400 bases. The N1 and N2 clusters are distinct. The N1 repeat has been cloned and the repeating organization confirmed with the cloned gene.
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Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.