Biomedical subjects

D T Brown

Publications and source records attributed to D T Brown.

At least 91 records · Page 5Linked to original sources

PubMed1991

Sindbis virus infection of a Chinese hamster ovary cell mutant defective in the acidification of endosomes.

Sindbis virus infection of a Chinese hamster ovary temperature-sensitive mutant cell line defective in its ability to acidify endosomes was examined to evaluate the importance of this step in the process of alphavirus penetration of host cells by measuring the time at which viral RNA synthesis was initiated. We found that RNA synthesis was initiated in these mutant cells at the same time after addition of virus at either permissive or nonpermissive temperature. We conclude that exposure to the acidic environment of an endosome is not a prerequisite for alphavirus infection.

Animals↗

PubMed1991

The effect of sucralfate on the growth of cariogenic streptococci.

Sucralfate, an aluminum salt of sucrose octasulfate, has been shown to be effective in reducing the discomfort of radiation therapy-induced oral mucositis. This study was done to determine whether sucralfate could be used as a nutritional source for dental caries-producing organisms. Three Streptococcus strains were cultured in a defined medium. Sucralfate powder was evaluated for its ability to be used as a carbohydrate food source by these organisms. The addition of sucralfate alone did not stimulate the organism's growth. The addition of sucralfate and glucose resulted in less growth than the addition of glucose alone. Increasing the sucralfate concentration from 1% to 10% in the glucose-containing cultures resulted in statistically significant growth inhibition (p less than 0.02). Sucralfate appears to have no cariogenic potential and may have some cariostatic potential.

Colony Count, Microbial↗

PubMed1991

Protein-protein interactions in an alphavirus membrane.

Using homobifunctional chemical cross-linkers with various span distances, we have determined the near-neighbor associations and planar organization of the E1 and E2 envelope glycoproteins which compose the icosahedral surface of Sindbis virus. We have found that E1-E2 heterodimers, which form the virus protomeric units, exist in two conformationally distinct forms, reflecting their nonequivalent positions in the icosahedron. Three of these heterodimers form the trimeric morphologic units (capsomeres) which are held together by central E1-E1 interactions. In addition, we present data which suggest that E2-E2 interactions organize the capsomeres into pentameric and hexameric geometric units and that E1-E1 interactions between capsomeres maintain the icosahedral lattice in mature virions.

Alphavirus↗

PubMed1991

Defective transport of Sindbis virus glycoproteins in End4 mutant Chinese hamster ovary cells.

Mutant V.24.1, a temperature-sensitive derivative of Chinese hamster ovary cells, defines the End4 complementation group of mutants selected for resistance to protein toxins and has defective lysosomes at the restrictive temperature (P. A. Colbaugh, M. Stookey, and R. K. Draper, J. Cell Biol. 108:2211-2219, 1989). We have investigated the biosynthesis of Sindbis virus envelope glycoproteins in V.24.1 cells. When the cells were infected at the restrictive temperature, the envelope glycoproteins E1 and E2 were undetectable on the cell surface and proteolytic processing of the precursor protein pE2 to envelope protein E2 did not occur. Protein retained intracellularly was sensitive to endoglycosidase H and, by immunofluorescence localization, appeared to accumulate in the endoplasmic reticulum. We conclude that the genetic defect in V.24.1 cells impairs the transport of Sindbis virus glycoproteins, apparently at the level of export from the endoplasmic reticulum.

Animals↗

PubMed1991

Proteolytic processing of the Sindbis virus membrane protein precursor PE2 is nonessential for growth in vertebrate cells but is required for efficient growth in invertebrate cells.

We have shown previously that processing of the Sindbis virus envelope protein precursor PE2 to envelope protein E2 is not required for virus maturation in cultured vertebrate fibroblast cells and that unprocessed PE2 can be incorporated into infectious virus in place of E2 (J. F. Presley and D. T. Brown, J. Virol. 63:1975-1980, 1989; D. L. Russell, J. M. Dalrymple, and R. E. Johnston, J. Virol. 63:1619-1629, 1989). To better understand the role of this processing event in the invertebrate vector portion of the alphavirus life cycle, we have examined the maturation of Sindbis virus mutants defective in PE2 processing in cultured mosquito cells. We found that although substantial amounts of structural proteins PE2, E1, and C were produced in infected mosquito (aedine) cell lines, very little infectious virus was released. When the period of infection was extended, plaque size variants appeared, some of which exhibited a restored ability to grow in mosquito cells. The nucleotide sequences of two such variants were determined. These variants contained point mutations that restored PE2 cleavage, indicating a genetic linkage between failure to cleave PE2 and failure to grow in mosquito cells.

Aedes↗

PubMed1990

P0 mRNA expression in cultures of Schwann cells and neurons that lack basal lamina and myelin.

Schwann cells of the peripheral nervous system depend on the presence of both axons and basal lamina to achieve a myelinating phenotype. Furthermore, removal of axonal influence results in the cessation of myelination and down-regulation of myelin protein expression by Schwann cells. Here we examine whether both axons and basal lamina are required by Schwann cells for the expression of mRNA encoding the major myelin glycoprotein, P0. Cultures of Schwann cells and neurons obtained from dorsal root ganglia of 15 day embryonic rat pups were grown for up to 20 days in vitro under conditions that either allowed or prohibited basal lamina and myelin formation. These cultures were assayed for the expression of P0 mRNA by using an S1 nuclease-protection assay. After 20 days in vitro, the cultures that did not assemble basal lamina and that were incapable of myelin formation expressed P0 mRNA at a level which was comparable to that seen in identically aged, myelinating cultures. Both the myelinating and nonmyelinating cultures demonstrated an appreciable increase in P0 mRNA when compared to the starting embryonic dorsal root ganglia Schwann cells. The latter had a low, but detectable, level of mRNa for this myelin glycoprotein. The cultures that were devoid of basal lamina and myelin showed a clear increase in P0 mRNA by 11-15 days in culture. This increase in expression depended on the presence of neurons/neurites, since Schwann cells which were grown in neuron-depleted cultures expressed little, if any, P0 mRNA. In contrast to the levels of P0 mRNA, the nonmyelinating cultures had a significantly lower amount of P0 glycoprotein than did the cultures which assemble myelin. This suggests that the nonmyelinating Schwann cells regulate the level of this glycoprotein at the translational and/or the posttranslational level. The data presented here suggest that myelin protein mRNA expression and myelin assembly by Schwann cells are separable events, with the former depending on one or more neuronal/axonal factors.

Basement Membrane↗

PubMed1990

A comparative study of temporomandibular symptoms following mandibular advancement by bilateral sagittal split osteotomies: rigid versus nonrigid fixation.

Rigid fixation to attach proximal and distal segments during bony healing of osteotomy sites has become increasingly popular. The effects of rigid fixation on the temporomandibular joints have been questioned. The purpose of this study was to evaluate the effects of rigid fixation after bilateral sagittal split osteotomies on temporomandibular dysfunction symptoms. Forty patients who had mandibular advancement surgery were evaluated for temporomandibular joint dysfunction. Twenty had received rigid fixation, and twenty had received nonrigid fixation. It was determined that there was no statistically significant difference in temporomandibular signs or symptoms between patients who were treated with rigid internal fixation for bilateral sagittal split osteotomies for mandibular advancement and those patients who were treated with nonrigid wire fixation.

Adolescent↗

PubMed1989

Intracellular transport and processing of Sindbis virus glycoproteins.

The intracellular transport and processing of Sindbis virus envelope glycoproteins were studied in cells infected with Sindbis virus using the mannose-specific enzyme, endoglycosidase H (endo H). In pulse/chase labeling experiments of hamster cells with [35S]methionine, Sindbis glycoproteins PE2 and E1 became endo H resistant in two steps at 12.5 and 20.0 min after a 5-min pulse, suggesting that the glycoproteins required this period of time to be transported to the Golgi compartments containing the enzymes which process the high mannose side chains acquired in the endoplasmic reticulum. E2 could be detected at the end of a 5-min pulse and the E2 produced early was found to be endo H sensitive. The rate at which PE2 was converted to E2, relative to the acquisition of endo H resistance, suggests the independence of this proteolytic event from cellular protein transport and raises the possibility that the proteolytic function is of viral origin.

Animals↗

PubMed1989

Effect of nalbuphine hydrochloride on the ventilatory and occlusion pressure responses to carbon dioxide in volunteers.

We studied the effect of nalbuphine on the ventilatory and occlusion pressure responses to carbon dioxide rebreathing in six healthy male volunteers (mean age 25.5 yr) in a single-blind laboratory study. On four separate days volunteers were assigned randomly to receive either placebo (0.9% sodium chloride) or three i.v. doses of nalbuphine (15, 30 and 60 mg 70 kg-1), followed 90 min later by naloxone 0.4 mg 70 kg-1. Duplicate rebreathing tests were performed and the mean intercept at PE'CO2 7 kPa and the slopes of the linear relationship between inspiratory minute ventilation (Vl) or occlusion pressure (P0.1) with PE'CO2 were measured. Nalbuphine significantly decreased the mean intercept of the Vl (P less than 0.01) and P0.1 (P less than 0.05) responses, but caused no changes in the slopes. No significant difference between the doses was noted, suggesting that an Effect maximum (E'max) for respiratory depression was reached with a dose of approximately 15 mg 70 kg-1. Naloxone was less effective in antagonizing the depression in Vl at the higher dose of nalbuphine. Similar P0.1 values were associated with the same inspiratory flow rate (1 litre s-1) before and after drug treatment, suggesting that nalbuphine acts centrally to depress ventilation. Sedation increased significantly following each dose of nalbuphine (P less than 0.001). No demonstrable difference between the doses was shown, suggesting an Effect maximum (E'max) for sedation was reached at about 15 mg 70 kg-1. Administration of nalbuphine was associated with pain at the injection site, dizziness, dreaming, nausea and vomiting. Cardiovascular stability was maintained in all subjects.

Adult↗

PubMed1989

Structure-activity relationships in differential nerve block at high and low frequency stimulation.

We have examined the in vitro sensitivities of A, B and C fibres in rabbit vagus nerves to local anaesthetic block with a range of local anaesthetic drugs. The nerves were maintained at 37 degrees C and pH 7.4 using an electrolyte solution equilibrated with 5% carbon dioxide. A fibres were the most, and C fibres the least, sensitive to block at low frequency stimulation (0.0167 Hz). Equipotent concentrations of the various drugs blocked C fibres at approximately the same rate. Rate of A fibre block was related to physicochemical properties of individual drugs. Low pKa and high lipid solubility favoured block of A fibres before C, whereas the reverse was true for high pKa and low solubility. Absolute potency was related to lipid solubility, but each type of local anaesthetic drug in clinical use has a different inherent potency. With stimulation at high frequencies (20 and 40 Hz), C fibres were more sensitive to use-dependent block than A fibres; this effect was more marked with drugs containing an amide than an ester linkage. Molecular size and lipid solubility may inter-react to govern the ability to produce use dependent block. Agents which contain an amide linking group, have a high pKa and are of low lipid solubility, may be used to produce differential C fibre block.

Action Potentials↗

PubMed1989

Form-determining functions in Sindbis virus nucleocapsids: nucleosomelike organization of the nucleocapsid.

Purified intact Sindbis virus nucleocapsids were treated at different pH values or with various concentrations of divalent cations, cation chelators, salt, or formamide. The resulting structures were examined by velocity sedimentation, electron microscopy, and protein-protein cross-linking. Changes in each of the test conditions led to alterations in the sedimentation profile of treated nucleocapsids. Appropriate concentrations of formamide or divalent cations generated beaded strandlike structures similar in morphology to those generated from adenovirus cores and nucleosomes. The capsid protein and RNA remained associated with each other at NaCl concentrations less than or equal to 1 M or after treatment of the structures with alkaline pH up to and including pH 10.7. Protein and RNA were dissociated by salt concentrations of greater than 1 M, suggesting that the arginine-rich, amino-terminal portion of the capsid protein is responsible for binding the RNA. Protein-protein cross-linking also indicated that the capsid proteins remained associated in small aggregates under some of the conditions that caused dissociation of the nucleocapsid and suggested the presence of more than one type of protein-protein interaction in the nucleocapsids. Collectively, these data suggest that, like histones and adenovirus core proteins, the Sindbis virus capsid protein serves to package segments of the genome into nucleoprotein beads which are capable of interacting with each other to form the nucleocapsid structure.

Capsid↗

PubMed1989

The proteolytic cleavage of PE2 to envelope glycoprotein E2 is not strictly required for the maturation of Sindbis virus.

The ionophore monensin has been shown previously to block the maturation of Sindbis virus as well as prevent the cleavage of pE2 to E2 when applied to cells in high concentration. We found that a moderate dose of monensin reduced virus titer and inhibited the cleavage of pE2 to E2. Under these conditions, pE2 appeared on the cell surface in a form susceptible to lactoperoxidase-mediated iodination. This pE2 was incorporated into virions, replacing E2. PE2-containing virions had a normal PFU-to-particle ratio, cosedimented with normal virus, and retained a normal morphology when negatively stained preparations were examined by electron microscopy. We conclude that the cleavage of pE2 to form E2 is not an absolute prerequisite for virus maturation. Recently, Russell et al. have reached a similar conclusion (D. L. Russell, J. M. Dalrymple, and R. E. Johnston, J. Virol. 63:1619-1629, 1989).

Animals↗

PubMed1989

The effects of family structure on institutionalized children's self-concepts.

This study examined the effects of family structure (intact, stepparent, and single parent) on children's self-concepts, using Parish and Taylor's Personal Attribute Inventory for Children. One hundred sixty-six institutionalized children in Grades 5 through 12 participated in the study. Although a trend was noted for students from single-parent families to check fewer positive adjectives, they did not differ significantly (.05 level) from children from intact families or stepfamilies.

Child↗

PubMed1988

Suppression of RNA synthesis by a specific antiviral activity in Sindbis virus-infected Aedes albopictus cells.

An antiviral protein is released by mosquito cells persistently infected with Sindbis virus. Differences in both sensitivity to and production of this virus-specific activity were apparent in three independently produced Aedes albopictus cell lines. This activity inhibits total viral RNA synthesis in a time-dependent manner. The antiviral effect is maximally realized when cells are treated with the activity 48 h before infections. These data suggest that the antiviral activity induces an antiviral state in treated cells which prevents the formation or efficient function of viral RNA-synthesizing complexes.

Aedes↗

PubMed1988

Effect of actinomycin D and cycloheximide on replication of Sindbis virus in Aedes albopictus (mosquito) cells.

Production of Sindbis virus in the presence of transcription and translation inhibitors was examined in three Aedes albopictus cell lines. Addition of cycloheximide to heat-resistant Sindbis virus (SVHR)-infected mosquito cells arrested viral RNA synthesis completely, in contrast to the effects of this drug on virus-infected vertebrate cells. Production of mature virus by both SVHR (a variant commonly used as a wild-type virus) and SBamr (a mutant which is resistant to the effects of 18 h of pretreatment of vertebrate cells with actinomycin D) in mosquito u4.4, C6-36, and C7-10 cells was inhibited by 2 h of pretreatment with actinomycin D. Pretreatment with this drug for 2 h slightly enhances virus production in vertebrate cells. Treatment of mosquito cells with actinomycin D resulted in shutoff of SVHR RNA synthesis. The mutant SBamr was able to overcome the effects of actinomycin D on viral RNA synthesis and produced both 26S and 49S RNAs, even though no viral structural proteins or mature particles were produced in the presence of the drug. This result suggests that, in the presence of actinomycin, the nonstructural genes of SBamr are translated sufficiently to allow for RNA synthesis but that 26S RNA may not be translated to an extent that allows significant virus production. These data demonstrate that host components are involved in at least two distinct steps in the production of Sindbis virus in mosquito cells: (i) production of viral RNA and (ii) synthesis of viral structural polypeptides.

Aedes↗

PubMed1988

Histone gene switching in murine erythroleukemia cells is differentiation specific and occurs without loss of cell cycle regulation.

We investigated the expression characteristics of the fully replication-dependent (FRD) and the partially replication-dependent (PRD) histone gene variants by measuring changes in steady-state mRNA levels during hexamethylene bisacetamide (HMBA)-induced differentiation of murine erythroleukemia (MEL) cells. Between 24 and 60 h after induction, there was a dramatic switch in histone gene expression, such that the ratio of PRD to FRD transcripts increased severalfold over that found in uninduced MEL cells. We demonstrated that this gene switching was not simply a partial or complete uncoupling of PRD gene expression from DNA synthesis. PRD and FRD transcript levels were regulated coordinately upon treatment of uninduced or induced MEL cells with inhibitors of DNA synthesis, protein synthesis, or both. Using several criteria, we were unable to detect any difference in PRD and FRD gene expression under any conditions except in cells undergoing differentiation. MEL cells were arrested at a precommitment stage of differentiation by induction with HMBA in the presence of dexamethasone (DEX). If DEX was subsequently removed, DNA synthesis resumed, the cells underwent commitment, and histone gene switching was observed. In contrast, if both DEX and HMBA were removed, DNA synthesis still resumed, but commitment did not occur and no gene switching was observed. These results imply that histone gene switching is intimately related to the differentiation process.

Animals↗

PubMed1988

Twenty-five years of respiratory intensive care.

During the years 1961 to 1986 the four-bedded Artificial Ventilation Unit of the Royal Infirmary of Edinburgh has treated 3046 patients with respiratory problems of all types. There has been a steady increase in the number of patients admitted each year to the present level of 180-190, a fall in mean length of stay from 15 to six days and the mortality rate has been reduced to 13%. The type of case admitted has shown considerable variation over the years with the most significant change being a large increase in the patients admitted post-operatively. The size of the unit has placed severe restrictions on the work that can be undertaken.

Adult↗

PubMed1987

Isolation and characterization of a mouse fully replication-dependent H1 gene within a genomic cluster of core histone genes.

We have used an oligonucleotide complementary to a sequence coding for the conserved central globular domain of H1s to screen a mouse genomic library for H1 genes. We then used a series of universal histone oligonucleotides to identify five different H1 genes which were linked to core histone genes. We characterized one of the H1 genes which was linked to an H2a, an H2b, an H3, and an H4 histone gene. This characterization involved: 1) sequencing of the coding region of the gene and several hundred base pairs of flanking region. 2) Comparison of this sequence to other H1 sequences from other organisms. This sequence analysis clearly showed that the gene coded for an H1 and identified H1 consensus sequences in the 5'- and 3'-flanking region. 3) Mapping of the 5'- and 3'-ends of the mRNA complementary to this gene by S1 nuclease analysis. 4) Identifying this gene and an adjacent H3 gene as being of the fully replication-dependent expression class, by measuring changes in the steady state levels of their mRNAs in the presence of hydroxyurea and during differentiation of murine erythroleukemia cells.

Amino Acid Sequence↗