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D T Brown

Publications and source records attributed to D T Brown.

At least 37 records · Page 2Linked to original sources
PubMed2000

The surface conformation of Sindbis virus glycoproteins E1 and E2 at neutral and low pH, as determined by mass spectrometry-based mapping.

Sindbis virus contains two membrane glycoproteins, E1 and E2, which are organized into 80 trimers of heterodimers (spikes). These trimers form a precise T=4 icosahedral protein lattice on the surface of the virus. Very little is known about the organization of the E1 and E2 glycoproteins within the spike trimer. To gain a better understanding of how the proteins E1 and E2 are arranged in the virus membrane, we have used the techniques of limited proteolysis and amino acid chemical modification in combination with mass spectrometry. We have determined that at neutral pH the E1 protein regions that are accessible to proteases include domains 1-21 (region encompassing amino acids 1 to 21), 161-176, and 212-220, while the E2 regions that are accessible include domains 31-84, 134-148, 158-186, 231-260, 299-314, and 324-337. When Sindbis virus is exposed to low pH, E2 amino acid domains 99-102 and 262-309 became exposed while other domains became inaccessible. Many new E1 regions became accessible after exposure to low pH, including region 86-91, which is in the putative fusion domain of E1 of Semliki Forest virus (SFV) (M. C. Kielian et al., J. Cell Biol. 134:863-872, 1996). E1 273-287 and region 145-158 were also exposed at low pH. These data support a model for the structure of the alphavirus spike in which the E1 glycoproteins are centrally located as trimers which are surrounded and protected by the E2 glycoprotein. These data improve our understanding of the structure of the virus membrane and have implications for understanding the protein conformational changes which accompany the process of virus-cell membrane fusion.

Capsid↗
PubMed2000

Sindbis virus glycoprotein E1 is divided into two discrete domains at amino acid 129 by disulfide bridge connections.

The E1 membrane glycoprotein of Sindbis virus contains structural and functional domains, which are conformationally dependent on the presence of intramolecular disulfide bridges (B. A. Abell and D. T. Brown, J. Virol. 67:5496-5501, 1993; R. P. Anthony, A. M. Paredes, and D. T. Brown, Virology 190:330-336, 1992). We have examined the disulfide bonds in E1 and have determined that the E1 membrane glycoprotein contains two separate sets of interconnecting disulfide linkages, which divide the protein into two domains at amino acid 129. These separate sets of disulfides may stabilize and define the structural and functional regions of the E1 protein.

Amino Acid Sequence↗
PubMed2000

A single deletion in the membrane-proximal region of the Sindbis virus glycoprotein E2 endodomain blocks virus assembly.

The envelopment of the Sindbis virus nucleocapsid in the modified cell plasma membrane involves a highly specific interaction between the capsid (C) protein and the endodomain of the E2 glycoprotein. We have previously identified a domain of the Sindbis virus C protein involved in binding to the E2 endodomain (H. Lee and D. T. Brown, Virology 202:390-400, 1994). The C-E2 binding domain resides in a hydrophobic cleft with C Y180 and W247 on opposing sides of the cleft. Structural modeling studies indicate that the E2 domain, which is proposed to bind the C protein (E2 398T, 399P, and 400Y), is located at a sufficient distance from the membrane to occupy the C protein binding cleft (S. Lee, K. E. Owen, H. K. Choi, H. Lee, G. Lu, G. Wengler, D. T. Brown, M. G. Rossmann, and R. J. Kuhn, Structure 4:531-541, 1996). To measure the critical spanning length of the E2 endodomain which positions the TPY domain into the putative C binding cleft, we have constructed a deletion mutant, DeltaK391, in which a nonconserved lysine (E2 K391) at the membrane-cytoplasm junction of the E2 tail has been deleted. This mutant was found to produce very low levels of virus from BHK-21 cells due to a defect in an unidentified step in nucleocapsid binding to the E2 endodomain. In contrast, DeltaK391 produced wild-type levels of virus from tissue-cultured mosquito cells. We propose that the phenotypic differences displayed by this mutant in the two diverse host cells arise from fundamental differences in the lipid composition of the insect cell membranes which affect the physical and structural properties of membranes and thereby virus assembly. The data suggest that these viruses have evolved properties adapted specifically for assembly in the diverse hosts in which they grow.

Amino Acid Sequence↗
PubMed2000

Very low levels of the mtDNA A3243G mutation associated with mitochondrial dysfunction in vivo.

We studied mitochondrial function in vivo in 2 brothers harboring the mitochondrial DNA A3243G mutation by using magnetic resonance spectroscopy. One brother presented with recurrent strokes and had a mitochondrial respiratory chain complex I defect, with 85% A3243G mutation in his quadriceps. The maximum rate of mitochondrial ATP production in his calf, measured in vivo, was reduced to 21% of the normal mean value. The second brother had mild exercise intolerance, normal muscle histochemistry, and normal respiratory chain activity in vitro. Despite a level of the A3243G mutation of only 5.95% (SD, 4.45; range, 0.7-16.1%) within single muscle fibers from the gastrocnemius muscle, the maximum rate of mitochondrial ATP production in his calf, measured in vivo, was reduced to 35% of the normal mean value. These findings suggest that there may not be a clear genetic threshold level for the expression of the A3243G mutation in skeletal muscle in vivo.

Adult↗
PubMed2000

The epidemiology of pathogenic mitochondrial DNA mutations.

During the past decade, there have been many descriptions of patients with neurological disorders due to mitochondrial DNA (mtDNA) mutations, but the extent and spectrum of mtDNA disease in the general population have not yet been defined. Adults with suspected mtDNA disease in the North East of England were referred to a single neurology center for investigation over the 10-year period from 1990 to 1999 inclusive. We defined the genetic defect in these individuals. For the midyear period of 1997, we calculated the minimum point prevalence of mtDNA disease in the adults of working age (> 16-<60 years old for female subjects and <65 years old for male subjects) and the minimum prevalence of adults and children (<60 years for female subjects, <65 years for male subjects) at risk of developing mtDNA disease. mtDNA defects caused disease in 6.57 per 100,000 individuals in the adult population of working age, and 7.59 per 100,000 unaffected adults and children were at risk of developing mtDNA disease. Overall, 12.48 per 100,000 individuals in the adult and child population either had mtDNA disease or were at risk of developing mtDNA disease. These results reflect the minimum prevalence of mtDNA disease and pathogenic mtDNA mutations and demonstrate that pathogenic mtDNA mutations are a common cause of chronic morbidity. These findings have resource implications, particularly for supportive care and genetic counseling.

Adolescent↗
PubMed1999

Effects of H1 histone variant overexpression on chromatin structure.

The importance of histone H1 heterogeneity and total H1 stoichiometry in chromatin has been enigmatic. Here we report a detailed characterization of the chromatin structure of cells overexpressing either H1(0) or H1c. Nucleosome spacing was found to change during cell cycle progression, and overexpression of either variant in exponentially growing cells results in a 15-base pair increase in nucleosome repeat length. H1 histones can also assemble on chromatin and influence nucleosome spacing in the absence of DNA replication. Overexpression of H1(0) and, to a lesser extent, H1c results in a decreased rate of digestion of chromatin by micrococcal nuclease. Using green fluorescent protein-tagged H1 variants, we show that micrococcal nuclease-resistant chromatin is specifically enriched in the H1(0) variant. Overexpression of H1(0) results in the appearance of a unique mononucleosome species of higher mobility on nucleoprotein gels. Domain switch mutagenesis revealed that either the N-terminal tail or the central globular domain of the H1(0) protein could independently give rise to this unique mononucleosome species. These results in part explain the differential effects of H1(0) and H1c in regulating chromatin structure and function.

3T3 Cells↗
PubMed1999

Overproduction of histone H1 variants in vivo increases basal and induced activity of the mouse mammary tumor virus promoter.

BALB/c 3T3 cell lines containing integrated copies of the MMTV promoter driving a reporter gene were constructed. Expression vectors in which either of two H1 variants, H10 or H1c, were under control of an inducible promoter were introduced into these lines. Surprisingly, overproduction of either variant resulted in a dramatic increase in basal and hormone-induced expression from the MMTV promoter. H1 overproduction also slowed the loss of MMTV promoter activity associated with prolonged hormone treatment. Transiently transfected MMTV reporter genes, which do not adopt a phased nucleosomal arrangement, do not display increased activity upon H1 overproduction. Thus the effects observed for stable constructs most likely represents a direct effect of H1 on a chromatin-mediated process specific to the nucleosomal structure of the integrated constructs. Induction of increased levels of acetylated core histones by treatment with trichostatin A also potentiated MMTV activity and this effect was additive to that caused by H1 overproduction. However, the effects of TSA treatment, in control or H1-overproducing cells, were eliminated by inhibiting protein synthesis. TSA treatment does not necessarily potentiate MMTV promoter activity by increasing core histone acetylation within the MMTV promoter but perhaps by altering the synthesis of an unlinked transcriptional regulator.

3T3 Cells↗
PubMed1998

Comparison of Sindbis virus-induced pathology in mosquito and vertebrate cell cultures.

We have compared Sindbis virus-induced cytopathology in vertebrate and mosquito (Aedes albopictus) cell cultures. It has been shown that vertebrate cells undergo apoptosis when infected by Sindbis virus and this was confirmed here using hamster cells (BHK). The occurrence of cell death in Sindbis virus-infected A. albopictus cells is a cell clone-specific phenomenon and, unlike in BHK cell cultures, mosquito cell death does not correlate with a large induction of apoptosis, as determined by assays testing for DNA fragmentation or reduced cellular DNA content. Cell cycle distribution changes were observed in Sindbis virus-infected BHK and C7-10 cell cultures, and the changes are distinct, both in the time of induction and the types of perturbations. In Sindbis virus-infected BHK cells, the major cell cycle profile change is the early accumulation of cells with sub-G1 DNA content and a corresponding reduction in the proportion of cells in G1 and G2/M. For Sindbis virus-infected C7-10 cells, the major perturbations are an increased proportion of cells showing G2/M or polyploid DNA content and a reduction in the proportion of G1 and S phase cells. These data suggest that the pathology induced in mosquito cell cultures by Sindbis virus infection may be distinct from the pathology which appears in vertebrate cell cultures.

Animals↗
PubMed1998

Laser assisted soldering: microdroplet accumulation with a microjet device.

BACKGROUND AND OBJECTIVE: We investigated the feasibility of a microjet to dispense protein solder for laser assisted soldering. STUDY DESIGN: Successive micro solder droplets were deposited on rat dermis and bovine intima specimens. Fixed laser exposure was synchronized with the jetting of each droplet. After photocoagulation, each specimen was cut into two halves at the center of solder coagulum. One half was fixed immediately, while the other half was soaked in phosphate-buffered saline for a designated hydration period before fixation (1 hour, 1, 2, and 7 days). After each hydration period, all tissue specimens were prepared for scanning electron microscopy (SEM). RESULTS: Stable solder coagulum was created by successive photocoagulation of microdroplets even after the soldered tissue exposed to 1 week of hydration. CONCLUSIONS: This preliminary study suggested that tissue soldering with successive microdroplets is feasible even with fixed laser parameters without active feedback control.

Animals↗
PubMed1998

Validation of continuous thermodilution cardiac output in critically ill patients with analysis of systematic errors.

PURPOSE: Bolus thermodilution cardiac output (BCO) measurements are affected by variations in injectate volume, rate, and temperature. These variations are eliminated when CO is measured by a continuous automated thermal technique (CCO). Further, CCO eliminates the need for fluid boluses, reduces contamination risk, requires no operator, and provides a continuous CO trend. We prospectively evaluated CCO versus BCO in a population of critically ill adults with low, normal, and high CO states. We sought to discern any systematic effects of temperature fluctuations or signal-to-noise-ratios (SNR) on disparities between BCO and CCO measurements and also sought to assess the relative cost effectiveness of the CCO system. MATERIALS AND METHODS: Pulmonary artery catheterizations were performed in a convenience sample of 20 patients over 6 months. BCO data were obtained using a standardized protocol. Three bolus injections of 5% dextrose were given when each CO was within 10% of the median before averaging; otherwise five boluses were given, with the high and low values eliminated before averaging. Injectates were administered randomly through the respiratory cycle and at 1-minute intervals. CCO measurements were recorded from a Vigilance monitor pre and post BCO measurements, yielding an average CCO value. Also recorded were pre- and post-core temperatures and SNR during the first CCO measurement. Cost data included estimates of operator time for BCO determinations as well as costs of Intellicath (Baxter-Edwards, Irvine, CA) pulmonary artery catheters, Vigilance (Baxter-Edwards, Irvine, CA) monitors, conventional catheters, and injectates. RESULTS: Of the 20 patients, 15 were mechanically ventilated. A total of 306 paired CO values were obtained for analysis. CCO ranged from 2.5 to 14.4 L/min and BCO from 2.4 to 13.3 L/min. Absolute differences between CCO and BCO measurements increased with increasing CO, but percentage differences did not. Of the paired values, 77% were within 1 L/min of one another. Temperature instability and SNR independently had weak correlations with CCO/BCO disparities. The Vigilance system had a slightly higher net cost than conventional BCO, although no economical value was assigned to the clinical usefulness of continuous, as opposed to intermittent, CO monitoring. CONCLUSIONS: Continuous CO is a reliable and cost-effective alternative to bolus thermodilution CO for critically ill patients in low, normal, and high CO states.

Adolescent↗
PubMed1998

"True normal" TMD control subjects: a rare clinical finding.

This study evaluated the accuracy of the clinical diagnosis of patients with temporomandibular joint dysfunction symptoms compared to a group of asymptomatic volunteers. The clinical examination and history questionnaire used during the evaluation of TMD patients were less accurate evaluating asymptomatic subjects than when combined with computerized joint vibration analysis. "True normal" control subjects were rarely found when these diagnostic modalities were combined in the TMD examination process. Most of the asymptomatic subjects had subclinical signs of TMD. The small sample size is significant, especially since large numbers of subjects appear to be necessary to obtain even small numbers of "true normals."

Adult↗
PubMed1997

Differential effect of H1 variant overproduction on gene expression is due to differences in the central globular domain.

The in vivo overproduction of two mouse histone H1 variants in homologous mouse fibroblasts has opposite effects on gene expression. Overproduction of H1(0) results in repression of transcript levels of all polymerase II genes tested. In contrast, overproduction of H1c results in elevated levels of transcripts. We created a series of chimeric H1 genes in which the regions encoding the three structural domains common to this family of these proteins were systematically switched. Overexpression of these genes in vivo resulted in the accumulation of large amounts of the chimeric H1 in chromatin. Analysis of the effects of overproduction of these proteins revealed that the differential effect of H1 variant overproduction on gene expression is due to differences in the central globular domain.

Amino Acid Sequence↗
PubMed1997

Prosthodontic considerations when using implants for orthodontic anchorage.

STATEMENT OF PROBLEM: The use of implants for orthodontic anchorage can produce superior preprosthetic tooth positions. Their use often requires a crown or prosthesis to be fabricated for use as a connection between the orthodontic devices and the implant. PURPOSE: In this article, factors that affect the design of prostheses required for orthodontic movement and examples of prosthesis designs and materials, based on the authors' experience, are presented.

Dental Implantation, Endosseous↗
PubMed1997

Characterization of the infection of Aedes albopictus cell clones by Sindbis virus.

We have investigated the infection of Aedes albopictus (mosquito) cell clones by Sindbis virus. Variation in the multiplicity of infection (MOI) from ranges of 50-0.00005 pfu/cell was determined to have no effect on the progression of the infection to high acute phase titer, suggesting that intracellular factors alone are responsible for the restriction of virus production seen as the infection enters the persistent phase: While persistently infected (over 1 year post infection) cell clones are morphologically indistinct from uninfected cells, they do display a uniform 30% reduction in growth rate compared with uninfected cells of the same clone. Using flow cytometry-based DNA content analysis, we found that persistent Sindbis virus infection induces distinct cytological effects on these cells, including an increase in apoptosis and polyploidy in one clone and cell cycle phase effects in another. Finally, the observation that the number of cells in persistently infected cell cultures which are productively infected closely approximates the number of cells dying by apoptosis prompted us to investigate the role that cell death may play in the maintenance of the persistent infection. Persistently infected cell cultures which were artificially induced into apoptosis by short 45 degrees C heat treatments do not display increased Sindbis virus production. This result does not support the hypothesis that infection sensitivity induced by random apoptosis in persistently infected cell cultures is responsible for the long-term maintenance of the persistent infection.

Acute Disease↗
PubMed1997

The formation of intramolecular disulfide bridges is required for induction of the Sindbis virus mutant ts23 phenotype.

The Sindbis virus envelope protein spike is a hetero-oligomeric complex composed of a trimer of glycoprotein E1-E2 heterodimers. Spike assembly is a multistep process which occurs in the endoplasmic reticulum (ER) and is required for the export of E1 from the ER. PE2 (precursor to E2), however, can transit through the secretory pathway and be expressed at the cell surface in the absence of E1. Although oligomer formation does not appear to be required for the export of PE2, there is evidence that defects in E1 folding can affect PE2 transit from the ER. Temperature-sensitive mutant ts23 of Sindbis virus contains two amino acid substitutions in E1, while PE2 and capsid protein have the wild-type sequence; however, at the nonpermissive temperature, both E1 and PE2 are retained within the ER and can be isolated in protein aggregates with the molecular chaperone GRP78-BiP. We previously demonstrated that the temperature sensitivity for ts23 was lost as oligomer formation took place at the permissive temperature, suggesting that temperature sensitivity is initiated early in the process of viral spike assembly (M. Carleton and D. T. Brown, J. Virol. 70:952-959, 1996). Experiments described herein investigated the defects in envelope protein maturation that occur in ts23-infected cells and which result in retention of both envelope proteins in the ER. The data demonstrate that in ts23-infected cells incubated at the nonpermissive temperature, E1 folding is disrupted early after synthesis, resulting in the rapid incorporation of both E1 and PE2 into disulfide-stabilized aggregates. Furthermore, the aberrant E1 conformation which is responsible for induction of the ts phenotype requires the formation of intramolecular disulfide bridges formed prior to E1 association with PE2 and the completion of E1 folding.

Animals↗
PubMed1997

Role of glycoprotein PE2 in formation and maturation of the Sindbis virus spike.

Sindbis virus envelope assembly is a multistep process resulting in the maturation of a rigid, highly ordered T=4 icosahedral protein lattice containing 80 spikes composed of trimers of E1-E2 heterodimers. Intramolecular disulfide bonds within E1 stabilize E1-E1 associations required for envelope formation and maintenance of the envelope's structural integrity. The structural integrity of the envelope protein lattice is resistant to reduction by dithiothreitol (DTT), indicating that E1 disulfides which stabilize structural domains become inaccessible to DTT at some point during virus maturation. The development of E1 resistance to DTT occurs prior to the completion of E1 folding and is temporally correlated with spike assembly in the endoplasmic reticulum. From these data we have predicted that in the final stages of spike assembly, E1 intramolecular disulfides, which stabilize the structural integrity of the envelope protein lattice, are buried within the spike and become inaccessible to the reductive activity of DTT. The spike is formed prior to the completion of E1 folding, and we have suggested that PE2 (the precursor to E2) may play a critical role in E1 folding after PE2-E1 oligomer formation has occurred. In this study we have investigated the role of PE2 in E1 folding, oligomer formation, and development of E1 resistance to both protease digestion and reduction by DTT by using a Sindbis virus replicon (SINrep/E1) which allows for the expression of E1 in the presence of truncated PE2. Through pulse-chase analysis of both Sindbis virus- and SINrep/E1-infected cells, we have determined that the folding of E1 into a trypsin-resistant conformation and into its most compact and stable form is not dependent upon association of E1 with PE2. However, E1 association with PE2 is required for oligomer formation, the export of E1 from the endoplasmic reticulum, and E1 acquisition of resistance to DTT.

Dithiothreitol↗
PubMed1997

Superinfection exclusion of alphaviruses in three mosquito cell lines persistently infected with Sindbis virus.

Three Aedes albopictus (mosquito) cell lines persistently infected with Sindbis virus excluded the replication of both homologous (various strains of Sindbis) and heterologous (Aura, Semliki Forest, and Ross River) alphaviruses. In contrast, an unrelated flavivirus, yellow fever virus, replicated equally well in uninfected and persistently infected cells of each line. Sindbis virus and Semliki Forest virus are among the most distantly related alphaviruses, and our results thus indicate that mosquito cells persistently infected with Sindbis virus are broadly able to exclude other alphaviruses but that exclusion is restricted to members of the alphavirus genus. Superinfection exclusion occurred to the same extent in three biologically distinct cell clones, indicating that the expression of superinfection exclusion is conserved among A. albopictus cell types. Superinfection of persistently infected C7-10 cells, which show a severe cytopathic effect during primary Sindbis virus infection, by homologous virus does not produce cytopathology, consistent with the idea that cytopathology requires significant levels of viral replication. A possible model for the molecular basis of superinfection exclusion, which suggests a central role for the alphavirus trans-acting protease that processes the nonstructural proteins, is discussed in light of these results.

Aedes↗