Biomedical subjects
Publications and source records attributed to D T Brown.
In most eukaryotes, histones, which are the major structural components of chromatin, are expressed as a family of sequence variants encoded by multiple genes. Because different histone variants can contribute to a distinct or unique nucleosomal architecture, this heterogeneity can be exploited to regulate a wide range of nuclear functions, and evidence is accumulating that histone variants do indeed have distinct functions.
STATEMENT OF PROBLEM: Two varieties of unalloyed titanium, Ti-6Al-4V and NiTi, commonly are used in medical and dental fields. Several other types of alloys for potential use in these fields have been developed, including Ti-4.5Al-3V-2Mo-2Fe and vanadium-free alloys (Ti-5Al-2.5Fe and Ti-5Al-3Mo-4Zr). The corrosion of these alloys under simulated physiologic conditions is not known. Purpose. This study compared the corrosion behaviors of 6 titanium materials through electrochemical polarization tests in 37 degrees C Ringer's solution. MATERIAL AND METHODS: The applied voltage was potentiostatically scanned from -0.6 to 1.0 V. From polarization curves, the corrosion rate (averaged over 3 samples) for each alloy was calculated and compared with that of other alloys. Analysis of variance (ANOVA) and the Student-Newman-Keuls multiple range test were performed at a 95% overall confidence level to identify statistically significance differences in corrosion rates. Surface oxide films were identified by electron diffraction, and the electrolyte medium was analyzed by atomic absorption spectrophotometry after each alloy was tested. RESULTS: Commercially pure titanium and Ti-5Al-2.5Fe were the most resistant to corrosion; Ti-5Al-3Mo-4Zr, Ti-6Al-4V, and NiTi were the least resistant to corrosion. NiTi exhibited pitting corrosion along with transpassivation. CONCLUSION: Electron diffraction patterns indicated that all titanium alloys were covered mainly with rutile-type oxide (TiO(2)) after corrosion tests. The oxides that formed on Ti-5Al-2.5Fe were identified as a mixture of TiO(2) and Ti(9)O(17), and those that formed on NiTi were identified as a mixture of TiO(2) and Ni(2)Ti(4)O.
Leber's hereditary optic neuropathy (LHON) is a common cause of bilateral optic nerve disease. The majority of LHON patients harbour one of three point mutations of the mitochondrial DNA (mtDNA) complex I, or NADH:ubiquinone oxidoreductase (ND) genes (G11778A in ND4, G3460A in ND1, T14484C in ND6). As a consequence, screening for these mutations has become part of the routine clinical investigation of young adults who present with bilateral optic neuropathy, and the absence of these mutations is interpreted as indicating there is a low likelihood that an optic neuropathy is LHON. However, there are many individuals who develop the clinical features of LHON but who do not harbour one of these primary LHON mutations. We describe two LHON pedigrees that harbour the same novel point mutation within the mtDNA ND6 gene (A14495G). This mutation was heteroplasmic in both families, and sequencing of the mitochondrial genome confirmed that the mutation arose on two independent occasions. This is the seventh mutation in the ND6 gene that causes optic neuropathy, indicating that this gene is a hot spot for LHON mutations. Protein modelling studies indicate that all of these pathogenic mutations lie within close proximity to one another in a hydrophobic cleft or pocket. This is the first evidence for a relationship between a specific disease phenotype and a specific structural domain within a mitochondrial respiratory chain subunit. These findings suggest that the mtDNA ND6 gene should be sequenced in all patients with LHON who do not harbour one of the three common LHON mutations.
It is widely held that the penetration of cells by alphaviruses is dependent on exposure to the acid environment of an endosome. The alphavirus Sindbis virus replicates in both vertebrate and invertebrate cell cultures. We have found that exposure to an acid environment may not be required for infection of cells of the insect host. In this work, we investigated the effects of two agents (NH(4)Cl and chloroquine), which raise the pH of intracellular compartments (lysosomotropic weak bases) on the infection and replication of Sindbis virus in cells of the insect host Aedes albopictus. The results show that both of these agents increase the pH of endosomes, as indicated by protection against diphtheria toxin intoxication. NH(4)Cl blocked the production of infectious virus and blocked virus RNA synthesis when added prior to infection. Chloroquine, in contrast to its effect on vertebrate cells, had no inhibitory effect on infectious virus production in mosquito cells even when added prior to infection. Treatment with NH(4)Cl did not prevent the penetration of virus RNA into the cell cytoplasm or translation of the RNA to produce a precursor to virus nonstructural proteins. These data suggest that while these two drugs raise the pH of endosomes, they do not block insect cell penetration. These data support previous results published by our laboratory suggesting that exposure to an acid environment within the cell may not be an obligatory step in the process of infection of cells by alphaviruses.
We measured the proportion of mutant mtDNA (mutation load) in 82 primary oocytes from a woman who harbored the A3243G mtDNA mutation. The frequency distribution of mutation load indicates that random drift is the principal mechanism that determines the level of mutant mtDNA within individual oocytes.
Recent observations in cultured human fibroblasts suggest that the accumulation of point mutations in the noncoding control region of mtDNA may be important in human aging. We studied the mtDNA control region in brain tissue from 31 normal elderly individuals, from 35 individuals who had Alzheimer disease, and from 47 individuals who had dementia with Lewy bodies. We found no evidence that these somatic mtDNA point mutations accumulate either in the brains of normal elderly individuals or in the brains of individuals with neurodegenerative disease.
The linker histone H1 is believed to be involved in chromatin organization by stabilizing higher-order chromatin structure. Histone H1 is generally viewed as a repressor of transcription as it prevents the access of transcription factors and chromatin remodelling complexes to DNA. Determining the binding properties of histone H1 to chromatin in vivo is central to understanding how it exerts these functions. We have used photobleaching techniques to measure the dynamic binding of histone H1-GFP to unperturbed chromatin in living cells. Here we show that almost the entire population of H1-GFP is bound to chromatin at any one time; however, H1-GFP is exchanged continuously between chromatin regions. The residence time of H1-GFP on chromatin between exchange events is several minutes in both euchromatin and heterochromatin. In addition to the mobile fraction, we detected a kinetically distinct, less mobile fraction. After hyperacetylation of core histones, the residence time of H1-GFP is reduced, suggesting a higher rate of exchange upon chromatin remodelling. These results support a model in which linker histones bind dynamically to chromatin in a stop-and-go mode.
We investigated the relationship between linker histone stoichiometry and the acetylation of core histones in vivo. Exponentially growing cell lines induced to overproduce either of two H1 variants, H1(0) or H1c, displayed significantly reduced rates of incorporation of [(3)H]acetate into all four core histones. Pulse-chase experiments indicated that the rates of histone deacetylation were similar in all cell lines. These effects were also observed in nuclei isolated from these cells upon labeling with [(3)H]acetyl-CoA. Nuclear extracts prepared from control and H1-overexpressing cell lines displayed similar levels of histone acetylation activity on chromatin templates prepared from control cells. In contrast, extracts prepared from control cells were significantly less active on chromatin templates prepared from H1-overexpressing cells than on templates prepared from control cells. Reduced levels of acetylation in H1-overproducing cell lines do not appear to depend on higher order chromatin structure, because it persists even after digestion of the chromatin with micrococcal nuclease. The results suggest that alterations in chromatin structure, resulting from changes in linker histone stoichiometry may modulate the levels or rates of core histone acetylation in vivo.
The diagnosis and treatment of temporomandibular disorders (TMD) has been surrounded by considerable controversy. Clinical research in the field to date has been inconsistent and methodologically limited and has not firmly established the role of treatment interventions. This large scale, multisite study attempts to answer some of the questions regarding TMD diagnosis and treatment. More than 6,500 patients are currently being analyzed and 1,212 have completed TMD therapy, providing the basis for comparison with 198 untreated patients. A wide variety of treatments have been administered from 60 practice sites, whose practitioners were carefully calibrated to maximize inter-rater reliability. A validated symptom measurement system, the TMJ Scale, was employed to assure uniform assessment of treatment outcomes. Treated patients reported statistically and clinically significant symptom improvement, while untreated patients reported unchanged symptom levels. Data presented here do not support the hypothesis that TMD patients improve spontaneously without treatment.
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.
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.
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.
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.
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.
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.
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.
This case study discusses the advantage of using both lateral and protrusive mandibular excursive movements to improve the accuracy of the joint vibration analysis rather than relying only on opening and closing movements of the mandible for analysis.
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.