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Biomedical subjects

R W Taylor

Publications and source records attributed to R W Taylor.

At least 19 recordsLinked to original sources
PubMed2001

Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease.

The selective manipulation of mitochondrial DNA (mtDNA) replication and expression within mammalian cells has proven difficult. One promising approach is to use peptide nucleic acid (PNA) oligomers, nucleic acid analogues that bind selectively to complementary DNA or RNA sequences inhibiting replication and translation. However, the potential of PNAs is restricted by the difficulties of delivering them to mitochondria within cells. To overcome this problem we conjugated a PNA 11mer to a lipophilic phosphonium cation. Such cations are taken up by mitochondria through the lipid bilayer driven by the membrane potential across the inner membrane. As anticipated, phosphonium-PNA (ph-PNA) conjugates of 3.4-4 kDa were imported into both isolated mitochondria and mitochondria within human cells in culture. This was confirmed by using an ion-selective electrode to measure uptake of the ph-PNA conjugates; by cell fractionation in conjunction with immunoblotting; by confocal microscopy; by immunogold-electron microscopy; and by crosslinking ph-PNA conjugates to mitochondrial matrix proteins. In all cases dissipating the mitochondrial membrane potential with an uncoupler prevented ph-PNA uptake. The ph-PNA conjugate selectively inhibited the in vitro replication of DNA containing the A8344G point mutation that causes the human mtDNA disease 'myoclonic epilepsy and ragged red fibres' (MERRF) but not the wild-type sequence that differs at a single nucleotide position. Therefore these modified PNA oligomers retain their selective binding to DNA and the lipophilic cation delivers them to mitochondria within cells. When MERRF cells were incubated with the ph-PNA conjugate the ratio of MERRF to wild-type mtDNA was unaffected, even though the ph-PNA content of the mitochondria was sufficient to inhibit MERRF mtDNA replication in a cell-free system. This unexpected finding suggests that nucleic acid derivatives cannot bind their complementary sequences during mtDNA replication. In summary, we have developed a new strategy for targeting PNA oligomers to mitochondria and used it to determine the effects of PNA on mutated mtDNA replication in cells. This work presents new approaches for the manipulation of mtDNA replication and expression, and will assist in the development of therapies for mtDNA diseases.

Binding Sites↗
PubMed2000

Mitochondrial DNA haplogroups and susceptibility to AD and dementia with Lewy bodies.

The authors analyzed the relationship between nuclear genetic risk factors (apolipoprotein E genotype) and mitochondrial DNA (mtDNA) sequence variants in pathologically proved cases of AD (n = 185), dementia with Lewy bodies (DLB; n = 84), and control subjects (n = 179). Specific European mtDNA haplogroups and the A4336G mutation were not associated with an increased risk of AD. mtDNA haplogroup H was overrepresented in the DLB patients when compared with control subjects. Additional studies are needed to clarify the significance of the association.

Aged↗
PubMed2000

Ionomycin, a carboxylic acid ionophore, transports Pb(2+) with high selectivity.

Studies utilizing phospholipid vesicle loaded with chelator/indicators for polyvalent cations show that ionomycin transports divalent cations with the selectivity sequence Pb(2+) > Cd(2+) > Zn(2+) > Mn(2+) > Ca(2+) > Cu(2+) > Co(2+) > Ni(2+) > Sr(2+). The selectivity of this ionophore for Pb(2+) is in contrast to that observed for A23178 and 4-BrA23187, which transport Pb(2+) at efficiencies that are intermediate between those of other cations. When the selectivity difference of ionomycin for Pb(2+) versus Ca(2+) was calculated from relative rates of transport, with either cation present individually and all other conditions held constant, a value of approximately 450 was obtained. This rose to approximately 3200 when both cations were present and transported simultaneously. 1 microM Pb(2+) inhibited the transport of 1 mM Ca(2+) by approximately 50%, whereas the rate of Pb(2+) transport approached a maximum at a concentration of 10 microM Pb(2+) when 1 mM Ca(2+) was also present. Plots of log rate versus log ionomycin or log Pb(2+) concentration indicated that the transporting species is of 1:1 stoichiometry, ionophore to Pb(2+), but that complexes containing an additional Pb(2+) may occur. The species transporting Pb(2+) may include H.IPb.OH, wherein ionomycin is ionized once and the presence of OH(-) maintains charge neutrality. Ionomycin retained a high efficiency for Pb(2+) transport in A20 B lymphoma cells loaded with Indo-1. Both Pb(2+) entry and efflux were observed. Ionomycin should be considered primarily as an ionophore for Pb(2+), rather than Ca(2+), of possible value for the investigation and treatment of Pb(2+) intoxication.

Aminoquinolines↗
PubMed2000

Overweight and obese children have low bone mass and area for their weight.

OBJECTIVES: To determine whether girls and boys categorized from body mass index (BMI) values as overweight or obese for their age have lower bone mineral content (BMC) or lower bone area in relation to total body weight than children of normal adiposity. DESIGN: Cross-sectional study in a university bone research unit. SUBJECTS: Two hundred girls and 136 boys aged 3-19 y recruited from the general population by advertisement. MEASUREMENTS: Total body BMC (g) and bone area (cm2) measured by dual energy X-ray absorptiometry (DXA) in relation to body weight (kg), lean tissue mass (kg) and fat mass (kg) in boys and girls of three different BMI percentile groupings: normal weight (BMI<85th percentile); overweight (85 to 94th BMI percentile); obese (> or =95th BMI percentile). RESULTS: Obese children had higher BMC, bone area, and fat mass for chronological age than those of normal body weight (P<0.001). In spite of this the observed values for age-adjusted total body BMC and bone area relative to body weight were each lower than predicted values, in both overweight and obese children (2.5-10.1% less, P<0.05) than in children of lower adiposity. CONCLUSION: In overweight and obese boys and girls there is a mismatch between body weight and bone development during growth: their bone mass and bone area are low for their body weight.

Adipose Tissue↗
PubMed2000

The quarternary molecular architecture of TetA, a secondary tetracycline transporter from Escherichia coli.

TetA, a tetracycline cation/proton antiporter, was expressed in Escherichia coli with a C-terminal tag of six histidines, solubilized in dodecyl maltoside and purified in a single step using Ni2+ affinity chromatography. Two-dimensional crystals were obtained after reconstitution of purified protein with lipids. Electron microscopy of negatively stained crystals revealed a trigonal symmetry, from which we infer that this secondary transporter has a trimeric structure. An overall molecular envelope can be described by a triangle of side approximately 100 A enclosing a central stain-filled depression. These dimensions are consistent with those obtained from projection views of single, isolated TetA particles that also display a trimeric architecture, confirming that the threefold symmetry is not simply a consequence of crystal-packing interactions. These data represent the first direct view of the quarternary arrangement of any antibiotic efflux pump. They are fully consistent with biochemical data on TetA, which indicate that it functions as a multimer and that the monomer consists of two domains, one of which plays the major part in oligomerization interactions.

Antiporters↗
PubMed2000

The perception of optimal profile in African Americans versus white Americans as assessed by orthodontists and the lay public.

This study was designed to assess the perceived optimal profiles of African Americans versus white Americans. A survey was conducted using profile silhouettes of 30 African American and 30 white patients, ranging in age from 7 to 17 years. Twenty white orthodontists, 18 African American orthodontists, 20 white laypersons, and 20 African American laypersons evaluated the profiles. The preference of each rater for each of the 60 profiles was scored on an attached visual analog scale. Eighteen cephalometric variables were measured for each profile, and statistical analyses were performed on the profiles that had a mean rating of 60 or greater from an analog scale of 0 to 100. The results show the following 6 cephalometric variables were significant: Z-angle, skeletal convexity at A-point, upper lip prominence, lower lip prominence, nasomental angle, and mentolabial sulcus. All raters preferred the African American sample to have a greater profile convexity than they preferred for the white sample. The raters preferred the African American sample with upper and lower lips that were more prominent compared with the white sample. However, only the choice of the African American orthodontists for the African American sample was significantly different for this parameter. The white orthodontists gave the highest mean scores for the profile chosen, whereas the African American laypersons gave the lowest scores.

Adolescent↗
PubMed2000

Evaluation of waist circumference, waist-to-hip ratio, and the conicity index as screening tools for high trunk fat mass, as measured by dual-energy X-ray absorptiometry, in children aged 3-19 y.

BACKGROUND: A central fat pattern has adverse health implications in both children and adults. Because adiposity tracks from childhood into adulthood, the ability of simple anthropometric techniques to correctly measure truncal adiposity in childhood needs to be assessed. OBJECTIVES: We sought to assess the validity of waist circumference, waist-to-hip ratio (WHR), and the conicity index as indicators of trunk fat mass in children and adolescents. DESIGN: Trunk fat mass (kg) was measured with dual-energy X-ray absorptiometry in 278 girls and 302 boys aged 3-19 y. Receiver operating characteristic (ROC) curves and areas under the curves (AUCs) for the ROCs were calculated to compare the relative abilities of the anthropometric measures to correctly identify children with high trunk fat mass (z score for our study population of > or =1). RESULTS: The 80th percentile for waist circumference correctly identified 89% of girls and 87% of boys with high trunk fat mass (sensitivity) and 94% of girls and 92% of boys with low trunk fat mass (specificity). Waist circumference performed significantly better as an index of trunk fat mass than did WHR or the conicity index, as shown by the AUCs in girls and boys, respectively: waist circumference AUCs = 0.97 and 0.97, conicity index AUCs = 0.80 and 0.81, and WHR AUCs = 0.73 and 0.71. Our cutoffs for high trunk fat mass and high waist circumference are provided for both sexes for each year of age. CONCLUSION: Waist circumference provides a simple yet effective measure of truncal adiposity in children and adolescents.

Abdomen↗
PubMed2000

In-vitro genetic modification of mitochondrial function.

Defects of mitochondrial (mt) DNA cause a diverse group of incurable, progressive diseases that often lead to severe disability and premature death. Most patients with pathogenic mtDNA defects have a mixture of mutant and wild-type mtDNA (heteroplasmy), and the clinical defect is only expressed when the percentage of mutant mtDNA exceeds a critical threshold. Since mtDNA is continually replicating and being turned over, we have proposed an approach to the treatment of these disorders that utilizes sequence-specific antigenomic peptide nucleic acids (PNAs) to hybridize and specifically inhibit the replication of mutant mtDNA under physiological conditions. By allowing the selective propagation of wild-type molecules, it may be possible to correct the cellular biochemical defect and to prevent the progression of disease. This paper summarizes the experimental progress in this area, including the cellular uptake of PNA molecules and their import into mitochondria both in vitro and in cell culture by the addition of a nuclear-encoded mitochondrial targeting sequence. The possibilities of extending this strategy to the treatment of mtDNA deletion disorders are discussed.

DNA Replication↗
PubMed2000

More broken bones: a 4-year double cohort study of young girls with and without distal forearm fractures.

Predictors of childhood fractures have not been investigated previously. This study was undertaken to determine whether a previous history of forearm fracture, low bone mineral density (BMD; both areal bone mineral density [aBMD, g/cm2] and volumetric bone mineral apparent density [BMAD, g/cm3]), or anthropometry, influence fracture risk in young girls. At baseline, two cohorts of girls, aged 3-15 years, were evaluated: 100 had recently broken a forearm (group 1) and 100 were fracture free (group 2). Four years later we restudied 170 of these girls (82 from group 1 and 88 from group 2). We now report the relationships of previous fracture history, baseline BMD (measured by dual-energy X-ray absorptiometry), baseline weight, and height to risk of new fracture. More new fractures occurred in group 1 (37 fractures in 24 girls) than in group 2 (8 fractures in 7 girls; p = 0.0007). The independent predictors for occurrence of a new fracture at any skeletal site in a multivariate model adjusting for age, weight, total body aBMD, and fracture history were previous fracture (hazard ratio [HR], 3.28; 95% CI, 1.41-7.64); age (HR per 1-year increase, 0.91; 95% CI, 0.84-0.99); total body aBMD (HR per 1 SD decrease, 1.92; 95% CI, 1.31-2.81); and body weight (HR per 1 SD increase, 1.49; 95% CI, 1.06-2.08). Girls with two risk factors together had substantially greater fracture risk: previous fracture and low spinal BMAD (HR, 9.4; 95% CI, 2.8-32.0), previous fracture and high body weight (HR, 10.2; 95% CI, 2.8-37.6), or previous fracture and low total body aBMD (HR, 13.0; 95% CI, 3.9-43.1). We conclude that previous forearm fracture, low total body aBMD, low spinal BMAD, and high body weight each increase risk of new fractures within 4 years in young girls. Interventions to reduce the risk of fractures, particularly forearm fractures, in girls warrant further study.

Absorptiometry, Photon↗
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↗
PubMed2000

Late-onset optic atrophy, ataxia, and myopathy associated with a mutation of a complex II gene.

Genetic defects affecting the mitochondrial respiratory chain are an important cause of neurological disease. Previously, we identified a family with complex II deficiency and late-onset neurodegenerative disease with progressive optic atrophy, ataxia, and myopathy. The affected family members are now shown to carry a C-to-T transition in one allele of the nuclear gene encoding the flavoprotein subunit of complex II. Mutation of the equivalent base in Escherichia coli generates an inactive enzyme unable to bind flavin adenine dinucleotide covalently. Compatible with these findings, our patients have an approximate 50% decrease in complex II and succinate dehydrogenase activity. These results suggest that genetic defects of nuclear-encoded subunits of the mitochondrial respiratory chain can result in late-onset neurodegenerative disease.

Age of Onset↗
PubMed1999

Nonrandom tissue distribution of mutant mtDNA.

Heteroplasmic mitochondrial DNA (mtDNA) defects are an important cause of inherited human disease. On a cellular level, the percentage of mutant mtDNA is the principal factor behind the expression of the genetic defect. Marked variation in the level of mutant mtDNA among tissues is thought to be responsible for the diverse clinical phenotypes associated with the same pathogenic mtDNA mutation. This study was designed to determine whether the percentage level of a pathogenic mtDNA molecule is determined by a purely random process. The tissue distribution of the A3243G MELAS point mutation was analyzed in five individuals who were members of a family with maternally inherited diabetes and deafness. The level of mutant mtDNA was measured in four tissues in three individuals and three tissues in two individuals. The highest level of mutant mtDNA occurred in skeletal muscle, followed by hair follicles, and then buccal mucosa, with the lowest levels in blood (leucocyte/platelet fraction). The probability of observing any strict hierarchy in family is 4.82 x 10(-5). These results indicate that the distribution of the A3243G mutation is not solely determined by random processes.

Adult↗
PubMed1999

Structural variation of type XII collagen at its carboxyl-terminal NC1 domain generated by tissue-specific alternative splicing.

This paper reports the identification of two structural variations in the NC1 domain of rat and mouse type XII collagen. The long NC1 domain encoding 74 amino acids showed homology to chicken type XII and XIV collagens. The short NC1 domain was composed of 19 amino acids. Through genomic DNA analyses, two alternative exons were identified, each of which contained the variable NC1 sequence. With the amino-terminal NC3 splicing alternatives, we propose here a new descriptive nomenclature: types XIIA-1 and XIIB-1 which include a long NC1 sequence encoded by exon 1 (from the 3'-end), and types XIIA-2 and XIIB-2 which include a short NC1 sequence encoded by exon 2. Types XIIA-1 and XIIB-1, the predominant transcripts in 15-day old mouse embryos, showed decreased expression in 17-day old embryos when type XIIB-2 expression was sustained at constant levels. In adult mice, type XIIB-1 associates with ligament and tendon, whereas type XIIB-2 is expressed in various other tissues. The long NC1 domain contains an extended acidic region (pI = 3.4) followed by a terminal basic region (pI = 13.8). Because the short NC1 domain lacks these features, structural variations in the type XII collagen NC1 domain suggests different functional roles in a tissue-specific fashion.

Alternative Splicing↗
PubMed1999

Peptide nucleic acid delivery to human mitochondria.

Peptide nucleic acids (PNAs) are synthetic polynucleobase molecules, which bind to DNA and RNA with high affinity and specificity. Although PNAs have enormous potential as anti-sense agents, the success of PNA-mediated gene therapy will require efficient cellular uptake and sub-cellular trafficking. At present these mechanisms are poorly understood. To address this, we have studied the uptake of biotinylated PNAs into cultured cell lines using fluorescence confocal microscopy. In human myoblasts, initial punctate staining was followed by the release of PNAs into the cytosol and subsequent localisation and concentration in the nucleus. To determine whether PNAs could also be used as therapeutic agents for mtDNA disease, we attempted to localise PNAs to the mitochondrial matrix. When attached to the presequence peptide of the nuclear-encoded human cytochrome c oxidase (COX) subunit VIII, the biotinylated PNA was successfully imported into isolated organelles in vitro. Furthermore, delivery of the biotinylated peptide-PNA to mitochondria in intact cells was confirmed by confocal microscopy. These studies demonstrate that biotinylated PNAs can be directed across cell membranes and to a specific sub-cellular compartment within human cells - highlighting the importance of these novel molecules for human gene therapy.

Animals↗
PubMed1999

An mtDNA mutation in the initiation codon of the cytochrome C oxidase subunit II gene results in lower levels of the protein and a mitochondrial encephalomyopathy.

A novel heteroplasmic 7587T-->C mutation in the mitochondrial genome which changes the initiation codon of the gene encoding cytochrome c oxidase subunit II (COX II), was found in a family with mitochondrial disease. This T-->C transition is predicted to change the initiating methionine to threonine. The mutation load was present at 67% in muscle from the index case and at 91% in muscle from the patient's clinically affected son. Muscle biopsy samples revealed isolated COX deficiency and mitochondrial proliferation. Single-muscle-fiber analysis revealed that the 7587C copy was at much higher load in COX-negative fibers than in COX-positive fibers. After microphotometric enzyme analysis, the mutation was shown to cause a decrease in COX activity when the mutant load was >55%-65%. In fibroblasts from one family member, which contained >95% mutated mtDNA, there was no detectable synthesis or any steady-state level of COX II. This new mutation constitutes a new mechanism by which mtDNA mutations can cause disease-defective initiation of translation.

Adult↗
PubMed1999

Lactose malabsorption and rate of bone loss in older women.

OBJECTIVES: to study the prevalence of lactose malabsorption with increasing age and to determine whether lactose malabsorbers consume less dietary calcium, have lower bone mineral density or display faster bone loss than lactose absorbers. DESIGN: 80 healthy Caucasian women aged 40-79 years (20 per decade) were studied for 1 year. METHODS: breath hydrogen exhalation was measured for 3 after a 50 g oral lactose challenge. Bone density was assessed in the radius, femoral neck, lumbar spine and total body by dual energy x-ray absorptiometry and dietary calcium intake was estimated by 4-day diet records and food-frequency questionnaires. RESULTS: lactose malabsorption rose with age (15% in those aged 40-59 years versus 50% in those aged 60-79; P < 0.01). Malabsorbers aged 70-79 years consumed significantly less calcium than lactose absorbers of this age (P < 0.05). Baseline total body calcium values were lower in lactose malabsorbers (n=26) than in lactose absorbers (n=54) but age-adjustment eliminated this difference. Bone change (% per year) was correlated with dietary calcium intake at the femoral neck and trochanter (P < 0.05) but was not statistically greater in malabsorbers than in absorbers. CONCLUSIONS: the ability to absorb lactose declines in the 7th decade. This may contribute to decreased dietary intakes of milk products and calcium in elderly women. However, lactose malabsorption without reduction in calcium intake has little effect on bone mineral density or the rate of bone loss.

Adult↗
PubMed1998

Diagnosis of mitochondrial disease: assessment of mitochondrial DNA heteroplasmy in blood.

Mitochondrial DNA (mtDNA) mutations are an important cause of neurological disease. The identification of causative mtDNA mutations may be particularly troublesome in blood where there are often low levels of mutant mtDNA. This is evident from a recent study in which heteroplasmic mtDNA mutations in cytochrome c oxidase genes were incorrectly thought to be linked to Alzheimer's disease. We wished to explore whether analysis of blood mtDNA, prepared by a number of DNA extraction procedures, influenced the diagnosis of mtDNA disease. DNA was extracted by different procedures from 4 patients with heteroplasmic mtDNA mutations, and the level of heteroplasmy investigated by radioactive PCR-RFLP analysis. Whilst there was no consistent decrease in the level of mtDNA heteroplasmy, we observed the coamplification of a novel mtDNA pseudogene from DNA samples extracted by a simple 'boiling' procedure using primers designed to screen for the tRNALeu(UUR) A3243G mutation. This pseudogene was readily amplified from DNA extracted from rho degrees (mtDNA-less) cells, confirming its nuclear location. We believe that mtDNA pseudogenes may therefore present significant difficulties in the accurate identification of pathogenic heteroplasmic mtDNA mutations in blood.

DNA, Mitochondrial↗