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Biomedical subjects

R G Roberts

Publications and source records attributed to R G Roberts.

At least 91 records · Page 5Linked to original sources
PubMed1992

Point mutations in the dystrophin gene.

Defining the range of mutations in genes that cause human disease is essential to determine the mechanisms of genetic variation and the function of gene domains and to perform precise carrier and prenatal diagnosis. The mutations in one-third of Duchenne muscular dystrophy patients remain unknown as they do not involve gross rearrangements of the dystrophin gene. The size and complexity of the gene have prohibited the systematic definition of point mutations. We have developed a method for the identification of these mutations by nested amplification, chemical mismatch detection, and sequencing of reverse transcripts of trace amounts of dystrophin mRNA from peripheral blood lymphocytes. Analysis of the entire coding region (11 kilobases) in seven patients has resulted in detection of a sequence change in each case that is clearly sufficient to cause the disease. All mutations should cause premature translational termination, and the resulting phenotypes are thus equivalent to those caused by frameshifting deletions. The results support a particular functional importance for the C-terminal region of dystrophin. Application of this approach to mutation detection will extend direct carrier and prenatal diagnosis to virtually every affected family.

Adolescent↗
PubMed1992

Effects of capsaicin on cutaneous vasodilator responses in humans.

The neurovascular responses to noxious electrically evoked axon reflex (EAR) stimulation and iontophoretic application of endothelium-dependent [acetylcholine (ACh)] and independent [sodium nitrite (NaNO2)] vasodilator substances were examined in human forearm skin using laser Doppler flowmetry, before and after repeated topical applications of the neurotoxin capsaicin (chronic capsaicin pre-treatment). Following 3 or 4 days of capsaicin pre-treatment the normal vasodilator response to acute application of capsaicin was significantly reduced as was the EAR response. There were, however, no significant changes in the vasodilator responses to either ACh or NaNO2, suggesting that the neurogenic axon reflex is in series with the endothelial and microvascular smooth muscle mechanisms of vasodilatation. Recovery of normal EAR occurred within 2-4 weeks of cessation of capsaicin pre-treatment.

Adult↗
PubMed1992

Determination of the exon structure of the distal portion of the dystrophin gene by vectorette PCR.

The structure of the 3' one-third of the dystrophin gene has not previously been established. We have used vectorette PCR on a yeast artificial chromosome containing part of the human dystrophin gene to determine that there are 20 exons in this region and to characterize adjacent intron sequences of each one. Combined with previous information on the remainder of the gene, this study shows that the coding sequence is distributed between 79 exons. We have used PCR between exons to measure the distances that separate the more closely clustered exons. Vectorette PCR products were used as probes on Southern blots to assign all the 3' exons to genomic HindIII fragments that are commonly detected in the analysis of dystrophin gene deletions. The results will be useful for determining the effect of genomic deletions on the translational reading frame, for setting up genomic PCR assays to confirm point mutations, for analyzing splice site mutations, and for investigating potential cis-acting elements involved in tissue-specific alternative splicing. Vectorette PCR using primers derived from cDNA sequence represents an efficient and widely applicable method for establishing gene structure and obtaining intron sequence flanking exons, starting from a genomic clone and a cDNA sequence.

Base Sequence↗
PubMed1992

Construction of a 2.6-Mb contig in yeast artificial chromosomes spanning the human dystrophin gene using an STS-based approach.

A sequence tagged site (STS)-based approach has been used to construct a 2.6-Mb contig in yeast artificial chromosomes (YACs) spanning the human dystrophin gene. Twenty-seven STSs were used to identify and overlap 34 YAC clones. A DNA fingerprint of each clone produced by direct Alu-PCR amplification of YAC colonies and the isolation of YAC insert ends by vectorette PCR were used to detect overlaps in intron 1 (280 kb) where no DNA sequence data were available, thereby achieving closure of the map. This study has evaluated methods for mapping large regions of the X chromosome and provides a valuable resource of the dystrophin gene in cloned form for detailed analysis of gene structure and function in the future.

Base Sequence↗
PubMed1992

Identification of geosmin as a volatile metabolite of Penicillium expansum.

Cultures of Penicillium expansum produce a musty, earthy odor. Geosmin [1,10-trans-dimethyl-trans(9)-decalol] was identified by gas chromatography-mass spectrometry from headspace samples of P. expansum cultures. Olfactory comparison of P. expansum cultures with a geosmin standard indicated geosmin is the primary component of the odor associated with P. expansum.

Naphthols↗
PubMed1992

Investigation of a female manifesting Becker muscular dystrophy.

Females manifesting Becker muscular dystrophy (BMD) are even more rarely observed than for the allelic condition Duchenne muscular dystrophy. The male proband has typical BMD with greatly raised CK activity and a myopathic muscle biopsy. His mother experienced walking difficulties from 35 years of age and has a myopathy with marked calf hypertrophy, a raised CK, and a myopathic muscle biopsy. Dystrophin analysis was undertaken on both the proband and his mother. Immunoblotting showed a protein of normal size but of reduced abundance in both. Immunocytochemical analysis in the proband indicated that the majority of the fibres showed weak dystrophin labelling and in his mother both dystrophin positive and dystrophin negative fibres were present. Non-random X inactivation at locus DXS255, was observed in DNA isolated from peripheral lymphocytes of the mother. Neither extended multiplex PCR performed on DNA from the proband nor analysis of lymphocyte derived mRNA showed a structural alteration in the dystrophin gene suggesting that an unusual mutation was responsible for BMD in this family.

Adult↗
PubMed1992

Confounding factors in non-invasive tests of neurovascular function in diabetes mellitus.

Disturbances of neurovascular function in the extremities may occur in patients with diabetes mellitus, exposure to toxic substances and chronic exposure to vibrating hand tools, as well as in Raynaud's phenomena. In these conditions symptoms of paraesthesia, finger numbness and blanching occur, so nerve conduction studies, vibration and temperature threshold measurements and neurovascular function tests are used for objective assessment of neurological dysfunction. The aim of the present study was to examine some factors which may confound quantitative neuro-vascular function measurements if used to assess neuropathy in diabetics. All subjects were consenting volunteers without exposure to known neurotoxic chemicals. The 5 groups were (a) healthy non-diabetic subjects not exposed to vibration (n = 10, mean age 52.3 yrs) (b) 2 insulin dependent and 8 non-insulin dependent diabetic subjects with a mean of 6 years treatment (n = 10, mean age 55.7 yrs) (c) maintenance employees exposed to high frequency pneumatic hand tools (n = 10, mean age 52.2 yrs) (d) subjects who were not diabetic or exposed to vibrating tools, but were being treated with the ACE-inhibitor enalapril 20 mg daily for hypertension (n = 5, mean age 54 yrs) (e) subjects who had smoked more than 10 cigarettes daily for at least 15 yrs (n = 10, mean age 51 yrs). Neurovascular tests included axon reflex responses measured by laser Doppler velocimeter evoked on the dorsum of the finger by iontophoresis of acetylcholine 16 mC in a circumferential chamber: cutaneous microvascular dilator responses to endothelial stimulation by iontophoretic application of the muscarinic agonist pilocarpine 16 mC and to direct nitrodilator sodium nitroprusside 16 mC. The skin temperature of the digits was held between 33 degrees and 34 degrees C during testing and dilator responses were measured as flux change by on-line computer analysis using 'Perisoft'. There was a significant reduction (P < 0.05) in the neurovascular responses of both diabetics and vibration--exposed subjects to acetylcholine and, in the case of vibration-exposed subjects, to pilocarpine, but nitroprusside responses were not significantly different. Our findings of reductions in neurovascular responses in diabetics and in subjects exposed to higher frequency vibration is consistent with recent epidemiological findings. Furthermore, subjects treated with an ACE-inhibitor (enalapril) showed significant reduction in acetylcholine-evoked axon reflex responses, while the test group of smokers showed a significant reduction in their dilator response to pilocarpine.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1991

Noxious heat hyperalgesia test instrument.

A simple feedback thermode has been developed to quantify cutaneous hyperalgesia to noxious heat. A noxious temperature set between 42 degrees C and 60 degrees C is applied to a test site by the subject. A resettable impedance timing circuit measures the contact duration to +/- 0.01s. Despite site-to-site, inter- and intra-subject variability, the test sensitivity is enhanced by comparing affected and corresponding normal skin in the same subject. This test strategy was used in 55 subjects in whom topical 0.025 to 0.05% capsaicin application provoked hyperalgesia on the forearm and foot. Capsaicin-treated sites showed a significant reduction of 52 degrees C withdrawal latency (0.51-0.77 x normal mean; P less than 0.05). The device has been tested clinically in patients with diabetic neuropathy who had hypersensitivity to pinprick on one foot. A significant 42% difference was found between corresponding contralateral sites (P less than 0.05). These data suggest that even mild hyperalgesia is detectable using this instrument. It provides an objective measure of hyperalgesia which can be used to assess changes in nociceptive acuity.

Capsaicin↗
PubMed1991

Direct detection of dystrophin gene rearrangements by analysis of dystrophin mRNA in peripheral blood lymphocytes.

Using 10 overlapping nested sets of primers and using peripheral blood lymphocyte (PBL) total RNA as template, we have developed a system, based on PCR, which allows the rapid production of double-stranded cDNA corresponding to the entire coding sequence of the dystrophin gene. The product can be visualized on native minigels by ethidium staining and directly sequenced after gel purification. We have used this system to analyze the structures of PBL dystrophin mRNA in 26 Duchenne, Becker, or intermediate muscular dystrophy patients who have gross rearrangements of the dystrophin gene. In each case, the effect that the genomic rearrangement has on the structure of the transcript--and, by inference, on the dystrophin protein--has been determined, and the results confirm the frameshift hypothesis. The study also identifies a series of alternatively spliced transcripts which are specific to the rearranged genotypes and which seem therefore to arise following the alteration in the context of the splice signal. The system has been used for unambiguous identification of carrier females. Furthermore, the rapid production of microgram quantities of dystrophin cDNA from a readily accessible tissue makes point-mutation screening a practical proposition.

Base Sequence↗
PubMed1991

Development and operation of the Wisconsin Research Network.

In 1987, the Wisconsin Academy of Family Physicians developed the Wisconsin Research Network (WReN) to support practice-based primary care research throughout Wisconsin. WReN has three objectives: to support the research efforts of individual physicians in community practices, to facilitate collaborative research among practicing physicians, and to provide academically based investigators with access to community practice sites. Due to a policy of actively encouraging membership, WReN has grown to 460 members during its 4-year history. Five WReN-supported papers have been published, and 22 state and national level presentations of WReN-supported research results have been made. Competitive grants totaling more than $2 million have been awarded to university-based investigators for studies using the resources of WReN. This paper describes the development, organization, and success of WReN as well as the challenges which must be addressed.

Family Practice↗
PubMed1990

Arterial segmental vasoconstriction in hypercholesterolaemic patients.

Regular, wave-like constriction in medium-sized arteries, arterial segmental vasoconstriction (ASV), has been observed at arteriography and described by many authors. We found ASV in arteriograms of the superficial femoral artery in 13 of 107 hypercholesterolaemic patients, enrolled in the Probucol Quantitative Regression Swedish Trial (PQRST). The arteriograms were digitized and studied with a quantitative computer-assisted technique. The frequency of ASV was higher than has been reported earlier in clinical materials, possibly because of an increased vasoreactivity in hypercholesterolaemia, as recently observed experimentally. The ASV patients were, on average, younger, had lower blood pressure and less atherosclerosis, than the non-ASV patients. ASV was not found in any of the 19 patients in the material who either had symptoms of peripheral vascular disease or arteriographically verified arterial occlusions. No significant correlations with smoking habits or serum cholesterol levels were found. A computer-based index of ASV and measurement of ASV wavelength are discussed.

Adult↗
PubMed1989

Detection of novel genetic markers by mismatch analysis.

Chemical mismatch detection has been used to identify previously unknown genomic sequence variations that represent a new source of markers for genetic analysis. The approach detects all types of sequence changes, and therefore overcomes the limitation of restriction analysis, which identifies only a small fraction of the available sequence variations. Three new markers identified at the 3' end of the human dystrophin gene result from variable numbers of exact tandem repeats of 4bp (two examples) or 5bp (one example). None of these would have been detected as restriction fragment length polymorphisms by established procedures.

Base Sequence↗