Breast cancer screening in women aged under 50.
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Adult↗
Biomedical subjects
S Fletcher
Publications and source records attributed to S Fletcher.
First myocardial infarction in patients of Indian subcontinent and European origin: comparison of risk factors, management, and long term outcome.
OBJECTIVE: To compare long term outcome after first myocardial infarction among British patients originating from the Indian subcontinent and from Europe. DESIGN: Matched pairs study. SETTING: Coronary care unit in central Leicester. SUBJECTS: 238 pairs of patients admitted during 1987-93 matched for age (within 2 years), sex, date of admission (within 3 months), type of infarction (Q/non-Q), and site of infarction. MAIN OUTCOME MEASURES: Incidence of angina, reinfarction, or death during follow up of 1-7 years. RESULTS: Patients of Indian subcontinent origin had a higher prevalence of diabetes (35% v 9% in patients of European origin, P < 0.001), lower prevalence of smoking (39% v 63%, P < 0.001), longer median delay from symptom onset to admission (5 hours v 3 hours, P < 0.01), and lower use of thrombolysis (50% v 66%, P < 0.001). During long term follow up (median 39 months), mortality was higher in patients of Indian subcontinent origin (unadjusted hazard ratio = 2.1, 95% confidence interval 1.3 to 3.4, P = 0.002). After adjustment for smoking, history of diabetes, and thrombolysis the estimated hazard ratio fell slightly to 2.0 (1.1 to 3.6, P = 0.02). Patients of Indian subcontinent origin had almost twice the incidence of angina (54% v 29%; P < 0.001) and almost three times the risk of reinfarction during follow up (34% v 12.5% at 3 years, P < 0.001). The unadjusted hazard ratio for reinfarction in patients of Indian subcontinent origin was 2.8 (1.8 to 4.4, P < 0.001). Adjustment for smoking, history of diabetes, and thrombolysis made little difference to the hazard ratio. Coronary angiography was performed with similar frequency in the two groups; triple vessel disease was the commonest finding in patients of Indian subcontinent origin and single vessel disease the commonest in Europeans (P < 0.001). CONCLUSIONS: Patients of Indian subcontinent origin are at substantially higher risk of mortality and of further coronary events than Europeans after first myocardial infarction. This is probably due to their higher prevalence of diffuse coronary atheroma. Their need for investigation with a view to coronary revascularisation is therefore greater. History of diabetes is an inadequate surrogate for ethnic origin as a prognostic indicator.
Coronary Angiography↗
Variation in the methylation profile and structure of Pax3 and Pax7 among different mouse strains and during expression.
Structural alterations within the myogenic and neurogenic developmental gene Pax7 which involve TaqI recognition sequences have previously been reported. These alterations are associated with differences in the efficiency of regrowth of damaged skeletal muscle. To identify other structural features of Pax genes which may influence skeletal muscle regrowth, variation in the structure and methylation status of Pax7 and the closely related gene Pax3 has been sought among different mouse strains and during gene expression using the restriction endonucleases MspI and HpaII. Following MspI digestion, RFLPs within Pax7 have been found which most likely reflect intron size variability within the paired box. Differences in the size of MspI and HpaII fragments hybridising with Pax7 and Pax3 region specific sub-probes indicate that the paired boxes are hypomethylated, whereas the region encoding the homeodomain of each gene is highly methylated in the spleen and other tissues from adult mice. In the skeletal muscle precursor cell line C2C12, which expresses Pax7 but not Pax3, the homeodomain encoding region of Pax7 is hypomethylated. In spleen cells, the Pax7 paired box is transcribed but the homeodomain encoding region is not. By contrast, both the paired box and the homeobox of Pax3 are hypermethylated in C2C12 cells indicating that generation of alternate transcripts from Pax genes may be controlled by DNA methylation. In contrast to Pax3, reference to the size of fragments hybridising with a Pax7 homeobox specific probe provides evidence for CpNpG methylation within and immediately downstream from the region encoding the homeodomain. Interestingly, CpNpG methylation remains when the Pax7 homeobox is expressed. Structural variation recognised by MspI digestion and differences in the methylation profile of Pax7 are not associated with the ability to regrow damaged skeletal muscle.
Animals↗
Use of the dog model for Duchenne muscular dystrophy in gene therapy trials.
Golden retriever muscular dystrophy (GRMD) is an excellent model for the study of the efficacy of gene therapy in dystrophin deficient myopathies for there are many similarities between affected dogs and Duchenne muscular dystrophy (DMD) in boys. GRMD is not caused by deletion mutation but results from a point mutation in the consensus splice acceptor in intron 6 of the canine dystrophin gene. As a result exon 7 is skipped during processing of the GRMD dystrophin messenger RNA. We have developed a rapid test which makes direct use of exon 7 specific genomic PCR products. We have undertaken preliminary experiments on gene therapy using the mini-gene and the full length gene alone and in combination with lipofectin and/or the bacterial beta-galactosidase reporter gene Lac Z. Following direct injection of the Lac Z plasmid, either alone or with lipofectin, about 50% of the sites showed expression when biopsied some 14 days later. The beta-galactosidase activity was present in muscle and granulation tissue but was never abundant. Pups injected intraperitoneally with Lac Z were found to have positive material in their mesenteric lymph nodes, liver and spleen. Those injected with Lac Z and lipofectin also had positive material in the diaphragm, intercostal muscles and abdominal muscles, but again only a small amount of positive material was present at any of the sites. In animals directly injected into the muscle with the dystrophin mini-gene, half had positive staining for dystrophin in biopsies taken 14 days later. Of the 6 sites in the muscles of animals given the mini-gene and lipofectin only one had fibres positive for dystrophin when examined 14 days later. Six pups were injected directly with full-length gene construct and when biopsies were taken 10 days later two of the animals had strongly stained peripheries to a small number of fibres.
Adenoviridae↗
Alternate Pax7 transcripts are expressed specifically in skeletal muscle, brain and other organs of adult mice.
Pax7 is associated with formation of skeletal muscle and the neural tube in developing embryos. Interestingly, in adult mice, rearrangements of Pax7 are associated with differences in the efficiency of skeletal muscle regrowth between mouse strains. The aim of this study was to investigate the possibility that Pax7 is expressed in skeletal muscle or other tissues from adult mice. Total RNA was isolated from adult mouse tissues and the polymerase chain reaction was performed on reverse transcribed mRNA using primers specific for regions that encode the paired and homeodomain of Pax7. At least four different Pax7 transcripts were found. A full-length transcript similar in sequence to that published previously was identified in skeletal muscle, brain and spleen cells of adult mice. Further putative full-length Pax7 transcripts, including one that contains a hexanucleotide insertion in the paired box and one in which approximately 10 bp have been deleted in the homeobox, were found to be expressed in skeletal muscle and brain of adult mice, respectively. A truncated Pax7 splice product comprising the paired box only was found to be expressed in most adult tissues except liver. Results of these studies demonstrate that there are alternate transcripts of Pax7, some of which are expressed exclusively in adult skeletal muscle and brain. It is possible that one of these transcripts may specify an alternate myogenic pathway involved in regeneration of damaged skeletal muscle in adult mice.
Alternative Splicing↗
Purification of 5-hydroxytryptamine3 receptors from porcine brain.
1. We demonstrate, for the first time, the purification of the 5-hydroxytryptamine3 (5-HT3) receptor from a native tissue source, pig cerebral cortex. 2. From a range of detergents, the non-ionic detergent Triton X-100 was demonstrated to exhibit the least inhibition of [3H]-(S)-zacopride binding to membrane bound 5-HT3 receptors from pig cerebral cortex at concentrations above its critical micellular concentration (CMC). This detergent was therefore selected to solubilize 5-HT3 binding sites from homogenates of pig cerebral cortex. Maximum yield (43.8 +/- 3.7%, mean +/- s.e.mean, n = 13) was obtained with Triton X-100 at 0.4% (22.1 x CMC). Radioligand binding studies with [3H]-(S)-zacopride indicated that the solubilized 5-HT3 receptor displayed near identical pharmacology to the membrane bound receptor (the correlation coefficient (r) between the pKi values of structurally unrelated compounds competing for [3H]-(S)-zacopride binding in the membrane bound and solubilized 5-HT3 receptor preparations was 0.99, Bmax = 20.7 +/- 4.2 fmol mg(-1) protein, Kd = 1.57 +/- 0.53 nM, mean +/- s.e.mean, n = 6). 3. Solubilized (0.4% Triton X-100) 5-HT3 receptors were affinity purified using Affi-Gel 15 coupled to the high affinity 5-HT3 receptor ligand GR119566X. Radioligand binding studies indicated that the pharmacological profile of the affinity purified 5-HT3 receptor, assessed using ligands with a range of affinities spanning 3 orders of magnitude, was similar to that in both crude homogenates (r = 0.85) and solubilized 5-HT3 receptor sites (r = 0.85) from pig brain. The specific activity for the purified 5-HT3 receptor overlapped the theoretical specific activity of the receptor (Bmax = 3.27 +/- 1.41 and 5.35 +/- 2.33 nmol mg(-1) protein, assessed by saturation and competition studies respectively, mean +/- s.e.mean, n = 3-4), which indicated a 60000-100000 fold purification of the membrane bound receptor. 4. Under non-reducing conditions, samples of the affinity purified protein failed to enter a 10% separating gel in SDS-PAGE analysis, indicating a molecular mass for the receptor complex of > 200 kDa. Further investigation of the non-reduced purified protein with a 7.5% separating gel gave a mass for the complex of approximately 279 kDa. Under reducing conditions, SDS-PAGE analysis of the affinity purified 5-HT3 receptor resulted in 3-6 silver stained bands at apparent molecular masses of 37, 44-50, 52, 57-61, 63 and 65-71 kDa (n = 12). Unlike protein bands at 45, 50, 60 and 66 kDa, the bands corresponding to proteins of 52, 57, 63 and 71 kDa consistently gave no reaction with an antiserum specific for the cloned A subunit of the 5-HT3 receptor in both a modified dot blot procedure and a Western blot procedure (n = 2-5). 5. We conclude that we have purified the 5-HT3 receptor from pig brain to homogeneity and suggest this may contain non-5-HT3-A receptor subunit(s).
Animals↗
Immunocytochemical detection of a reduction in 1,25 dihyroxyvitamin D3 receptor expression in uraemic parathyroid tissue.
BACKGROUND: The vitamin D receptor is expressed in nuclei of parathyroid cells and plays an important role in the regulation of cell growth and parathyroid hormone secretion. A number of studies have shown reduced receptor expression in uraemic parathyroid glands but the relationship between receptor expression and renal function has not been identified. METHODS: This study used archive parathyroid tissue from patients with primary adenomas, dialysis patients with diffuse hyperplasia secondary to uraemia and normal parathyroid tissue. An immunocytochemical assay with a vitamin D receptor antibody was used to identify parathyroid cells expressing receptor in their nuclei. RESULTS: In patients with a serum creatinine < or = 110 mumol/l, 81.94% +/- 6.5 of parathyroid cells expressed vitamin D receptor. This fell to a mean of 49.3% in two patients with a serum creatinine between 110 and 300 mumol/l and to 40.17% +/- 8.6 in dialysis patients. CONCLUSION: Vitamin D receptor expression in parathyroid glands is reduced in renal failure.
Adenoma↗
Acute small-bowel obstruction following mesenteric perforation by CAPD catheter.
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Aged↗
Assessment of renal osteodystrophy in dialysis patients: use of bone alkaline phosphatase, bone mineral density and parathyroid ultrasound in comparison with bone histology.
Bone biopsies were studied in 73 patients to determine if a two-site radioimmunometric assay for serum bone alkaline phosphatase (BAP), total serum alkaline phosphatase (ALP), serum intact parathyroid hormone (iPTH), hand X-rays, regional bone mineral density (BMD) measurements and parathyroid enlargement detected by ultrasonography could accurately predict renal osteodystrophy. In the patients studied 57 had hyperparathyroid bone disease, 4 mixed renal osteodystrophy, 3 adynamic bone disease, 1 osteomalacia and 8 normal histology. Serum BAP, ALP and iPTH correlated positively with mineral apposition rate, osteoblastic, osteoid and eroded surface. In the diagnosis of hyperparathyroid bone disease serum iPTH was the most sensitive investigation, detecting 81% of patients at a level > 100 pg/ml but with a specificity of only 66%. Serum BAP was more sensitive, 70% at a level of > 10 ng/ml, than serum total ALP, 30% at a level of 300 IU/l, with similar specificities, 92 and 100%, respectively. Ultrasound detection of an enlarged parathyroid gland had a sensitivity of 64% and a specificity of 100% for the diagnosis of hyperparathyroid bone disease. Hand X-rays had a poor sensitivity, 47%, but a high specificity, 92%, for the detection of hyperparathyroid bone disease. The majority of patients had regional BMD values within the normal reference range and this test was of poor discriminatory value. The non-invasive markers were unable to distinguish between patients with low turnover, mild hyperparathyroidism and patients with normal histology. In conclusion the measurement of serum iPTH is a useful screening tool for the detection of hyperparathyroid bone disease which can be confirmed by the finding of a raised serum BAP or parathyroid enlargement. For definitive diagnosis, however, the gold standard remains bone biopsy and at present one cannot recommend any non-invasive method as an adequate substitute.
Adult↗
Sudden deepening of pigmentation during haemodialysis due to severe haemolysis.
A 37-year-old woman with chronic renal failure developed pain in the right flank during haemodialysis followed by rapid and dramatic deepening of pigmentation. Investigation indicated that the cause was severe haemolysis most probably due to mechanical damage to red blood cells passing through a stenosis in a dialysis blood line. Severe haemolysis should be suspected in subjects who suffer rapidly increasing pigmentation during dialysis. Such severe haemolysis should be recognized and treated rapidly since it may produce life-threatening systemic problems.
Acute Disease↗
Distribution and characterisation of the [3H](S)-zacopride labelled 5-HT3 receptor in pig forebrain.
The present study demonstrates the presence and distribution of [3H](S)-zacopride labelled 5-HT3 (5-hydroxytryptamine3) receptors in pig forebrain. The pharmacological characterisation of 5-HT3 receptor recognition sites in homogenates of pig cerebral cortex provides further evidence for inter-species variation in the pharmacology of the 5-HT3 receptor.
Animals↗
Insertion/deletion polymorphism in the angiotensin-converting enzyme gene and risk of and prognosis after myocardial infarction.
OBJECTIVES: We sought to prospectively investigate whether genetic variation at the angiotensin-converting enzyme gene locus defined by an insertion (I)/deletion (D) polymorphism influences the risk of myocardial infarction or prognosis after infarction, or both. BACKGROUND: It has been suggested that the deletion allele of the angiotensin-converting enzyme gene, and specifically the DD genotype, may increase the risk of myocardial infarction, although previous studies have produced conflicting reports. No studies have yet examined the effect of I/D polymorphism on survival after infarction. METHODS: Angiotensin-converting enzyme genotypes in 684 patients with myocardial infarction recruited at the time of the acute event through coronary care units in two centers were compared with those of 537 control subjects recruited from the base populations. All patients were followed up to assess the impact of the angiotensin-converting enzyme genotype on prognosis. RESULTS: We found no difference (p = 0.89) in the genotype distribution between patients and control subjects (patients DD 31%, ID 47%, II 22%; control subjects DD 30%, ID 48%, II 22%). The odds ratio for myocardial infarction for DD compared with II/ID genotype adjusted for age, gender and center was 1.16 (95% confidence interval [CI] 0.82 to 1.65, p = 0.44). The study had 90% power to detect a 1.5-fold increase in risk of myocardial infarction associated with the DD genotype. For one center, data were available for other risk factors (hypertension, diabetes, angina, previous myocardial infarction, smoking, body mass index, total and high density lipoprotein cholesterol) in both patients and control subjects. In a stepwise logistic regression analysis the odds ratio for DD versus ID/II genotypes remained nonsignificant (1.44, 95% CI 0.84 to 2.46, p = 0.20) for these subjects. Over a median follow-up period of 15 months (range 3 to 22), 155 patients (22.7%) died. There was no difference in mortality between subjects with the DD genotype and those with ID/II genotypes. (21.8% vs. 23.1%, p = 0.25). Likewise, there was no difference in the distribution of survival times in the two groups (p = 0.62). The study had 70% power to detect a 1.5-fold increase in mortality during follow-up associated with the DD genotype. CONCLUSIONS: We conclude that in the groups studied, genetic variation at the angiotensin-converting enzyme gene locus defined by I/D polymorphism does not significantly influence either the risk of or the short- to medium-term prognosis after myocardial infarction.
Aged↗
Calcium phosphate transfection and cell-specific expression of heterologous genes in primary fetal rat hepatocytes.
In order to study transcriptional regulation of hepatic genes during development, a method for transfer of fusion genes to primary cultures of fetal hepatocytes was required. The aim of this study was to assess currently available transfection methods and optimize the best method for use with cultured fetal hepatocytes. The Rous sarcoma virus 5' long terminal repeat controlling transcription of the beta-galactosidase reporter gene (pRSV lac Z II) was used to assess electroporation, lipofection, DEAE-dextran and calcium phosphate transfection in cultured primary fetal hepatocytes. The success of transfection was determined by histochemical detection and quantitation of beta-galactosidase activity. Results showed that calcium phosphate transfection was optimal for fetal hepatocytes with respect to beta-galactosidase activity and cell survival. For maximum transfection of cells, 10 micrograms/ml DNA, HEPES buffered saline transfection buffer at pH 7.05 and a 24 hr expression period for the reporter gene were employed. Glycerol shock did not increase transfection efficiency significantly. The method was simplified by adding calcium chloride solution to DNA diluted in transfection buffer and the resulting co-precipitate added directly to the medium covering the cells. Transfection 24 hr after initial culture and a precipitate incubation time of 20 hr were optimal. The suitability of this method was confirmed with a liver-specific promoter controlling beta-galactosidase and chloramphenicol acetyltransferase expression. In conclusion this study shows that a modified calcium phosphate transfection method is most effective for transferring DNA to primary cultured fetal hepatocytes. It is concluded that this method is appropriate for use with fetal hepatocytes and will facilitate studies of gene regulation during liver development.
Animals↗
[3H]L-655,708, a novel ligand selective for the benzodiazepine site of GABAA receptors which contain the alpha 5 subunit.
A compound (L-655,708) has been identified which has at least 50-fold selectivity for the benzodiazepine site on GABAA receptors containing an alpha 5 subunit over those containing an alpha 1, alpha 2, alpha 3 or alpha 6 subunit in combination with beta 3 and gamma 2. The compound was radiolabelled with tritium and investigated as a novel radioligand which recognizes the benzodiazepine site of GABAA receptors which contain the alpha 5 subunit. [3H]L-655,708 labels one saturable and specific population of binding sites in rat hippocampus with a Kd of 2.4 +/- 0.7 nM and a Bmax of 256 +/- 42 fmol/mg protein. The pharmacology of the binding site labelled was consistent with that of receptors present in cells transfected with alpha 5, beta 2 and gamma 2 and with receptors immunoprecipitated from rat brain with an alpha 5-selective antiserum. It is concluded that [3H]L-655,708 is the first radioligand to date which is selective for any BZ2 subtype of the GABAA receptor and should provide a valuable tool for elucidating the structure and function of the alpha 5-containing GABAA receptor subtype.
Animals↗
Oliguria in a renal graft: the role of ureteric catheterization.
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Adult↗
Onboard acoustic recording from diving northern elephant seals.
This study was the first phase in a long-term investigation of the importance of low-frequency sound in the aquatic life of northern elephant seals, Mirounga angustirostris. By attaching acoustic recording packages to the backs of six translocated juveniles, the aim was to determine the predominant frequencies and sound levels impinging on them, and whether they actively vocalize underwater on their return to their rookery at Ano Nuevo, California, from deep water in Monterey Bay. All packages contained a Sony digital audio tape recorder encased in an aluminum housing with an external hydrophone. Flow noise was minimized by potting the hydrophone in resin to the housing and orienting it posteriorly. The diving pattern of four seals was recorded with a separate time-depth recorder or a time-depth-velocity recorder. Good acoustic records were obtained from three seals. Flow noise was positively correlated with swim speed, but not so high as to mask most low-frequency sounds in the environment. Dominant frequencies of noise impinging on the seals were in the range 20-200 Hz. Transient signals recorded from the seals included snapping shrimp, cetacean vocalizations. boat noise, small explosive charges, and seal swim strokes, but no seal vocalizations were detected. During quiet intervals at the surface between dives, the acoustic record was dominated by respiration and signals that appeared to be heartbeats. This study demonstrates the feasibility of recording sounds from instruments attached to free-ranging seals, and in doing so, studying their behavioral and physiological response to fluctuations in ambient sounds.
Acoustics↗
Minocycline-induced chronic interstitial nephritis?
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Acne Vulgaris↗
Mutation segregation and rapid carrier detection of X-linked muscular dystrophy in dogs.
OBJECTIVES: To use exon 7-specific genomic polymerase chain reaction (PCR) products to identify the genotypes of normal, affected, and carrier female dogs in pedigrees segregating Golden Retriever muscular dystrophy (GRMD), and to confirm the concordant segregation of the mutation in all carrier and affected dogs presently available. DESIGN: The GRMD mutation is found in the consensus splice acceptor site in intron 6 of the canine dystrophin gene. PCR cycle-sequencing and restriction fragment length polymorphism/PCR were used for determination of the pattern of segregation of the point mutation which causes GRMD. ANIMALS: Normal, clinically affected, and obligate carrier dogs in pedigrees of GRMD. PROCEDURE: DNA from blood was amplified, using PCR and primers that bracket all of exon 7 of the canine dystrophin gene as well as 100 base pairs of intron on either side. PCR products were either cycle-sequenced directly or submitted to a second round of PCR, using 1 of the original primers coupled with a mutagenic restriction fragment length polymorphism-primer, which thus creates an artificial restriction site. Digestion with Stu I detected the normal allele. To detect the affected allele, Sau96 I was used to digest the 310-base pair exon 7 genomic fragment directly. CONCLUSIONS: Simple, clear diagnosis of carrier status was possible using these methods. This mutation is passed through all carrier and affected dogs in both United States GRMD colonies and the colony in Australia. CLINICAL RELEVANCE: Rapid, accurate diagnosis of carrier and affected dogs will enhance study of this homologue of Duchenne muscular dystrophy.
Alleles↗