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S Fletcher

Publications and source records attributed to S Fletcher.

At least 55 records · Page 3Linked to original sources
PubMed1999

Development of a snapback method of single-strand conformation polymorphism analysis for genotyping Golden Retrievers for the X-linked muscular dystrophy allele.

OBJECTIVE: To develop a snapback method of single-strand conformation polymorphism (SSCP) analysis for genotyping Golden Retrievers for the X-linked muscular dystrophy allele. ANIMALS: 20 Golden Retriever puppies from a colony with X-linked muscular dystrophy. PROCEDURE: DNA spanning the canine dystrophin mutation was amplified by means of a polymerase chain reaction (PCR), using a primer modified to have an additional sequence at the 5' terminus. The primer was designed so that 1 terminus of the single-stranded PCR product could anneal to the normal sequence flanking the region of the mutation in the allele but not in the mutant allele. True disease status of the dogs was determined by means of a PCR and restriction digest protocol. RESULTS: Snapback SSCP analysis allowed for accurate and unambiguous genotyping of unaffected, carrier, and affected dogs, whereas conventional SSCP analysis, using the unmodified primer, did not. Creatine kinase activities measured within 24 hours after birth were not consistent with genotype. CONCLUSION AND CLINICAL RELEVANCE: Snapback SSCP analysis provided a simple, fast, and accurate method for genotyping Golden Retrievers for the mutation known to cause X-linked muscular dystrophy.

Animals↗
PubMed1999

Parallel syntheses of disulfide inhibitors of the thioredoxin redox system as potential antitumor agents.

We have reported previously that unsymmetrical disulfide inhibitors of the human thioredoxin/thioredoxin reductase redox system (hTrx/TR) possess antitumor activity. We have broadened the search for more potent inhibitors and evaluated a large range of mono- and bis-disulfide compounds, prepared using parallel syntheses. Reaction of isothioisourea-HCI salts (R') or bis-salts (R) with aromatic or aryl thiols (R") in wells of 96-well plates produced >450 derivatives with the structures R"SSR' and R"SSRSSR". The excellent yield and purity of the disulfides provided sufficient material for evaluations of enzyme inhibition and cytotoxicity. Selection criteria based on the IC50 values for hTrx/TR inhibition and for cytotoxicities of the disulfides identified agents for subsequent scale-up syntheses and in vivo evaluations of antitumor activity. These scale-up studies confirmed the original activities of agents synthesized in the plates and validated the parallel synthetic approach. Structure-activity information derived from the hTrx/TR IC50 data allow for a number of generalizations. The most potent inhibitors of the Trx system contained two heteroatoms ortho to the disulfide moiety in an aromatic functionality. The thioalkylating moieties had greatest activity with one branch point alpha to the disulfide. In the absence of branching, more potent inhibition was observed with the electron withdrawing functionalities. Bis-disulfides showed patterns of activity which depended on chain length, with optimum activity observed when the disulfide units were separated by 3.9 A, a similar distance to that separating the thioredoxin active site cysteine residues. From the agents selected for scale-up syntheses, three disulfide compounds were studied for their antitumor activity in vivo against human tumor xenografts in scid mice. One of the analogues discovered through the combinatorial syntheses/screening for Trx inhibition, 1-phenylethyl 2-imidazolyl disulfide, N1 (ProlX agent PX-C5), has demonstrated excellent in vivo activity against the MCF-7 human breast cancer and the HL-60 human leukemia, thus validating this approach for novel drug discovery.

Animals↗
PubMed1998

High-level dystrophin expression after adenovirus-mediated dystrophin minigene transfer to skeletal muscle of dystrophic dogs: prolongation of expression with immunosuppression.

Replication-deficient adenovirus vectors (AdV) have been successfully used to transfer a truncated human dystrophin cDNA to skeletal muscle of dystrophin-deficient mdx mice. A dystrophin-deficient golden retriever dog model (GRMD) has been identified, which, unlike the mouse model, leads to a clinicopathological phenotype similar to that of Duchenne muscular dystrophy (DMD). We show for the first time that high-level dystrophin expression in skeletal muscle of GRMD dogs can be achieved by AdV-mediated gene transfer. However, a humoral and cellular immune response of the host against antigens of viral and transgene origin (similar to that occurring in mdx mice after AdV-mediated dystrophin gene transfer) leads to a decline of dystrophin expression over a 2-month period. Immunosuppression by cyclosporin significantly prolonged transgene expression. The GRMD model may help to solve the open questions pertaining to dystrophin gene transfer such as systemic delivery and improvement of muscle function before human trials for gene replacement therapy in DMD may be considered.

Adenoviridae↗
PubMed1998

Direct dystrophin and reporter gene transfer into dog muscle in vivo.

Bacterial beta-galactosidase cDNA was injected without lipofectin into 41 sites in dog muscle and expression was seen in 22 of them. The cDNA and lipofectin was injected into 35 similar sites and expression was seen in 21. Expression was seen in a maximum of 2.5% of muscle fibers and 23.21% of nonmuscle cells. A total of 106 muscle sites were injected with the minigene with and without lipofectin. In 4 of the 45 sites injected with the minigene without lipofectin human dystrophin was expressed around the periphery of 0.3% of the fibers. Bacterial beta-galactosidase cDNA was injected into the peritoneal cavity of 4 pups, 2 of which also received lipofectin. In all 4, expression was seen in liver, spleen, and mesenteric lymph node. In the 2 pups that received lipofectin, expression was also seen in the diaphragm, intercostal, and abdominal muscles of 1 and in the diagphragm and intercostal muscles of the other. These experiments show that human dystrophin transgene expression can be obtained in dog muscle. However, other methods will be required to increase the degree of expression before gene therapy trials can be undertaken.

Animals↗
PubMed1998

Snapback SSCP analysis: engineered conformation changes for the rapid typing of known mutations.

Several approaches may be applied to detect known mutations, including restriction enzyme cleavage, allele-specific oligonucleotide (ASO) hybridization or amplification, dideoxy fingerprinting, and direct DNA sequencing. All these approaches require several extra steps after PCR and may involve radioactive isotopes, time-consuming hybridization, template purification, or digestion steps. The ease and simplicity of the SSCP test make it a popular choice for mutation detection, but a significant limitation is that some DNA changes will not alter the overall conformation of either single strand and are thus not amenable to SSCP typing. We describe Snapback SSCP to genotype normal and mdx mice (an animal model of Duchenne muscular dystrophy) that previously could not be differentiated by conventional SSCP analysis. A snapback primer was designed with additional bases at the 5' terminus, which were complementary to the normal sequence flanking the mdx mutation and used under the original amplification conditions. Each single strand of these snapback PCR products now had one terminus capable of re-annealing or "snapping back" to the normal sequence but not the mdx mutation. In this manner, a conformation change was engineered into the normal strand that could be readily distinguished from the mdx allele on a SSCP gel. This approach could be applied to the routine screening of other known mutations that are not amenable to detection by simple SSCP analysis.

Animals↗
PubMed1998

Evidence that porcine native 5-HT3 receptors do not contain nicotinic acetylcholine receptor subunits.

The present investigation utilised monoclonal antibodies directed against subunits of the nicotinic acetylcholine receptor in immunoblot and immunoprecipitation studies, which failed to demonstrate that the native 5-hydroxytryptamine3 (5-HT3) receptor complex purified from porcine brain contains the alpha1, alpha3, alpha4, alpha5, alpha7 or beta2 subunits of the nicotinic acetylcholine receptor.

Animals↗
PubMed1998

Desperately seeking subunits: are native 5-HT3 receptors really homomeric complexes?

The 5-HT3 receptor complex is a ligand-gated ion channel, and is therefore likely to comprise multiple subunits in common with other members of this superfamily. To date, however, only one 5-HT3 receptor subunit, plus an alternatively spliced variant, have been identified. In this article, Stephanie Fletcher and Nicholas Barnes review some of the extensive data in the literature that suggest the presence of other 5-HT3 receptor subunits. This is particularly relevant given the recent demonstration that the 5-HT3 receptor purified from pig brain contains a non-5-HT3A-like protein(s).

Allosteric Regulation↗
PubMed1998

Pax7 includes two polymorphic homeoboxes which contain rearrangements associated with differences in the ability to regenerate damaged skeletal muscle in adult mice.

Pax7 is a paired-type homeobox gene which has previously been shown to play an important role in skeletal muscle formation. It is expressed in skeletal muscle of the limbs during embryogenesis and in adulthood. The aims of this study were firstly to determine the degree of polymorphism of Pax7 amongst inbred laboratory mice using Southern blotting and Pax7 regional specific sub-probes. Secondly, functional studies were performed on mice with each of the different structural forms of Pax7 to determine whether they were associated with differences in the ability to regenerate damaged skeletal muscle. Four different allelic forms of Pax7 have now been identified in laboratory mice indicating that the previously reported DNA sequence of Pax7 is not applicable to all laboratory mice. Hybridisation patterns of TaqI digested DNA representing each of the different Pax7 alleles with the Pax7 specific sub-probes suggested that in contrast to previous findings, Pax7 is associated with two highly polymorphic homeoboxes. The presence of two homeoboxes in BALB/c mice has been confirmed by DNA sequencing. Results of functional studies have also shown that the ability to regenerate damaged skeletal muscle in adult mice is strongly associated with the presence of a 0.15-kb TaqI fragment derived from one of the homeoboxes.

Alleles↗
PubMed1998

Assessment of ultrasound guided percutaneous ethanol injection and parathyroidectomy in patients with tertiary hyperparathyroidism.

BACKGROUND: Tertiary hyperparathyroidism continues to cause significant morbidity in patients with chronic renal failure. This is frequently resistant to medical management and may ultimately require a surgical parathyroidectomy. Recent studies have reported upon the technique of percutaneous ethanol ablation for both primary and tertiary hyperparathyroidism. In this study we report on a 5 year experience using ethanol injection and compare the results with surgical parathyroidectomy. METHODS: A prospective study in 39 patients with tertiary hyperparathyroidism, 25 were dialysis dependent and 14 had a functioning renal allograft. Twenty-two patients underwent percutaneous fine needle ethanol injection (PFNEI) and 17 underwent surgical parathyroidectomy. RESULTS: A > 30% reduction in intact parathyroid hormone (iPTH) was achieved in 11 of 22 patients undergoing PFNEI after a mean of 1.8 +/- 1.4 injections per gland. In four patients, symptomatic hyperparathyroidism recurred and they required further PFNEI or surgical parathyroidectomy at 17, 28, 46, and 48 months later. There was no significant reduction in iPTH in 11 patients following PFNEI after a mean of 2.5 +/- 1.3 injections per gland. They all required a subsequent surgical parathyroidectomy for symptomatic hyperparathyroidism. Four patients developed a laryngeal nerve palsy following PFNEI, two of which were permanent. Seventeen patients underwent successful surgical parathyroidectomy as a primary procedure. CONCLUSION: Whilst PFNEI is successful in primary hyperparathyroidism, when typically only one adenoma is present, the effectiveness of PFNEI is unpredictable and the long term results are poor compared with those of surgical parathyroidectomy in tertiary hyperparathyroidism. The procedure is not without complications and makes subsequent surgery more difficult. Therefore it can only be recommended for patients with a known single parathyroid gland such as patients in whom hyperparathyroidism has recurred following a previous surgical subtotal parathyroidectomy and who are unsuitable for further surgery.

Administration, Cutaneous↗
PubMed1997

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↗
PubMed1997

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↗
PubMed1997

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↗
PubMed1997

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↗
PubMed1997

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↗
PubMed1997

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↗