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Biomedical subjects

S Fletcher

Publications and source records attributed to S Fletcher.

At least 19 recordsLinked to original sources
PubMed2001

Somatic colocalization of rat SK1 and D class (Ca(v)1.2) L-type calcium channels in rat CA1 hippocampal pyramidal neurons.

In hippocampal neurons, the firing of a train of action potentials is terminated by generation of the slow afterhyperpolarization (AHP). Recordings from hippocampal slices have shown that the slow AHP likely results from the activation of small-conductance calcium-activated potassium (SK) channels by calcium (Ca(2+)) entry through L-type Ca(2+) channels. However, the relative localization of these two channel subtypes is not known. The cloning and characterization of three subtypes of SK channel has suggested that SK1 may underlie generation of the slow AHP. Using a novel antibody directed against rat SK1 (rSK1), it has been determined that the rSK1 channel is primarily in the soma of hippocampal CA1 neurons. In conjunction with antibodies directed against C (Ca(v)1.2) and D (Ca(v)1.3) class L-type Ca(2+) channel alpha1 subunits, it was observed that rSK1 channels were selectively colocalized with D class L-type channels. This colocalization supports the functional coupling of L-type and SK channels previously observed in cell-attached patches from hippocampal neurons. However, it appears contrary to the slow rise and decay of the slow AHP. Induction of delayed facilitation of L-type Ca(2+) channels in cell-attached patches from hippocampal neurons evoked delayed opening of coupled SK channels. Generation of ensemble currents produced waveforms identical to the ionic current underlying the slow AHP (I(sAHP)). Therefore, these data indicate that the slow AHP is somatic in origin, resulting from delayed facilitation of D class L-type Ca(2+) channels colocalized with rSK1 channels.

Action Potentials↗
PubMed2001

Dystrophin expression in muscle following gene transfer with a fully deleted ("gutted") adenovirus is markedly improved by trans-acting adenoviral gene products.

Helper-dependent adenoviruses (HDAd) are Ad vectors lacking all or most viral genes. They hold great promise for gene therapy of diseases such as Duchenne muscular dystrophy (DMD), because they are less immunogenic than E1/E3-deleted Ad (first-generation Ad or FGAd) and can carry the full-length (Fl) dystrophin (dys) cDNA (12 kb). We have compared the transgene expression of a HDAd (HDAdCMVDysFl) and a FGAd (FGAdCMV-dys) in cell culture (HeLa, C2C12 myotubes) and in the muscle of mdx mice (the mouse model for DMD). Both vectors encoded dystrophin regulated by the same cytomegalovirus (CMV) promoter. We demonstrate that the amount of dystrophin expressed was significantly higher after gene transfer with FGAdCMV-dys compared to HDAdCMVDysFl both in vitro and in vivo. However, gene transfer with HDAdCMVDysFl in the presence of a FGAd resulted in a significant increase of dystrophin expression indicating that gene products synthesized by the FGAd increase, in trans, the amount of dystrophin produced. This enhancement occurred in cell culture and after gene transfer in the muscle of mdx mice and dystrophic golden retriever (GRMD) dogs, another animal model for DMD. The E4 region of Ad is required for the enhancement, because no increase of dystrophin expression from HDAdCMVDysFl was observed in the presence of an E1/E4-deleted Ad in vitro and in vivo. The characterization of these enhancing gene products followed by their inclusion into an HDAd may be required to produce sufficient dystrophin to mitigate the pathology of DMD by HDAd-mediated gene transfer.

Adenoviridae↗
PubMed2001

Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease arising from defects in the dystrophin gene, typically nonsense or frameshift mutations, that preclude the synthesis of a functional protein. A milder, allelic version of the disease, Becker muscular dystrophy, generally arises from in-frame deletions that allow synthesis of a shorter but still semifunctional protein. Therapies to introduce functional dystrophin into dystrophic tissue through either cell or gene replacement have not been successful to date. We report an alternative approach where 2'-O-methyl antisense oligoribonucleotides have been used to modify processing of the dystrophin pre-mRNA in the mdx mouse model of DMD. By targeting 2'-O-methyl antisense oligoribonucleotides to block motifs involved in normal dystrophin pre-mRNA splicing, we induced excision of exon 23, and the mdx nonsense mutation, without disrupting the reading frame. Exon 23 skipping was first optimized in vitro in transfected H-2K(b)-tsA58 mdx myoblasts and then induced in vivo. Immunohistochemical staining demonstrated the synthesis and correct subsarcolemmal localization of dystrophin and gamma-sarcoglycan in the mdx mouse after intramuscular delivery of antisense oligoribonucleotide:liposome complexes. This approach should reduce the severity of DMD by allowing a dystrophic gene transcript to be modified, such that it can be translated into a Becker-dystrophin-like protein.

Animals↗
PubMed2001

The spread of transgene expression at the site of gene construct injection.

Seven 2-day-old golden retriever pups were given focal intramuscular injections of a first generation adenovirus-dystrophin minigene construct and adenovirus-beta-galactosidase construct as a 2:1 mixture into the left anterior tibial muscle. The spread of transgene expression within the anterior tibial muscle was compared with the spread of methylene blue dye after identical injection into the contralateral muscle. Transgene expression 5-7 days after intramuscular injection was shown to extend between 5.8 and 11.6 mm along the biopsied muscle length (range of biopsy lengths 11.1-12.2 mm). The level of transgene expression at 2-2.5-mm intervals from the site of injection was significantly related to the distance from the site of injection (dystrophin, P = 0.009; beta-galactosidase, P = 0.015). The spread of methylene blue dye within the anterior tibial muscle < or =24 h after identical intramuscular injection demonstrated a similar pattern to the transgene expression, with dye staining measured between 5.5 and 8.5 mm along the muscle sample length (range of biopsy lengths 5.6-15.6 mm). The greatest transgene expression and dye staining was measured 2-2.5 mm proximal to the site of injection with a maximum of 23% of muscle fibers expressing the dystrophin transgene, 95.2% expressing the beta-galactosidase transgene, and 98% of the tissue section stained with methylene blue dye. These results suggest transgene expression after focal intramuscular injection is relatively localized around the site of injection. Further research is required to develop techniques that will provide transgene expression throughout the length and breadth of a muscle.

Adenoviridae↗
PubMed2001

Cryptic splicing involving the splice site mutation in the canine model of Duchenne muscular dystrophy.

Golden retriever muscular dystrophy arises from a mutation in the acceptor splice site of intron 6 of the dystrophin gene. Skipping of exon 7 disrupts the mRNA reading frame and results in premature termination of translation. We are using this animal model to evaluate treatments for Duchenne muscular dystrophy, including gene repair induced by chimeric oligonucleotides. After injection of golden retriever muscular dystrophy (GRMD) muscle with a chimeric oligonucleotide to repair the lesion, immunostaining revealed a modest increase in the number of dystrophin-positive fibres at the injection sites. Dystrophin gene transcripts containing exon 7 were detected by reverse transcription-polymerase chain reaction, suggesting that low levels of splice site correction may have occurred. However, DNA sequencing of these apparently normal dystrophin gene transcripts revealed that the first five bases of exon 7 were missing. It will be important to be aware of this phenomenon with respect to further gene correction studies in the canine model.

Alternative Splicing↗
PubMed2001

Nanocomposite electrodes made of carbon nanofibers and black wax. Anodic stripping voltammetry of zinc and lead.

Nanocomposite electrodes offer exciting new possibilities in electroanalytical chemistry. In this preliminary study, nanocomposite electrodes made of carbon nanofibers and black wax were characterized and investigated as novel substrates for metal deposition and stripping processes. Carbon nanofibers were grown from ethylene-hydrogen gas mixtures over Fe-Ni-Cu (85:10:5) nanoparticle catalysts at 600 degrees C and then embedded in Apiezon black wax under vacuum at 140 degrees C. The resulting nanocomposite electrodes showed (i) good conductivity, (ii) a wide potential window in aqueous solutions, (iii) low background currents, (iv) near steady state voltammetric responses with substantial Faradaic currents and (v) sharply peaked fast scan metal stripping responses. Zinc is a notoriously difficult metal to determine in aqueous solutions, because its deposition and stripping are accompanied by hydrogen evolution at extreme negative potentials. It therefore provided a challenging test for our new nanocomposite electrode. Although numerous complications associated with the hydrogen evolution process could not be eliminated, remarkably clear voltammograms could be obtained even at scan rates of 40 V s(-1).

Journal Article↗
PubMed2001

Evaluation of a short interspersed nucleotide element in the 3' untranslated region of the defective dystrophin gene of dogs with muscular dystrophy.

OBJECTIVES: To determine the distribution of a 231-base pair (bp) element in the dystrophin gene 3' untranslated region (UTR) in a colony of Golden Retrievers with muscular dystrophy and other unrelated dogs and to estimate the frequency of recombination for the canine dystrophin gene. ANIMALS: 77 dogs from the Golden Retriever Muscular Dystrophy (GRMD) colony at the Murdoch Veterinary School and 30 unrelated dogs from the Murdoch University Veterinary Clinic. PROCEDURE: Samples of blood or hair from dogs were used for amplification of DNA, using primers to the canine dystrophin 3' UTR. RESULTS: The DNA from affected dogs generated a larger PCR product than that obtained from clinically normal dogs. Products were cloned and sequenced, and the difference in size was found to be attributable to a 231-bp short interspersed nucleotide element (SINE). The SINE was found in all affected dogs in the colony but not in most unaffected puppies in the colony. Eighteen of 19 dogs in the colony were heterozygous for the GRMD mutation, and 7 of 30 unrelated dogs also were heterozygous for the SINE. CONCLUSION AND CLINICAL RELEVANCE: Evidence of recombination between the GRMD mutation and the SINE was observed in only 4 dogs (2 sets of littermates) in the GRMD colony. Incidence of this SINE in a few unrelated dogs suggests that this particular insertion into the dystrophin gene may have been a recent event. The SINE in the dystrophin 3' UTR did not have an apparent influence on dystrophin mRNA concentrations.

3' Untranslated Regions↗
PubMed2001

Test of canine sperm function in vitro using primary homologous oocytes with fluorescence and confocal microscopy.

In vitro capacitated canine spermatozoa can interact with both mature and immature homologous oocytes in vitro, but it is difficult to determine whether spermatozoa have actually penetrated the oocyte or have simply bound to the zona pellucida. The aim of the first part of this study was to determine accurately the location of spermatozoa in relation to the oocyte by comparing observations made using confocal and fluorescence microscopy. The fluorescence technique had a sensitivity of 66%, and correctly identified 98% of the spermatozoa that had penetrated or bound oocytes and could discriminate between penetrated and bound spermatozoa with a sensitivity and specificity of 97% and 100%, respectively. This finding demonstrates that evaluation of the assay by fluorescence microscopy is reliable for detecting the presence and location of spermatozoa in relation to homologous oocytes. The aim of the second part of the study was to attempt to simplify the assay using fluorescence microscopy and immature oocytes. No significant difference was found between sperm interaction with mature and immature oocytes, which demonstrates that culturing oocytes before the assay has no benefit, and that the assay can be performed quickly and easily using non-cultured oocytes to provide rapid evidence of the fertilizing potential of spermatozoa.

Animals↗
PubMed2000

Prompt Optical Observations of Gamma-Ray Bursts.

The Robotic Optical Transient Search Experiment (ROTSE) seeks to measure simultaneous and early afterglow optical emission from gamma-ray bursts (GRBs). A search for optical counterparts to six GRBs with localization errors of 1 deg2 or better produced no detections. The earliest limiting sensitivity is mROTSE>13.1 at 10.85 s (5 s exposure) after the gamma-ray rise, and the best limit is mROTSE>16.0 at 62 minutes (897 s exposure). These are the most stringent limits obtained for the GRB optical counterpart brightness in the first hour after the burst. Consideration of the gamma-ray fluence and peak flux for these bursts and for GRB 990123 indicates that there is not a strong positive correlation between optical flux and gamma-ray emission.

Journal Article↗
PubMed2000

Cardiovascular disease and outcome of acute stroke: influence of pre-existing cardiac failure.

BACKGROUND AND AIMS: Whilst a number of variables, mostly a consequence of a stroke, are known to predict mortality of acute stroke there is limited information on the significance of pre-existing cardiovascular variables on stroke mortality. We have investigated the influence of pre-existing cardiovascular factors in one cohort of stroke patients. METHODS: We studied 295 patients, mean age 74+/-10 (range 34-96) years; 133 males, presenting with acute stroke for pre-existing cardiovascular disease (CVD) defined as hypertension, atrial fibrillation (AF), ischaemic heart disease (IHD) and cardiac failure (CF). In addition, data were collected on epidemiological and neurological variables known to influence stroke mortality. The most significant of the cardiovascular factors was further investigated against all the other cardiovascular groups together and against those without any CVD. Outcome was measured as their influence on acute phase and 3-month mortality. RESULTS: There was no significant difference in 3-month mortality with hypertension (P=0.62) and IHD (P=0.33) but there was a significant higher mortality in patients with AF (P=0.05) and CF (P <0.001). CF was more significant than all other CVD (hypertension+AF+IHD) together without the failure (P<0.001); odds ratio of 4.5 (95% CI 2.28-9.07). Partial correlation coefficient revealed CF to be an independent significant variable to influence stroke mortality when controlled with AF, stroke syndromes, age, incontinence, pyrexia, dysphagia and Glasgow coma score. CONCLUSIONS: Pre-existing CF has an adverse influence on stroke mortality independent of other known factors. Cardiovascular factors without failure do not have such an effect except the marginal effect of AF.

Adult↗
PubMed2000

Gene therapy and molecular approaches to the treatment of hereditary muscular disorders.

Gene therapy for inherited muscle disease is an active area of research and development. Initial emphasis has been on gene replacement but alternative approaches are increasingly being considered in order to overcome difficulties, such as the immune rejection of transduced cells, the need for appropriate and tissue-specific control of expression, and the requirement for systemic spread in some conditions. However, the most significant obstacles to the clinical success of gene therapy are still the lack of efficiency and accuracy of gene medicine delivery.

Animals↗
PubMed2000

Prognostic value of CT scan features in acute ischaemic stroke and relationship with clinical stroke syndromes.

The aim of this prospective study was to investigate the value of CT scan in prognosis of acute ischaemic stroke patients as a variable additional to clinical stroke syndromes to develop a simple classification of CT scan features to provide a practical approach to prognosticate and manage such patients. One hundred and eight nine patients admitted with ischaemic stroke were investigated with a CT scan. CT scan features were classified into two groups: large infarct, LI (across more than one lobe) and non-large infarcts (N-LI) for all other features. Patients were also studied for clinical syndromes, analysing results for total anterior circulation syndrome (TACS). Outcome was measured as early post stroke Barthel index, acute phase and 3-month mortality. Patients with LI had features of severe strokes and had a lower Barthel index (p < 0.001), lower Glasgow coma score (p < 0.001), more association with incontinence (p < 0.001), pyrexia (p = 0.007) and dysphagia (p < 0.001). LI patients required higher level of care in acute wards and had a higher length of stay (p = 0.01). Both the LI and TACS individually had a significantly higher mortality (p < 0.001) and similar positive predictive value, sensitivity and specificity for 3-month mortality. While the combined factor of 'large infarct and TACS' provided the highest likelihood ratio (3.1) for mortality, the factor of 'large infarct or TACS' was the most sensitive (85%) to identify majority of patients at a risk of mortality. N-LI patients had a better prognosis. Classification of cerebral infarcts into large and non-large categories identifies patients who require higher level of care in acute wards and have a higher mortality. Combined factor of 'large infarct and/or TACS' identifies the majority of patients at risk of 3-month mortality as compared to either variable taken individually. CT scan features are complimentary to clinical syndromes for managing acute stroke patients.

Adult↗
PubMed2000

T-ceIl recognition of discrete regions of the thrombolytic drug streptokinase.

Streptokinase (SK) is a bacterial protein used clinically as a thrombolytic agent in humans. Administration of SK causes a rapid increase in the frequency of anti-SK T cells and the titre of specific anti-SK antibodies that, on subsequent administration of SK, may neutralize the activity of the drug or elicit allergic-type reactions. By locating and modifying the immunogenic T-cell epitopes within the SK protein, it is possible that an agent with reduced immunogenicity but equal efficacy may be produced. We have investigated the T-cell epitopes within SK using nine non-overlapping, recombinant peptide fragments of SK. We investigated the proliferative T-cell response of peripheral blood mononuclear cells obtained from patients before and 6 days after administration of SK for myocardial infarction. We also examined the response of cultured anti-SK T-cell lines derived from patients 6 days after treatment with SK. Before administration of SK, peripheral blood mononuclear cells from six of nine patients showed a proliferative response to SK. The response was significantly higher 6 days after administration of SK (P = 0.0004). Cultured T-cell lines showed similar proliferative responses to clinical-grade SK and recombinant SK. Marked differences in T-cell responses were apparent in response to each recombinant SK fragment (P = 0.04). The mean proliferative response exceeded background to only two peptides, peptide 2 (P = 0.04) and peptide 3 (P = 0.009). Peptide 3, representing amino acids 100-150 of mature SK, was recognized preferentially in the majority of assays. Marked variation in the T-cell response to SK following treatment with this agent was observed between subjects. Despite these differences, peptides 2 and 3 induced T-cell proliferation at a level significantly above background in the majority of subjects. These epitopes may represent a region of enhanced immunogenicity within SK.

Analysis of Variance↗
PubMed1999

Autoradiographic localization of the [3H]-(S)-zacopride labelled 5-HT3 receptor in porcine brain.

Using the technique of in vitro receptor autoradiography, we have determined the distribution of the [3H]-(S)-zacopride labelled 5-HT3 receptor in porcine brain. Highest densities of 5-HT3 receptor-associated [3H]-(S)-zacopride binding were detected in areas of porcine spinal cord, nodose ganglion, trigeminal nerve nucleus, area postrema and cerebral cortex, with relatively lower levels in other brain regions (e.g. hippocampus, caudate-putamen). The distribution of [3H]-(S)-zacopride binding in porcine forebrain provides further evidence for inter-species differences with respect to the differential expression of the 5-HT3 receptor in the forebrain.

Animals↗