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J Tinsley

Publications and source records attributed to J Tinsley.

17 recordsLinked to original sources
PubMed2002

Prevention of pathology in mdx mice by expression of utrophin: analysis using an inducible transgenic expression system.

Duchenne muscular dystrophy results from the absence of dystrophin, a cytoskeletal protein. Previously, we have shown in a transgenic mouse model of the disease (mdx) that high levels of expression of the dystrophin-related protein, utrophin can prevent pathology. We developed a new transgenic mouse model where muscle specific utrophin expression was conditioned by addition of tetracycline in water. Transgene expression was turned on at different time points: in utero, at birth, 10 and 30 days after birth. We obtained moderate levels of expression, variable from fibre to fibre (mosaicism) but sufficient to induce a correct localization of the dystro-sarcoglycan complex. Histology revealed a reduction of necrotic foci and of the percentage of centronucleated fibres, which remained still largely above the normal level. Isometric force was not improved but the resistance to eccentric contractions was significantly stronger. When utrophin expression was activated 30 days after birth, improvements were marginal, suggesting that the age at which utrophin therapy is initiated could be an important factor. Our results also provide an unexpected insight into the pathogenesis of the dystrophinopathies. We observed a complete normalization of the characteristics of the mechano-sensitive/voltage-independent Ca(2+) channels (occurrence, open probabilities and Ca(2+) currents), while the classical markers of dystrophy were still abnormal. These observations question the role of increased Ca(2+) channel activity in initiating the dystrophic process. The new model shows that utrophin therapy, initiated after birth, can be effective, but the extent of correction of the various symptoms of dystrophinopathy critically depends on the amount of utrophin expressed.

Animals↗
PubMed1999

Role of phospholipase C, protein kinase C, and calcium in VEGF-induced venular hyperpermeability.

We previously demonstrated that vascular endothelial growth factor (VEGF)-elicited increase in the permeability of coronary venules was blocked by the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). The aim of this study was to delineate in more detail the signaling pathways upstream from NO production in VEGF-induced venular hyperpermeability. The apparent permeability coefficient of albumin (Pa) and endothelial cytosolic Ca2+ concentration ([Ca2+]i) were measured in intact perfused porcine coronary venules using fluorescence microscopy. VEGF (10(-10) M) induced a two- to threefold increase in Pa, which was blocked by a monoclonal antibody directed against the VEGF receptor Flk-1/KDR, the phospholipase C (PLC) antagonist U-73122, or the protein kinase C (PKC) antagonist bisindolylmaleimide (BIM). In 12 venules that displayed the [Ca2+]i response to bradykinin (10(-6) M) and ionomycin (10(-6) M), only 4 vessels responded to VEGF with a transient increase in [Ca2+]i. Furthermore, Western blot analysis of cultured human umbilical vein endothelial cells showed that VEGF increased tyrosine phosphorylation of PLC-gamma and serine phosphorylation of endothelial constitutive NO synthase (ecNOS). The hyperphosphorylation of PLC-gamma was greatly attenuated by the KDR receptor antibody and U-73122, but not by BIM or L-NMMA. In contrast, U-73122 and BIM were able to inhibit VEGF-elicited serine phosphorylation of ecNOS. The results suggest that VEGF induces venular hyperpermeability through a KDR receptor-mediated activation of PLC. In turn, ecNOS is activated by PLC-mediated PKC and/or cytosolic Ca2+ elevation stimulation.

Animals↗
PubMed1998

Expression of full-length utrophin prevents muscular dystrophy in mdx mice.

Duchenne muscular dystrophy (DMD) is a lethal, progressive muscle wasting disease caused by a loss of sarcolemmal bound dystrophin, which results in the death of the muscle fiber leading to the gradual depletion of skeletal muscle. The molecular structure of dystrophin is very similar to that of the related protein utrophin. Utrophin is found in all tissues and is confined to the neuromuscular and myotendinous junctions in mature muscle. Sarcolemmal localization of a truncated utrophin transgene in the dystrophin-deficient mdx mouse significantly improves the dystrophic muscle phenotype. Therefore, up-regulation of utrophin by drug therapy is a plausible therapeutic approach in the treatment of DMD. Here we demonstrate that expression of full-length utrophin in mdx mice prevents the development of muscular dystrophy. We assessed muscle morphology, fiber regeneration and mechanical properties (force development and resistance to stretch) of mdx and transgenic mdx skeletal and diaphragm muscle. The utrophin levels required in muscle are significantly less than the normal endogenous utrophin levels seen in lung and kidney, and we provide evidence that the pathology depends on the amount of utrophin expression. These results also have important implications for DMD therapies in which utrophin replacement is achieved by delivery using exogenous vectors.

Animals↗
PubMed1997

Expression of truncated utrophin leads to major functional improvements in dystrophin-deficient muscles of mice.

Dystrophin-deficient mice (mdx) expressing a truncated (trc) utrophin transgene show amelioration of the dystrophic phenotype. Here we report a multifunctional study demonstrating that trcutrophin expression leads to major improvements of the mechanical performance of muscle (that is, force development, mechanical resistance to forced lengthenings and maximal spontaneous activity) and of the maintenance of the intracellular calcium homeostasis. These are two essential functions of muscle fibers, known to be impaired in mdx mouse muscles and Duchenne muscular dystrophy (DMD) patients. Our results bring strong support to the hypothesis that muscle wasting in dystrophin-deficient DMD patients could be prevented by upregulation of utrophin.

Animals↗
PubMed1992

Characterization of a 4.8kb transcript from the Duchenne muscular dystrophy locus expressed in Schwannoma cells.

The 14kb dystrophin transcript from the Duchenne muscular dystrophy (DMD) locus, which encodes a 427kDa protein, is differentially spliced at the amino terminal end giving rise to alternative transcripts expressed in muscle and brain. Here we present evidence for a 4.8kb transcript from the DMD locus which is ubiquitously expressed but is particularly abundant in Schwannoma cells where dystrophin could not be detected. The hybridisation of Western blots with dystrophin antibodies also identifies a protein of approximately 80kDa of variable abundance in different human and mdx tissues. Immunocytochemistry studies confirm the expression of this protein in nerve cells, a tissue in which full length dystrophin is not detected. Sequencing of the 5' end of a clone isolated from a rat Schwannoma cDNA library, shows that the 4.8kb transcript shares exons with the carboxy terminal end of dystrophin but the 5' untranslated region is not contained within the dystrophin transcript. We propose that the 4.8kb gene product be called apodystrophin-1 as its expression is distinct from the dystrophin 14kb mRNA but it is transcribed from the same locus.

Amino Acid Sequence↗
PubMed1983

Expression of the human interferon-beta gene cloned in phage M13 mp7.

The single-stranded DNA phage, M13 mp7 was used in the construction of an expression vector containing the coding sequence for mature interferon-beta (IFN-beta). Two clones expressed a fused polypeptide showing the biological and physicochemical properties of IFN-beta, despite the fact that the N-terminal amino acid sequence had been changed; 10(6) I.U./l of culture were produced with a molecular weight of 20 000.

Amino Acid Sequence↗
PubMed1981

The effect of 5-fluorouracil and 5-fluorocytosine on the development of the filarial nematodes Brugia pahangi and Dirofilaria immitis.

5-fluorouracil and 5-fluorodeoxyuridine at 30 mg/kg body weight daily for four days inhibit microfilarial production in Brugia pahangi in the jird. Disruption of intrauterine embryogenesis was observed in treated female worms but the compounds were not macrofilaricidal or microfilaricidal under the conditions employed. 5-fluorocytosine possessed no filaricidal or embryostatic activity. The inhibition of microfilaria production by 5-fluorouracil was temporary and larval production was resumed within nine weeks. The compound also inhibited the development of B. pahangi and Dirofilaria immitis larvae in the mosquito Aedes aegypti, when administered to cages of mosquitoes as a 0.01 or 0.001% solution in a 10% aqueous sucrose solution on cotton wool wicks. The development of infective larvae of B. pahangi in the jird was inhibited by 5-fluorouracil.

Animals↗