Spinal muscular atrophy (SMA) is, after Duchenne muscular dystrophy, the most common neuromuscular disorder in childhood. The gene responsible for childhood SMA has been mapped to the q11. 2 – q13. 3 region of chromosome 5. We have extended our linkage studies of SMA In the French - Canadian population to Include microsatellite markers at the D5S125, D5S351, D5S435, JK53CA1/ 2 and MAPI B locl. These markers span about 4 cM of the SMA candidate region. We observed significant evidence for linkage between SMA and all the markers tested. The analysis of recombinant chromosomes provide evidence for the following genetic order: D5S125-D5S435-MAP1B-3'-JK53CA1/2 and places D5S351 proximal to JK53CA1/2. Furthermore, we confirm the current localization of the SMA gene distal to D5S435. Finally, we provide demonstration of significant linkage disequilibrium between childhood-onset SMA and four of the five marker loci, D5S125, D5S435, D5S351 and JK53CA1/2. Analysis of SMA-region haplotypes suggests that there may be a predominant SMA allele that is present on about 17% of SMA chromosomes in this sample of the French - Canadian population. We conclude that the observed linkage disequilibrium is likely due to genetic drift among regions of Quebec, consistent with this population's early history.
The vast majority of drugs associated with acquired QT prolongation are known to interact with hERG. Due to the awareness of the potential danger of such QT drugs, the regulatory authorities issued recommendations for the establishment of cardiac safety testing during preclinical drug development. Traditionally, lead compounds in late stage preclinical studies were tested for hERG binding using electrophysiology. These are laborious methods, requiring significant skill in the end-user to perform a successful assay. Furthermore, many researchers wish to test lead compounds for safety earlier in the process of drug development. This requires a higher throughput type of assay. Here we describe methods for the automation of hERG screening using Invitrogen’s fluorescence polarization based Predictor™ assay using the Precision™ Automated Pipetting System for the serial dilution of known inhibitors of hERG to generate IC50 data. FP-based IC50 data will be compared to electrophysiology and radiometric data.
The chemokine receptor CXCR4 has multiple critical functions in normal physiologies including embryonic development of the cardiovascular, hemopoietic and central nervous systems. It has also been implicated in disease pathologies such as HIV infection, cancer metastasis, leukemia progression and rheumatoid arthritis, and is fueling the search for small molecule CXCR4 antagonists as a means for intervention 1 . Here we demonstrate the suitability of the Tag-lite HTRF technology for CXCR4 ligand binding assays for high throughput screening applications. Automated assay performance and pharmacology will be presented.
Elevated levels of circulating prorenin, the precursor of renin, have been reported to precede the appearance of microvascular complications in diabetes mellitus. Although several studies using animal models have attempted to address the link between elevated prorenin and the tissue remodeling and damage associated with both hypertension and diabetes mellitus, the results have been contradictory, and the mechanism whereby prorenin might contribute to these pathologies remains a subject of debate. To directly test the role of prorenin in these pathologies, we generated transgenic mice with selective increases (13- to 66-fold) in circulating native or active site-mutated prorenin. Systolic blood pressure was either unchanged or increased (+25 mm Hg) in native prorenin-expressing mice, whereas the mice expressing active site-mutated prorenin showed no significant differences in systolic blood pressure compared with control animals. There was no increase in cardiac fibrosis or renal glomerular sclerosis in any of the transgenic animals tested, even at an advanced age (18 months). Captopril (an angiotensin-converting enzyme inhibitor) rapidly normalized blood pressure of hyperproreninemic mice, whereas infusion of the putative antagonist of the prorenin receptor (handle region peptide) had no effect. These results suggest that the primary consequence of chronic elevations in circulating prorenin is an increase in blood pressure and do not support a role for prorenin as the primary causative agent in cardiac fibrosis or renal glomerular injury. The lack of effect seen with active site-mutated prorenin and the efficacy of angiotensin-converting enzyme inhibition are also consistent with prorenin acting through the generation of angiotensin II to raise blood pressure.
Elevated levels of circulating prorenin, the precursor of renin, have been reported to precede the appearance of microvascular complications in diabetes mellitus. Although several studies using animal models have attempted to address the link between elevated prorenin and the tissue remodeling and damage associated with both hypertension and diabetes mellitus, the results have been contradictory, and the mechanism whereby prorenin might contribute to these pathologies remains a subject of debate. To directly test the role of prorenin in these pathologies, we generated transgenic mice with selective increases (13- to 66-fold) in circulating native or active site-mutated prorenin. Systolic blood pressure was either unchanged or increased (+25 mm Hg) in native prorenin-expressing mice, whereas the mice expressing active site-mutated prorenin showed no significant differences in systolic blood pressure compared with control animals. There was no increase in cardiac fibrosis or renal glomerular sclerosis in any of the transgenic animals tested, even at an advanced age (18 months). Captopril (an angiotensin-converting enzyme inhibitor) rapidly normalized blood pressure of hyperproreninemic mice, whereas infusion of the putative antagonist of the prorenin receptor (handle region peptide) had no effect. These results suggest that the primary consequence of chronic elevations in circulating prorenin is an increase in blood pressure and do not support a role for prorenin as the primary causative agent in cardiac fibrosis or renal glomerular injury. The lack of effect seen with active site-mutated prorenin and the efficacy of angiotensin-converting enzyme inhibition are also consistent with prorenin acting through the generation of angiotensin II to raise blood pressure.
