Bisulfite genomic sequencing of microdissected cells

Mapping of methylation patterns in CpG islands has become an important tool for understanding tissue-specific gene expression in both normal and pathological situations. However, the inherent cellular heterogeneity of any given tissues can affect the outcome and interpretation of molecular studies. In order to analyse genomic DNA methylation on a pure cell population from tissue sample, we have developed a simple technique of single-cell microdissection from cryostat sections which can be combined with bisulfite-mediated sequencing of 5-methylcytosine. We report here our results on the methylation status of the androgen receptor gene studied by bisulfite genomic sequencing on purified cells isolated from human testis.     

Assessing release protocols for Canada lynx reintroduction in colorado

Reintroductions are a common strategy to restore ecosystem integrity, especially when top predators are involved. Reintroductions are often time consuming, expensive, and controversial, and thus understanding what aspects are important for a successful program is needed. Focusing on the example of the reintroduction of Canada lynx (Lynx canadensis) to Colorado, we investigated how different release protocols (RP) affected mortality within the first year post-release. We found that average monthly mortality in the study area during the first year decreased with time in captivity from 0.205 (95% CI = 0.069, 0.475) for lynx having spent up to 7 days in captivity to 0.028 (95% CI = 0.012, 0.064) for lynx spending >45 days in captivity before release. Our results also suggest that keeping lynx in captivity beyond 5–6 weeks accrued little benefit in terms of monthly survival. We found that, on a monthly average basis, lynx were as likely to move out (P = 0.196, SE = 0.032) as well as back onto (P = 0.143, SE = 0.034) the reintroduction area during the first year after release. Mortality was 1.6 times greater outside of the study area, suggesting that permanent emigration and differential mortality rates on and off reintroduction areas should be factored into sample size calculations for an effective reintroduction effort. A post-release monitoring plan is critical to providing information to assess aspects of RP and to improve survival of individuals. Future lynx and other carnivore reintroductions may use our results to help design reintroduction programs including both the release and post-release monitoring protocols. © 2011 The Wildlife Society.     

Analysis of Kinetic Intermediates in Single-Particle Dwell-Time Distributions

Many biological and chemical processes proceed through one or more intermediate steps. Statistical analysis of dwell-time distributions from single molecule trajectories enables the study of intermediate steps that are not directly observable. Here, we discuss the application of the randomness parameter and model fitting in determining the number of steps in a stochastic process. Through simulated examples, we show some of the limitations of these techniques. We discuss how shot noise and heterogeneity among the transition rates of individual steps affect how accurately the number of steps can be determined. Finally, we explore dynamic disorder in multistep reactions and show that the phenomenon can obscure the presence of rate-limiting intermediate steps.     

Les injections intracaverneuses de prostaglandine E1: un reel progres dans l’efficacite et la securite des injections intracaverneuses

Intracavernosal injections of vaso-active agents were a major breakthrough in the investigation and treatment of impotence. However the first generation agents (papaverine ± phentolamine, phenoxybenzamine) result in a too high rate of complications (prolonged erection, priapism, corporeal fibrosis,), what requires strict precautions, making their use cumbersome and even stressing. A tremendous development of the pharmacological research arises in order to find agents not resulting in those risks. This article reviews the litterature concerning one of them, Prostaglandin E1 (PGE1), and compares its effects in near 4000 patients to those of papaverine ±phentolamine in over 5000. Experimental data suggests that PGE1 might act in normal erection, and its local tolerance might be better than that of Papaverine, specially as regards the risk of fibrosis. The studies without intraindividual comparison done in men tend to confirm this better tolerance: during the workup period, PGE1 induces prolonged erection in 2.7% of men (requiring treatment in only 0.3%) compared to 9.5% with papaverine and 5.3 % with papaverine + phentolamine (p<0.001); during auto-intracavernosal therapy, the rate of prolonged erections is 0.8% with PGE1 compared to 8.7% with papaverine, and the rate of fibrosis is 0.4% versus 3.5 to 5.4% with papaverine (p<0.001). PGE1 induces slightly more erections compatible with penetration than papaverine ± phentolamine (77% vs 73 %). However it induces pain in 23% of the cases, either at the time of injection, or, more specifically, during erection. This pain is intense in only 3.9% of the cases. General tolerance is excellent. The studies with intra-individual comparison confirm the statistically significant reduction of the risk of prolonged erection requiring treatment (1.7% with PGE1 versus 4.3% with papaverine and 6% with the combination, p<0.05) and the increase in efficacy (75% versus 64% with the combination), though papaverine is superior to PGE1 in a minority of the cases. By reducing the risks, PGE1 allows directly using the effective dosage as regards the diagnostic injections. Its higher effectiveness reduces the false-negative rate in the psychogenic patients, and widens the possibilities of the intracavernosal therapy.     

The role of insulin receptor kinase domain autophosphorylation in receptor-mediated activities. Analysis with insulin and anti-receptor antibodies.

The role of specific tyrosine autophosphorylation sites in the human insulin receptor kinase domain (Tyr1158, Tyr1162, and Tyr1163) was analyzed using in vitro mutagenesis to replace tyrosine residues individually or in combination. Each of the three single-Phe, the three possible double-Phe a triple-Phe and a triple-Ser mutant receptors, stably expressed in Chinese hamster ovary cells, were compared with the wild-type receptor in their ability to mediate stimulation of receptor kinase activity, glycogen synthesis, and DNA synthesis by insulin or the human-specific anti-receptor monoclonal antibody 83-14. At a concentration of 0.1 nM insulin which produced approximately half-maximal responses with wild-type receptor, DNA synthesis and glycogen synthesis mediated by the three single-Phe mutants ranged from 52 to 88% and from 32 to 79% of the wild-type receptor, respectively. The corresponding figures for the double-Phe mutants averaged 15 and 6%, whereas the triple-mutants were unresponsive in both assays. The level of biological function approximately paralleled the insulin-stimulated tyrosine kinase activity in the intact cell as estimated by tyrosine phosphorylation of the insulin receptor and its endogenous substrate pp 185/IRS-1. Interestingly, all mutants showed a marked decrease in insulin-stimulated receptor internalization. Anti-receptor antibody stimulated receptor kinase activity and mimicked insulin action in these cells. In general, the impairment of the metabolic response was greater and impairment of the growth response was less when antibody was the stimulus. These experiments show that the level and specific sites of autophosphorylation are critical determinants of receptor function. The data are consistent with a requirement for the receptor tyrosine kinase either as an obligatory step or a modulator, in both metabolic and growth responses, and demonstrate the important role of the level of insulin receptor kinase domain autophosphorylation in regulating insulin sensitivity.