University of Saskatchewan

About: University of Saskatchewan is a education organization based out in Saskatoon, Saskatchewan, Canada. It is known for research contribution in the topics: Population & Health care. The organization has 25021 authors who have published 52579 publications receiving 1483049 citations. The organization is also known as: USask.

Laser-induced gene expression in specific cells of transgenic zebrafish

Abstract: Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.

High-Precision Laser Spectroscopy D/H and 18O/16O Measurements of Microliter Natural Water Samples

Abstract: Newly available gas analyzers based on off-axis integrated cavity output spectroscopy (OA-ICOS) lasers have been advocated as an alternative to conventional isotope-ratio mass spectrometers (IRMS) for the stable isotopic analysis of water samples In the case of H2O, OA-ICOS is attractive because it has comparatively low capital and maintenance costs, the instrument is small and field laboratory portable, and provides simultaneous D/H and 16O/18O ratio measurements directly on H2O molecules with no conversion of H2O to H2, CO, or H2/CO2−water equilibration required Here we present a detailed assessment of the performance of a liquid-water isotope analyzer, including instrument precision, estimates of sample memory and sample mass effects, and instrumental drift We provide a recommended analysis procedure to achieve optimum results using OA-ICOS Our results show that, by using a systematic sample analysis and data normalization procedure routine, measurement accuracies of ± 08‰ for δD and ±01‰ δ18O ar

The Gasotransmitter Role of Hydrogen Sulfide

Abstract: A novel concept of "gasotransmitter" arrived recently Gasotransmitters are small molecules of endogenous gases with important physiological functions Their production and metabolism are enzymatically regulated, and their effects are not dependent on specific membrane receptors Following the identification of nitric oxide and carbon monoxide as gasotransmitters, hydrogen sulfide (H(2)S) may be qualified as the third gasotransmitter Recent studies have shown that H(2)S is generated from vascular smooth muscle cells (SMCs), catalyzed by specific H(2)S-generating enzyme At physiologically relevant concentrations, H(2)S relaxes vascular tissues, an effect mediated by the activation of ATP-sensitive K(+) (K(ATP)) channels in vascular SMCs H(2)S directly alters the activity of K(ATP) channels without the involvement of second messengers Furthermore, the endogenous production of H(2)S in the cardiovascular system is likely regulated by nitric oxide, whereas the vasorelaxant effect of nitric oxide is inhibited by H(2)S It is anticipated that future studies will better reveal the molecular mechanisms underlying the effect of H(2)S on K(ATP) channel proteins, the interaction of H(2)S and other gasotransmitters in cardiovascular system, the endogenous stimulators and inhibitors of H(2)S metabolism, the role of H(2)S in the regulation of heart function, and the abnormal H(2)S production and action under various pathophysiological conditions

Activation of KATP channels by H2S in rat insulin-secreting cells and the underlying mechanisms.

Abstract: H2S is an important gasotransmitter, generated in mammalian cells from L-cysteine metabolism. As it stimulates K(ATP) channels in vascular smooth muscle cells, H2S may also function as an endogenous opener of K(ATP) channels in INS-1E cells, an insulin-secreting cell line. In the present study, K(ATP) channel currents in INS-1E cells were recorded using the whole-cell and single-channel recording configurations of the patch-clamp technique. K(ATP) channels in INS-1E cells have a single-channel conductance of 78 pS. These channels were activated by diazoxide and inhibited by gliclazide. ATP (3 mm) in the pipette solution inhibited K(ATP) channels in INS-1E cells. Significant amount of H2S was produced from INS-1E cells in which the expression of cystathinonie gamma-lyase (CSE) was confirmed. After INS-1E cells were transfected with CSE-targeted short interfering RNA (CSE-siRNA) or treated with DL-propargylglycine (PPG; 1-5 mm) to inhibit CSE, endogenous production of H2S was abolished. Increase in extracellular glucose concentration significantly decreased endogenous production of H2S in INS-1E cells, and increased insulin secretion. After transfection of INS-1E cells with adenovirus containing the CSE gene (Ad-CSE) to overexpress CSE, high glucose-stimulated insulin secretion was virtually abolished. Basal K(ATP) channel currents were significantly reduced after incubating INS-1E cells with a high glucose concentration (16 mm) or lowering endogenous H2S level by CSE-siRNA transfection. Under these conditions, exogenously applied H2S significantly increased whole-cell K(ATP) channel currents at concentrations equal to or lower than 100 microm. H2S (100 microm) markedly increased open probability by more than 2-fold of single K(ATP) channels (inside-out recording) in native INS-1E cells (n = 4, P < 0.05). Single-channel conductance and ATP sensitivity of K(ATP) channels were not changed by H2S. In conclusion, endogenous H2S production from INS-1E cells varies with in vivo conditions, which significantly affects insulin secretion from INS-1E cells. H2S stimulates K(ATP) channels in INS-1E cells, independent of activation of cytosolic second messengers, which may underlie H2S-inhibited insulin secretion from these cells. Interaction among H2S, glucose and the K(ATP) channel may constitute an important and novel mechanism for the fine control of insulin secretion from pancreatic beta-cells.