No abstract

Primary nonfunction of transplanted islets results in part from their sensitivity to reactive oxygen species (ROS) generated during the isolation and transplantation process. Our aim was to examine whether coexpression of antioxidant enzymes to detoxify multiple ROS increased the resistance of mouse islets to oxidative stress and improved the initial function of islet grafts. Islets from transgenic mice expressing combinations of human copper/zinc superoxide dismutase (SOD), extracellular SOD, and cellular glutathione peroxidase (Gpx-1) were subjected to oxidative stress in vitro. Relative viability after hypoxanthine/xanthine oxidase treatment was as follows: extracellular SOD ؉ Gpx-1 ؉ Cu/Zn SOD > extracellular SOD ؉ Gpx-1 > extracellular SOD > wild type. Expression of all three enzymes was the only combination protective against hypoxia/ reoxygenation. Islets from transgenic or control wildtype mice were then transplanted into streptozotocininduced diabetic recipients in a syngeneic marginal islet mass model, and blood glucose levels were monitored for 7 days. In contrast to single-and double-transgenic grafts, triple-transgenic grafts significantly improved control of blood glucose compared with wild type. Our results indicate that coexpression of antioxidant enzymes has a complementary beneficial effect and may be a useful approach to reduce primary nonfunction of islet grafts. Diabetes 54:2109 -2116, 2005

Recently, we described a rare cell type within the adult murine pituitary gland with progenitor cell hallmarks (PCFCs). PCFCs are contained exclusively within a subpopulation of cells that import fluorescent ␤-Ala-Lys-N-AMCA (7-amino-4-methylcoumarin-3-acetic acid). Herein, we investigate the utility of cell surface molecules angiotensin-converting enzyme (ACE) and stem cell antigen-1 (Sca-1) to further enrich for PCFCs. ACE and Sca-1 were expressed on 61% and 55% of AMCA ؉ CD45 ؊ CD31 ؊ cells, respectively, and coexpressed on 38%. ACE

Ischemia-reperfusion injury limits the survival of muscle involved in tissue trauma or transfers during microsurgical reconstruction. Priming stresses such as ischemic preconditioning or mild hyperthermia have frequently been associated with improved survival of ischemic-reperfused cardiac muscle, such protection coinciding with induction of the stress-related heat shock protein 70 (Hsp70). Little is known about the response of skeletal muscle to priming stresses. This review summarizes the current knowledge on the use of priming stresses as protective strategies against the consequences of ischemia-reperfusion in cardiac and skeletal muscle and the potential role of Hsp70.

ABSTRACT

The survival of pancreatic islet beta-cell xenografts and allografts may be affected by damaging reactive oxygen and nitrogen species generated during hypoxia-reoxygenation. Peroxynitrite, which is formed from superoxide and nitric oxide, appears to be an important mediator of beta-cell destruction. The intracellular antioxidant enzymes glutathione peroxidase-1 (Gpx-1) and copper-zinc superoxide dismutase (CuZn SOD) detoxify peroxynitrite and superoxide, respectively. The aim of this study was to examine whether enhanced expression of Gpx-1 and/or CuZn SOD protected NIT-1 mouse insulinoma cells from hypoxia-reoxygenation injury. Stable transfectants expressing human Gpx-1 or CuZn SOD were isolated and tested for their resistance to hydrogen peroxide (H(2)O(2)) and menadione, which generates superoxide intracellularly. Clones expressing one or both enzymes were subjected to hypoxia in glucose-free medium for 18 h, followed by reoxygenation in complete medium for 1.5 h. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) reduction assay. Increases of up to two fold in Gpx or total SOD activity protected NIT-1 cells from H(2)O(2) and menadione. Expression of Gpx-1 significantly increased NIT-1 survival following hypoxia-reoxygenation (viability 65 +/- 9% vs. control 15 +/- 3%, P < 0.001) but CuZn SOD expression had no effect (15 +/- 1%). Expression of both enzymes was no more protective (60 +/- 6%) than expression of Gpx-1 alone. Genetic manipulation of islet beta cells to increase expression of Gpx-1 may protect them from oxidative injury associated with the transplantation procedure.

The ability of heat stress to improve the survival of ischemic‐reperfused skeletal muscle in vivo was investigated. Ischemia‐reperfusion was applied using the rat hindlimb tourniquet model. The viability of ischemic‐reperfused muscle (11 ± 1%) was increased by prior mild heat stress (86 ± 2%). To investigate whether heat shock protein 70 (Hsp 70) expression in the muscle of the heated limb was responsible for this protection, the survival of Hsp 70–expressing transduced myoblasts and myocytes was measured after exposure to mediators of ischemia‐reperfusion injury. Survival was improved in Hsp 70–positive myoblasts but not in myocytes, suggesting that the mechanism of protection conferred by heat stress in vivo cannot be explained by the expression of Hsp 70 in myocytes and may involve a more complex mechanism. In conclusion, prior heat stress is effective in protecting mature skeletal muscle in vivo against necrosis after ischemia‐reperfusion and has potential for use in microsurgical procedures requiring tourniquet applications. © 2000 John Wiley & Sons, Inc. Muscle Nerve 23: 1847–1855, 2000

Few details are available on the heterogeneity of glycosaminoglycans (GAGs) in healing fetal wound tissue. We used a sensitive assay for hexosamines to examine changes occurring in the development of normal sheep skin and of wound healing tissue in PVA sponges inserted subcutaneously at different stages of gestation. It was assumed that glucosamine was derived mainly from hyaluronan and galactosamine mainly from dermatan sulphate and chondroitin sulphate. Hexosamine-containing tissue infiltrating the sponges was deposited more rapidly in the first week than in the second week. Three days after wounding, approximately 70% of the total GAGs in wound tissue was hyaluronan. The proportion of hyaluronan then fell progressively and by the 14th day contributed 57% to the total GAGs. In uninjured skin the contribution of hyaluronan to the total GAGs fell progressively with increasing fetal maturity, the level being 70% at 75 days gestation, but only 35-40% in newborn or adult skin. At no stage of development was there a sudden change in GAG composition suggestive of a transition from regeneration to scar formation. It is concluded that hyaluronan may play an important role in the biochemical sequence leading to collagen fibrillogenesis and mature scar formation.