Inactivation of bacterial spores and viruses in biological material using supercritical carbon dioxide with sterilant.

The purpose of this study was to validate supercritical carbon dioxide (SC-CO(2)) as a terminal sterilization method for biological materials, specifically acellular dermal matrix. In this study, bacterial spores, Bacillus atrophaeus, were inoculated onto porcine acellular dermal matrix to serve as a "worst case" challenge device. The inactivation of the spores by SC-CO(2) with peracetic acid (PAA) sterilant was analyzed as a function of exposure times ranging from 1 to 30 min. A linear inactivation profile for the Bacillus atrophaeus spores was observed, and a SC-CO(2) exposure time of 27 min was determined to achieve a sterility assurance level of 10(-6). The inactivation of viruses was also studied using Encephalomyocarditis (EMC) viruses. After 15 min of exposure to SC-CO(2) with PAA sterilant, more than a 6 log(10) reduction was observed for EMC viruses. Biochemical and biomechanical evaluations showed that the SC-CO(2) treatment with PAA sterilant did not cause significant changes in porcine acellular matrix's susceptibility to collagenase digestion, tensile or tear strength, indicating limited alteration of the tissue structure following SC-CO(2) sterilization.

Muscle-specific overexpression of wild type and R225Q mutant AMP-activated protein kinase gamma3-subunit differentially regulates glycogen accumulation.

AMP-activated protein kinase (AMPK) is a heterotrimeric complex that works as an energy sensor to integrate nutritional and hormonal signals. The naturally occurring R225Q mutation in the gamma3-subunit in pigs is associated with abnormally high glycogen content in skeletal muscle. Because skeletal muscle accounts for most of the body's glucose uptake, and gamma3 is specifically expressed in skeletal muscle, it is important to understand the underlying mechanism of this mutation in regulating glucose and glycogen metabolism. Using skeletal muscle-specific transgenic mice overexpressing wild type gamma3 (WTgamma3) and R225Q mutant gamma3 (MUTgamma3), we show that both WTgamma3 and MUTgamma3 mice have 1.5- to 2-fold increases in muscle glycogen content. In WTgamma3 mice, increased glycogen content was associated with elevated total glycogen synthase activity and reduced glycogen phosphorylase activity, whereas alterations in activities of these enzymes could not explain elevated glycogen in MUTgamma3 mice. Basal, 5-aminoimidazole-AICAR- and phenformin-stimulated AMPKalpha2 isoform-specific activities were decreased only in MUTgamma3 mice. Basal rates of 2-DG glucose uptake were decreased in both WTgamma3 and MUTgamma3 mice. However, AICAR- and phenformin-stimulated 2-DG glucose uptake were blunted only in MUTgamma3 mice. In conclusion, expression of either wild type or mutant gamma3-subunit of AMPK results in increased glycogen concentrations in muscle, but the mechanisms underlying this alteration appear to be different. Furthermore, mutation of the gamma3-subunit is associated with decreases in AMPKalpha2 isoform-specific activity and impairment in AICAR- and phenformin-stimulated skeletal muscle glucose uptake.

Functional role of AMP-activated protein kinase in the heart during exercise.

AMP-activated protein kinase (AMPK) plays a critical role in maintaining energy homeostasis and cardiac function during ischemia in the heart. However, the functional role of AMPK in the heart during exercise is unknown. We examined whether acute exercise increases AMPK activity in mouse hearts and determined the significance of these increases by studying transgenic (TG) mice expressing a cardiac-specific dominant-negative (inactivating) AMPKalpha2 subunit. Exercise increased cardiac AMPKalpha2 activity in the wild type mice but not in TG. We found that inactivation of AMPK did not result in abnormal ATP and glycogen consumption during exercise, cardiac function assessed by heart rhythm telemetry and stress echocardiography, or in maximal exercise capacity.

Cloning and characterization of mouse 5'-AMP-activated protein kinase gamma3 subunit.

Naturally occurring mutations in the regulatory gamma-subunit of 5'-AMP-activated protein kinase (AMPK) can result in pronounced pathological changes that may stem from increases in muscle glycogen levels, making it critical to understand the role(s) of the gamma-subunit in AMPK function. In this study we cloned the mouse AMPKgamma3 subunit and revealed that there are two transcription start sites, which result in a long form, gamma3L (AF525500) and a short form, gamma3S (AF525501). AMPKgamma3L is the predominant form in mouse and is specifically expressed in mouse skeletal muscle at the protein level. In skeletal muscle, AMPKgamma3 shows higher levels of expression in fast-twitch white glycolytic muscle (type IIb) compared with fast-twitch red oxidative glycolytic muscle (type IIa), whereas gamma3 is undetectable in soleus muscle, a slow-twitch oxidative muscle with predominantly type I fibers. AMPKgamma3 can coimmunoprecipititate with both alpha and beta AMPK subunits. Overexpression of gamma3S and gamma3L in mouse tibialis anterior muscle in vivo has no effect on alpha1 and alpha2 subunit expression and does not alter AMPKalpha2 catalytic activity. However, gamma3S and gamma3L overexpression significantly increases AMPKalpha1 phosphorylation and activity by approximately 50%. The increase in AMPKalpha1 activity is not associated with alterations in glycogen accumulation or glycogen synthase expression. In conclusion, the gamma3 subunit of AMPK is highly expressed in fast-twitch glycolytic skeletal muscle, and wild-type gamma3 functions in the regulation of alpha1 catalytic activity, but it is not associated with changes in muscle glycogen concentrations.