Inactivation of vitamin B\u3csub\u3e6\u3c/sub\u3e by hydroxyurea in Aspergillus culture media

We found that sublethal levels of Hydroxyurea (HU), an inhibitor of ribonucleotide reductase, inhibited the growth of pyroA4 mutants of Aspergillus nidulans. Growth inhibition increased with the age of the HU-containing medium, and was remediated by supplementation with additional vitamin B6, indicating that HU leads to the inactivation of vitamin B6. HU inactivated vitamin B6 in the presence or absence of media components. These results demonstrate a direct affect of HU on vitamin B6, and they complicate the interpretation of results from experiments involving HU and fungal strains auxotrophic for vitamin B6

Mediation of the Translocation of nNOSμ During Unloading-Induced Atrophy of Skeletal Muscle via NOX2 Inhibition

Mechanical unloading results in detachment of the mu-splice variant of neuronal nitric oxidase synthase (nNOSμ) from the dystrophin-glycoprotein complex and sarcolemma and translocation to the cytosol. We recently found that reactive oxygen species (ROS) play a role in nNOSμ translocation during unloading and muscle atrophy. NOX2, an isoform of NADPH oxidase and source of ROS, may play a causal role in nNOSμ translocation. The purpose of the study was to determine the effectiveness of NOX2 peptidyl inhibition in reducing the translocation of nNOSμ from the sarcolemma and subsequently soleus CSA. Adult male Fisher 344 rats were randomly assigned to one of three groups: CON (control), HU-S (hind limb unloaded with gp91ds-tat scramble) and HU-G (hind limb unloaded with gp91ds-tat). The hind limb unloading period was 7 days. Mean body weights for CON (353.26 g ± 15.47), HU-S (305.14 g ± 18.18) and HU-G (306.34 g ± 16.84) at the beginning of the experiment were not significantly different. Muscle mass/body mass ratio for the gastrocnemius complex (gastrocnemius, plantaris and soleus) was significantly reduced in HU-S rats (10.08 mg/g ± 0.24) but was maintained in HU-G rats (10.88 mg/g ± 0.47). SMASH analysis revealed that average soleus CSA in HU-G rats (3293.08 μm2 ± 46.82) decreased significantly less than HU-S rats (2606.66 μm2 ± 33.46) compared with ambulatory controls (p \u3e 0.0001). Immunofluorescence and staining of nNOS activity with NADPH Diaphorase of soleus tissue showed considerable loss of sarcolemmal nNOSμ in the HU-S group while the HU-G group revealed substantial maintenance of nNOSμ at the sarcolemma. The results of this study suggest that NOX2 inhibition via gp91ds-tat is effective in reducing the translocation of nNOSμ from the sarcolemma to the cytosol and maintaining CSA of the soleus with mechanical unloading via the inhibition of NOX2

Treatment with EUK-134 Enhance Anabolic Akt/mTOR/p70S6kinase Pathways, Protecting Against Muscle Atrophy in the Rat Soleus in a 7 Day Hindlimb Unloading

Muscle atrophy occurs with mechanical unloading, disease and aging. Mechanical unloading occurs with bedrest, microgravity, and casting/immobilization, all of which elicit the unloading-induced phenotype: (a) reduction of muscle fiber cross-section area (CSA) and (b) a shift from slow-twitch to fast-twitch muscle fibers. Unloading-induces muscle fiber atrophy appears to be a consequence of both decrease in contractile protein synthesis combined with increased protein catabolism. Oxidative stress has been cited as potential contributing factor to the unloading phenotype, although the mechanisms remain unresolved and controversial. We recently reported that a superoxide dismutase/catalase mimetic (EUK-134) reduced muscle atrophy, fiber-type shift, and pro-catabolic signaling. The purpose of this study was to test the hypothesis that EUK-134 would attenuate loss in a canonical, pro-anabolic signaling pathway involving phosphorylation of Akt, mTOR, and p70S6K and would ameliorate muscle fiber atrophy after seven-days hindlimb unloading. Male Fisher-344 rats were divided into three groups: ambulatory control group (CON, N=11), 7 days of hindlimb unloading plus saline injections (HU, n=11), or 7 days of hindlimb unloading plus 3 mg/kg/day EUK-134 (HU-EUK, n=9). The soleus muscles from both hindlimbs were dissected for histochemistry and immunoflorescence analyses. The soleus muscle of the HU group exhibited a decrease in CSA compared to the CON group, while soleus muscle fiber CSA from HU-EUK group was greater than the HU group, but lower than CON. Furthermore, the HU group displayed a partial shift from slow-twitch to fast-twitch, an effect attenuated by EUK-134. The ratio of phosphorylated Akt to total Akt was significant lower in the HU group compared to the CON group. However, the p-Akt/Akt was significantly higher in the HU-EUK group vs. HU group, but lower than the CON. The ratio of phosphorylated m-TOR to total m-TOR from the HU group was not different than controls. However, the ratio of p-mTOR/mTOR was a significantly higher in the EUK group compared to the CON group. Moreover, the HU group displayed a decrease in phosphorylation of p70S6Kinase, while but treatment with EUK-134 enhanced phosphorylation of p70S6Kinase. Therefore, our data are consistent with the hypothesis that EUK-134 protects anabolic signaling in the unloaded rat soleus

Resistance exercise during hindlimb unloading in rats mitigates disuse-induced bone loss

Disuse-related bone loss affects various populations of individuals including astronauts, bedridden patients, those with spinal cord injuries, and, to not as drastic a degree, the increasing population living sedentary lifestyles. The lack of mechanical stimulation on bones can lead to bone loss and an increased risk of low bone mass, osteoporosis, and fractures. Therefore, the goal of this study was to determine the effects of resistance exercise on bone during a period of disuse in skeletally mature rodents. Male rats were placed into three different groups, hindlimb unloading, (HU), hindlimb unloading with resistance training (HU+RE), and cage control (CC), for 4 weeks. Resistance exercise was accomplished by jump training 3 days a week with progressive increases in weights added to a vest placed on the rat (10% - 40% body weight in added weight). The right tibia of each rat was harvested and bone mass parameters were measured using peripheral quantitative computed tomography (pQCT) scans of the proximal metaphysis and midshaft tibia. At the proximal tibia, HU rat means were significantly lower than those of CC in total mineral content, cortical content, and cancellous density (p\u3c0.05). HU+RE had significantly higher total bone content, trabecular content, cancellous density, and cortical content than the HU group (p\u3c0.01). Total content was significantly different between CC and HU+RE (p\u3c0.05). No differences were seen among group means for any variable at the midshaft tibia (100% cortical bone). Resistance exercise can mitigate bone loss during a period of disuse, especially in the cancellous-rich compartment of metaphyseal bone. This highlights the importance of resistance training during periods of disuse, as well as demonstrating the importance of regular loading of bone for individuals leading sedentary lifestyles