The Root Extract of Gentiana macrophylla Pall. Alleviates Cardiac Apoptosis in Lupus Prone Mice

The roots of the perennial herb Gentiana macrophylla Pall. (GM) are known as Qinjiao, which has been used for centuries to treat systemic lupus erythematosus (SLE). However, little is known about the effects of GM on cholesterol-aggravated cardiac abnormalities in SLE, and the mechanisms thereof. This study investigates whether GM exhibits anti-apoptotic effects, focusing on the left ventricle (LV) of NZB/W F1 mice fed with high-cholesterol diet. The morphology and apoptotic status of ventricular tissues were determined by microscopy and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Levels of apoptotic biomarkers were determined by immunoblotting. The results thus obtained revealed that GM significantly reduced the cholesterol-aggravated apoptosis of LV in NZB/W F1 mice by suppressing both intrinsic and extrinsic apoptotic pathways. Additionally, GM significantly increased the cardiac insulin-like growth factors (IGF)-1 survival signaling and anti-apoptotic proteins in LV tissues. Accordingly, GM is considered to be beneficial in alleviating cholesterol-aggravated cardiac damage in SLE, and therefore constitute an alternative treatment for SLE patients with cardiac abnormalities.     

Akt suppresses DLK for maintaining self-renewal of mouse embryonic stem cells

Mouse embryonic stem cells (ES cells) can proliferate indefinitely. To identify potential signals involved in suppression of self-renewal, we previously screened a kinase/phosphatase expression library in ES cells, and observed that inhibition of Dual Leucine zipper-bearing Kinase (DLK) increased relative cell numbers. DLK protein was detected in both the pluripotent and differentiated states of mouse ES cells while DLK kinase activity increased upon differentiation. Overexpression of DLK in mouse ES cells displayed reductions in relative cell/colony numbers and Nanog expression, suggesting a suppressive role of DLK in self-renewal. By examining protein sequences of DLK, we identified 2 putative Akt phosphorylation sites at S584 and T659. Blocking PI3K/Akt signaling with LY-294002 enhanced DLK kinase activity dramatically. We found that Akt interacts with and phosphorylates DLK. Mutations of DLK amino acid residues at putative Akt phosphorylation sites (S584A, T659A, or S584A and T659A) diminished the level of DLK phosphorylation. While the mutated DLKs (S584A, T659A, or S584A and T659A) were expressed, a further reduction in cell/colony numbers and Nanog expression appeared in mouse ES cells. In addition, these mutant DLKs (S584A, T659A, or S584A and T659A) exhibited more robust kinase activity and cell death compared to wild type DLK or green fluorescence (GFP) controls. In summary, our results show that DLK functions to suppress self-renewal of mouse ES cells and is restrained by Akt phosphorylation.     

Involvement of the residues of GSKIP, AxinGID, and FRATtide in their binding with GSK3β to unravel a novel C-terminal scaffold-binding region

The specificity and regulation of GSK3β are thought to involve in the docking interactions at core kinase domain because of the particular amino acid residues. Recent X-ray diffraction studies illuminated the relative binding residues on AxinGID and FRATtide for GSK3β docking and appeared that GSK3β Val267Gly (V267G) and Tyr288Phe (Y288F) could distinguish the direct interaction between AxinGID and FRATtide. In order to explore the mode that involved the binding of GSKIP to GSK3β and compare it with that of AxinGID and FRATtide, we pinpointed the binding sites of GSKIP to GSK3β through the single-point mutation of four corresponding sites within GSK3β (residues 260–300) as scaffold-binding region I (designated SBR-I_260–300). Our data showed that these three binding proteins shared similar binding sites on GSK3β. We also found that the binding of GSK3β V267G mutant to GSKIP and AxinGID, but not that of Y288F mutant (effect on FRATtide), was affected. Further, based on the simulation data, the electron-density map of GSKIPtide bore closer similarity to the map AxinGID than to that of FRATtide. Interestingly, many C-terminal helix region point-mutants of GSK3β L359P, F362A, E366K, and L367P were able to eliminate the binding with FRATtide, but not AxinGID or GSKIP. In addition, CABYR exhibited a unique mode in binding to C-terminal helix region of GSK3β. Taken together, our data revealed that in addition to the core kinase domain, SBR-I_260–300, another novel C-terminus helix region, designated SBR-II_339–383, also appeared to participate in the recognition and specificity of GSK3β in binding to other specific proteins.     

Effects of insulin-like growth factor 1 on muscle atrophy and motor function in rats with brain ischemia

Although insulin-like growth factor 1 (IGF 1) has been used in immobilizated muscles to prevent muscle atrophy, its effects on muscle atrophy after brain ischemia are not known. This study aimed to determine the effects of IGF 1 on preventing muscle atrophy in rats with brain ischemia. Middle cerebral artery occlusion (MCAO) was used to induce the brain ischemia. In the first part of the study, rats were assigned to sham control, ischemic control, and ischemia with different dosages of IGF 1 injection groups to determine the optimal dosage of IGF 1 on preventing muscle atrophy after brain ischemia. In the second part of the study, rats were assigned to sham control, ischemic control, ischemia with IGF 1, or with IGF 1 receptor inhibitor (AG1024) injection groups to determine the specificity of IGF 1 on preventing muscle atrophy after brain ischemia. IGF 1 or AG1024 was injected locally to calf muscles and anterior tibialis (TA) starting from one day after brain ischemia and injections were carried out every other day for 4 times. Muscle weight and myosin heavy chain (MHC) expression in both red (red gastrocnemius and soleus) and white (white gastrocnemius and TA) muscles were significantly decreased after brain ischemia. With at least moderate-dosage (200 ng/100 microl PBS) IGF 1 injection, the muscle weight and MHC protein could be restored in both red and white muscles resulting in better motor performance. However, the high-dose injection of IGF 1 (400 ng/100 microl PBS) did not result in further effects. IGF 1 increased the expression of p-Akt, but such effects were prevented by AG1024 resulting in muscle atrophy and poor motor function. In conclusion, peripheral application of IGF 1 not only prevented muscle atrophy but also enhanced motor function in rats with brain ischemia. The IGF 1-induced PI3K/Akt pathways are important for preventing muscle atrophy induced by brain ischemia.     

Effect of mild intermittent hypoxia on glucose tolerance, muscle morphology and AMPK-PGC-1alpha signaling

The main goal of this study was to investigate the long-term effect of daily 8-hour mild intermittent hypoxia (14-15% O2) on glucose tolerance and muscle morphology of Sprague-Dawley rats. The involvement of AMPK-PGC-1alpha-VEGF signaling pathways in the skeletal muscle was also determined during the first 8 hours of hypoxia. We found that mRNA levels of VEGF and PGC-1alpha were significantly increased above control after 8-h mild hypoxia without a change in AMPK phosphorylation. After 8 weeks of mild intermittent hypoxia treatment, plasma glucose and insulin levels in oral glucose tolerance test (OGTT), epididymal fat mass, and body weight were significantly lower compared to the control group. While soleus muscle weight was not changed, capillary and fiber densities in the hypoxia group were 33% and 35% above the control suggesting reorganization of muscle fibers. In conclusion, our data provide strong evidence that long-term mild intermittent hypoxia decreases the diffusion distance of glucose and insulin across muscle fibers, and decreases adiposity in rats. These changes may account for the improved glucose tolerance observed following the 8-week hypoxia treatment, and provides grounds for investigating the development of a mild non-pharmacological intervention in the treatment of obesity and type 2 diabetes.     

Reassortment and concerted evolution in banana bunchy top virus genomes

The nanovirus Banana bunchy top virus (BBTV) has six standard components in its genome and occasionally contains components encoding additional Rep (replication initiation protein) genes. Phylogenetic network analysis of coding sequences of DNA 1 and 3 confirmed the two major groups of BBTV, a Pacific and an Asian group, but show evidence of web-like phylogenies for some genes. Phylogenetic analysis of 102 major common regions (CR-Ms) from all six components showed a possible concerted evolution within the Pacific group, which is likely due to recombination in this region. The CR-M of additional Rep genes is close to that of DNA 1 and 2. Comparison of tree topologies constructed with DNA 1 and DNA 3 coding sequences of 14 BBTV isolates showed distinct phylogenetic histories based on Kishino-Hasegawa and Shimodaira-Hasegawa tests. The results of principal component analysis of amino acid and codon usages indicate that DNA 1 and 3 have a codon bias different from that of all other genes of nanoviruses, including all currently known additional Rep genes of BBTV, which suggests a possible ancient genome reassortment event between distinctive nanoviruses.     

Parasympathetic nervous activity mirrors recovery status in weightlifting performance after training

Heart rate variability (HRV) and parasympathetic power are closely related to the well-being and health status in humans. The main goal of the study was to determine whether these measures can reflect recovery status after weight training. After a 10-day detraining period, 7 weightlifters were challenged with a 2-hour weight training which elicited approximately fourfold increases in circulating muscle creatine kinase level and protracted pain feeling (p < 0.05). Weightlifting performance was then evaluated 3, 24, 48, and 72 hours after training to determine the degree of recovery from fatigue. Heart rate variability, circulating dehydroepiandrostendione sulfate (DHEA-S), and muscle damage markers were measured before each performance test. An electrocardiogram was recorded for 5 minutes continuously at rest in seated positions. After training, weightlifting performance of the subjects decreased below baseline in paralleled with suppressed parasympathetic power (high-frequency [HF] HRV), whereas sympathetic power (normalized low-frequency HRV) was slightly elevated at 3 hours of recovery (p < 0.05). Both weightlifting performances and parasympathetic power returned to baseline values in 24 hours and further increased above baseline during 48-72 hours of recovery in a similar fashion (p < 0.05). Circulating DHEA-S level dropped at 24 hours (p < 0.05) and returned to normal values by 48 hours. Muscle pain increased at 3 hours after training and remained higher than baseline values for the 72-hour recovery period (p < 0.05). Our data suggest that parasympathetic power, indicated by HF HRV, is able to reflect the recovery status of weightlifters after training.     

Identification and characterization of the nuclear import and export signals of the mammalian Ste20-like protein kinase 3

Mst3, a human Ste20-like protein kinase, has been recently demonstrated to undergo a caspase-mediated cleavage during apoptosis. The proteolytic cleavage of the C-terminus of Mst3 caused nuclear translocation of its kinase domain. This work provides evidence that Mst3 may contain a bipartite-like nuclear localization sequence (NLS) at the C-terminus of its kinase domain (residues 278-292). The removal of NLS from the kinase domain of Mst3 led to the cytoplasmic accumulation of EGFP-Mst3(Delta277). The presence of nuclear exporting signals in the Mst3 was also demonstrated by leptomycin B-treatment and serial deletion of the C-terminal regulatory domain of Mst3. A nuclear export signal was also postulated to be in the regions of amino acids 335-386. In conclusion, Mst3 contains both NLS and NES signals, which may cooperate to control the subcellular distribution of Mst3.     

Glucose tolerance and insulin sensitivity following an one-week volleyball competition

The purpose of the study was to compare glucose tolerance and insulin sensitivity between trained (TR) and competition (CP) states, in relation to cortisol and testosterone levels. Sixteen highly trained volleyball players voluntarily participated in this study. The first testing session (TR state) occurred 1 week before the start of national level volleyball CP, and the second testing session (CP state) occurred next morning after the 1-week CP. Fasted serum sample was used for measuring cortisol and testosterone. Subjects were then orally challenged with 75 g of glucose solution for determinations of oral glucose tolerance test (OGTT) and insulin response. Under both fasted and glucose challenged conditions, glucose levels of CP were not different from TR state, whereas insulin levels of CP were significantly elevated above TR (50 min: from 78.8 +/- 8.7 to 96.6 +/- 8.1 microU/ml, P < 0.05; 80 min: from 62.8 +/- 7.0 to 82.0 +/- 7.3; P < 0.05). Muscle creatine kinase (CK) level in blood was significantly increased above TR, suggesting greater muscle damage by CP. Serum leptin level, percent fat mass, and body weight were not different between two states. CP significantly increased serum cortisol level without significantly change in testosterone level. The new finding of the study was that volleyball CP reduced the whole-body insulin sensitivity significantly compared to TR state. The greater level of insulin concentration under CP state appears to be associated with elevated serum cortisol level. Despites the benefit of increased physical activity on metabolic function is widely recognized, physiological stress associated with CP can result in attenuation of systemic insulin sensitivity compared TR state.