To investigate the role of p38 mitogen activated protein kinase ( p38 MAPK) in the regulation of cytosolic phospholipase A2 ( cPLA2 ) expression and degradation of membrane phospholipids in myocardium in early stage of burn rats.Wistar rats were randomized into normal group (n = 8), burn(n =40) , burn and SB203580(n = 16), burn and isotonic saline( n = 16) groups, with 8 rats at each time-points. There were 5 time-points in burn group, and 2 time-points in other groups. The rats in the latter 3 groups were inflicted with 40% TBSA full-thickness burns, and those in burn and SB203580, burn and isotonic saline groups were administered with SB203580 (p38 MAPK inhibitor) or isotonic saline, respectively. The levels of cPLA2 mRNA and membrane phospholipids in myocardium were detected with RT-PCR. In the same experiment, the effect of SB203580 on cPLA2 expression in rat myocardial cells was determined after hypoxia and burn serum treatment in vitro.The level of myocardial cPLA2 mRNA in burn group at each time-point was obviously higher than those in normal group (0. 280 +/- 0. 020) , and it reached the peak value at 3 PBH. In contrast, the level of cardiac membrane phospholipids was lowered immediately after burns, and it reached the lowest level at 6 PBH [(0. 052 +/- 0. 017) mg phosphorus/mg protein]. Herein, a significant negative correlation was showed between the levels of cPLA2 mRNA and cardiac membrane phospholipids ( r = - 0. 53, P < 0. 05). Administration of SB203580, however, inhibited the increased activity of p38 MAP kinase, suppressed the upregulation of cPLA2(72% and 51% of those in burn and saline group, P <0. 01) , and markedly increased the levels of membrane phospholipids in myocardium at 6 and 12 PBH. In addition, treatment of cardiac myocytes with SB203580 also abolished the upregulation of cPLA2 mRNA elicited by hypoxia and burn serum challenge.p38 MAP kinase play an important role in the burn-induced degradation of cardiac membrane phospholipids in rat through the upregulation of myocardial expression of cPLA2 mRNA in the myocardial cells.
Bismaleimide matrix composites (abbreviated as MT700/802) reinforced by domestic carbon fiber (MT700) were prepared by autoclave process. The properties of tensile, compressive and in-plane shear for quasi-isotropic laminates were comprehensively studied at room temperature and at -55 °C. The experimental results showed that 90° compressive strength and 0° in-plane shear strength at -55 °C were improved by 10% than those at room temperature, however, both of 0° and 90° tensile strength and modulus at -55 °C were reduced by near 9% than those at room temperature. Meanwhile, a component with local variable thickness was developed, and it was tested under the working condition of tensile and shear load. The results exhibited that the tensile failure load of the component run at 31.45 kN and the shear failure load was more than 54.11 kN.
The energy status of a cell plays a key role in its survival, and the exposure of eukaryotic cells to the hypoxia that accompanies the depletion of intracellular ATP triggers specific systemic adaptive responses. AMP-activated protein kinase (AMPK) has emerged as a key regulator of energy metabolism in the heart and plays a critical role in inducing these responses. However, the specific mechanism responsible for AMPK activation in cardiomyocytes at very early stages of hypoxia remain unclear. The goals of this study were to assess the relative contribution to AMPK activation of phosphorylation by AMPK kinase (AMPKK) and of positive allosterism due to AMP:ATP ratios in the early stages of hypoxia. Our results demonstrated that, compared with normoxic controls, neither intracellular AMP concentrations nor AMP:ATP ratios significantly increased within 1h of hypoxia onset. In contrast, a SAMS peptide phosphorylation assay and an immunoblot analysis revealed significant increases in both AMPK activity and ACC phosphorylation within 5min of hypoxic treatment. Furthermore, exposure of cardiomyocytes to hypoxia significantly increased AMPK phosphorylation within 5min, by 3- to 4-fold compared with controls (P<0.01), while overall levels of AMPKα protein did not differ between aerobic and anoxic cardiomyocytes. We also observed increased AMPKK activity in anoxic cardiomyocytes, through use of an α(312) substrate. Taken together, our findings demonstrate that in the early stage of hypoxia in cardiomyocytes, increases in AMPK activity occur prior to and independently of increases in AMP concentration or in the AMP:ATP ratio. Instead, under these circumstances, AMPK is primarily activated by phosphorylation of the conserved Thr-172 residue in its activation loop by its upstream kinase AMPKK.
Keratinocyte migration is an early event in the wound healing process. Although we previously found that CD9 downregulation is required for the keratinocyte migration during wound repair, the mechanism of how CD9 expression is regulated remains unclear. Here, we observed the effect of hypoxia (2% O2) on CD9 expression and keratinocyte migration. CD9 expression was downregulated and keratinocyte migration was increased under hypoxic conditions. In addition, CD9 overexpression reversed hypoxia-induced cell migration. We also found that hypoxia activated the p38/MAPK pathway. SB203580, a p38/MAPK inhibitor, increased CD9 expression and inhibited keratinocyte migration under hypoxia, while MKK6 (Glu) overexpression decreased CD9 expression and promoted hypoxic keratinocyte migration. Our results demonstrate that hypoxia regulates CD9 expression and CD9-mediated keratinocyte migration via the p38/MAPK pathway.
To investigate the influence of peroxidative injury in rat myocardium on the mitochondrial DNA (mtDNA) during early postburn stage.Thirty-six Sprague-Dawley (SD) rats were employed in the study and were randomly divided into sham scald (SS) and scald groups. The rats in scald groups were inflicted with 30% TBSA III degree scalding and were further divided into 1, 3, 6, 12 and 24 post-scald hour (PSH) groups. The mtDNA deletion was determined by semi-quantitative PCR. The rat myocardial tissue samples were harvested and homogenized and the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) were determined.There was no mtDNA deletion in the rat myocardium in SS group. Partial or complete large fragment (4.8 kb) mtDNA deletion in the rat myocardium was identified at 1, 3 and 24 PSHs (P < 0.05 or 0.01). The SOD activity in the rat myocardium significantly decreased at 1 PSH, reaching the lowest level (76.90 +/- 8.30 U/mg) at 6 PSH, but the MDA content increased evidently at 1 PSH, peaking [(3.17 +/- 0.80) nmol/mg] at 6 PSH (P < 0.05).Peroxidative injury to the rat myocardium during early postburn stage might be the principal cause of the 4834 bp deletion of mtDNA in rat myocardium.
Thyristor controlled series compensation (TCSC) is expected to provide various benefits to power systems, including transient stability improvement, subsynchronous resonance (SSR) mitigation, power swings damping, fault current reduction, etc. Grasping the characteristics of TCSC installations and ending effective firing control strategies are essential to implement these advantages. This paper studies two important aspects of TCSC characteristics, the time constant of TCSC dynamic response and impacts of reference signals for thyristor firing angles on TCSC dynamic response. Closed-loop impedance control is proposed to speed the dynamic response of TCSC. A "two-stage" firing control method is presented to accomplish smooth switching between the capacitive region and inductive region. Both digital simulations with EMTDC/PSCAD software and physical experiments are employed in the study.
Vehicular ad hoc networks (VANETs), which are a class of Mobile ad hoc networks, have recently been developed as a standard means of communication among moving vehicles. Congestion is one of the key issues which can restrict the Network performance, and it is no exception for VANETs. Congestion control mechanisms include three phases: congestion detection, congestion notification and rate adjustment. Congestion detection is the first stage of congestion control mechanisms, and it is also the content what this paper focuses on. Because the available resources are limited, and the network topology and node density change over time, applying conventional congestion detection protocols to VANETs can be problematic. To address this challenge, this paper proposes an aggregate parameter based on weights for congestion detection, which can monitor the network performance from the four aspects: Messages Delivered Ratio (MDR), Average Delay (AD), Throughput and Overhead Ratio (OR), so as to achieve the purpose of the congestion detection. In addition, we introduce the method of normalizing the above four parameters, which make the aggregate parameter more accurate. Finally, simulation results show that the aggregate parameter is correct and effective.
This study aimed to elucidate the biological function and upstream regulatory mechanism of CELSR1 in glioma.We evaluated the expression of CELSR1 in glioma by TCGA_GEPIA tool, RT-qPCR, and Western blot assays. CCK-8, wound healing, and transwell invasion assays were, respectively, performed to detect the effect of CELSR1 on cell proliferation, migration, and invasion. The upstream regulatory miRNAs of CELSR1 were predicted by TargetScan and validated by luciferase activity reporter assay.CELSR1 is overexpressed in glioma (P<0.05). CELSR1 promoted glioma cell proliferation, migration and invasion (P<0.01). CELSR1 was a direct target of miR-199a-5p. miR199a-5p mimics significantly inhibited CELSR1 mRNA and protein expression (P<0.01). miR199a-5p mimics reversed the effects of CELSR1 on glioma cell behaviors (P<0.01).CELSR1 acts as an oncogene promoting glioma cell proliferation, migration, and invasion, which is regulated by miR199a-5p.
Tetraspanin CD9 has been implicated in various cellular and physiological processes, including cell migration. In our previous study, we found that wound repair is delayed in CD9-null mice, suggesting that CD9 is critical for cutaneous wound healing. However, many cell types, including immune cells, endothelial cells, keratinocytes and fibroblasts undergo marked changes in gene expression and phenotype, leading to cell proliferation, migration and differentiation during wound repair, whether CD9 regulates kerationcytes migration directly remains unclear. In this study, we showed that the expression of CD9 was downregulated in migrating keratinocytes during wound repair in vivo and in vitro. Recombinant adenovirus vector for CD9 silencing or overexpressing was constructed and used to infect HaCaT cells. Using cell scratch wound assay and cell migration assay, we have also demonstrated that downregulation of CD9 promoted keratinocyte migration in vitro, whereas CD9 overexpression inhibited cell migration. Moreover, CD9 inversely regulated the activity and expression of MMP-9 in keratinocytes, which was involved in CD9-regulated keratinocyte migration. Importantly, CD9 silencing-activated JNK signaling was accompanied by the upregulation of MMP-9 activity and expression. Coincidentally, we found that SP600125, a JNK pathway inhibitor, decreased the activity and expression of MMP-9 of CD9-silenced HaCaT cells. Thus, our results suggest that CD9 is downregulated in migrating keratinocytes in vivo and in vitro, and a low level of CD9 promotes keratinocyte migration in vitro, in which the regulation of MMP-9 through the JNK pathway plays an important role.
Objective: To develop a monoclonal antibodies against Rabies virus phosphoprotein (P), which has conserved domains, and such regions are potentially interesting targets for the diagnosis. Methodes: In the study, a rabies virus p gene coding for the rabies virus phosphoprotein was amplified by PCR and cloned into the expression vector pET32a (+). The recombinant protein was expressed in Escherichia coli, purified with a HisTrap FF crude prepacked column and used to produce mouse monoclonal antibodies (Mabs). The anti-P Mabs were purified and their specificity was tested by indirect ELISA. One specific Mab was further tested by Western blotting against recombinant P and immunofluorescence analysis on cell monolayers infected with rabies virus. Results: The recombinant viral phosphoprotein was successfully expressed as a 53.8 kDa fusion protein which corresponded to the whole protein of the rabies virus. Of the Mabs that were generated, one (4B5) was shown to be highly specific, and could be used to detect recombinant P and native P in the whole virus. Conclusion: The use of a P protein obtained from heterologous expression in E. coli allowed the development of a Mab that interacted specifically with the P protein. This Mab should be useful for future studies with the rabies virus.
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