Background: Bupivacaine is an amide type local anesthetic which is widely used for epidural anesthesia and nerve blockade in patients. However, local administration of bupivacaine could cause neuron injury showing transient neurologic symptoms. α-Lipoic acid (LA) was shown to protect nerve cells from substance-induced injury. We hypothesized that LA administration could attenuate bupivacaine-induced neurotoxicity. Methods: To evaluate our hypothesis, we treated mouse neuroblastoma N2a cells with LA 30 min before the cells were exposed to bupivacaine. We evaluated cellular injury by examination of cell viability, morphology changes, nuclear condensation, and Annexin V staining. We also examined the levels of intracellular reactive oxygen species (ROS) and activation of PI3K/Akt signaling pathway. In a separate experiment, we determined the effect of Akt inhibition on cell viability in the presence of LA and bupivacaine. Results: Bupivacaine treatment significantly induced cell injury as evidenced by decreased cell viability, increased nuclear condensation and Annexin V staining. Administration of LA significantly attenuated bupivacaine-induced cell injury. In addition, LA treatment increased the levels of phospho-Akt and phospho-GSK3β and attenuated bupivacaine decreased the levels of ROS. More significantly, pharmacological inhibition of Akt abolished the LA-induced protection from bupivacaine-caused cell injury. Conclusions: Our findings suggest that pretreatment of neuroblastoma cells with LA protected neural cells from bupivacaine-induced injury. The mechanisms involve activation of the PI3K/Akt signaling pathway.

We examined the role of Tollip in the hypertrophic response of cardiomyocytes. C57BL/6 mice were subjected to transverse aortic constriction (TAC) for 2 weeks and age-matched sham surgical operated mice served as control. TAC significantly reduced the association of Tollip with IRAK-1 by 66.4 percent and increased NF-kappaB binding activity by 86.5 percent and the levels of phosphop38 by 114.6 percent in the myocardium compared with sham control, respectively. In vitro experiments showed that IL-1beta stimulation also significantly reduced the association of Tollip with IRAK-1 and increased NFkappaB binding activity in neonatal cardiomyocytes. Tollip overexpression by transfection of cardiac myocytes significantly attenuated the IL-1beta-induced hypertrophic response of cardiac myocytes as evidenced by reduced cell size (16.4 percent) and decreased ANP expression (33.3 percent). Overexpression of Tollip also reduced NFkappaB binding activity by 30.7 percent and phospho-p38 by 47.1 percent, respectively. The results suggest that Tollip could be a negative regulator during the development of cardiac hypertrophy. The negative regulation of cardiac hypertrophy by Tollip may involve downregulation of the MyD88-dependent NF-kappaB activation pathway.

Objective: We have previously demonstrated that nuclear factor kappa B (NFκB) activation is needed for the development of cardiac hypertrophy in vivo. NFκB is a downstream transcription factor in the Toll-like receptor (TLR)-mediated signaling pathway; therefore, we investigated a role of TLR4 in cardiac hypertrophy in vivo. Methods: TLR4-deficient mice (C.C3H-Tlr4 lps-d, n = 6), wild-type (WT) genetic background mice (BALB/c, n = 6), TLR4-deleted strain (C57BL/10ScCr, n = 8), and WT controls (C57BL/10ScSn, n = 8) were subjected to aortic banding for 2 weeks. Age-matched surgically operated mice served as controls. In a separate experiment, rapamycin (2 mg/kg, daily) was administered to TLR4-deficient mice and WT mice immediately following aortic banding. The ratio of heart weight/body weight (HW / BW) was calculated, and cardiac myocyte size was examined by FITC-labeled wheat germ agglutinin staining of membranes. NFκB binding activity and the levels of phospho-p70S6K in the myocardium were also examined. Results: Aortic banding significantly increased the ratio of HW / BW by 33.9% (0.601 ± 0.026 vs. 0.449 ± 0.004) and cell size by 68.4% in WT mice and by 10.00% (0.543 ± 0.011 vs. 0.495 ± 0.005) and by 11.8% in TLR4-deficient mice, respectively, compared with respective sham controls. NFκB binding activity and phospho-p70S6K levels were increased by 182.6% and 115.2% in aortic-banded WT mice and by 78.0% and 162.0% in aortic-banded TLR4-deficient mice compared with respective sham controls. In rapamycin-treated aortic-banded mice, the ratio of HW / BW was increased by 18.0% in WT mice and by 3.5% in TLR4-deficient mice compared with respective sham controls. Conclusion: Our results demonstrate that TLR4 is a novel receptor contributing to the development of cardiac hypertrophy in vivo and that both the TLR4-mediated pathway and PI3K/Akt/mTOR signaling are involved in the development of cardiac hypertrophy in vivo.

We examined whether blocking the MyD88 mediated pathway could protect myocardium from ischemia/reperfusion (I/R) injury by transfecting Ad5-dnMyD88 into the myocardium of rats (n = 8) 3 days before the hearts were subjected to ischemia (45 min) and reperfusion (4 h). Ad5-GFP served as control (n = 8). One group of rats was (n = 8) subjected to I/R without transfection. Transfection of Ad5-dnMyD88 significantly reduced infarct size by 53.6% compared with the I/R group (15.1 ± 3.02 vs 32.5 ± 2.59) while transfection of Ad5-GFP did not affect I/R induced myocardial injury (35.4 ± 2.59 vs 32.5 ± 2.59). Transfection of Ad5-dnMyD88 significantly inhibited I/R-enhanced NFκB activity by 50% and increased the levels of phospho-Akt by 35.6% and BCL-2 by 81%, respectively. Cardiac myocyte apoptosis after I/R was significantly reduced by 59% in the Ad5-dnMyD88 group. The results demonstrate that both inhibition of the NFκB activation pathway and activation of the Akt signaling pathway may be responsible for the protective effect of transfection of dominant negative MyD88.

No description available.

Abscisic acid is an important plant hormone in the responses to biotic and abiotic stresses, which also regulates various growth and developmental processes in plants. Three major components-receptors (PYRs), the PP2C type phosphatases and the SnRK2 subtype kinases form a double negative regulatory system: PYR/PYL/RCARs inhibit the activity of PP2Cs while PP2Cs inhibit that of SnRK2s in ABA signaling pathway. The results of my studies showed that ABA would directly affect the interaction between SnRK2.2 and ABI1 in absence of PYRs. Furthermore, ABA can inhibit the catalytic activity of the SnRK2.2 kinase. These findings indicated that ABA may directly interact with SnRK2.2. Posttranslational modifications play a key role in signal transduction. Phosphorylation is the most important posttranscriptional modification in ABA signal transduction. To dissect new components in ABA signaling network in plants, proteomics studies were carried out in Arabidopsis for identifying ABA- responsive phosphoproteins. Ten phosphoproteins, ATPB, ATPC1, FBA1, CTIMC, GGAT1, GAPC1, GAPC2, GAPA1 and ALDH11A3, were identified by 2-DE proteomic approach and LC-MS/MS analysis. These proteins are likely to be the potential phosphorylated targets of SnRK2s in ABA signaling network. Lysine acetylation (LysAc) also emerges as one of the important posttranslational modifications for protein regulation in plants. Eleven lysine acetylated proteins with altered acetylation in response to ABA were identified in Arabidopsis using proteomic approach. The increased LysAc of Rubisco and the decreased Rubisco activity by ABA treatment indicates the acetylation of Rubisco caused by ABA resulted in the inhibition of Rubisco activity. ABA has also been shown to exist and function in both lower animals and mammalians. The medical application of ABA has become a new area of investigation. To explore the role of protein phosphorylation in ABA-mediated function in mammalians, phosphoproteomic study was conducted in mouse 3T3-L1 cells. Ten phosphoproteins with significant changes in serine/threonine phosphorylation in response to ABA were identified. These results suggest these phosphoproteins are involved in ABA signaling network in mouse cells. The significance of the function of SFRS1, ANXA1 and Galectin-3 on human diseases indicated that ABA could be a potential treatment for some human diseases, such as cancer.

Cryphonectria parasitica is the causal agent of chestnut blight, which devastated the American Chestnut tree population in the early 20th century. The discovery of hypoviruses that reduce the severity of the chestnut blight infection offers the potential for biological control. However, the spread of the hypoviruses is hampered by a diverse genetically controlled nonself-recognition system, vegetative incompatibility (vic). CPVIB-1 was identified as a transcription regulator playing an important role in the programmed cell death response to this stimulus. In this study, we have found that CPVIB-1 is ubiquitin-decorated which might lead to its degradation in the proteasome pathway. RNA-Seq and ChIP-Seq were used to further explore the downstream targets of CPVIB-1 that mediate the various metabolic changes that lead to the altered phenotype of the Δcpvib-1 mutant. Due to inaccuracies in the prior annotation, we performed a genome re-annotation to improve the accuracy using a MAKER2-two-pass pipeline. To validate the improvement a second pipeline, PEPA, was developed to compare quality metrics between the old and new annotations. Approximately 1/3 of the original annotations from 2009 were found to be inaccurate. Experimental confirmation by testing 27 predicted genes using a diagnostic PCR protocol to differentiate between prior and new transcript structures showed that over 80 % of tested genome locations supported for the new annotation. Using rapamycin treatment to mimic stimulation of the vic response and applying the RNA-seq and ChIP-seq data to this new information, we found that CPVIB-1 is related to TOR signaling pathways, promoting autophagy and the proteasome pathway, but repressing carbon metabolism, protein and lipid biosynthesis. In depth analysis of CPVIB-1-bound DNA targets showed that this protein is a member of the GAGA regulator family, a group of multifaceted transcription factors with diverse roles in gene activation and repression, maintenance of mitosis, and cell development. Following treatment with rapamycin the recognition sequence bound by CPBVIB-1 was altered leading to the regulation of different suite of genes with diverse metabolic functions. Ultimately, we have developed a revised model of TOR signaling pathway where TORC1 and TORC2 signaling pathways are connected by the action of CPVIB1.

No description available.

No description available.

No description available.