NGF Attenuates High Glucose-Induced ER Stress,Preventing Schwann Cell Apoptosis by Activating the PI3K/Akt/GSK3β and ERK1/2 Pathways

Diabetic peripheral neuropathy (DPN) is one of the most common and troublesome complications of diabetes mellitus. It has been demonstrated that nerve growth factor (NGF) exerts a pivotal role in the regulation of neuronal growth and the promotion of DPN recovery. However, the exact molecular mechanisms are not well understood. Recent studies have indicated that as a novel therapeutic target, endoplasmic reticulum (ER) stress participates in the onset and progression of DPN. In the present study, it has been demonstrated that NGF prevents the sciatic nerve from degeneration and demyelination in DPN rats. Thus, RSC 96 cells, which retain the characteristic features of Schwann cells (SCs), were cultured in medium containing 30 mM glucose (high glucose, HG) to mimic SCs in DPN mice. The 50-ng/ml dose of NGF was identified to be the optimal concentration for treating an excessive ER stress level under HG conditions for 24 h. We found that NGF treatment significantly inhibits HG-induced ER stress and subsequently suppresses ER-related apoptosis. Further, NGF administration also activates the upstream signaling pathway of ER stress, PI3K/Akt/GSK3β signaling and ERK1/2 signaling. Co-treatment with the PI3K inhibitor LY294002 or ERK1/2 inhibitor U0126 significantly reverses the protective role of NGF on HG-induced excessive ER stress and subsequent apoptosis. These observations suggest that the neuroprotective role of NGF in DPN is mediated by the inhibition of excessive ER stress via the activation of the PI3K/Akt/GSK3β and ERK1/2 signaling pathways.     

Amphotericin B: How Much Is Enough?

The total curative dose of amphotericin B for any given fungal infection or specific patient is not precisely known. Prior to the availability of lipid formulations of amphotericin B (LFAB), dosing of amphotericin B was dictated by its associated toxicity. The unique pharmacokinetic features of each LFAB have led to differences in their recommended doses. Most published data have evaluated doses of 3–5 mg/kg/day, although niches exist for both higher and lower doses. Low-dose LFAB allows for intravenous broad-spectrum antifungal coverage without drug-drug interactions and with reduced toxicity. High-dose LFAB demonstrates increased fungal clearance in animal models, although this has not translated to improved clinical outcomes for most invasive fungal infections. Although available data do not demonstrate significant benefit associated with high-dose therapy, for liposomal amphotericin B, the data also demonstrate no significant harm. As such, the use of high-dose liposomal amphotericin B for salvage therapy may be a consideration.     

Characterization of 5-enolpyruvylshikimate 3-phosphate synthase gene from Camptotheca acuminata

5-enolpyruvylshikimate 3-phosphate synthase (EPSPS; 3-phosphoshikimate 1-carboxyvinyl-transferase; EC 2.5.1.19) is a critical enzyme in the shikimate pathway. The full-length EPSPS cDNA sequence (CaEPSPS, GenBank accession number: AY639815) was cloned and characterized for the first time from woody plant, Camptotheca acuminata, using rapid amplification of cDNA ends (RACE) technique. The full-length cDNA of CaEPSPS was 1778 bp containing a 1557 bp ORF (open reading frame) encoding a polypeptide of 519 amino acids with a calculated molecular mass of 55.6 kDa and an isoelectric point of 8.22. Comparative and bioinformatic analyses revealed that CaEPSPS showed extensive homology with EPSPSs from other plant species. CaEPSPS contained two highly conserved motifs owned by plant and most bacteria EPSPSs in its N-terminal region. Phylogenetic analysis revealed that CaEPSPS belonged to dicotyledonous plant EPSPS group. Tissue expression pattern analysis indicated that CaEPSPS was constitutively expressed in leaves, stems and roots, with the lower expression being found in roots. The coding sequence of CaEPSPS gene was successfully subcloned in a plasmid-Escherichia coli system (pET-32a), and the cells containing the plasmid carrying the CaEPSPS gene exhibited enhanced tolerance to herbicide glyphosate, compared to the control.     

MARCH5 restores endothelial cell function against ischaemic/hypoxia injury via Akt/eNOS pathway

MARCH5 is a critical regulator of mitochondrial dynamics, apoptosis and mitophagy. However, its role in cardiovascular system remains poorly understood. This study aimed to investigate the role of MARCH5 in endothelial cell (ECs) injury and the involvement of the Akt/eNOS signalling pathway in this process. Rat models of myocardial infarction (MI) and human cardiac microvascular endothelial cells (HCMECs) exposed to hypoxia (1% O₂) were used in this study. MARCH5 expression was significantly reduced in ECs of MI hearts and ECs exposed to hypoxia. Hypoxia inhibited the proliferation, migration and tube formation of ECs, and these effects were aggravated by knockdown of MARCH5 but antagonized by overexpressed MARCH5. Overexpression of MARCH5 increased nitric oxide (NO) content, p-eNOS and p-Akt, while MARCH5 knockdown exerted the opposite effects. The protective effects mediated by MARCH5 overexpression on ECs could be inhibited by eNOS inhibitor L-NAME and Akt inhibitor LY294002. In conclusion, these results indicated that MARCH5 acts as a protective factor in ischaemia/hypoxia-induced ECs injury partially through Akt/eNOS pathway.     

PKC pathway associated with the expression of an A-type K+ channel induced by TGF-beta1 in rat vascular myofibroblasts

Our previous study indicated that TGF-beta1 induced the expression of a transient outward K+ channel (A-type) during the phenotypic transformation of vascular fibroblasts to myofibroblasts. Here, we studied the relevant signal transduction pathway using whole cell recording and a quantitative RT-PCR technique. Results indicate that the protein kinase C (PKC) agonist phorbol-12-myristate-13-acetate (PMA, 1 microM) could mimic the effect of TGF-beta1 (20 ng/ml) on the expression of an A-type K+ channel and induced a similar A-type K+ current. Moreover, a PKC inhibitor, bisindolylmaleimide I (1 microM), could abrogate the effect of TGF-beta1 on K(V)4.2 expression. This result suggests that a PKC pathway may be involved in the expression of an A-type K+ channel induced by TGF-beta1 in rat vascular myofibroblasts.     

Heteronuclear nuclear magnetic resonance assignments, structure and dynamics of SUMO-1, a human ubiquitin-like protein

The structure of a ubiquitin-like protein, small ubiquitin-related modifier-1 (SUMO-1), was earlier determined using homonuclear nuclear magnetic resonance (NMR) spectroscopy, since the spectral quality of the protein was not suitable for heteronuclear NMR data collection. In this study, a slightly different construct of the SUMO-1 gene was used for protein over-expression. The protein purified from this construct showed high spectral qualities, therefore, multi-dimensional heteronuclear NMR data for a dynamic study and structural determination were acquired. The structure of SUMO-1 obtained in this study differs in several respects from the structure obtained from homonuclear NMR data. Furthermore, structural differences were observed between the new SUMO-1 and ubiquitin structures. These differences may be important for SUMO-1-specific recognition in cells. Additionally, relaxation parameters indicate that SUMO-1 undergoes highly anisotropic tumbling in solution and that the long amino (N)-terminal sequence of SUMO-1 is highly dynamic with increasing flexibility towards the end.     

Protein Interactions and Regulation of EscA in Enterohemorrhagic E. coli

Infections caused by enterohemorrhagic Escherichia coli (EHEC) can lead to diarrhea with abdominal cramps and sometimes are complicated by severe hemolytic uremic syndrome. EHEC secretes effector proteins into host cells through a type III secretion system that is composed of proteins encoded by a chromosomal island, locus for the enterocyte effacement (LEE). EspA is the major component of the filamentous structure connecting the bacteria and the host''s cells. Synthesis and secretion of EspA must be carefully controlled since the protein is prone to polymerize. CesAB, CesA2, and EscL have been identified as being able to interact with EspA. Furthermore, the intracellular level of EspA declines when cesAB, cesA2, and escL are individually deleted. Here, we report a LEE gene named l0033, which also affects the intracellular level of EspA. We renamed l0033 as escA since its counterpart in enteropathogenic E. coli has been recently described. Similar to CesAB, EscL, and CesA2, EscA interacts with EspA and enhances the protein stability of EspA. However, EscA is also able to interact with inner membrane-associated EscL, CesA2, and EscN, but not with cytoplasmic CesAB. In terms of gene organizations, escA locates in LEE3. Expression of EscA is faithfully regulated via Mpc, the first gene product of LEE3. Since Mpc is tightly regulated to low level, we suggest that EscA is highly synchronized and critical to the process of escorting EspA to its final destination.     

RETRACTED ARTICLE: The Critical Role of SRPK1 in EMT of Human Glioblastoma in the Spinal Cord

Up to now, the serine-arginine protein kinase 1 (SRPK1) has been suggested as an important signal mediator, which is implicated in the development of cancers. Unfortunately, some molecular pathways in SRPK1-mediated epithelial-mesenchymal transition (EMT) in human spinal glioblastoma have been not elucidated. In this work, we detected the expression of SRPK1 in human spinal glioblastoma tissues and GBM cell lines and analyzed the relevant molecular proteins using in vitro experiments, including RT-PCR, gene silencing, and Western blot. In this study, RT-PCR and Western blot revealed that the expression of SRPK1 mRNA and protein became higher in all six spinal glioblastoma specimens; however, its expression was low in matched normal specimens. We also demonstrated SRPK1 expression facilitated the proliferation of U87 and U251 cells and inhibited the apoptosis in U87 and U251 cells. Also, SRPK1 promoted the expression of EMT-regulating markers, involving N-cadherin, Snail, and MMP9 and decreased the expression of mesenchymal marker E-cadherin. Moreover, knockdown of SRPK1 significantly inhibited the expression levels of p-Akt rather than t-Akt. In conclusion, knockdown of SRPK1 inhibited glioblastoma cell proliferation, invasion, and EMT process via suppressing p-Akt signaling pathway. This study also lays a new foundation for the clinically biological treatment.