The Potential Neuroprotection Mechanism of GDNF in the 6-OHDA-Induced Cellular Models of Parkinson’s Disease

The glial cell line-derived neurotrophic factor (GDNF) potential as a therapeutic agent for the treatment of Parkinson’s Disease (PD) has been extensively explored. However, the mechanism of the GDNF neuroprotective effects is still unclear. In this study, the neuroprotective mechanism of the GDNF in the PD cellular models, which was obtained by the 6-hydroxydopamine (6-OHDA)-induced dopaminergic (DA) cell line MN9D damage was investigated by microarray. Interestingly, 54 constitutively increased or decreased genes were detected, 17 of which have not been reported previously. The expression of 5 up-regulated and 5 down-regulated genes which displayed the most obvious changes compared to the no GDNF treatment cells and was previously proven to be related to cell survival was validated by real-time PCR and western blot. Moreover, the up-regulated gene Ager and down-regulated gene Ccnl2 which were related to the PI-3K/Akt signaling pathway, but not researched in the neuron-cells, were investigated by overexpression and RNA interference. Overexpression of Ager or knockdown the expression of Ccnl2 decreased the damage to MN9D cells caused by 6-OHDA and reduced their apoptosis. All these results suggested that the protective effects of the GDNF on the 6-OHDA damaged MN9D cells could be understood by enhancing the expression of the apoptosis inhibiting genes and decreasing the expression of the apoptosis promoting genes. Thus, this study might provide a number of specific candidates and potential targets to investigate the protective mechanism of GDNF in DA neurons.     

Cloning,characterization and expression of a cDNA encoding a granulin-like polypeptide in Ciona savignyi

Previous study in our laboratory confirmed that a novel polypeptide, CS5931 derived from Ciona savignyi possesses potent antitumor activity. In the present study, the full length cDNA of CS5931 precursor, termed Cs-pgrn-1 was cloned. The complete cDNA sequence of this gene consists of 685 bp containing an open reading frame (ORF) of 522 bp (173 amino acid residues). In silico analysis revealed that the polypeptide consists of two identical domains, similar with granulin (GRN) found in other species, and each of the domain encodes a polypeptide identical with CS5931. Phylogenetic analysis confirmed that CS5931 shares high homology with Ciona intestinalis GRN and is conserved during evolution. The polypeptide also shows high similarity with human GRN A, B, and C. Prediction of 3D protein structure revealed the 3D structure of CS5931 is very similar with human GRN A. The CS5931 was expressed using a prokaryotic expression system and the purified polypeptide inhibited the growth of several tumor cell lines in vitro via apoptotic pathway. Our study revealed that CS5931 has the potential to be developed as a novel antitumor agent.     

Vascular endothelial growth factor regulates primate choroid-retinal endothelial cell proliferation and tube formation through PI3K/Akt and MEK/ERK dependent signaling

Vascular endothelial growth factor (VEGF) is a hypoxia-induced angiogenic protein that exhibits a broad range of biological and pathological effects in wet age-related macular degeneration and proliferative diabetic retinopathy. However, its specific mechanism is still not fully understood. Here, we examined the effects of VEGF on choroid-retinal endothelial cells (RF/6A) proliferation and tube formation, and the underlying signal pathways responsible in this process. RF/6A cells were pretreated with MEK inhibitor or PI3K inhibitor, and then incubated in a hypoxia chamber. Real-time PCR and Western blot analysis were carried out to explore VEGF expression on mRNA and protein levels. Hypoxia inducible factor-1α (HIF-1α) and VEGFR2 expression levels were also investigated in the presence and absence of hypoxic conditions. CCK-8 analysis and tube formation assay were tested under hypoxia, exogenous recombinant VEGF, and different signal pathway inhibitors, respectively. Mean while, the PI3K/Akt and MEK/ERK pathways in this process were also investigated. Our results showed that VEGF, HIF-1α, VEGFR2, p-ERK, and p-Akt were up-regulated in RF/6A cells under hypoxic conditions. MEK inhibitor (PD98059) and PI3K inhibitor (LY294002) decreased ERK and Akt activity, respectively, and reduced VEGF expression. VEGF-induced RF/6A proliferation and tube formation requires MEK/ERK and PI3K/Akt signaling, and both of the two pathways were needed in regulating VEGF expression. These suggest that VEGF plays an important role in RF/6A proliferation and tube formation, and MEK/ERK and PI3K/Akt pathway may be responsible for this process.     

Association of two variants in the interleukin-6 receptor gene and premature coronary heart disease in a Chinese Han population

Two novel single nucleotide polymorphisms (SNPs; rs7529229 and rs2228145) in the interleukin-6 receptor (IL6R) gene have recently been associated with coronary heart disease (CHD) in a European population. We sought to replicate this finding and to investigate associations of these two SNPs with the severity and clinical phenotypes of premature CHD in a Chinese Han population. A total of 418 patients were studied, including 187 cases with coronary stenosis ≥50 % or acute myocardial infarction (males < 55 years and females < 65 years) and 231 controls without documented CHD. A ligase detection reaction was performed to detect rs7529229 and rs2228145. There were no differences between the controls and premature CHD groups in the frequencies for the three genotypes and alleles of rs7529229 and rs2228145 (all P > 0.05), nor did they differ between the two groups when grouped by gender (all P > 0.05). There were also no associations between these two SNPs and the severity of coronary lesions or clinical phenotypes of premature CHD (all P > 0.05). Our results do not support an association between rs7529229 or rs2228145 with premature CHD in the Chinese Han population. Further studies are warranted to elucidate the role of these two SNPs in the development of atherosclerosis and CHD.     

Overexpression of β-NGF promotes differentiation of bone marrow mesenchymal stem cells into neurons through regulation of AKT and MAPK pathway

Bone marrow stromal stem cells (BMSCs) are fibroblastic in shape and capable of self-renewal and have the potential for multi-directional differentiation. Nerve growth factor (NGF), a homodimeric polypeptide, plays an important role in the nervous system by supporting the survival and growth of neural cells, regulating cell growth, promoting differentiation into neuron, and neuron migration. Adenoviral vectors are DNA viruses that contain 36 kb of double-stranded DNA allowing for transmission of the genes to the host nucleus but not inserting them into the host chromosome. The present study aimed to investigate the induction efficiency and differentiation of neural cells from BMSCs by β-NGF gene transfection with recombinant adenoviral vector (Ad-β-NGF) in vitro. The results of immunochemical assay confirmed the induced cells as neuron cells. Moreover, flow cytometric analysis, Annexin-V-FITC/PI, and BrdU assay revealed that chemical inducer β-mercaptoethanol (β-met) triggered apoptosis of BMSCs, as evidenced by inhibition of DNA fragmentation, nuclear condensation, translocation of phospholipid phosphatidylserine, and activation of caspase-3. Furthermore, the results of western blotting showed that β-met suppressed AKT signaling pathway and regulated the MAPKs during differentiation of BMSCs. In contrast, Ad-β-NGF effectively induced the differentiation of BMSCs without causing any cytopathic phenomenon and apoptotic cell death. Moreover, Ad-β-NGF recovered the expression level of phosphorylated AKT and MAPKs in cells exposed to chemical reagents. Taken together, these results suggest that β-NGF gene transfection promotes the differentiation of BMSCs into neurons through regulation of AKT and MAPKs signaling pathways.     

C-reactive protein reduces protein S-nitrosylation in endothelial cells

Glycodelin A (GdA) is a dimeric glycoprotein synthesized by the human endometrium under progesterone regulation. Based on the high sequence similarity with β-lactoglobulin, it is placed under the lipocalin superfamily. The protein is one of the local immunomodulators present at the feto-maternal interface which affects both the innate as well as the acquired arms of the immune system, thereby bringing about successful establishment and progression of pregnancy. Our previous studies revealed that the domain responsible for the immunosuppressive activity of glycodelin lies on its protein backbone and the glycans modulate the same. This study attempts to further delineate the apoptosis inducing region of GdA. Our results demonstrate that the stretch of amino acid sequence between Met24 to Leu105 is necessary and sufficient to inhibit proliferation of T cells and induce apoptosis in them. Further, within this region the key residues involved in harboring the activity were shown to be present between Asp52 and Ser65.