Effect of ATF3-deletion on apoptosis of cultured retinal ganglion cells

AIM：To investigate the effect of activating transcription factor-3（ATF3）-deletion on apoptosis of cultured retinal ganglion cells（RGCs）.METHODS：Three ATF3 siR NA（ATF3-rat-651, ATF3-rat-319, ATF3-rat-520） were constructed, and were transiently transfected into RGC-5 cells. Quantitative real-time polymerase chain reaction（PCR） was used to examine ATF3 expression and the most effective ATF3 siR NA was selected for further studies. Flow cytometry was applied to investigate the effects of ATF3 deletion on RGC-5 apoptosis under elevated hydrostatic pressure. Quantitative realtime PCR and Western blot were performed to validate differentially expressed genes and proteins in ATF3-knockdown RGC-5 cells.RESULTS：ATF3 specific siR NA effectively down-regulated ATF3 expression and significantly inhibited cell apoptosis in RGC-5 cells. Quantitative real-time PCR and Western blot confirmed that ATF3 knockdown remarkably decreased Jun-B and increased c-Jun at both m RNA and protein levels in RGC-5 cells.CONCLUSION：ATF/cA MP-response element-binding family of transcription factors may be involved in the development of glaucoma and could be novel treatment targets for glaucoma.

Inhibition on Apoptosis Induced by Elevated Hydrostatic Pressure in Retinal Ganglion Cell-5 via Laminin Upregulating β1-integrin/Focal Adhesion Kinase/Protein Kinase B Signaling Pathway

Background：Glaucoma is a progressive optic neuropathy characterized by degeneration of neurons due to loss of retinal ganglion cells （RGCs）.High intraocular pressure （HIOP）,the main risk factor,causes the optic nerve damage.However,the precise mechanism of HIOP-induced RGC death is not yet completely understood.This study was conducted to determine apoptosis of RGC-5 cells induced by elevated hydrostatic pressures,explore whether laminin is associated with apoptosis under pressure,whether laminin can protect RGCs from apoptosis and affirm the mechanism that regulates the process of RGCs survival.Methods：RGC-5 cells were exposed to 0,20,40,and 60 mmHg in a pressurized incubator for 6,12,and 24 h,respectively.The effect of elevated hydrostatic pressure on RGC-5 cells was measured by Annexin V-fluorescein isothiocyanate/propidium iodide staining,3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay,and Western blotting of cleaved caspase-3 protein.Location and expression oflaminin were detected by immunofluorescence.The expression of β 1-integrin,phosphorylation of focal adhesion kinase （FAK） and protein kinase B （PKB,or AKT） were investigated with real-time polymerase chain reaction and Western blotting analysis.Results：Elevated hydrostatic pressure induced apoptosis in cultured RGC-5 cells.Pressure with 40 mmHg for 24 h induced a maximum apoptosis.Laminin was declined in RGC-5 cells after exposing to 40 mmHg for 24 h.After pretreating with laminin,RGC-5 cells survived from elevated pressure.Furthermore,β1-integrin and phosphorylation of FAK and AKT were increased compared to 40 mmHg group.Conclusions：The data show apoptosis tendency of RGC-5 cells with elevated hydrostatic pressure.Laminin can protect RGC-5 cells against high pressure via β 1-integrin/FAK/AKT signaling pathway.These results suggest that the decreased laminin of RGC-5 cells might be responsible for apoptosis induced by elevated hydrostatic pressure,and laminin or activating β1-integrin/FAK/AKT pathway might be potential treatments to prevent RGC loss in glaucomatous optic neuropathy.

Isolation and characterization of a crude oil degrading bacteria from formation water:comparative genomic analysis of environmental Ochrobactrum intermedium isolate versus clinical strains

In this study, we isolated an environmental clone of Ochrobactrum intermedium, strain 2745-2, from the formation water of Changqing oilfield in Shanxi, China, which can degrade crude oil. Strain 2745-2 is aerobic and rod-shaped with optimum growth at 42 ℃ and pH 5.5. We sequenced the genome and found a single chromosome of 4800175 bp, with a G＋C content of 57.63%. Sixty RNAs and 4737 protein-coding genes were identified： many of the genes are responsible for the degradation, emulsification, and metabolizing of crude oil. A comparative genomic analysis with related clinical strains （M86, 229E, and LMG3301T） showed that genes involved in virulence, disease, defense, phages, prophages, transposable elements, plasmids, and antibiotic resistance are also present in strain 2745-2.