Indirubin-3-Oxime Prevents H2O2-Induced Neuronal Apoptosis via Concurrently Inhibiting GSK3ß and the ERK Pathway.

Oxidative stress-induced neuronal apoptosis plays an important role in many neurodegenerative disorders. In this study, we have shown that indirubin-3-oxime, a derivative of indirubin originally designed for leukemia therapy, could prevent hydrogen peroxide (H2O2)-induced apoptosis in both SH-SY5Y cells and primary cerebellar granule neurons. H2O2 exposure led to the increased activities of glycogen synthase kinase 3ß (GSK3ß) and extracellular signal-regulated kinase (ERK) in SH-SY5Y cells. Indirubin-3-oxime treatment significantly reversed the altered activity of both the PI3-K/Akt/GSK3ß cascade and the ERK pathway induced by H2O2. In addition, both GSK3ß and mitogen-activated protein kinase inhibitors significantly prevented H2O2-induced neuronal apoptosis. Moreover, specific inhibitors of the phosphoinositide 3-kinase (PI3-K) abolished the neuroprotective effects of indirubin-3-oxime against H2O2-induced neuronal apoptosis. These results strongly suggest that indirubin-3-oxime prevents H2O2-induced apoptosis via concurrent inhibiting GSK3ß and the ERK pathway in SH-SY5Y cells, providing support for the use of indirubin-3-oxime to treat neurodegenerative disorders caused or exacerbated by oxidative stress.

[Construction and identification of a mouse spermatocyte-derived cell line with a stable expression of PIAS-NY].

OBJECTIVE: To construct a lentiviral expression vector of the PIAS-NY gene, and establish a mouse spermatocyte-derived cell line with a stable overexpression of PIAS-NY. METHODS: PIAS-NY was synthesized, amplified by PCR and cloned into the lentiviral vector expression plasmid pGC-FU. After digestion and sequencing, pGC-FU-PIAS-NY, pHelper 1.0 and pHelper 2.0 were co-transfected into 293T cells. Then the lentiviral particles were used to transfect the mouse spermatocyte-derived cells. The expression of the PIAS-NY protein was detected by Western blot. RESULTS: We successfully constructed the lentiviral expression vector pGC-FU-PIAS-NY and established a mouse spermatocyte-derived cell line with a stable overexpression of PIAS-NY. CONCLUSION: The construction of the lentiviral expression vector pGC-FU-PIAS-NY and the obtainment of stably transfected mouse spermatocyte-derived cells have paved the way for further studies on the roles of the PIAS-NY gene in spermatogenesis.

[Preparation and application of rabbit polyclonal antibody against mouse Tex33].

Objective To prepare rabbit-antimouse testis expressed 33 (Tex33) polyclonal antibody and detect the expression of Tex33 during mouse spermatogenesis. Methods Tex33 open reading frame was amplified by PCR from mouse testis cDNA. The PCR product was finally sub-cloned into pET-30a vector. The prokaryotic expression plasmid pET-30a-Tex33 was constructed and then transformed into E. coli BL21. Tex33 prokaryotic protein was induced by IPTG and then purified by Ni-NTA affinity chromatography. Tex33 polyclonal antibody was obtained from adult male New Zealand white rabbits immunized with purified Tex33 protein. ELISA, Western blotting and immunofluorescence assays were used to determine the potency and specificity. Results pET-30a-Tex33 recombinant vector was successfully constructed. After induced by IPTG, the recombinant Tex33 protein could be expressed effectively. The titer of polyclonal antibody was 1:1 000 000. Western blotting showed that the antibody could recognize Tex33 protein in mouse testis. Immunofluorescence assay revealed that Tex33 gene was expressed in spermatids and sperms of adult mouse testis. And Tex33 was located on the acrosome and flagellum of spermatozoa. Conclusion We have prepared rabbit-antimouse Tex33 polyclonal antibody with high specificity and found that Tex33 gene was expressed in mouse testis.

[Preparation and application of rabbit anti-mouse Setd8 polyclonal antibody].

Objective: To purify the recombinant Setd8 protein and prepare rabbit anti-mouse Setd8 polyclonal antibody. Methods: The recombinant plasmid p ET-30a-Setd8 was constructed by double enzyme digestion and linkage,and then transformed into E. coli BL21. The expression of the target protein was induced by IPTG and the expression product was purified by Ni-NTA affinity chromatograph. The purified protein was used to immunize New Zealand white rabbits to produce polyclonal antibody. The titer and specificity of the antibody were identified by ELISA,Western blotting and immunohistochemistry. Results: The prokaryotic expression vector p ET-30a-Setd8 was constructed successfully. After induced by IPTG,the recombinant Setd8 protein was expressed effectively in E. coli BL21. Polyclonal antibody against Setd8 was generated by immunizing rabbits with the routine method. ELISA showed that the titer of rabbit anti-Setd8 antiserum was 1 ∶ 1 000 000.Western blotting demonstrated that the polyclonal antibody could recognize the native mouse Setd8 protein. Immunohistochemistry revealed that Setd8 protein recognized by the polyclonal antibody was mainly distributed in the nucleus of spermatogonia in adult mouse testis. Conclusion: Using the prokaryotic expression vector p ET-30a-Setd8,we have prepared successfully the polyclonal antibody with high affinity and specificity.

[Preparation of anti-BRDT-NY polyclonal antibody and expression of BRDT-NY protein in digestive tract tumors].

AIM: To prepare the mouse polyclonal antibody against human BRDT-NY prokaryotic protein and analyze the expression of BRDT-NY protein in digestive tract tumors. METHODS: The N-terminal amino acids of BRDT-NY protein was amplified by PCR and cloned into the expression vector pET28a(+). The recombinant plasmid pET28a+-BRDT-NY was transformed into E.coli BL21 and induced to express the recombinant protein with IPTG. We immunized BALB/c mice with the purified BRDT-NY protein for preparing the specific polyclonal antibody. The titer of the antibody was analyzed by ELISA. The expression of BRDT-NY protein in digestive tract tumors was detected by immunohistochemistry. RESULTS: Recombinant human BRDT-NY protein was expressed in BL21 and purified successfully by Ni-affinity chromatography. Two months after the mice were immunized with the purified BRDT-NY protein, we obtained the specific polyclonal antibody of high titer 1:100 000. Immunohistochemical analysis revealed that BRDT-NY protein was highly expressed in digestive tract tumors. CONCLUSION: The successful preparation of mouse polyclonal antibody against BRDT-NY lays a foundation for the research on the role of BRDT-NY protein in the pathology of human digestive tract tumors.