Objective To explore the regulatory role of transient receptor potential channel 6(TRPC6)on podocyte autophagy under the influence of homocysteine(Hcy)in mouse kidney.Methods Mouse renal podocytes were divided into control group and Hcy groups(stimulated by Hcy at 40,60,80 and 100 μmol/L for 48 h).The level of TRPC6 mRNA was assessed using quantitative reverse transcription polymerase chain reaction(qRT-PCR)to identify the optimal Hcy concentration for subsequent experiments.Western blotting was employed to evaluate the expression levels of autophagy-related proteins LC3 Ⅱ and p62,as well as the expression levels of podocyte structural proteins Nephrin and Podocin.The expression levels of TRPC6 mRNA and protein in both groups were determined using qRT-PCR,Western blotting and immunofluorescence.Transfections of cells with TRPC6 overexpression or interference were set as follows:(1)control group(untreated),negative control group of TRPC6 overexpression,and TRPC6 overexpression group;(2)control group(untreated),negative control group of TRPC6 interference,and TRPC6 interference group(si-1,si-2,si-3).The expression level of TRPC6 was detected using qRT-PCR.The cells after overexpressing or interfering of TRPC6 were further set as follows:(1)control group(untreated),Hcy group(80 μmol/L Hcy added),TRPC6 overexpression control+Hcy group,TRPC6 overexpression+Hcy group;(2)control group(untreated),Hcy group,TRPC6 interference control+Hcy group,and TRPC6 interference+Hcy group.The expression levels of p62,LC3 Ⅱ,and TRPC6 proteins were detected using Western blotting.Results qRT-PCR detection results showed that compared with control group,the expression level of TRPC6 mRNA in Hcy group increased with the increase of Hcy concentration,with the highest expression level observed at 80 μmol/L Hcy.Therefore,80 μmol/L Hcy was selected as the optimal concentration for intervention.At this time,the expression level of autophagy-related protein LC3 Ⅱ increased,and the expression level of p62 decreased(P<0.05).Western blotting results showed that compared with control group,the expression levels of podocyte-related proteins Nephrin and Podocin in Hcy group were significantly decreased(P<0.05).qRT-PCR results showed that compared with control group,the expression level of TRPC6 mRNA in Hcy group was significantly increased(P<0.05).Compared with negative control group for TRPC6 overexpression,both mRNA and protein expression levels of TRPC6 in TRPC6 overexpression group were significantly higher(P<0.05).Compared with negative control group for TRPC6 interference,both mRNA and protein expression levels of TRPC6 in TRPC6 interference group were significantly decreased(P<0.05).Western blotting results showed that compared with negative control group for TRPC6 overexpression,the expression level of autophagy-related protein LC3 Ⅱ in TRPC6 overexpression+Hcy group was significantly increased,and the expression level of p62 was significantly decreased(P<0.05).Compared with TRPC6 negative control+Hcy group for TRPC6 interference+Hcy,the expression level of autophagy-related protein LC3 Ⅱ in TRPC6 interference+Hcy group was significantly decreased,and the expression level of p62 was significantly increased(P<0.05).Conclusion Hcy can induce autophagy of renal podocytes.Inhibiting the expression of TRPC6 can significantly reduce the autophagy damage to podocytes.

Cangumycins A-F (1-6), six new angucyclinone analogues, together with two known ones (7 and 8), were isolated from the fermentation broth of a soil-derived Streptomyces sp. KIB-M10. Structures of these compounds were elucidated via a joint use of spectroscopic analyses and single-crystal X-ray diffractions. Among them, cangumycins E (5) and F (6) share a C-ring cleaved backbone, and cangumycins B (2) and E (5) exhibit potent immunosuppressive activity (IC 8.1 and 2.7 μmol·L, respectively) against human T cell proliferation at a non-cytotoxic concentration.

BACKGROUNDSurvivin is a rather specific gene in tumor tissue. We transfected dendritic cells (DCs) with recombinant adenovirus (Ad) containing survivin gene and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and tested the inducing effect of the transfected DCs on cytotoxic T lymphocytes (CTL) to kill leukemic cells.

METHODSAfter derived from the peripheral, DCs was assayed by mixed leukocyte reaction (MLR) tests. Lactate dehydrogenase (LDH) release test was used to evaluate cytotoxicity of CTL.

RESULTSExpression of survivin in transfected DCs was confirmed by Western blotting analysis. GM-CSF expression was confirmed by enzyme-linked immunosorbent assay (ELISA). In MLR assay, DCs coinfected with Ad-survivin and Ad-GM-CSF induced higher allogeneic lymphocyte reaction than control DCs at ratios of 1:5, 1:10, 1:50 and 1:100. DCs coinfected with Ad-survivin and Ad-GM-CSF had much higher activity of CTL to HL-60 cells than DCs infected with Ad-survivin only, Ad-GM-CSF only, or control DCs. Levels of interleukin-12 (IL-12) and interferon gamma (IFN-gamma) in lymphocyte supernatants containing DCs coinfected with Ad-survivin and Ad-GM-CSF were significantly higher than those in the control group.

CONCLUSIONDCs coinfected with Ad-survivin and Ad-GM-CSF induce much higher anti-leukemic response in vitro than those infected with either factor. Therefore, adenovirus vectors containing survivin and GM-CSF genes may be promising vaccine candidates for leukemia therapy.

Adenoviridae ; genetics ; Cytotoxicity, Immunologic ; Dendritic Cells ; physiology ; ultrastructure ; Genetic Therapy ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; HL-60 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; Interferon-gamma ; biosynthesis ; Interleukin-12 ; biosynthesis ; Leukemia ; therapy ; Lymphocyte Activation ; Microtubule-Associated Proteins ; genetics ; Neoplasm Proteins ; genetics ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection

This study was purposed to investigate the immunological effects of modified dendritic cells (DCs) in inducing cytotoxic T cells (CTLs) effect against lymphoma cells. The DCs were derived from human peripheral blood and transfected with recombined adenovirus vector carrying survivin gene, Western blot was used to detect the expression of survivin, the lactate dehydrogenase (LDH) release test was used to determine the cytotoxicity of CTLs, the mixed lymphocyte reaction (MLR) was used to measure the ability to proleferate allo-lymphocyte by DCs, ELISA was used to assay IL-12 level in supernatant. The results showed that the expression of survivin in transfected dentritic cells was confirmed by Western blot analysis. In MLR assay, DCs transfected with Ad-survivin could induce higher allogeneic lymphocytes reaction at the ratios of 1:5, 1:10, 1:50 and 1:100. DCs transfected with Ad-survivin had much higher activity of CTL to CA46 cells than control DCs. The levels of IL-12 of supernatants containing DCs transfected with Ad-survivin were significantly higher than that in the control group. It is concluded that DCs transfected with Ad-survivin can induce CTL response in vitro against lymphoma cells.
Adenoviridae ; genetics ; metabolism ; Apoptosis ; immunology ; Dendritic Cells ; immunology ; Genetic Vectors ; genetics ; Humans ; Inhibitor of Apoptosis Proteins ; Interleukin-12 ; metabolism ; Lymphoma ; immunology ; Microtubule-Associated Proteins ; genetics ; immunology ; Recombination, Genetic ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Tumor Cells, Cultured

OBJECTIVETo study the effect of peroxisome proliferators activated receptors (PPAR) alpha, gamma ligand on ATP-binding cassette transporter A1 (ABCA1) and caveolin-1 expressions and cholesterol, ox-LDL contents in human monocyte derived foam cells.

METHODMalondialdehyde (MDA) was measured by TBARS method, ox-LDL detected by ELISA method, cholesterol measured by fluorescence spectrophotometric method, ABCA1, caveolin-1 mRNA and protein expressions determined by RT-PCR and Western blot, in human monocytes, foam cells [human monocyte-derived macrophage induced by myristate acetate (PMA) further treated with 50 mg/L ox-LDL for 24 h], foam cells plus 10 micromol/L pioglitazone for 48 h, foam cells plus 5 micromol/L clofibrate for 48 h.

RESULTThe intracellular total cholesterol (TC), free cholesterol (FC), cholesteryl ester (CE), ox-LDL and lipid peroxide were significantly increased and the membrane expressions of ABCA1, caveolin-1 were down-regulated in foam cells compared to monocytes (all P < 0.05) and these changes were significantly attenuated by cotreatment with PPARalpha, gamma ligand.

CONCLUSIONThe anti-atherosclerosis effects of PPARalpha, gamma ligand are related to reducing cholesterol contents and up-regulating ABCA1, caveolin-1 expressions in foam cells.

ATP Binding Cassette Transporter 1 ; ATP-Binding Cassette Transporters ; metabolism ; Caveolin 1 ; metabolism ; Cell Line ; Cholesterol ; genetics ; metabolism ; Foam Cells ; metabolism ; Gene Expression ; Humans ; Malondialdehyde ; metabolism ; Monocytes ; metabolism ; PPAR alpha ; metabolism ; PPAR gamma ; metabolism

The study was aimed to construct the recombinant adenovirus vectors containing human survivin gene, and to investigate their expression in transfected dendritic cells. Full length cDNA encoding survivin was obtained by PCR amplification from plasmid pcDNA3.0-survivin. The PCR product was restricted, and then inserted into pShuttle-CMV. The plasmids of pShuttle-CMV-survivin were linearized with PmeI, and the fragment containing survivin was ligated with pShuttle-CMV and transfected into E. coli BJ5183. After homologous recombination in bacteria, the extracted plasmid from the positive bacteria were linearized with PacI, transfected into HEK293 cells with liposome Lipofectamine 2000. Then, the harvested adenovirus supernatants were transfected into dendritic cells. The results showed that the recombinant adenovirus-survivin was constructed successfully and its titer was about 2.65 x 10(9) pfu/ml. The expression of survivin in transfected dendritic cells was confirmed by Western blot analysis. It is concluded that the recombinant adenovirus vector containing human survivin was constructed successfully, which may provide preliminary laboratory evidence for anti-leukemia immunotherapy.
Adenoviridae ; genetics ; metabolism ; Dendritic Cells ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Viral ; Genetic Vectors ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Neoplasm Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection