Molecular and functional characterization of ferredoxin NADP(H) oxidoreductase from Gracilaria chilensis and its complex with ferredoxin

BACKGROUND: Ferredoxin NADP(H) oxidoreductases (EC 1.18.1.2) (FNR) are flavoenzymes present in photosynthetic organisms; they are relevant for the production of reduced donors to redox reactions, i.e. in photosynthesis, the reduction of NADP+ to NADPH using the electrons provided by Ferredoxin (Fd), a small FeS soluble protein acceptor of electrons from PSI in chloroplasts. In rhodophyta no information about this system has been reported, this work is a contribution to the molecular and functional characterization of FNR from Gracilaria chilensis, also providing a structural analysis of the complex FNR/Fd. METHODS: The biochemical and kinetic characterization of FNR was performed from the enzyme purified from phycobilisomes enriched fractions. The sequence of the gene that codifies for the enzyme, was obtained using primers designed by comparison with sequences of Synechocystis and EST from Gracilaria. 5'RACE was used to confirm the absence of a CpcD domain in FNRPBS of Gracilaria chilensis. A three dimensional model for FNR and Fd, was built by comparative modeling and a model for the complex FNR: Fd by docking. RESULTS: The kinetic analysis shows KMNADPH of 12.5 M and a kcat of 86 s-1, data consistent with the parameters determined for the enzyme purified from a soluble extract. The sequence for FNR was obtained and translated to a protein of 33646 Da. A FAD and a NADP+ binding domain were clearly identified by sequence analysis as well as a chloroplast signal sequence. Phycobilisome binding domain, present in some cyanobacteria was absent. Transcriptome analysis of Gch revealed the presence of two Fd; FdL and FdS, sharing the motif CX5CX2CX29X. The analysis indicated that the most probable partner for FNR is FdS. CONCLUSION: The interaction model produced, was consistent with functional properties reported for FNR in plants leaves, and opens the possibilities for research in other rhodophyta of commercial interest.

Biological effect of down-regulating of MTRR gene on cisplatin-resistant ovarian cancer SKOV3 cells in vitro and in vivo studies / 中华妇产科杂志

Objective To study the biological effects of down-regulatingof methionine synthase reductase (MTRR) gene on cisplatin resistant ovarian cancer SKOV3/DDP cell in vitro and in vivo. Methods (1) Establishing the cell line of MTRR down-regulated. Four short hairpin RNA (shRNA) for MTRR gene (U6-GFP-Neo-homo-1106, U6-GFP-Neo-homo-1931, U6-GFP-Neo-homo-419, U6-GFP-Neo-homo-1460) were designed respectively. Western blot was used to detect the interference efficiency and selected the most efficient shRNA. The MTRR 1106 was selected as the best silencing effect of interference fragment and then therecombinant plasmid vector pSicoR-1106 was constructed and transfected into SKOV3/DDP cells.The stably transfected cells was obtained by screening of flow cytometry(FCM).Fluorescence quantitative reverse transcription (RT)-PCR and western blot were used to detect the expression of MTRR mRNA and protein. (2) Study in vitro: recombinant plasmid expression vector pSicoR-1106, pSicoR-NC and packaging plasmid were respectively transfected into 293T cell. SKOV3/DDP cells were transfected by viral supernatant. The experiment was divided into three groups, namely SKOV3/DDP-MTRRi (down-regulated MTRR group), SKOV3/DDP-NC (negative control group), and the SKOV3/DDP (blank control group). The cell growth curves and half maximal inhibitory concentration (IC50) of cisplatin were made by methyl thiazolyl tetrazolium (MTT) method. Three groups cells were treated with different concentration of cisplatin (0, 1, 2 and 4 μg/ml). The clonogenicity efficiency was observed by clony formation test. The cell cycles were measured by FCM . (3) Study in vivo: three groups cells were subcutaneously inoculated into the nude mice to develop a tumor model. Mice were injected intraperitoneally with cisplatin at 2.5 mg/kg (once every 2 days, in 21 rounds), then the tumor growth was observed. The expression of MTRR and proliferation-related Ki-67 antigen by immunohistochemistry in xenograft tumors were measured. Results (1) Results showed that U6-GFP-Neo-homo-1106 was the best shRNA with interference effect to MTRR. The recombinant plasmid pSicoR-1106 was constructed and transfected into SKOV3/DDP. The MTRR mRNA and protein were down-regulated after transfected. This result showed that MTRR down-regulated SKOV3/DDP cell line was constructed successfully. (2) The cell growth curves showed that the growth of SKOV3/DDP-MTRRi cells were significantly decreased compared with that in the SKOV3/DDP-NC cells and SKOV3/DDP cells (P<0.05). The IC50 of SKOV3/DDP-MTRRi, SKOV3/DDP-NC and SKOV3/DDP were 4.01, 7.90, and 8.91 μg/ml, respectively. The IC50 of SKOV3/DDP-MTRRi was significantly lower than that in control cell groups (P<0.05).Clony formation tests showed that the clony numbers of varied concentration of cisplatin of SKOV3/DDP-MTRRi were significantly less than those of SKOV3/DDP-NC cells and SKOV3/DDP cells (P<0.05). FCM showed that when the cisplatin concentration rose to 4 μg/ml, the G0/G1 phase cell ratio in SKOV3/DDP-MTRRi cells group was (72.8±5.0)%, which was significantly higher than those in the SKOV3/DDP-NC cells group and SKOV3/DDP cells group [(64.4±2.5)%and (64.3±3.0)%], respectively (all P<0.05).(3) Six weeks after nude mice intraperitoneal injection with cisplatin, the tumor volume of SKOV3/DDP-MTRRi, SKOV3/DDP-NC and SKOV3/DDP were respectively (97 ± 32), (168 ± 45), and (173 ± 32) mm3, the tumor weight were (0.36±0.17), (1.08±0.17), and (1.11±0.20) g, in which tumor volume and weight of SKOV3/DDP-MTRRi were significantly less than those of SKOV3/DDP-NC cells and SKOV3/DDP cells (all P<0.05). In three groups tumor tissue, positive rates of MTRR were respectively 2/8, 5/8, and 7/8, the positive rates of Ki-67 were respectively1/8, 6/8, and 7/8, in which SKOV3/DDP-MTRRi was significantly lower those SKOV3/DDP-NC cells and SKOV3/DDP cells (all P<0.05). Conclusion The growth and cisplatin resistance of ovarian cancer cells could be decreased by down-expressing of MTRR gene in vitro and in vivo.

Effect of MTRR gene on apoptosis and autophagy pathways in multi-resistant epithelial ovarian cancer / 中华妇产科杂志

Objective To explore the effect of down-regulated methionine synthase reductase (MTRR) gene on the apoptosis and autophagy pathway, and offer a possible approach for the MTRR to reverse the multi-resistant ovarian cancer. Methods (1) The experiment was divided into 3 groups, SKOV3/DDP-MTRRi (down-regulated MTRR group), SKOV3/DDP-NC (negative control group), and SKOV3/DDP (blank control group). Different concentration of cisplatin (0, 1, 2, and 4 μg/ml) treated on 3 groups cells. The apoptosis rate was measured by flow cytometry (FCM). Autophagy was detected by immunofluorescence. Autophagy microtubule associated protein light chain 3β(LC3B) and p62 were detected by western blot. The formation of autophagosome of cells was observed by transmission electron microscope. (2) Detection of autophagy and apoptosis of SKOV3/DDP-MTRRi induced by rapamycin. The experiment was divided into 4 groups included rapamycin group (5 nmol/L rapamycin), rapamycin+cisplatin group (5 nmol/L rapamycin+4μg/ml cisplatin), cisplatin group (4μg/ml cisplatin) and blank control group. LC3B and p62 protein were detected by western blot. The survival rate cells were detected by methyl thiazolyl tetrazolium (MTT) method. The apoptosis rate was measured by FCM. (3) The 3 groups cells (SKOV3/DDP, SKOV3/DDP-NC and SKOV3/DDP-MTRRi) induced by a certain concentration of cisplatin (4 μg/ml) after 48 hours, then detecting the protein expression of caspase, Bcl-2 family in apoptosis pathway and the key proteins in phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) autophagy pathways by western blot, getting the time when the proteins′expression changed. Results (1) The 3 groups cells (SKOV3/DDP, SKOV3/DDP-NC, and SKOV3/DDP-MTRRi) induced by a certain concentration of cisplatin (4 μg/ml) after 48 hours, apoptosis and autophagy of 3 groups of cells were gradually increased with the increased concentration of cisplatin. The apoptosis rate of SKOV3/DDP-MTRRi cells [(26.2 ± 1.4)%] were significantly increased compared with the SKOV3/DDP-NC cells or SKOV3/DDP cells [(14.8 ± 2.4)%, (14.2 ± 2.4)%;all P0.05), but cleaved caspase-3, cleaved caspase-7, cleaved caspase-9, and cleaved PARP were significantly increased in SKOV3/DDP-MTRRi cells (P<0.05). For the autophagy pathway, the expression of phosphorylated Akt (p-Akt) and phosphorylated mammalian target of rapamycin (p-mTOR) were significantly increased (P<0.05), but Akt and mTOR had no significant variation. The expression of phosphatase and tensin homologue deleted on chromosome ten (PTEN) was significantly decreased (P<0.05). Conclusions MTRR silencing significantly increase cisplatin-induced apoptosis and reduce the autophagy induced by cisplatin in SKOV3/DDP cells. Down-regulation of MTRR enhanced the chemosensitivity of cisplatin-resistant ovarian cancer cells may be by activating caspase and Bcl-2 apoptosis family and inhibiting the PI3K/Akt autophagy pathway.

Association of methionine synthase reductase gene polymorphism with unexplained recurrent spontaneous abortion / 中华妇产科杂志

Objective To explore the relationship between the polymorphism of methionine synthase reductase(MTRR) A66G and the susceptibility to unexplained repeated spontaneous abortion (URSA).Methods Total of 200 Henan Han couples with URSA (URSA group) and 76 Henan Han healthy couples without URSA (control group)were enrolled in this study.Their MTRR A66G genotypes were determined by PCR restriction fragment length polymorphism (PCR-RFLP).Results (1) The allele frequencies of MTRR A66G:the frequencies of allele A and allele G in URSA group were 76.5％ (153/200)in husband and 72.8％ (146/200) in wife,23.5％ (47/200) in husband and 27.2％ (54/200) in wife,respectively.The frequencies of allele A and allele G in control group were 78.9％ (60/76) in husband and 78.3％ (59/76) in wife,21.1％ (16/76) in husband and 21.7％ (16/76) in wife,respectively.The frequencies of allele A and allele G were not significantly different between female and male subjects within the same experimental group (P ＞ 0.05),and also there were not significantly different between the same gender subjects at URAS and control groups(P ＞ 0.05).(2) The genotype frequencies of MTRR A66G:the frequencies of genotype AA,AG and GG in URSA group were 57.0％ (114/200) in husband and 52.0％ (104/200) in wife,39.0％ (78/200) in husband and 41.5％ (83/200) in wife,4.0％ (8/200) in husband and 6.5％ (13/200) in wife,prepectively.The frequencies of genotype AA,AG and GG in control group were 59.2％ (45/76) in husband and 59.2％ (50/76) in wife,39.5％ (30/76) in husband and 38.2％ (29/76) in wife;1.3 ％ (1/76) in husband and 2.6％ (2/76) in wife,prepectively.The frequencies of genotype AA,AG and GG were not significantly different between female and male subjects within the same group (P ＞ 0.05),and also there were not significantly different between the same gender subjects at URSA and control groups (P ＞0.05).(3) Combined genotype of couples:the combined genotype frequencies of GG + GG,GG + AG,GG +AA,AG + AG,AG + AA and AA + AA in URSA group were 1.0％ (2/200),2.5％ (5/200),6.0％ (12/200),20.0％ (40/200),38.0％ (76/200),and 32.5 ％ (65/200),prepectively ; the combined genotype frequencies in control group were 0,1.3％ (1/76),2.6％ (2/76),17.1％ (13/76),42.1％ (32/76),36.8％ (28/76),prepectively.The combined genotype analysis between the two groups were also not significantly different (P ＞ 0.05).Conclusion The polymorphism of MTRR A66G gene was not associated with the susceptibility to URSA (P ＞ 0.05),and so it was not the inherited genetic risk factor of URSA.