A homologue of vitamin K epoxide reductase in Solanum lycopersicum is involved in resistance to osmotic stress.

Vitamin K epoxide reductase (VKOR) exists widely in the chloroplasts of higher plants and plays important roles in redox regulation. However, investigations of plant VKOR function have primarily focused on VKOR from Arabidopsis, and knowledge concerning this function is therefore quite limited. In this study, Solanum lycopersicum was used to study the function of VKOR. Transcription level analysis revealed that SlVKOR (S. lycopersicum VKOR) expression was upregulated by salt or drought stress. To further investigate the function of SlVKOR in plants, we generated sense and antisense transgenic S. lycopersicum homozygotes at T2 generation plants. Compared with wild-type (WT) plants, the leaf disks of the SlVKOR overexpression plants retained a much higher percentage of chlorophyll after salt or drought treatment, whereas the antisense transgenic plants displayed an opposite response. The overexpressed plants displayed lower levels of H2O2 and superoxide anion radical (O2(·-)) than WT plants, whereas antisense plants accumulated considerably more H2O2 and O2(·-). The activities of reactive oxygen scavenger enzymes, including superoxide dismutase, peroxidase, ascorbate peroxidase and catalase, were consistent with the accumulation of reactive oxygen species. Based on these results, we suggest that SlVKOR is involved in resistance to salt or drought stress.

Genome-wide characterization and analysis of F-box protein-encoding genes in the Malus domestica genome.

The F-box protein family is a large family that is characterized by conserved F-box domains of approximately 40-50 amino acids in the N-terminus. F-box proteins participate in diverse cellular processes, such as development of floral organs, signal transduction and response to stress, primarily as a component of the Skp1-cullin-F-box (SCF) complex. In this study, using a global search of the apple genome, 517 F-box protein-encoding genes (F-box genes for short) were identified and further subdivided into 12 groups according to the characterization of known functional domains, which suggests the different potential functions or processes that they were involved in. Among these domains, the galactose oxidase domain was analyzed for the first time in plants, and this domain was present with or without the Kelch domain. The F-box genes were distributed in all 17 apple chromosomes with various densities and tended to form gene clusters. Spatial expression profile analysis revealed that F-box genes have organ-specific expression and are widely expressed in all organs. Proteins that contained the galactose oxidase domain were highly expressed in leaves, flowers and seeds. From a fruit ripening expression profile, 166 F-box genes were identified. The expressions of most of these genes changed little during maturation, but five of them increased significantly. Using qRT-PCR to examine the expression of F-box genes encoding proteins with domains related to stress, the results revealed that F-box proteins were up- or down-regulated, which suggests that F-box genes were involved in abiotic stress. The results of this study helped to elucidate the functions of F-box proteins, especially in Rosaceae plants.

Thylakoid membrane oxidoreductase LTO1/AtVKOR is involved in ABA-mediated response to osmotic stress in Arabidopsis.

Arabidopsis lumen thiol oxidoreductase 1 (LTO1) - the At4g35760 gene product - was previously found to be related to reactive oxygen species (ROS) accumulation. Here, we show that ROS accumulated in a mutant Arabidopsis line (lto1-2, mutant of LTO1/AtVKOR) under osmotic stress at a higher level than that observed in wild-type and transgenic complemented plants of the lto1-2 mutant (lto1-2C, transgenic complemented plants of lto1-2). Because ROS accumulation in osmotic stress is triggered by abscisic acid (ABA), an ABA-responsive gene, Annexin 1 (AnnAt1), was selected to study the response. Osmotic stress or exogenous ABA can significantly upregulate the transcription of AnnAt1 in wild-type and lto1-2C plants. Only a slight change in the transcriptional abundance of AnnAt1 was observed under osmotic stress in the lto1-2 mutant, but exogenous ABA application could increase the expression of AnnAt1, which suggested that exogenous ABA had a partial complementation role. Because the transcription of AnnAt1 is regulated by ABRE (ABA-responsive elements) binding proteins (AREBs)/ABRE binding factors (ABFs), the expression of AREBs/ABFs was also analyzed. The transcription of AREBs/ABFs in the lto1-2 mutant was not induced by osmotic stress but was significantly upregulated by exogenous ABA, which significantly differs from the wild-type and lto1-2C plant responses. Similarly, the expression of another ABA-responsive gene, RD29B (responsive to desiccation stress gene 29B), in the lto1-2 mutant was also upregulated by exogenous ABA. The partial complementation of mutants by ABA indicated that the ABA signal transduction pathway was not significantly affected in the lto1-2 mutant. Taken together, these results suggest that LTO1 is involved in ABA-mediated response to osmotic stress, possibly by affecting the biosynthesis of endogenous ABA.

The identification of novel and differentially expressed apple-tree genes under low-temperature stress using high-throughput Illumina sequencing.

Low temperature is a major environmental constraint on the production of apples worldwide. An apple rootstock with high cold tolerance was selected to identify genes related to stress tolerance. The transcriptional profiles of the genes in the leaves were examined after various intervals of exposure to cold stress. We obtained three libraries of 14,219, 11,176 and 16,116 tag-mapped predicted coding sequences at three time points (0, 1 and 6 h) during cold stress. In the two time periods, which were from 0 to 1 h and from 1 to 6 h, 139 and 1,085 genes were upregulated, and 1,499 and 381 genes were downregulated, respectively. These groups included a large number of unknown genes. The distribution of genes indicated cold adaptation in the plant. Most of the differential expression genes (DEGs) had temporal specificity and significantly different expression changes at different time points. The classification of DEGs by GO category and KEGG pathway analysis revealed that the DEGs are involved in numerous biological pathways, including metabolism, plant-pathogen interaction and signal transduction. Eleven randomly selected tag-mapped genes were examined by qRT-PCR. The results of the qRT-PCR were in accordance with the transcriptional profiles. The most upregulated gene (MDP0000198054) from 0 to 1 h encodes a dehydration-responsive element-binding protein/C-repeat factor (DREB/CBF). In this study, MDP0000198054 and related genes involved in the cold stress response were discussed. These results could provide new insights into the stress tolerance mechanisms of apple rootstocks.

An obesity-like gene MdTLP7 from apple (Malus × domestica) enhances abiotic stress tolerance.

Tubby-like proteins (TLPs) are found in a broad range of multicellular organisms. In mammals, genetic mutation of tubby or other TLPs can result in certain disease phenotypes related to animal specific characters: obesity, retinal degeneration, hearing loss, et al. Plants also harbor a large number of TLP genes, but the information in plants is far more limited. We identified a highly up-regulated obesity-like gene, MdTLP7, in our previous study of apple differential gene expression profile under chilling, indicating its possible role in plant abiotic stress tolerance. cDNA of MdTLP7 was amplified and expressed in Escherichia coli. In the solid and solution medium, the rate of growth and the quantity of the cell carrying MdTLP7 gene were significantly more than that of empty vector under salt and temperature stresses. To identify the functional region, serial deletion from both N-terminus and C-terminus of MdTLP7 was performed. In 415 amino acid polypeptide chain of MdTLP7, a middle conservative fragment (120-310 amino acid residues) played vital roles in stress tolerance. This fragment was involved in ß barrel of Tubby domain according to the model of Tubby domain. All above results suggested MdTLP7 confers stress-tolerance to E. coli cell against abiotic stresses.

Reproductive performance after surgery for endometriosis: predictive value of the revised American Fertility Society classification and the endometriosis fertility index.

BACKGROUND/AIMS: To determine whether the revised American Fertility Society (rAFS) classification and endometriosis fertility index (EFI) predict pregnancy rates (PRs) in patients with surgically confirmed endometriosis attempting natural conception. METHODS: We retrospectively assessed 194 women with endometriosis who underwent laparoscopic surgery; 161 women completed the follow-up. Pregnancy outcomes, rAFS stages and EFI scores were documented. Cumulative PRs were compared using Kaplan-Meier survival analysis. RESULTS: The cumulative PR 36 months after surgery was 46.6% (stage I, 53.6%; stage II, 36.0%; stage III, 51.7%, and stage IV, 41.7%; log-rank test, χ(2) = 4.143, p = 0.246). In the 1st year, PRs significantly differed between patients with rAFS stage IV and those with stages I-III (Pearson's χ(2) test, χ(2) = 6.024, p = 0.014). Significant differences in cumulative PRs were observed among EFI scores (group 1, EFI score 0-3, 8.3%; group 2, EFI score 4-7 41.2%, and group 3, EFI score 8-10 60.9%; log-rank test, χ(2) = 16.254, p < 0.001). CONCLUSIONS: EFI scores, but not rAFS stage, predict PRs in patients with endometriosis-associated infertility. EFI scores may be used to guide postoperative treatment.

The Juvenile-Hormone-Responsive Factor AmKr-h1 Regulates Caste Differentiation in Honey Bees.

Honey bees are typical model organisms for the study of caste differentiation, and the juvenile hormone (JH) is a crucial link in the regulatory network of caste differentiation in honey bees. To investigate the mechanism of JH-mediated caste differentiation, we analyzed the effect of the JH response gene AmKr-h1 on this process. We observed that AmKr-h1 expression levels were significantly higher in queen larvae than in worker larvae at the 48 h, 84 h, and 120 h larval stages, and were regulated by JH. Inhibiting AmKr-h1 expression in honey bee larvae using RNAi could lead to the development of larvae toward workers. We also analyzed the transcriptome changes in honey bee larvae after AmKr-h1 RNAi and identified 191 differentially expressed genes (DEGs) and 682 differentially expressed alternative splicing events (DEASEs); of these, many were related to honey bee caste differentiation. Our results indicate that AmKr-h1 regulates caste differentiation in honey bees by acting as a JH-responsive gene.

Lycium barbarum polysaccharide alleviates DSS-induced chronic ulcerative colitis by restoring intestinal barrier function and modulating gut microbiota.

PURPOSE: This study examined the protective effects and mechanism of Lycium barbarum polysaccharides (LBP) in the context of intestinal barrier function and intestinal microbiota in mice with dextran sulfate sodium (DSS)-induced chronic ulcerative colitis (UC). METHODS: C57BL/6J male mice were assigned to a standard normal diet without DSS (control group), a normal diet with DSS (DSS group, 2% DSS given discontinuously for 3 weeks) or a normal diet supplemented with LBP (1% dry feed weight, LBP group, 2% DSS given discontinuously for 3 weeks) for a total of 8 weeks, at which point colonic tissues and caecal contents were collected. RESULTS: LBP exerted a significant effect against colitis by increasing body weight, colon length, DAI and histopathological scores. LBP inhibited proinflammatory cytokines (IL-1ß, IL-6, iNOS and TNF-α) expression, improved anti-inflammatory cytokine (IL-10) expression, promoted the expression of tight junction proteins (Occludin and ZO-1) via nuclear factor erythroid 2-related factor 2 (Nrf2) activation and decreased Claudin-2 expression to maintain the intestinal mucosal barrier. In addition, the abundances of some probiotics (Ruminococcaceae, Lactobacillus, Butyricicoccus, and Akkermansia) were decreased with DSS treatment but increased obviously with LBP treatment. And LBP reduced the abundance of conditional pathogens associated with UC (Mucispirillum and Sutterella). Furthermore, LBP improved the production of short-chain fatty acids (SCFAs), including acetic acid, propionic acid, butyric acid and isobutyric acid. CONCLUSION: LBP can alleviate DSS-induced UC by regulating inflammatory cytokines and tight junction proteins. Moreover, LBP promotes probiotics, suppresses conditional pathogens and increases SCFAs production, showing a strong prebiotic effect.

Insulin resistance in school-aged girls with overweight and obesity is strongly associated with elevated white blood cell count and absolute neutrophil count.

Background: The primary objective of the study was to discuss the sex differences in insulin resistance-induced changes in metabolic and inflammatory markers in school-aged children with overweight and obesity. Methods: A cross-sectional study of 800 children aged seven and twelve years was performed. Questionnaires, anthropometric data and fasting blood samples were collected. Results: Children with overweight and obesity showed statistically significant differences in multiple metabolic and inflammatory markers compared with children with normal BMI. The correlation coefficient (r) between white blood cell count, absolute neutrophil count, fasting plasma insulin, HOMA-IR, HOMA-ß, triglyceride, HDL-C, triglyceride/HDL ratio, alanine transaminase, serum uric acid, systolic blood pressure and BMI were higher in all children, but the linear relationships between white blood cell count, absolute neutrophil count and BMI were stronger in girls with overweight and obesity than in boys with overweight and obesity. Subsequently, HOMA-IR was shown to be more strongly associated with increased white blood cell count and absolute neutrophil count in school-aged girls with overweight and obesity by partial correlation analysis and the multiple linear regression analysis. Conclusions: Elevated white blood cell count and absolute neutrophil count in children with overweight and obesity, especially girls, can serve as markers of insulin resistance.

Endowing magnesium with the corrosion-resistance property through cross-linking polymerized inorganic sol-gel coating.

The design of a highly adhesive, defect-free and low-temperature sol-gel coating for the protection of magnesium alloys is desirable yet challenging. In this study, a novel SiO2-based sol-gel coating is developed by a ring-opening addition reaction. Notably, the integration of individual sol clusters endows the sol-gel coating with a smooth and compact surface morphology, and eliminates the potential corrosion site of the low-temperature-prepared sol-gel coating. Besides, the as-obtained sol-gel coating exhibits excellent metallic adhesion nature. Most importantly, it increases the overall impedance modulus by 27 times than that of the conventional strategy and decreases the corrosion rate from 3.8 ± 0.5 mg cm-2 per day (commercial chromate conversion coating) to 0.5 ± 0.2 mg cm-2 per day.

The anxiolytic-like effects of estazolam on a PTSD animal model.

Post-traumatic stress disorder (PTSD) is a serious psychiatric disorder. Estazolam has been shown to produce anxiolytic-, hypnotic-, amnestic-, and sedative-like effects. However, few studies are concerned about its anti-PTSD-like effects. The anti-PTSD-like effects of estazolam were evaluated by single prolonged stress animal model. After exposure to single prolonged stress, rats (Sprague-Dawley, male, 8 weeks) were administered by estazolam (0.5, 1 and 2 mg/kg, i.p.) from day 2 to 13 once daily. The behavioral assessments were performed during treatment with drugs. After the behavioral evaluation, the role of allopregnanolone in the anti-PTSD-like effects of estazolam was also evaluated via astrocyte cells and brain tissues (e.g. prefrontal cortex, hippocampus, and amygdala). The PTSD-like behavioral deficits were significantly blocked by estazolam (1 and 2 mg/kg, i.p.) without affecting locomotor activity. Consistently, the levels on allopregnanolone were increased by estazolam (1 and 2 mg/kg, i.p.) in prefrontal cortex, hippocampus, and amygdala. The levels of allopregnanolone were increased by sertraline (1 µmoL/L) and estazolam (4 µmoL/L), while the effects were antagonized by trilostane (1 µmoL/L) and finasteride (1 µmoL/L) in astrocyte cells, respectively. Collectively, the anxiety-like behavior deficits were ameliorated by estazolam in the single prolonged stress animal model that was associated with biosynthesis of allopregnanolone.

The anxiolytic-like effects of ginsenoside Rg3 on chronic unpredictable stress in rats.

The present study is to evaluate the anxiolytic-like activities underlying ginsenoside Rg3 (GRg3). The anxiolytic-like activities were induced by GRg3 (20 and 40 mg/kg, i.g), evidenced by blocking the decreased time and entries in the open arms in elevated plus maze test and by reversing the increased latency to feed in novelty-suppressed feeding test. In addition, the decreased levels on progesterone, allopregnanolone, serotonin (5-HT) in the prefrontal cortex and hippocampus of chronic unpredictable stress (CUS) were blocked by GRg3 (20 and 40 mg/kg, i.g). Furthermore, the increased corticotropin releasing hormone, corticosterone and adrenocorticotropic hormone were blocked by GRg3 (20 and 40 mg/kg, i.g). Collectively, the anxiolytic-like effects produced by GRg3 were associated with the normalization of neurosteroids biosynthesis, serotonergic system as well as HPA axis dysfunction.

The effects of PK11195 on the MCF-7 and T47D were associated with the allopregnanolone biosynthesis, which was mediated by Translocator Protein 18 KDa.

Breast cancer is currently the most common malignancy affecting women worldwide. It had been shown that the allopregnanolone biosynthesis was associated with tumorigenesis and PK11195, the Translocator Protein 18 KDa (TSPO) antagonist, had the effects of the allopregnanolone biosynthesis. However, little is known about the association between the effects of PK11195 on the breast cancer and the allopregnanolone biosynthesis. To evaluate this, the breast cancer cell lines MCF-7 and T47D were cultured. Cell viability and proliferation were determined by CCK-8 assay. The IC50 of PK11195 on the MCF-7 and T47D were 5.4 nM and 6 nM. The cell viability and proliferation of AC-5216 (TSPO selective ligand, 3 and 6 nM) was blocked by PK11195 (5.4 nM and 6 nM). Moreover, we evaluated the role of allopregnanolone biosynthesis in the effects of TSPO on breast cancer. Enzyme-Linked ImmunoSorbent Assay (ELISA) was used in the measurement of the allopregnanolone level. We found that the allopregnanolone level was increased by AC-5216 (3 and 6 nM) and the increase was reversed by PK11195 (5.4 nM and 6 nM, resepectively) in MCF-7 and T47D. The TSPO mRNA level was determined by real time polymerase chain reaction (PCR). The TSPO mRNA level were increased by AC-5216 (6 nM), which the increases were reversed by PK11195 (5.4 nM and 6 nM, resepectively) in MCF-7 and T47D. Collectedly, it firstly indicated that the effects of PK11195 on MCF-7 and T47D were associated with the decrease of allopregnanolone biosynthesis, which was mediated by TSPO.

Genome-Wide Identification and Expression Analysis of the Tubby-Like Protein Family in the Malus domestica Genome.

Tubby-like proteins (TLPs), which have a highly conserved ß barrel tubby domain, have been found to be associated with some animal-specific characteristics. In the plant kingdom, more than 10 TLP family members were identified in Arabidopsis, rice and maize, and they were found to be involved in responses to stress. The publication of the apple genome makes it feasible to systematically study the TLP family in apple. In this investigation, nine TLP encoding genes (TLPs for short) were identified. When combined with the TLPs from other plant species, the TLPs were divided into three groups (group A, B, and C). Most plant TLP members in group A contained an additional F-box domain at the N-terminus. However, no common domain was identified other than tubby domain either in group B or in group C. An analysis of the tubby domains of MdTLPs identified three types of conserved motifs. Motif 1 and 2, the signature motifs in the confirmed TLPs, were always present in MdTLPs, while motif 3 was absent from group B. Homology modeling indicated that the tubby domain of most MdTLPs had a closed ß barrel, as in animal tubby domains. Expression profiling revealed that the MdTLP genes were expressed in multiple organs and were abundant in roots, stems, and leaves but low in flowers. An analysis of cis-acting elements showed that elements related to the stress response were prevalent in the promoter sequences of MdTLPs. Expression profiling by qRT-PCR indicated that almost all MdTLPs were up-regulated at some extent under abiotic stress, exogenous ABA and H2O2 treatments in leaves and roots, though different MdTLP members exhibited differently in leaves and roots. The results and information above may provide a basis for further investigation of TLP function in plants.

Efficacy of adoptive cellular therapy in patients with gastric cancer: a meta-analysis.

AIM: To systemically evaluate the efficacy and safety of adoptive cellular therapy for the treatment of gastric cancer (GC). MATERIALS & METHODS: We performed a systemic review and meta-analysis of nine eligible trials with GC and evaluated the effect of adoptive cellular therapy on the overall survival (OS) rate, T-cell subsets and adverse events. RESULTS: Overall, 829 patients were involved in the analysis. Adoptive cellular therapy significantly improved the OS rate compared with the control group. Meanwhile, we observed greatly increased percentages of CD3(+), CD4(+) and CD4(+)/CD8(+) in cellular therapy groups. CONCLUSION: Adoptive cellular therapy combined with adjuvant therapy resulted in significantly better OS rates, progression-free survival and T-lymphocyte responses in patients with GC.

C-Jun NH2-Terminal Kinase and p38 Inhibition Suppresses Prostaglandin E2-Stimulated Aromatase and Estrogen Receptor Levels in Human Endometriosis.

CONTEXT: Endometriosis is an estrogen-dependent disease. P38 and C-jun NH2-terminal kinase (JNK) inhibitors may have a therapeutic effect on endometriosis through regulation of prostaglandin E2 (PGE2)-induced estrogen metabolism. OBJECTIVE: The objective of this study was to determine whether the activated MAPKs signaling pathway observed in human ectopic endometrial stromal cells (ESCs) from ovarian endometriomas influences levels of aromatase and estrogen receptor ß (ERß) protein regulated by PGE2. In turn, the effects of inhibiting MAPKs in the presence of PGE2 on estrogen production were investigated in vitro and in vivo. RESULTS: Expression of aromatase and ERß regulated by PGE2 were much higher in ESCs than eutopic ESCs from the same person. Activation of p38, JNK, ERK 1/2 and ERK 5 MAPKs by PGE2 were observed in ESCs, where PGE2-stimulated aromatase and ERß expression mainly through p38 and JNK pathway. P38 and JNK inhibition or small interfering RNA knockdown blocked PGE2-induced aromatase and ERß expression. PGE2 enhanced binding of downstream p38 and JNK transcription factors activating transcription factor-2 and c-Jun to aromatase and ERB promoter regions in ESCs. Moreover, treatment of endometriosis xenografts with inhibitors of p38 and JNK abrogated PGE2-amplified estradiol synthesis and xenograft growth. CONCLUSIONS: PGE2 activates p38 and JNK signaling pathways, further stimulating c-Jun and activating transcription factor-2 binding to aromatase and ERB promoter regions with elevated estradiol production. Inhibition of JNK and P38 may be a potential method of treating human endometriosis.

Metformin Suppresses Prostaglandin E2-Induced Cytochrome P450 Aromatase Gene Expression and Activity via Stimulation of AMP-Activated Protein Kinase in Human Endometriotic Stromal Cells.

BACKGROUND: Cytochrome P450 aromatase (encoded by the CYP19A1/aromatase gene) plays a critical physiologic role in endometriosis. Metformin is known to suppress prostaglandin E2 (PGE2)-induced CYP19A1 messenger RNA (mRNA) expression in human endometriotic stromal cells (ESCs). However, the possible mechanism behind this suppression remains to be determined. METHODS: In this study, ESCs were cultured with metformin, PGE2, and adenosine monophosphate (AMP)-activated protein kinase (AMPK) inhibitors. Expression of CYP19A1 mRNA and aromatase activity were measured by quantitative polymerase chain reaction and aromatase activity assay, respectively. The binding of the cyclic AMP response element-binding (CREB) protein to CYP19A1 promoter II (PII) was assessed by chromatin immunoprecipitation assay. RESULTS: We demonstrated that metformin downregulated the expression of aromatase mRNA (32%) and activity (25%) stimulated by PGE2 (4.18-fold and 2.14-fold) in ESCs via stimulation of AMPK. Following PGE2 treatment, there was a marked increase in CREB binding to aromatase PII, while metformin attenuated the above-mentioned stimulation by 67%. CONCLUSION: Metformin could inhibit PGE2-induced CYP19A1 mRNA expression and aromatase activity via AMPK activation and inhibition of CREB to CYP19A1 PII in human ESCs. The results of the present study suggest that metformin may have unique therapeutic potential as an antiendometriotic drug in the future.

Key amino acids of arabidopsis VKOR in the activity of phylloquinone reduction and disulfide bond formation.

Many proteins in chloroplast are regulated through the disulfide bond/thiol transformation to realize their activities. A homologue of VKOR (Vitamin K epoxide reductase) in Arabidopsis chloroplast is found to catalyze the disulfide bond formation in vivo and to mediate the specific phylloquinone reduction in vitro. It is also called LTO1 (Lumen Thiol Oxidoreductase 1). Investigations about functions and essential amino acid residues of AtVKOR have important theoretical significance to clarify the chloroplast redox regulation mechanism. In this study, several amino acids in the VKOR domain of AtVKOR were identified to be involved in binding of phylloquinone. Site-directed mutagenesis was used to study the function of these positions. The results suggested that residues Ser77, Leu87, Phe137 and Leu141 were quite important in the binding and catalyzing the reduction of phylloquinone. These residues were also involved in the electron transferring and disulfide bond formation of substrate proteins by motility assays in vivo, suggesting that the binding of phylloquinone not only affected the delivery of electrons to phylloquinone but also affected the whole electron transfer process. The conserved cysteines in the AtVKOR domain also played critical roles in phylloquinone reduction. When each of the four conserved cysteines was mutated to alanine, the mutants lost reduction activity entirely, suggesting that the four conserved cysteines played crucial roles in the electron transfer process.

Metformin inhibits StAR expression in human endometriotic stromal cells via AMPK-mediated disruption of CREB-CRTC2 complex formation.

CONTEXT: Endometriosis is an estrogen-dependent disease affecting reproductive women. Metformin could have a therapeutic effect on endometriosis through regulation of local estrogen production. OBJECTS: The aim of this study was to investigate the molecular and cellular mechanism by which metformin regulates StAR expression in human endometriotic stromal cells (ESCs). METHODS: ESCs derived from ovarian endometriomas were cultured with metformin and prostaglandin E2 (PGE2). StAR mRNA was measured by quantitative PCR; pregnenolone, progesterone, and estrogen production were measured by ELISA kits; steroidogenic acute regulatory protein (StAR), AMP-activated protein kinase, cAMP response element binding protein (CREB), and CREB-regulated transcription coactivator 2 (CRTC2) protein expression were measured by Western blot assay; and CRTC2 translocation and its association with CREB were assessed by coimmunoprecipitation assay and CRTC2-CREB complex binding by a chromatin immunoprecipitation assay. RESULTS: 1) StAR mRNA levels in ESCs are 264 times higher than those in endometrial cells. 2) Metformin downregulates the StAR mRNA expression (maximum 31.7%) stimulated by PGE2 (2.4-fold) in ESCs. 3) PGE2 induces CRTC2 translocation and enhances its association with CREB to form a transcription complex that binds to the StAR promoter region. 4) Metformin prevents the nuclear translocation of CRTC2 by increasing AMP-activated protein kinase phosphorylation. This inhibits transcription of StAR by disrupting formation of the CREB-CRTC2 complex, involved in activation of the StAR promoter cAMP response element. CONCLUSIONS: We have demonstrated a detailed mechanistic analysis of StAR expression regulated by metformin in ESCs. Our data highlight a role for CRTC2 in the mechanism by which metformin inhibits StAR expression.

Identification of potential targets for thylakoid oxidoreductase AtVKOR/LTO1 in chloroplasts.

The Arabidopsis thylakoid membrane bimodular oxidoreductase, AtVKOR, could catalyze disulfide bond formation, and its direct functional domain (thioredoxin-like domain) is located in the thylakoid lumen according to the topological structure. Many proteins have one or several disulfide bonds in the thylakoid lumen, including photosynthetic chain components. A yeast two-hybrid assay was used to identify potential targets for the AtVKOR, and a Trx-like domain was constructed into a BD vector as bait. Twenty-two thylakoid lumenal proteins with disulfides were selected. The cDNAs encoding these proteins were constructed into an AD vector. Eight proteins were identified from the hybrid results to interact with AtVKOR, including HCF164, cytochrome c6A, violaxanthin deepoxidase, embryo sac development arrest 3 protein (EDA3), two members pentapeptide repeat proteins (TL17 and TL20.3), and two FK-506 binding proteins (FKBP13 and FKBP20-2). The BIACORE system was used to demonstrate that the recombinant HCF164 and Trx-like domain of AtVKOR could interact directly in vitro. The KD value for binding HCF164 to AtVKOR was calculated as 2.5×10(-6) M. These results suggest that AtVKOR can interact with partial thylakoid lumenal proteins and indicates AtVKOR plays an important role in regulating the thylakoid lumen redox.