Prolyl Isomerization-Mediated Conformational Changes Define ATR Subcellular Compartment-Specific Functions.

ATR is a PI3K-like kinase protein, regulating checkpoint responses to DNA damage and replication stress. Apart from its checkpoint function in the nucleus, ATR actively engages in an antiapoptotic role at mitochondria following DNA damage. The different functions of ATR in the nucleus and cytoplasm are carried out by two prolyl isomeric forms of ATR: trans- and cis-ATR, respectively. The isomerization occurs at the Pin1 Ser428-Pro429 motif of ATR. Here, we investigated the structural basis of the subcellular location-specific functions of human ATR. Using a mass spectrometry-based footprinting approach, the surface accessibility of ATR lysine residues to sulfo-NHS-LC-biotin modification was monitored and compared between the cis- and the trans-isomers. We have identified two biotin-modified lysine residues, K459 and K469, within the BH3-like domain of cis-ATR that were not accessible in trans-ATR, indicating a conformational change around the BH3 domain between cis- and trans-ATR. The conformational alteration also involved the N-terminal domain and the middle HEAT domain. Moreover, experimental results from an array of complementary assays show that cis-ATR with the accessible BH3 domain was able to bind to tBid while trans-ATR could not. In addition, both cis- and trans-ATR can directly form homodimers via their C-terminal domains without ATRIP, while nuclear (trans-ATR) in the presence of ATRIP forms dimer-dimer complexes involving both N- and C-termini of ATR and ATRIP after UV. Structural characteristics around the Ser428-Pro429 motif and the BH3 domain region are also analyzed by molecular modeling and dynamics simulation. In support, cis conformation was found to be significantly more energetically favorable than trans at the Ser428-Pro429 bond in a 20-aa wild-type ATR peptide. Taken together, our results suggest that the isomerization-induced structural changes of ATR define both its subcellular location and compartment-specific functions and play an essential role in promoting cell survival and DNA damage responses.

Expression and clinical significance of CXCL17 and GPR35 in endometrial carcinoma.

Endometrial carcinoma is one of the most common gynecologic malignancies. CXCL17-CXCR8 (GPR35) axis is reported to play an indispensability role in tumors. Our purpose is to screen possible prognostic and immune-related factors in endometrial carcinoma by detecting the mRNA and protein expression of CXCL17 and CXCR8. We use the qRT-PCR method to test the mRNA expression of CXCL17 and CXCR8 in 35 pairs of endometrial carcinoma and adjacent tissue. The protein expression of CXCL17 and CXCR8 in 30 cases of normal proliferative endometrium, 30 cases of endometrial atypical hyperplasia and 50 cases of endometrial carcinoma was detected by tissue microarray immunohistochemistry. There was no significant difference in the positive expression rate between endometrial adenocarcinoma tissue and endometrial atypical hyperplasia tissue (P > 0.05). But significantly better than normal proliferative tissue (P < 0.001). Correlation analysis of CXCR8 and CXCL17 in endometrial carcinoma showed a positive correlation (r = 0.9123, P < 0.0001). For patients with endometrial cancer, the overall survival (OS) of patients with high CXCL17 expression was significantly higher than that low CXCL17 expression (log-rank test, P < 0.0001), whereas CXCR8 had no statistical significance. But the expression of CXCR8 is an independent prognostic factor of OS in endometrial carcinoma patients. Our study showed that CXCL17 and CXCR8 may be involved in the occurrence and development of endometrial cancer. High expression of CXCL17 may be used as a biomarker for predicting survival. Because CXCL17 and CXCL18 are related to lymphocytes and immune regulation, they are expected to become potential targets for immunotherapy.

Electroacupuncture inhibits IL-17/IL-17R and post-receptor MAPK signaling pathways in a rat model of chronic obstructive pulmonary disease.

OBJECTIVE: Interleukin (IL)-17, as a T-helper 17 cell (Th17) cytokine, plays a key role in chronic obstructive pulmonary disease (COPD) pathophysiology including chronic inflammation and airway obstruction, which lead to decreased pulmonary function. The aim of this study was to investigate the effect of acupuncture on IL-17, its receptor (IL-17R) and the mitogen-activated protein kinase (MAPK) signaling pathway, in a rat model of COPD. METHODS: The COPD model was induced in Sprague Dawley rats by exposure to cigarette smoke for 12 weeks. The model rats were treated with electroacupuncture (EA) at BL13 and ST36. The lung function and histology of the rats were observed. IL-17, tumor necrosis factor (TNF)-α, and IL-10 were detected by enzyme-linked immunosorbent assay (ELISA) in bronchoalveolar lavage fluid (BALF) and in plasma. The leukocytes and macrophages in the BALF were counted. The expression levels of IL-17R were assayed in lung tissue by real-time polymerase chain reaction (PCR), western blotting, and immunohistochemistry. MAPK signaling pathway molecules including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK)1/2 and p38, and their phosphorylated forms, were observed in the lung by western blotting. RESULTS: Compared with the control group rats, lung function decreased and there was a severe inflammatory infiltration of the pulmonary parenchyma in the COPD rats. EA effectively improved lung function and alleviated the inflammatory infiltration in the lungs of COPD rats. EA also reversed the elevated total leukocyte and macrophage counts, the high levels of IL-17 and TNF-α, and the low IL-10 content in COPD rats. Meanwhile, EA downregulated the increased mRNA and protein expression of IL-17R, and significantly inhibited the elevated levels of phosphorylated JNK, ERK1/2, and p38 in the lungs of COPD rats. CONCLUSION: Our results suggest that the protective effects of acupuncture therapy on the lungs of COPD rats are likely related to inhibition of IL-17/IL-17R and the post-receptor MAPK signaling pathways.

LncRNA Gas5 acts as a ceRNA to regulate PTEN expression by sponging miR-222-3p in papillary thyroid carcinoma.

Accumulating evidence demonstrates that the long non-coding RNA Growth Arrest-Specific 5 (Gas5) has practical significance in cancer progression and metastasis. However, its role and function in papillary thyroid carcinoma (PTC) remains unknown. In this study, we aimed to explore the potential involvement of Gas5 in papillary thyroid carcinogenesis and to highlight the emerging roles of ceRNAs in the biological regulation of PTC cells. The results suggested that Gas5 was markedly downregulated in both PTC tissues and PTC cell lines. Over-expression of Gas5 remarkably suppressed PTC cells proliferation in vitro and inhibited the growth of tumor cells in vivo likewise. Furthermore, Gas5 was identified as a target of miR-222-3p which was aberrantly high in PTC cells. Enhanced expression of miR-222-3p promoted the proliferation of PTC cells while knocking down miR-222-3p could inhibit it. The advanced effects of miR-222-3p on the proliferation of PTC cells could be partly reversed by the upregulation of Gas5 expression. Furthermore, we validated that Gas5 increased the protein level of the PTEN, one of miR-222-3p's targets, which further activated PTEN/AKT pathway. Taken together, our study identified a tumor suppressive role of Gas5 in PTC cells acting as a ceRNA, effectively becoming a sink for miR-222-3p, modulating the expression of PTEN, which lead to PTEN/AKT pathway activation and proliferation suppression. This finding may offer a new potential therapeutic strategy for PTC.

[Effect of application of cow manure and green manure on corn yield and soil physical-chemical properties in land restoration area]. / 施用牛粪和绿肥对土地整治区玉米产量和土壤理化性质的影响.

The effects of cow manure and green manure on maize yield, soil respiration and soil physical-chemical properties in land restoration area was evaluated through field experiments. The results indicated that the maize yield and thousand-grain mass with cow manure were increased by 7.2%-29.9% and 2.5%-18.2%, respectively compared with the application of chemical fertilizer (CF), while the soil active organic carbon and organic matter contents of cow manure were 5.3%-34.6% and 8.0%-17.6% higher than that obtained in CF. The maize yield and thousand-grain mass were increased by 10.8%-15.6% and 4.5%-8.4% with application of green manure, respectively compared with CF. The content of active organic carbon in green manure was 14.1%-48.6% higher than that detected in CF. In the second year, the content of organic matter in green manure treatment was 7.2% higher than that of CF. The soil respiration rates under cow manure and green manure treatments increased by 20.0%-69.3% compared with CF. CF and green manure could improve the soil bulk density and increase the aggregate ratios of ＜0.01 mm and 0.05-1 mm fractions, respectively. On the other hand, the cow manure and green manure could decrease the soil total porosity and the capillary porosity. In conclusion, the application of cow manure and green manure in land restoration region could increase maize yield during the two consecutive seasons, which showed a positive response to improvement of soil physical-chemical properties.

Electroacupuncture at Feishu (BL13) and Zusanli (ST36) down-regulates the expression of orexins and their receptors in rats with chronic obstructive pulmonary disease.

OBJECTIVE: Inflammation and lung function decline are the main pathophysiological features of chronic obstructive pulmonary disease (COPD). Acupuncture can improve lung function in patients with COPD, but the underlying mechanisms are not well understood. Orexins (OXs), which are found in peripheral plasma, are neuropeptides that regulate respiration and their levels are related to COPD. Therefore, we hypothesized that acupuncture might alter OXs, reduce lung inflammation and improve lung function in COPD. METHODS: COPD was induced in rats by exposure to cigarette smoke for 8 weeks and injecting with lipopolysaccharide twice. Electroacupuncture (EA) was performed at Feishu (BL13) and Zusanli (ST36) for 30 min/d for 2 weeks. Rat lung function and morphology were assessed after EA. The levels of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in bronchoalveolar lavage fluid (BALF) and orexin A and B levels in the lung tissue were detected by enzyme-linked immunosorbent assay. OX receptor mRNA levels and immunopositive cells were assessed with real-time polymerase chain reaction and immunohistochemical methods, respectively. The relationships among lung function, cell factors, and OX levels were analyzed by Pearson correlation analyses. RESULTS: Compared with the control group, lung function was significantly decreased in the rats with COPD (P<0.05). There were increases in TNF-α and IL-1ß levels in BALF (P<0.05 and P<0.01, respectively), orexin A level in lung tissue (P<0.01; but not orexin B) and mRNA expressions of OX (OXR1) and OX 2 (OXR2) in lung tissue (P<0.05 and P<0.01, respectively); the integrative optical densities (IODs) of both receptors were greater in the COPD group (P<0.05). For rats with COPD subjected to EA, lung function was improved (P<0.05). There were notable decreases in TNF-α and IL-1ß levels (P<0.05 and <0.01, respectively) in BALF. Orexin A, but not orexinB, levels in lung tissue also decreased (P<0.01), as did mRNA expression of OX1R and OX2R in lung tissue (P<0.05 and P<0.01, respectively). Receptor IODs were also reduced after EA treatment (P<0.05). Furthermore, orexin A levels and ratio of forced expiratory volume in 0.3 s to forced vital capacity were strongly negatively correlated (P<0.01), and orexin A was positively correlated with TNF-α and IL-1ß (P<0.001 and P<0.05, respectively). CONCLUSION: EA at Zusanli and Feishu improved lung function of rats with COPD and had an anti-inflammatory effect, which may be related to down-regulation of OXA and its receptors.

[Nutrient spatial variability of tobacco soil restoration area and fertility suitability level evaluation].

By using geographic information system technology (GIS) and geostatistics methods, this paper studied the spatial variability of soil properties and available nutrients in the new regulation area units located in Qingjiangyuan modern tobacco agriculture science and technology park (Enshi, Hubei), suburb of Enshi City and the Baiyang base of Lichuan City, and further evaluation of the soil fertility suitability index (SFI) was carried out by use fuzzy mathematics. The results indicated that the effects of land restoration on the soil available phosphorus content variability and spatial distribution were very obvious, possibly due to the landform characteristics and restoration extent. The effect of land restoration on soil pH was small, however, serious soil acidification was detected in the soil sampled from Baiyang (pH < 5.5). Low SFI was found in 77.6%, 17.1% and 31.4% of the soils taken from the suburb, Baiyang and Qingjiangyuan, respectively. In conclusion, attentions should be paid on soil acidification in Baiyang, soil fertility and equalization in the suburb, and soil fertility in the region of Qingjiangyuan with low SFI.

The impact of dietary iodine intake on lipid metabolism in mice.

The present study has been designed to investigate the impact of dietary iodine intake on lipid metabolism in mice, including iodine deficiency and iodine excess. Different amounts of iodine mixed in the drinking water were continuously administered to mice. The body weights and the levels of urinary iodine were measured 8 months after the treatment. Thyroid hormones in the serum were detected by chemiluminescence immunoassay. Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol and low-density lipoprotein cholesterol (LDL-C) were determined enzymatically by automatic analyzer. Results showed that the urine iodine concentrations paralleled the amounts of iodine intakes. No statistical differences of body weights among different groups were found. The levels of thyroid hormones were dramatically decreased in iodine deficiency while no significant differences were found between iodine excess groups and normal iodine group. In iodine deficiency groups, the levels of TG, TC, and LDL were increased at varying degrees. In iodine excess groups, the levels of TG in the male mice and the levels of TC in the female mice were much lower than normal iodine group. In conclusion, dietary iodine intake may affect the metabolism of serum lipids. Hypothyroid function induced by iodine deficiency may be responsible for the changes of lipids. Higher iodine intake might benefit lipid metabolism.

[The effects of iodine/selenium on the function of antigen presentation of peritoneal macrophages in rats].

OBJECTIVE: To observe the effects of iodine/selenium on the function of antigen presentation of peritoneal macrophages in rats and explore the immunological mechanisms of iodine/ selenium's role in pathogenesis of autoimmune thyroid diseases (AITD). METHODS: Female Lewis rats were randomly divided into four groups including (1) low selenium and normal iodine group (L(sE)N(I)) (2) low selenium and high iodine group (L(Se)H(I)) (3) normal selenium and normal iodine group (N(Se)N(I) ) (4) normal selenium and high iodine group (N(Se)H(I)). All rats were fed by a special diet with lower selenium and iodine in it and drunk ion-free water containing different levels of iodine and selenium for 3 months. Peritoneal macrophages of each group and OVA allergized T cells were prepared and cultured together. Then the function of antigen presentation were estimated by detecting the levels of IL-2 in the culture supernatant. The levels of the expression of co-stimulator CD86 in the spleen of each group were determined by RT-PCR. RESULTS: The level of IL-2 in the supernatant in N(Se)H(I) (43.22 +/- 3.27) pg/ml was much stronger than N(Se)N(I) [the level of IL-2 was (25.74 +/- 2.45) pg/ml, P < 0.05]. The level of IL-2 in L(Se)N(I) (15.79 +/- 2.13) pg/ml was significantly lower than N(Se)N(I) (P < 0.05). The expression of CD86 mRNA in N(Se)H(I) (CD86/beta-actin: 0.52 +/- 0.10) were higher than N(Se)N(I) (CD86/beta-actin: 0.35 +/- 0.04), P < 0.05. CONCLUSIONS: High iodine could promote the presentation function of macrophages to a higher state than normal. Therefore, high iodine intake might become an importantly inducing factor in thyroid autoimmunity. Low selenium could weaken the ability of recognizing and presenting OVA antigen of peritoneal macrophages which might destroy immunological homeostasis and thus the low selenium intake might also become an inducer of AITD.

[Influence of long-term treatment with MPA on the biological character of endometrial carcinoma Ishikawa cell].

The study was undertaken to investigate the effect of long-term treatment with MPA on the growth and invasiveness of endometrial carcinoma Ishikawa cells and the expression of the subtype of estrogen receptor and progesterone receptor. The human endometrial carcinoma Ishikawa cells were continually exposed to 2.5 micromol/L step-wise increase of MPA. MTT assay, flow cytometry and Matrigel invasion assay were used to detect the effect of MPA on the growth, cell cycle distribution and the invasion capability of the cells respectively. RT-PCR was performed to detect the changes of CyclinD1, MMP2 and MMP9 over different time treatment of MPA. Immunocytochemistry was used to examine the expression of estrogen receptor subtype and progesterone receptor subtype. Endometrial carcinoma Ishikawa cells became resistant to the inhibitory effect of MPA over ten months MPA treatment. The cells resistant to the growth inhibitory effect were named progestin-resistant Ishikawa cells and the cells which the progestin-resistant originated from were named the parent Ishikawa cells. The effects of MPA shifted from growth and invasiveness inhibitory effects on the parent Ishikawa cells to stimulatory effect on the progestin-resistant Ishikawa cells. Consistent with the phenomena, the treatment with MPA on Ishikawa cells resulted in statistically significant decreases of CyclinD1, MMP2 and MMP9 gene expression, reversely, the treatment with MPA on the progestin-resistant Ishikawa cell resulted in statistically significant increases of CyclinD1, MMP2, MMP9 gene expression. Immunocytochemical analysis showed reduced ERalpha and PR-B expression and increased ERbeta expression in the progestin-resistant Ishikawa cells compared with parental Ishikawa cells. From the experiment, it was concluded that the prolonged treatment with MPA on Ishikawa cell could give rise of the resistant effect of MPA, the effect of MPA on the growth and invasiveness capability of endometrial carcinoma shifted from inhibitory to stimulatory. The imbalance of ER subtype and PR subtype might contribute to the mechanisms involved in the progesterone resistance over long-term MPA treatment.

[Experimental study on effects of iodine deficiency and excess on thyroid autoimmunity].

OBJECTIVE: To observe the effects of iodine on the level of CD4/CD8 cells and the production of thyroglobulin autoantibody (TGAb) and thyroid peroxidase autoantibody (TPOAb) in Wistar rats and to investigate the role of iodine in thyroid autoimmunity. METHODS: Rat models with different iodine intakes including low iodine (LI,), normal iodine (NI,), 5 times normal iodine (5HI), 10 times normal iodine (10HI), 50 times normal iodine (50HI) and 100 times normal iodine (100HI) were established. The amount of iodine intake per rat per day in every group was about < 1, 6.15, 30.75, 61.50, 307.50, 615.00 microg separately. The levels of CD4 and CD8 immune cells in peripheral blood were measured by using flow cytometry. Radioimmunoassay (RIA) was used to determine the titers of TGAb and TPOAb in the serum. RESULTS: In peripheral blood, the level of CD4 cells in LI group was (57.9 +/- 4.3)%, being much higher than in NI group (51.2 +/- 4.9)%. When the level of CD8 cells in 100HI group was (18.4 +/- 3.1)% showing significantly lower than in NI group (26.5 +/- 4.1)%, thus making the ratio of CD4/CD8 cells in the above two groups (LI: 2.4 +/- 0.40 and 100 HI: 2.7 +/- 0.4) higher than in NI group (1.9 +/- 0.3). As comparing with NI group (2099 +/- 220) CPM, the level of TGAb in LI group (1510 +/- 221) CPM was significantly decreased; while in 50HI group (3986 +/- 286) and 100HI group (3550 +/- 378) CPM, the levels of TGAb were both increased, and the levels of TPOAb in 10HI group (2066 +/- 184) CPM and in 50HI group (2141 +/- 163) CPM were both distinctly lower than in NI group (2372 +/- 245) CPM. CONCLUSIONS: Iodine might exert influence on the level of CD4/CD8, and thus the production of thyroid antibodies might directly or indirectly take part in the process of thyroid autoimmunity. Both low iodine and 100 times normal iodine intakes might activate the immune state on some degrees. The effects of iodine on immune responses of TG and TPO antigen in thyroid autoimmunity might not be completely the same.

[Expression of glucose transporter-1 and its correlation with basic fibroblast growth factor and proliferating cell nuclear antigen in epithelial ovarian neoplasm].

OBJECTIVE: To study the expression of glucose transporter-1 (GLUT1) and its correlation with basic fibroblast growth factor (bFGF) and proliferating cell nuclear antigen (PCNA) in epithelial ovarian neoplasm. METHODS: Streptavidin-peroxidase complex technique was used to examine the expression of GLUT1, bFGF and PCNA protein in six cases of normal ovarian tissue, 20 cases of benign epithelial tumors, seven cases of borderline tumor and 44 cases of epithelial ovarian carcinoma. RESULTS: In normal ovary and benign ovarian tumor, GLUT1 was not detected, but in borderline ovarian tumor and cancer, the positive expression ratio of GLUT1 was 6/7 and 91% (40/44), respectively. The intensity of GLUT1 in ovarian epithelial neoplasm was significantly higher than in borderline tumors. The staining intensity of GLUT1 was significantly correlated with the histological grade of the tumor (r(S) = 0.499, P = 0.001), and was positively correlated with the clinical stage, cancer invasion and lymph node metastasis. GLUT1 staining was intense in cytoplasmic membrane, and was stronger in areas far away from blood vessels and near the necrotic center. GLUT(1) expression level did not show any association with histology type (P = 0.513). bFGF positive rate in tumor was 57% (25/44). The staining intensity of GLUT1 was significantly higher in bFGF positive group than in bFGF negative group (P < 0.01). The mean score of PCNA was significantly higher in cancer tissues with strong GLUT1 staining(+++) than that with moderate(++) and low(+) GLUT1 staining (P < 0.05). And the score of PCNA was also significantly higher in bFGF positive group than in bFGF negative group (P < 0.01). CONCLUSIONS: (1) The expression of GLUT1 may be closely related to malignant transformation of ovarian epithelial tumors. It is also correlated with clinical stage, cancer invasion and lymph node metastasis, and may be a useful predictor of poor prognosis. (2) bFGF could enhance the expression of GLUT1. Both of them play important roles in the carcinogenesis and progression of ovarian epithelial carcinoma.