Identification of key residues involved in the dimerization of the secretory Na(+)-K(+)-2Cl(-) cotransporter NKCC1

The "secretory" Na(+)-K(+)-2Cl(-) cotransporter, NKCC1, belongs to the SLC12 gene family of electroneutral cation-chloride cotransporters. A number of these proteins, including NKCC1 itself, exist as homodimers in the membrane, suggesting that this may be a common feature of the SLC12 family. We have previously demonstrated that replacing the C-terminus of NKCC1 with that of its close homologue NKCC2 produced a fully functional chimeric protein that formed homodimers but did not dimerize with NKCC1. Here we employ a novel co-immunoprecipitation assay to study the dimerization interaction of NKCC1 using additional NKCC1/NKCC2 C-terminal chimeras and point mutants. Our results indicate that the substitution of a number of regions of the C-terminus of NKCC1 with the corresponding sequence from NKCC2 results in weakened dimerization with wild-type NKCC1, demonstrating that various residues play a role in this interaction. Most interestingly, however, we find that the replacement of a single NKCC1 residue, G812, with cysteine, the corresponding amino acid in NKCC2, results in a point mutant that displays no significant dimerization with the wild-type protein. In addition to this effect on heterodimer formation, we also find that G812 mutants can nevertheless form homodimers but that this interaction can be weaker than that observed for wild-type NKCC1. We demonstrate that our results are consistent with at least one established mechanism of protein dimer formation, that of "domain swapping", as well as with a recently reported crystal structure of the C-terminus of a bacterial SLC12 homologue.     

Acclimation of hydrogen peroxide enhances salt tolerance by activating defense-related proteins in Panax ginseng C.A. Meyer

The effect of exogenously applied hydrogen peroxide on salt stress tolerance was investigated in Panax ginseng. Pretreatment of ginseng seedlings with 100 μM H₂O₂ increased the physiological salt tolerance of the ginseng plant and was used as the optimum concentration to induce salt tolerance capacity. Treatment with exogenous H₂O₂ for 2 days significantly enhanced salt stress tolerance in ginseng seedlings by increasing the activities of ascorbate peroxidase, catalase and guaiacol peroxidase and by decreasing the concentrations of malondialdehyde (MDA) and endogenous H₂O₂ as well as the production rate of superoxide radical (O₂ ^?). There was a positive physiological effect on the growth and development of salt-stressed seedlings by exogenous H₂O₂ as measured by ginseng dry weight and both chlorophyll and carotenoid contents. Exogenous H₂O₂ induced changes in MDA, O₂ ^?, antioxidant enzymes and antioxidant compounds, which are responsible for increases in salt stress tolerance. Salt treatment caused drastic declines in ginseng growth and antioxidants levels; whereas, acclimation treatment with H₂O₂ allowed the ginseng seedlings to recover from salt stress by up-regulation of defense-related proteins such as antioxidant enzymes and antioxidant compounds.     

Association of BLM and BRCA1 during Telomere Maintenance in ALT Cells

Fifteen percent of tumors utilize recombination-based alternative lengthening of telomeres (ALT) to maintain telomeres. The mechanisms underlying ALT are unclear but involve several proteins involved in homologous recombination including the BLM helicase, mutated in Bloom''s syndrome, and the BRCA1 tumor suppressor. Cells deficient in either BLM or BRCA1 have phenotypes consistent with telomere dysfunction. Although BLM associates with numerous DNA damage repair proteins including BRCA1 during DNA repair, the functional consequences of BLM-BRCA1 association in telomere maintenance are not completely understood. Our earlier work showed the involvement of BRCA1 in different mechanisms of ALT, and telomere shortening upon loss of BLM in ALT cells. In order to delineate their roles in telomere maintenance, we studied their association in telomere metabolism in cells using ALT. This work shows that BLM and BRCA1 co-localize with RAD50 at telomeres during S- and G2-phases of the cell cycle in immortalized human cells using ALT but not in cells using telomerase to maintain telomeres. Co-immunoprecipitation of BRCA1 and BLM is enhanced in ALT cells at G2. Furthermore, BRCA1 and BLM interact with RAD50 predominantly in S- and G2-phases, respectively. Biochemical assays demonstrate that full-length BRCA1 increases the unwinding rate of BLM three-fold in assays using a DNA substrate that models a forked structure composed of telomeric repeats. Our results suggest that BRCA1 participates in ALT through its interactions with RAD50 and BLM.     

Diagnostic test accuracy for detecting Schistosoma japonicum and S. mekongi in humans: A systematic review and meta-analysis

BackgroundMost of national schistosomiasis elimination programmes in Asia are relying on stool examination, particularly Kato Katz stool examination technique for regular transmission monitoring. However, the Kato-Katz technique has shown low sensitivity for the detection of light-intensity infections, and therefore highly sensitive diagnostic tools are urgently required to monitor prevalence of infection in low transmission settings. The objective of this systematic review was to evaluate and synthesize the performance of diagnostic tests for detecting Schistosoma japonicum and S. mekongi infection in people living in endemic areas.Methodology/Principal findingsWe comprehensively searched these nine electronic databases and other resources until July 2019, with no language or publication limits: PubMed, EMBASE, MEDLINE, Web of Science, BIOSIS Citation Index, HTA, CINAHL PLUS, The Cochrane Library, and PsycINFO. We included original studies that assessed diagnostic performance using antibody, antigen, and molecular tests with stool examination test as a reference standard. Two reviewers independently extracted a standard set of data and assessed study quality. We estimated the pooled estimates of sensitivity and specificity for each index test. We used diagnostic odds ratio to determine the overall accuracy and hierarchical summary receiver operating characteristics (HSROC) curve to assess the index tests performance.Fifteen studies (S. japonicum [n = 13] and S. mekongi [n = 2]) testing 15,303 participants were included in the review. Five studies reported performance of enzyme-linked immunosorbent assay (ELISA), seven studies reported indirect hemagglutination assay (IHA), and four studies reported polymerase chain reaction (PCR) for detecting S. japonicum. The pooled sensitivity and specificity were 0.93 (95% CI: 0.84–0.98) and 0.40 (95% CI: 0.29–0.53) for ELISA, 0.97 (95% CI: 0.90–0.99) and 0.66 (95% CI: 0.58–0.73) for IHA, and 0.89 (95% CI: 0.71–0.96) and 0.49 (95% CI: 0.29–0.69) for PCR respectively. A global summary indicated the best performance for IHA, closely followed by ELISA. We were unable to perform meta-analysis for S. mekongi due to insufficient number of studies.Conclusions/SignificanceIHA showed the highest detection accuracy for S. japonicum. Further studies are needed to determine the suitable diagnostic methods to verify the absence of transmission of S. mekongi and also to compare detection accuracy against more sensitive reference standards such as PCR.     

Interactions of anthropometric indices,rs9939609 FTO gene polymorphism and breast cancer: A case-control study

Contradictory results were reported on the effect of fat mass- and obesity-associated (FTO) gene and anthropometric measurements on breast cancer (BC). This study aimed to assess the interactions between rs9939609 polymorphism of FTO gene, anthropometric indices and BC risk in Iranian women. This case-control study was performed on 540 women including 180 women with BC and 360 healthy women in Tehran, Iran. Physical activity and dietary intakes were assessed by validated questionnaires. Data on sociodemographic and pathologic factors of the participants as well as their blood samples were collected. The rs9939609 FTO gene polymorphism was genotyped using the tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR). No significant association was found between BC and risk allele of FTO rs9939609 polymorphism after adjustments for the confounders. However, there was a significant association between rs9939609 polymorphism risk allele and BC risk in females with overweight, even after adjusting for age, family history of BC, abortion, BMI and the number of pregnancies (P < .05). The association was disappeared after further adjustments for lifestyle factors including smoking, alcohol consumption, calorie and macronutrients intake, and physical activity. The FTO gene polymorphism was associated with the risk of BC in overweight individuals. This association was influenced by environmental factors including diet, alcohol consumption and smoking. Future studies are required to confirm the association between the FTO gene and BC in overweight females and to identify the underlying mechanisms.     

Prediction of Genes Involved in Lung Cancer with a Systems Biology Approach Based on Comprehensive Gene Information

Biochemical Genetics - Over the past few years, hundreds of genes have been reported in relation to lung cancer. Systems biology studies can help validate this association and find the most valid...     

The interaction effects of aerobic exercise training and vitamin D supplementation on plasma lipid profiles and insulin resistance in ovariectomized rats

Purpose

The purpose of this study was to determine the interaction effects of aerobic exercise training and vitamin D supplementation on indices of obesity and plasma lipid profiles in ovariectomized (OVX) rats.

Methods

Forty female Wistar rats were divided into 5 groups: aerobic training (3 days/week for 8 weeks; AT; n = 8), aerobic training and vitamin D supplementation (OVX + AT + Vit D; n = 8), vitamin D supplementation (OVX + Vit D; n = 8), ovariectomized control (OVX + C, n = 8) and SHAM (n = 8). After blood sampling, visceral fat was taken from the abdominal cavity and weighed immediately. Data was statistically analyzed by One-way ANOVA and Repeated measure ANOVA tests with a 0.05 significance level.

Results

Body weight, visceral fat, BMI and food intake decreased significantly in OVX + AT + Vit D (P < 0.001); whereas these variables increased significantly in OVX + C (P < 0.001) and SHAM (P < 0.023) groups. At the end of two-months of follow-up, we observed significant differences in TC, TG, HDL-C, LDL-C, glucose, insulin, and HOMA-IR in all groups.

Conclusion

It seems that aerobic training with vitamin D, due to the involvement of muscle mass and exposure to dynamic pressure on the bones and muscles, increased energy expenditure, stimulated insulin exudation and glucose homeostasis, decreased insulin resistance and improved the lipid profile in ovariectomized rats.     

Interrelationship between calmodulin (CaM) and H2O2 in abscisic acid-induced antioxidant defense in the seedlings of Panax ginseng

Calmodulin (CaM), the predominant Ca(2+) receptors, is one of the best-characterized Ca(2+) sensors in all eukaryotes. In this study the role of CaM and the possible interrelationship between CaM and hydrogen peroxide (H(2)O(2)) in abscisic acid (ABA) induced antioxidant defense were investigated in the seedling of Panax ginseng. Treatment of ABA (100 μM) and H(2)O(2) (10 mM) increased the expression of Panax ginseng calmodulin gene (PgCaM) and significantly enhanced the expression of the antioxidant marker genes such as superoxide dismutase, ascorbate peroxidase, glutathione reductase and the activities of chloroplastic and cytosolic antioxidant enzymes. Pretreatments with two CaM antagonists, trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide hydrochloride (W7) and inhibitor or scavenger, diphenyleneiodonium chloride, and dimethylthiourea of reactive oxygen species almost completely suppressed the up-regulation of antioxidant and PgCaM gene. Moreover, H(2)O(2) production and CaM content was almost completely inhibited by pretreatments with two CaM antagonists. In addition, the expressions of PgCaM gene under different biotic stress were analyzed at different time intervals. Thus it may suggests that CaM are involved in ABA-induced increased expression of PgCaM which triggers H(2)O(2) production through activating trans-plasma membrane NADPH oxidase, resulting in up-regulation of defense related antioxidant gene and also plays a pivotal role in defense response against pathogens.