Toxicity of common biocides used in aquaculture to embryos and larvae of Brachymystax tsinlingensis Li.

Brachymystax tsinlingensis Li is a threatened fish species endemic to China. With the problems of environmental factors and seeding breeding diseases, it is important to further improve the efficiency of seeding breeding and the basis of resource protection. This study investigated the acute toxicity of copper, zinc and methylene blue (MB) on hatching, survival, morphology, heart rate (HR) and stress behaviour of B. tsinlingensis. Eggs (diameter: 3.86 ± 0.07 mm, weight: 0.032 ± 0.004 g) of B. tsinlingensis were selected randomly from artificial propagation and developed from eye-pigmentation-stage embryos to yolk-sac stage larvae (length: 12.40 ± 0.02 mm, weight: 0.03 ± 0.001 g) and exposed to different concentrations of Cu, Zn and MB for 144 h in a series of semi-static toxicity tests. The acute toxicity tests indicated that the 96-h median lethal concentration (LC50 ) values of the embryos and larvae were 1.71 and 0.22 mg l-1 for copper and 2.57 and 2.72 mg l-1 for zinc, respectively, whereas the MB LC50 after 144-h exposure for embryos and larvae were 67.88 and 17.81 mg l-1 , respectively. The safe concentrations of copper, zinc and MB were 0.17, 0.77 and 6.79 mg l-1 for embryos and 0.03, 0.03 and 1.78 mg l-1 for larvae, respectively. Copper, zinc and MB treatments with concentrations greater than 1.60, 2.00 and 60.00 mg l-1 , respectively, led to a significantly low hatching rate and significantly high embryo mortality (P < 0.05), and copper and MB treatments with concentrations greater than 0.2 and 20 mg l-1 led to significantly high larvae mortality (P < 0.05). Exposure to copper, zinc and MB resulted in developmental defects, including spinal curvature, tail deformity, vascular system anomalies and discolouration. Moreover, copper exposure significantly reduced the HR of larvae (P < 0.05). The embryos exhibited an obvious change in behaviour, converting from the normal behaviour of emerging from the membrane head first to emerging tail first, with probabilities of 34.82%, 14.81% and 49.07% under copper, zinc and MB treatments, respectively. The results demonstrated that the sensitivity of yolk-sac larvae to copper and MB was significantly higher than that of embryos (P < 0.05) and that B. tsinlingensis embryos or larvae might be more resistant to copper, zinc and MB than other members of the Salmonidae family, which benefits their resource protection and restoration.

The interaction of MC3R and MC4R with MRAP2a in rainbow trout (Oncorhynchus mykiss).

Melanocortin 3 and 4 receptors are two important neural G protein-coupled receptors that regulate energy homeostasis in vertebrates. Melanocortin receptor accessory protein 2 (MRAP2) is also involved in the regulation of food intake and body weight as a variable regulator of melanocortin receptors. Rainbow trout (Oncorhynchus mykiss) is a valuable cold-water fish cultured worldwide. In the rainbow trout model, we cloned and identified mrap2a, a paralog of mrap2. Rainbow trout mrap2a consisted of a 690 bp ORF and was expected to encode a putative protein of 229 amino acids. The qPCR results showed that rainbow trout mrap2a was expressed at high levels in brain tissue similar to mc3r and mc4r. In addition, co-immunoprecipitation verified that MRAP2a interacts with MC3R and MC4R in vitro and that MRAP2a is involved in and regulates the constitutive activity and signaling of MC3R and MC4R. MRAP2a reduced constitutive and agonist-stimulated cAMP levels of MC3R; furthermore, MRAP2a increased constitutive ERK1/2 activation but reduced ligand-induced stimulation at high levels of expression. For MC4R, MRAP2a showed decreased cAMP basal activity but increased agonist-stimulated cAMP signaling and increased ACTH ligand sensitivity. However, MRAP2a failed to affect MC4R constitutive activity and agonist-induced ERK1/2 signaling. Undoubtedly, our study will have great significance for revealing the conserved role of MC4R and MC3R signaling in teleost fish, especially in cold-water fish growth and energy homeostasis.

Simultaneous determination of thirteen aminoalcohol-diterpenoid alkaloids in the lateral roots of Aconitum carmichaeli by solid-phase extraction-liquid chromatography-tandem mass spectrometry.

Aminoalcohol-diterpenoid alkaloids have been reported as the cardioactive components in the lateral roots of Aconitum carmichaeli (Fuzi) according to recent studies. Determination of these effective components is of great significance for quality control purposes for Fuzi. Here we report, for the first, the development and validation of a new method to determine the 13 aminoalcohol-diterpenoid alkaloids in Fuzi by using a simple and accurate solid-phase extraction-liquid chromatography-tandem mass spectrometry. The chromatographic analysis was performed on an ODS column with methanol-0.1 % formic acid (80 : 20, v/v) as the mobile phase. The quantification was performed using MS/MS detection in the positive ion mode with multiple reaction monitoring. Linearity was observed within a range of concentrations of 20-2,000 ng/mL. For all the analytes, the r value was greater than 0.9990. The limit of detection and the limit of quantitation were less than 0.5 ng/mL and 2.0 ng/mL, respectively. The intraday and interday precisions were less than 5% and 10%, respectively. The accuracy was within the range of 90 to 105%. This method was successfully applied to determine the 13 aminoalcohol-diterpenoid alkaloids in Fuzi from different origins and with different processing methods.

Profiling the dynamics of abscisic acid and ABA-glucose ester after using the glucosyltransferase UGT71C5 to mediate abscisic acid homeostasis in Arabidopsis thaliana by HPLC-ESI-MS/MS.

The HPLC-MS/MS method was developed to profile the dynamics of abscisic acid (ABA) and ABA-glucose ester (ABA-GE) after cloning glycosyltransferase enzyme family gene AtUGT71C5 into Arabidopsis thaliana. By constructing over-expression lines (OE) and down-expression lines (DN), we acquired mutant strains to analyze the function of AtUGT71C5. The multiple-reaction monitoring (MRM) was used for quantitative determination in negative mode. The transition was m/z 263.1→153.0 for ABA ([M-H]+), m/z 425.1→263.0 for ABA-GE ([M-H]+), and m/z 321.0→152.0 for chloramphenicol. The linear range was 0.8684-217.1 ng/mL for ABA and 0.3920-196.0 ng/mL for ABA-GE. The accuracy was 88.0-109.0% for ABA and 86.6-113.0% for ABA-GE; the inter-day and intra-day precisions were less than 5.4% for ABA and 8.9% for ABA-GE, respectively. This method is simple and sensitive enough for determination of ABA and ABA-GE in A. thaliana leaves. All the evidence confirmed the speculation that AtUGT71C5 can mediate abscisic acid homeostasis.

A novel role of ribonuclease inhibitor in regulation of epithelial-to-mesenchymal transition and ILK signaling pathway in bladder cancer cells.

Human ribonuclease inhibitor (RI) is a cytoplasmic acidic protein possibly involved in biological functions other than the inhibition of RNase A and angiogenin activities. We have previously shown that RI can inhibit growth and metastasis in some cancer cells. Epithelial-mesenchymal transition (EMT) is regarded as the beginning of invasion and metastasis and has been implicated in the metastasis of bladder cancer. We therefore postulate that RI regulates EMT of bladder cancer cells. We find that the over-expression of RI induces the up-regulation of E-cadherin, accompanied with the decreased expression of proteins associated with EMT, such as N-cadherin, Snail, Slug, vimentin and Twist and of matrix metalloprotein-2 (MMP-2), MMP-9 and Cyclin-D1, both in vitro and in vivo. The up-regulation of RI inhibits cell proliferation, migration and invasion, alters cell morphology and adhesion and leads to the rearrangement of the cytoskeleton in vitro. We also demonstrate that the up-regulation of RI can decrease the expression of integrin-linked kinase (ILK), a central component of signaling cascades controlling an array of biological processes. The over-expression of RI reduces the phosphorylation of the ILK downstream signaling targets p-Akt and p-GSK3ß in T24 cells. We further find that bladder cancer with a high-metastasis capability shows higher vimentin, Snail, Slug and Twist and lower E-cadherin and RI expression in human clinical specimens. Finally, we provide evidence that the up-regulation of RI inhibits tumorigenesis and metastasis of bladder cancer in vivo. Thus, RI might play a novel role in the development of bladder cancer through regulating EMT and the ILK signaling pathway.

[Spectroscopic investigation on the interaction between astragalin from lotus leaf and DNA].

The interaction between astragalin (AST) from lotus leaf and deoxyribonucleic acid (DNA) in Tris-HCl buffer (pH = 7.4) was investigated by the application of fluorescence spectroscopy and ultraviolet absorption spectroscopy, the effects of ionic strength and anion quencher KI on the fluorescence intensity of AST from lotus leaf and the system of AST-DNA were explored, and the competitive binding to DNA between AST from lotus leaf and Neutral Red(NR)dye was also studied. The results demonstrated that AST could bind to DNA and the formed complex quenched the intrinsic fluorescence of AST from lotus leaf through static quenching mechanism. The quenching rate constants of biomolecule(Kq)of the reaction of DNA with AST from lotus leaf were calculated to be 3.120 X 10(12) and 2.630 X 10(12) L x mol(-1) x s(-1) by Stern-Volumer equation, the corresponding binding constants (Kd) were computed to be 3.412 x 10(12) and 1.762 x 10(4) L x mol(-1) and the number of binding sites(n) was counted to be 1.007 and 0.962 between AST from lotus leaf and DNA at 298 and 308 K, respectively. When bound to DNA, the AST from lotus leaf showed hypochromic effect and red shift in the absorption spectra. It was also found that different ionic strength had little or no effect on the fluorescence intensity of AST and AST-DNA, but the fluorescence intensity of AST-DNA quenched by anionic quencher KI was much less than that of free AST. AST could be intercalated into DNA and displaced the NR from the NR-DNA complex. It was showed that AST from lotus leaf could combine with DNA in the mode of intercalation.

[Autophosphorylation of erythrocyte insulin receptors in patients with polycystic ovary syndrome].

OBJECTIVE: To investigate the tyrosine autophosphorylation of insulin receptors in patients with polycystic ovary syndrome (PCOS). METHODS: Forty patients with PCOS and 20 age- and body mass index matched healthy women were included in the study. The patients with PCOS were classified as HI-PCOS (n=20) or non-HI-PCOS (n=20) based on the fasting insulin level (>or< or =15 mIU/L). 1) Serum gonadotropins and ovarian steroids were determined. Oral glucose tolerance tests (OGTT) and insulin releasing tests were performed to calculate the insulin resistance index (HOMA-IR). 2) Blood samples were obtained at five time-points during the OGTT (Fasting and 30 min, 60 min, 120 min, and 180 min after taking 75 g glucose orally). The erythrocytes were isolated and the autophosphorylation insulin receptors (APIR) and total insulin receptors (TIR) were measured by enzymatic immunoassay. 3) The in vivo autophosphorylation of insulin receptors was indicated by the APIR/TIR ratio. RESULTS: The HI-PCOS patients had lower APIR/TIR ratio than the non-HI-PCOS patients and healthy controls at the 60th minute after OGTT (P<0.05). No differences in other time-points were significant. CONCLUSION: The autophosphorylation of insulin receptors in HI-PCOS patients decrease, which might be a mechanism for insulin resistance in patients with PCOS.