LC-MS/MS separation and quantitation of ribavirin in chicken and comparison of different mass spectrometric platforms.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the analysis of ribavirin in chicken. Samples was extracted with 0.1% formic acid and purified by Hypercarb cartridge prior to LC-MS/MS analysis. The eluates were evaporated to dryness, reconstituted in 1 mL 5mM ammonium acetate containing 5% acetonitrile (v/v) and 0.1% (v/v) formic acid. Chromatographic separation was performed on a Hypercarb analytical column under a gradient elution program with acetonitrile and 0.1% (v/v) formic acid in 5 mM ammonium acetate at a flow rate of 0.6 ml/min. The intraday and interday accuracy ranged from - 7.83 - 1.39%, and - 6.38 - 2.25%, with precisions between 1.34 - 3.88%% and 1.10 - 4.67%. The limits of detection (LODs) and limits of quantitation (LOQs) of ribavirin was 0.1 ng/mL and 0.5 ng/mL, respectively. The method was validated for linearity, accuracy, precision, matrix effect and stability. Application of the method confirmed 3 ribavirin positive samples out of 50 commercial chicken samples, with concentrations of ribavirin ranging from 0.9 µg/kg to 5.8 µg/kg a, respectively. Additionally, both AB Sciex 5500 and Agilent 6945B were proven to be suitable in ribavirin separation and quantification. The described method is suitable for the determination of ribavirin in chicken in analytical practice to monitor illegal addition of this kind of anti-viral drug.

Biological active ingredients of Astragali Radix and its mechanisms in treating cardiovascular and cerebrovascular diseases.

BACKGROUND: With the rising age of the global population, the incidence rate of cardiovascular and cerebrovascular diseases (CCVDs) is increasing, which causes serious public health burden. The efforts for new therapeutic approaches are still being sought since the treatment effects of existing therapies are not quite satisfactory. Chinese traditional medicine proved to be very efficient in the treatment of CCVDs. Well described and established in Chinese medicine, Astragali Radix, has been commonly administered in the prophylaxis and cure of CCVDs for thousands of years. PURPOSE: This review summarized the action mode and mechanisms of Astragali Radix phytochemicals on CCVDs, hoping to provide valuable information for the future application, development and improvement of Astragali Radix as well as CCVDs treatment. METHODS: A plenty of literature on biological active ingredients of Astragali Radix used for CCVDs treatment were retrieved from online electronic PubMed and Web of Science databases. RESULTS: This review highlighted the effects of five main active components in Astragali Radix including astragaloside Ⅳ, cycloastragenol, astragalus polysaccharide, calycosin-7-O-ß-d-glucoside, and calycosin on CCVDs. The mechanisms mainly involved anti-oxidative damage, anti-inflammatory, and antiapoptotic through signaling pathways such as PI3K/Akt, Nrf2/HO-1, and TLR4/NF-κB pathway. In addition, the majority active constituents in AR have no obvious toxic side effects. CONCLUSION: The main active components of Astragali Radix, especially AS-IV, have been extensively summarized. It has been proved that Astragali Radix has obvious therapeutic effects on various CCVDs, including myocardial and cerebral ischemia, hypertension, atherosclerosis, cardiac hypertrophy, chronic heart failure. CAG possesses anti-ischemia activity without toxicity, indicating a worthy of further development. However, high-quality clinical and pharmacokinetic studies are required to validate the current studies.

A quantitative method for detecting meat contamination based on specific polypeptides.

OBJECTIVE: This study was aimed to establish a quantitative detection method for meat contamination based on specific polypeptides. METHODS: Thermally stable peptides with good responses were screened by high resolution liquid chromatography tandem mass spectrometry. Standard curves of specific polypeptide were established by triple quadrupole mass spectrometry. Finally, the adulteration of commercial samples was detected according to the standard curve. RESULTS: Fifteen thermally stable peptides with good responses were screened. The selected specific peptides can be detected stably in raw meat and deep processed meat with the detection limit up to 1% and have a good linear relationship with the corresponding muscle composition. CONCLUSION: This method can be effectively used for quantitative analysis of commercial samples.

Hydrogen Peroxide Is Involved in ß-Cyclodextrin-hemin Complex-Induced Lateral Root Formation in Tomato Seedlings.

Although previous results showed that ß-cyclodextrin-hemin complex (ß-CDH) could induce tomato lateral root (LR) formation, the corresponding downstream messengers are still not fully understood. In this report, similar to the inducing effects of exogenously applied hydrogen peroxide (H2O2), we discovered that ß-CDH elicited RBOH1 transcript upregulation, endogenous H2O2 accumulation, and thereafter tomato LR development. Above responses were sensitive to dimethylthiourea (DMTU) and ascorbic acid (AsA), two membrane-permeable scavengers of H2O2, showing that accumulation of H2O2 and LR formation were significantly blocked. The test with diphenyleneiodonium (DPI; the inhibitor of NADPH oxidase) revealed that H2O2 mainly produced by NADPH oxidase, might be involved in LR formation triggered by ß-CDH. qPCR combined with pharmacological and anatomical analyses showed that ß-CDH-modulated several marker genes responsible for LR formation, such as CYCA3;1, CYCA2;1, CYCD3;1, and CDKA1 (four cell cycle regulatory genes), ARF7 and RSI-1 (two auxin signaling genes), LAX3 (an auxin influx carrier), IAA14 (encoding a member of the Aux/IAA protein family), PIN3 and PIN7 (two auxin efflux carriers), isocitrate dehydrogenase [NADP], NADH-cytochrome b5 reductase 1, and L-ascorbate oxidase homolog genes (two reactive oxygen species-associated genes and one LR formation-related gene), were causally related to above H2O2 signaling. Particularly, representative proteins related to H2O2 metabolism and lateral rooting, were specifically induced in ß-CDH-treated tomato seedlings. Overall, the results clearly suggested a vital role of H2O2 in the ß-CDH-induced tomato LR formation, and ß-CDH-elicited H2O2-related target proteins responsible for LR formation might be, at least partially, regulated at transcriptional and translational levels.

Linking hydrogen-enhanced rice aluminum tolerance with the reestablishment of GA/ABA balance and miRNA-modulated gene expression: A case study on germination.

Although previous results showed that exogenous hydrogen (H2) alleviated aluminum (Al) toxicity, the detailed mechanism remains unclear. Here, we reported that the exposure of germinating rice seeds to Al triggered H2 production, followed by a decrease of GA/ABA ratio and seed germination inhibition. Compared to inert gas (argon), H2 pretreatment not only strengthened H2 production and alleviated Al-induced germination inhibition, but also partially reestablished the balance between GA and ABA. By contrast, a GA biosynthesis inhibitor paclobutrazol (PAC) could block the H2-alleviated germination inhibition. The expression of GA biosynthesis genes (GA20ox1 and GA20ox2) and ABA catabolism genes (ABA8ox1 and ABA8ox2), was also induced by H2. Above results indicated that GA/ABA might be partially involved in H2 responses. Subsequent results revealed that compared with Al alone, transcripts of miR398a and miR159a were decreased by H2, and expression levels of their target genes OsSOD2 and OsGAMYB were up-regulated. Whereas, miR528 and miR160a transcripts were increased differentially, and contrasting tendencies were observed in the changes of their target genes (OsAO and OsARF10). The transcripts of Al-tolerant gene OsSTAR1/OsSTAR2 and OsFRDL4 were up-regulated. Above results were consistent with the anti-oxidant defense, decreased Al accumulation, and enhanced citrate efflux. Together, our results provided insight into the mechanism underlying H2-triggered Al tolerance in plants.

Nitric oxide is involved in methane-induced adventitious root formation in cucumber.

Our previous studies revealed that methane (CH4 ) induces adventitious rooting in cucumber. However, the corresponding molecular mechanism is still elusive. In this work, we discovered that CH4 triggered the accumulation of nitric oxide (NO) and thereafter cucumber adventitious rooting, mimicking the inducing effects of sodium nitroprusside (SNP) and NONOate (two NO-releasing compounds). Above mentioned responses were sensitive to NO scavenger(s), showing that the accumulation of NO and adventitious root development were respectively impaired. Inhibitor test and biochemical analysis suggested that endogenous NO mainly produced by mammalian NO synthase-like enzyme and diamine oxidases (DAO), might be required for adventitious root formation elicited by CH4 . Molecular evidence confirmed that CH4 -mediated induction of several marker genes responsible for adventitious root development, including CsDNAJ-1, CsCDPK1, CsCDPK5, cell division-related gene CsCDC6, and two auxin signaling genes, CsAux22D-like and CsAux22B-like, was casually dependent on NO signaling. The possible involvement of S-nitrosylation during the mentioned CH4 responses was preliminarily illustrated. Taken together, through pharmacological, anatomical and molecular approaches, it is suggested that NO might be involved in CH4 -induced cucumber adventitious rooting, and CH4 -eliciated NO-targeted proteins might be partially modulated at transcriptional and post-translational levels. Our work may increase the understanding of the mechanisms underlying CH4 -elicited root organogenesis in higher plants.

Mutation of Arabidopsis HY1 causes UV-C hypersensitivity by impairing carotenoid and flavonoid biosynthesis and the down-regulation of antioxidant defence.

Previous pharmacological results confirmed that haem oxygenase-1 (HO-1) is involved in protection of cells against ultraviolet (UV)-induced oxidative damage in soybean [Glycine max (L.) Merr.] seedlings, but there remains a lack of genetic evidence. In this study, the link between Arabidopsis thaliana HO-1 (HY1) and UV-C tolerance was investigated at the genetic and molecular levels. The maximum inducible expression of HY1 in wild-type Arabidopsis was observed following UV-C irradiation. UV-C sensitivity was not observed in ho2, ho3, and ho4 single and double mutants. However, the HY1 mutant exhibited UV-C hypersensitivity, consistent with the observed decreases in chlorophyll content, and carotenoid and flavonoid metabolism, as well as the down-regulation of antioxidant defences, thereby resulting in severe oxidative damage. The addition of the carbon monoxide donor carbon monoxide-releasing molecule-2 (CORM-2), in particular, and bilirubin (BR), two catalytic by-products of HY1, partially rescued the UV-C hypersensitivity, and other responses appeared in the hy1 mutant. Transcription factors involved in the synthesis of flavonoid or UV responses were induced by UV-C, but reduced in the hy1 mutant. Overall, the findings showed that mutation of HY1 triggered UV-C hypersensitivity, by impairing carotenoid and flavonoid synthesis and antioxidant defences.

Characterization of a wheat heme oxygenase-1 gene and its responses to different abiotic stresses.

In animals and recently in plants, heme oxygenase-1 (HO1) has been found to confer protection against a variety of oxidant-induced cell and tissue injuries. In this study, a wheat (Triticum aestivum) HO1 gene TaHO1 was cloned and sequenced. It encodes a polypeptide of 31.7 kD with a putative N-terminal plastid transit peptide. The amino acid sequence of TaHO1 was found to be 78% similar to that of maize HO1. Phylogenetic analysis revealed that TaHO1 clusters together with the HO1-like sequences in plants. The purified recombinant TaHO1 protein expressed in Escherichia coli was active in the conversion of heme to biliverdin IXa (BV), and showed that the V(max) was 8.8 U·mg(-1) protein with an apparent K(m) value for hemin of 3.04 µM. The optimum Tm and pH were 35 °C and 7.4, respectively. The result of subcellular localization of TaHO1 showed that the putative transit peptide was sufficient for green fluorescent protein (GFP) to localize in chloroplast and implied that TaHO1 gene product is at least localized in the chloroplast. Moreover, we found that TaHO1 mRNA could be differentially induced by the well-known nitric oxide (NO) donor sodium nitroprusside (SNP), gibberellin acid (GA), abscisic acid (ABA), hydrogen peroxide (H(2)O(2)) and NaCl treatments. Therefore, the results suggested that TaHO1 might play an important role in abiotic stress responses.

Evidence of Arabidopsis salt acclimation induced by up-regulation of HY1 and the regulatory role of RbohD-derived reactive oxygen species synthesis.

In Arabidopsis thaliana, a family of four genes (HY1, HO2, HO3 and HO4) encode haem oxygenase (HO), and play a major role in phytochrome chromophore biosynthesis. To characterize the contribution of the various haem oxygenase isoforms involved in salt acclimation, the effects of NaCl on seed germination and primary root growth in Arabidopsis wild-type and four HO mutants (hy1-100, ho2, ho3 and ho4) were compared. Among the four HO mutants, hy1-100 displayed maximal sensitivity to salinity and showed no acclimation response, whereas plants over-expressing HY1 (35S:HY1) exhibited tolerance characteristics. Mild salt stress stimulated biphasic increases in RbohD transcripts and production of reactive oxygen species (ROS) (peaks I and II) in wild-type. ROS peak I-mediated HY1 induction and subsequent salt acclimation were observed, but only ROS peak I was seen in the hy1-100 mutant. A subsequent test confirmed the causal relationship of salt acclimation with haemin-induced HY1 expression and RbohD-derived ROS peak II formation. In atrbohD mutants, haemin pre-treatment resulted in induction of HY1 expression, but no similar response was seen in hy1-100, and no ROS peak II or subsequent salt acclimatory responses were observed. Together, the above findings suggest that HY1 plays an important role in salt acclimation signalling, and requires participation of RbohD-derived ROS peak II.