Search

Objective: To construct recombinant expression plasmid with an antibacterial peptide (attacin) mature peptide gene from Musca domestica and express an antibacterial peptide attacin in Pichia pastoris., Methods: The coding region of attacin mature peptide was cloned into the expression vector pPIC9K. The recombinant plasmid was linearized by digestion with Sac I and transformed into P. pastoris GS115 by electroporation. The insert clones containing multiple copies were selected with geneticin, pha, enotype and PCR. The expression of P. pastoris GS115/pPIC9K-attacin were induced with methanol. The supernatant of the expressed protein was analyzed by SDS-PAGE and Western blotting. The anti-bacterial activity of expression product was analyzed by agarose diffusion assay., Results: The expression plasmid was constructed and transformed into P. pastoris GS115. SDS-PAGE and Western blotting analysis indicated that the recombinant containing recombinant plasmid pPIC9K-attacin expressed a Mr 22000 protein. The expression product inhibited the growth of E. coli K12D31., Conclusion: The attacin gene from Musca domestica has been expressed in P. pastoris.

BACKGROUND Xanthoceras sorbifolia Bunge is a valuable oilseed tree that has linoleic acid-rich seed oil. Microsomal oleate desaturase ( FAD2; EC 1.3.1.35) is responsible for the conversion of oleic acid to linoleic acid during fatty acid synthesis. In this study, XsFAD2 was cloned from developing embryos of X. sorbifolia. RESULTS XsFAD2 contained three histidine boxes, a C-terminal endoplasmic reticulum retrieval motif, and five putative transmembrane domains representing the characteristics of membrane-bound fatty acid desaturase. XsFAD2 expression in yeast cells resulted in linoleic acid (18:2) and palmitolinoleic acid (16:2) production, confirming the biological activity of the enzyme encoded by XsFAD2. These fatty acids are not normally present in wild-type yeast. Phylogenetic analysis indicated that XsFAD2 is located in a subgroup of FAD2 enzymes specifically or highly expressed in developing seeds. The expression level of XsFAD2 in seeds was much higher than those in leaves and petals. Furthermore, XsFAD2 expression pattern correlated well with linoleic acid accumulated in seeds. CONCLUSION Results suggested that XsFAD2 is responsible for the high linoleic acid content in X. sorbifolia seed oil. This study provides insight on the regulation mechanism of fatty acid synthesis in X. sorbifolia seeds and a valuable gene for improving the oil quality in oilseed trees. © 2013 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Abstract: Xanthoceras sorbifolia is an excellent model system for studying triacylglycerol (TAG) biosynthesis in woody oilseed plants due to the high amount of seed oil, which is important for food and industrial uses. TAG is the major form of stored lipids in seeds and diacylglycerol acyltransferase (DGAT; EC 2. 3. 1. 20) catalyzes the final and critical step of TAG synthesis. Here, two novel DGAT genes, designated XsDGAT1 and XsDGAT2, were cloned from developing X. sorbifolia embryos. Sequence analysis showed that XsDGAT1 had little sequence homology to XsDGAT2. Heterologous expression of XsDGAT1 and XsDGAT2 in TAG-deficient yeast mutants restored TAG synthesis, confirming their biological activity. Expression of the two genes in wild-type Arabidopsis led to TAG synthesis and an increase in total seed oil in transgenic plants, with XsDGAT1 appearing to contribute to TAG synthesis at a greater level. Comparison of the expression patterns revealed that both XsDGAT1 and XsDGAT2 were expressed in the examined tissues and had similar spatiotemporal expression patterns with higher expression in embryos than in leaves and petals. The expression patterns of both XsDGAT1 and XsDGAT2 correlated with oil accumulation in developing X. sorbifolia embryos. These data suggest that XsDGAT1 and XsDGAT2 are both responsible for TAG synthesis in X. sorbifolia seeds. [Copyright &y& Elsevier]

Background/Aims: Water channels, also named aquaporins (AQPs), play crucial roles in cellular water homeostasis. Methods: RT-PCR indicated the mRNA expression of AQPs 1-5, 7, 9, and 11-12, but not AQPs 0, 6, 8, and 10 in the 2∼8-cell stage human embryos. AQP3 and AQP7 were further analyzed for their mRNA expression and protein expression in the oocyte, zygote, 2-cell embryo, 4-cell embryo, 8-cell embryo, morula, and blastocyst from both human and mouse using RT-PCR and immunofluorescence, respectively. Results: AQP3 and AQP7 were detected in all these stages. Knockdown of either AQP3 or AQP7 by targeted siRNA injection into 2-cell mouse embryos significantly inhibited preimplantation embryo development. However, knockdown of AQP3 in JAr spheroid did not affect its attachment to Ishikawa cells. Conclusion: These data demonstrate that multiple aquaporins are expressed in the early stage human embryos and that AQP3 and AQP7 may play a role in preimplantation mouse embryo development. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Our previous work indicated that TWEAK is associated with various types of cutaneous vasculitis (CV). Herein, we investigate the effects of TWEAK on vascular injury and adhesion molecule expression in CV mice. We showed that TWEAK priming in mice induced a local CV. Furthermore, TWEAK priming also increased the extravasation of FITC-BSA, myeloperoxidase activity and the expression of E-selectin and ICAM-1. Conversely, TWEAK blockade ameliorated the LPS-induced vascular damage, leukocyte infiltrates and adhesion molecules expression in LPS-induced CV. In addition, TWEAK treatment of HDMECs up-regulated E-selectin and ICAM-1 expression at both mRNA and protein levels. TWEAK also enhanced the adhesion of PMNs to HDMECs. Finally, western blot data revealed that TWEAK can induce phosphorylation of p38, JNK and ERK in HDMECs. These data suggest that TWEAK acted as an inducer of E-selectin and ICAM-1 expression in CV mice and HDMECs, may contribute to the development of CV. [ABSTRACT FROM AUTHOR]

Objective: To investigate the possible roles of cyclin B1/cyclin-dependent kinase (cdc2) and Polo-like kinase 1 (Plk1) in the pathogenesis of endometriosis.Design: A case-control study.Setting: University hospital.Patient(s): Patients with or without endometriosis were diagnosed by pathological examination or laparoscopy. The patients with the following criteria within the past 6 months were excluded: endocrine or inflammatory diseases, pregnancy or lactation, hormonal therapy, and neoplasm in the uterine cavity.Intervention(s): Eutopic and ectopic endometria were obtained at the time of surgery. Blood was collected on the same day as surgery.Main Outcome Measure(s): The mRNA/protein expression and localization of cyclin B1, cdc2, and Plk1 in endometrium, and serum levels of E(2) and P.Result(s): The expression levels of cyclin B1 and Plk1, but not cdc2, in ectopic endometria were significantly higher than in eutopic endometria. The immunohistochemical staining of cyclin B1 and Plk1 was detected in the nuclei of ectopic and eutopic endometrial cells. Furthermore, ectopic endometrial expression levels of cyclin B1 or Plk1 were positively correlated with serum E(2) levels.Conclusion(s): Cyclin B1 and Plk1 may play important roles in the pathogenesis of endometriosis by mediating ectopic endometrial cell proliferation under regulation of ovarian hormones. [ABSTRACT FROM AUTHOR]

The growth differential factor-9 (GDF-9) gene, an oocyte-specific factor, was screened in 106 Chinese women with diminished ovarian reserve (DOR), and three single-nucleotide polymorphisms, c.G169A, c.C447T and c.G546A, were detected. We found GDF-9 c.G546A, but not c.G169A or c.C447T, to be correlated with the poor ovarian stimulation and in vitro fertilization outcomes in women with DOR. [ABSTRACT FROM AUTHOR]

The ectopic expression of Arabidopsis gene RESISTANCE TO POWDERY MILDEW 8.1 (R1Y4 plant line) triggers a hypersensitive response (HR) providing defense potentially effective against different pathogens. In order to understand the regulation of RPW8.1- mediated signaling, an approach based on ethyl methanesulfonate (EMS) mutagenesis was adopted to identify mutants that enhance or impair RPW8.1 -mediated cell death. Here we describe the identification of mutants b10-6 and b9-1, point mutations that were induced in the At2g37630 locus resulting in an amino acid substitution or a premature stop codon in ASYMMETRIC LEAVES 1 (AS1), respectively. Loss-of-function in AS1 altered the transcription of RPW8.1 , RPW8.1 -dependent necrotic lesions in leaves and resistance to powdery mildew. In the mutant b10-6, RPW8.1 -mediated cell death and disease resistance was compromised, however. in b9-1 RPW8.1 -mediated cell death and resistance to powdery mildew was slightly enhanced. Expression of AS1-eYFP within b10-6 background suppressed RPW8.1 -mediated cell death and disease resistance in the short-day condition, but largely restored the phenotypes of b10-6 to that of R1Y4 in the long-day condition. These data imply that proper expression of AS1 is required for full function of RPW8.1 in triggering cell death and resistance to powdery mildew. [ABSTRACT FROM AUTHOR]

Abstract The development of D-glucaric acid (GA) production in recombinant cells has leapt forward in recent years, and higher throughput screening and selection of better-performing recombinant cells or biocatalysts is in current demand. A biosensor system which converts GA concentration into fluorescence signal in Escherichia coli was developed in 2016, but its application has rarely been reported. Herein, an effective high-throughput screening approach independent of special-purpose devices such as microfluidic platforms was established and tentatively applied. In this one-pot two-strain system, GA producers—bacterial or yeast cells containing the GA biosynthetic pathway—were sorted with the help of another E. coli strain acting as a GA biosensor. The identification of highly active mutants of myo-inositol oxygenase through this system validates its effectiveness in sorting E. coli cells. Subsequently, accurate ranking of the GA synthesis capacity of a small library of Saccharomyces cerevisiae strains containing distinct GA synthesis pathways demonstrated that this optimized one-pot two-strain system may also be used for eukaryotic producer strains. These results will assist in research into metabolic engineering for GA production and development of biosensor applications. Graphical abstract fx1 Highlights • An effective rapid screening approach based on D-glucaric acid (GA) biosensor in E. coli or yeast. • GA producers were sorted with the help of another E. coli biosensor cell. • D82Y and S173N mutations result in increased activity of MIOX. • Fine-tuning the cofactor balance could increase the GA production of yeast. [ABSTRACT FROM AUTHOR]

AbstractGambogic acid (GA), the main active component of gamboge, is well known for its marked antitumor effect in vitroand in vivo. The aim of this study was to assess the natural interaction between GA and chemotherapeutic agents, 5-fluorouracil (5-FU), oxaliplatin (Oxa), and docetaxel (Doc), which are widely used in gastric cancer treatment. This study also investigated the effect of GA on cell apoptosis and drug-associated gene expression for further mechanism research. Synergistic interaction on human gastric cancer BGC-823 cells and MKN-28 cells was evaluated using the combination index (CI) method. The double staining method with Annexin-V-FITC and PI was employed to distinguish the apoptotic cells from others. Expression of drug-associated genes, that is, thymidylate synthase (TS), excision repair cross-complementing (ERCC1), BRCA1, tau, and β-tubulin III, was measured by real-time quantitative RT-PCR. This study found that GA had a synergistic effect on the cytotoxity of chemotherapeutic agents against both cell lines. The combination of GA and chemotherapeutic agents could also induce apoptosis in a synergistic manner. The mRNA levels of TS, ERCC1, BRCA1, tau, and β-tubulin IIIwere suppressed at 0.009, 0.075, 0.140, 0.267, and 0.624-fold, respectively, when cells were exposed to GA at the concentration of 0.25 μM. These data suggest that GA has a promising role in enhancing the efficacy of 5-FU, Oxa, and Doc in the treatment of gastric cancer. The potential mechanism would be their synergistic effects on apoptosis induction and the downregulation of chemotherapeutic agent-associated genes. [ABSTRACT FROM AUTHOR]

Abstract: Caffeine is the most widely consumed psychoactive substance in the world. It can elevate the level of glucocorticoid which is involved in metabolism regulation, stress response, and immune function. However, the specific mechanism has yet to be elucidated. Glucocorticoid is steroid hormone synthesized in adrenal cortex and the key rate-limiting step in its biosynthesis is mediated by steroidogenic acute regulatory protein (StAR). This study was designed to investigate the direct effects and inheritable epigenetic mechanisms of caffeine on cortisol production and StAR expression in human adrenocortical cells. The human adrenocortical cell line NCI-H295A was cultured with 0.4–40μM caffeine. There was a significant increase of the cortisol production in cells. In both acutely and chronically caffeine-treated cell groups, mRNA and protein expressions of StAR were stimulated in a dose-dependent manner. DNA methylation detection via bisulfite-sequencing PCR (BSP) uncovered a single site CpG demethylation at nt −682 within the StAR promoter region. Then we investigated how long the increased StAR expression and the single CpG demethylation could last. The caffeine was withdrawn after 48h of treatment and then the cells were continually subcultured for up to 5 and 10 passages, respectively. The results showed that the StAR expression at post-caffeine passage 10 still increased, as compared with that in the control. The caffeine-induced demethylation at nt −682 in StAR promoter underwent a similar time course as StAR expression does. The present study reveals the direct effect and possible inheritable epigenetic mechanism of caffeine on steroidogenesis in human adrenocortical cells and has implications for our understanding of the consumption of caffeine. [Copyright &y& Elsevier]