RESUMO
With potent herbicidal activity, biocatalysis synthesis of L-glufosinate has drawn attention. In present research, NAP-Das2.3, a deacetylase capable of stereoselectively resolving N-acetyl-L-glufosinate to L-glufosinate mined from Arenimonas malthae, was heterologously expressed and characterized. In Escherichia coli, NAP-Das2.3 activity only reached 0.25 U/L due to the formation of inclusive bodies. Efficient soluble expression of NAP-Das2.3 was achieved in Pichia pastoris. In shake flask and 5 L bioreactor fermentation, NAP-Das2.3 activity by recombinant P. pastoris reached 107.39 U/L and 1287.52 U/L, respectively. The optimum temperature and pH for N-acetyl-glufosinate hydrolysis by NAP-Das2.3 were 45 °C and pH 8.0, respectively. The Km and Vmax of NAP-Das2.3 towards N-acetyl-glufosinate were 25.32 mM and 19.23 µmol mg-1 min-1, respectively. Within 90 min, 92.71% of L-enantiomer in 100 mM racemic N-acetyl-glufosinate was converted by NAP-Das2.3. L-glufosinate with high optical purity (e.e.P above 99.9%) was obtained. Therefore, the recombinant NAP-Das2.3 might be an alternative for L-glufosinate biosynthesis.
Assuntos
Reatores Biológicos , Pichia , Proteínas Recombinantes/química , Pichia/genética , Pichia/metabolismo , FermentaçãoAssuntos
Metabolismo Energético , Natação/fisiologia , Acelerometria , Fenômenos Biomecânicos , HumanosRESUMO
BACKGROUND: Endometrial cancer is one of the most common gynecological malignancies and has exhibited an increasing incidence rate in recent years. Cancer stem cells (CSCs), which are responsible for tumor growth and chemoresistance, have been confirmed in endometrial cancer. However, it is still challenging to identify endometrial cancer stem cells to then target for therapy. METHODS: Flow cytometry was used to identify the endometrial cancer stem cells. Sphere formation assay, western blotting, qRT-PCR assay, cell viability assay, xenograft assay and immunohistochemistry staining analysis were utilized to evaluate the effect of SPARC-related modular calcium binding 2 (SMOC-2) on the cells proliferation and drug resistance. Cell viability assay, qRT-PCR assay, immunofluorescence staining, Co-IP assay and luciferase reporter gene assay were performed to explore the possible molecular mechanism by which SMOC-2 activates WNT/ß-catenin pathway. FINDINGS: We found the expression of SPARC-related modular calcium binding 2 (SMOC-2), a member of SPARC family, was higher in endometrial CSCs than that in non-CSCs. SMOC-2 was also more highly expressed in spheres than in monolayer cultures. The silencing of SMOC-2 suppressed cell sphere ability; reduced the expression of the stemness-associated genes SOX2, OCT4 and NANOG; and enhanced chemosensitivity in endometrial cancer cells. By co-culture IP assay, we demonstrated that SMOC-2 directly interacted with WNT receptors (Fzd6 and LRP6), enhanced ligand-receptor interaction with canonical WNT ligands (Wnt3a and Wnt10b), and finally, activated the WNT/ß-catenin pathway in endometrial cancer. SMOC-2 expression was closely correlated with CSC markers CD133 and CD44 expression in endometrial cancer tissue. INTERPRETATION: Taken together, we conclude that SMOC-2 might be a novel endometrial cancer stem cell signature gene and therapeutic target for endometrial cancer. FUND: National Natural Science Foundation of China, Scientific and Technological Innovation Act Program of Shanghai Science and Technology Commission, Scientific and Technological Innovation Act Program of Fengxian Science and Technology Commission, Natural Science Foundation of Shanghai.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias do Endométrio/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Modelos Animais de Doenças , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Camundongos , Gradação de Tumores , Estadiamento de Neoplasias , Paclitaxel/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cervical cancer is an infectious cancer and the most common gynecologic cancer worldwide. E6/E7, the early genes of the high-risk mucosal human papillomavirus type, play key roles in the carcinogenic process of cervical cancer. However, little was known about its roles in modulating tumor microenvironment, particular extracellular matrix (ECM). In this study, we found that E6/E7 could regulate multiple ECM proteins, especially collagen triple helix repeat containing 1 (CTHRC1). CTHRC1 is highly expressed in cervical cancer tissue and serum and closely correlated with clinicopathological parameters. CTHRC1 promotes cervical cancer cell migration and invasion in vitro and metastasis in vivo. E6/E7 regulates the expression of CTHRC1 in cervical cancer by E6/E7-p53-POU2F1 (POU class 2 homeobox 1) axis. Futhermore, CTHRC1 activates Wnt/PCP signaling pathway. Take together, E6/E7-p53-POU2F1-CTHRC1 axis promotes cervical cancer cell invasion and metastasis and may act as a potential therapeutic target for interventions against cervical cancer invasion and metastasis.
Assuntos
Polaridade Celular , Proteínas da Matriz Extracelular/metabolismo , Fator 1 de Transcrição de Octâmero/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Via de Sinalização Wnt , Movimento Celular/genética , Proliferação de Células/genética , Proteínas da Matriz Extracelular/sangue , Proteínas da Matriz Extracelular/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica , Curva ROC , Regulação para Cima/genética , Neoplasias do Colo do Útero/sangue , Neoplasias do Colo do Útero/genéticaRESUMO
The LiNbO3 crystals doped with Cr3+ ion (Cr:LN) and co-doped with Cr3+ and Zn ions (Zn:Cr:LN) were grown by the Bridgman method. The absorption coefficient and concentration of Cr3+ in crystals were measured by UV/Visible spectra and inductively coupled plasma (ICP) spectrometry, respectively. The effective distribution coefficients (k) of Cr3+ in the crystals were calculated. The results indicated that the k for Cr:LN crystal decreased from 3.75 to 2.49 as the incorporating concentration of Cr3+ increased from 0.1 to 0.5 mol%; the introduction of ZnO in Cr:LN induced the reduction of k value effectively. However, the k value increased from 1.85 to 2.25 as the incorporating concentration of ZnO increased from 3 to 6 mol%. The variation of the k value was explained by the suppressing effect of ZnO on the incorporation of Cr3+ ions and the distribution of Cr3+ in LN.
Assuntos
Compostos de Cromo/análise , Nióbio/química , Óxidos/química , Espectrofotometria Ultravioleta/métodos , Espectrofotometria/métodos , Cromo/análise , Cromo/química , Compostos de Cromo/química , Cristalização , Cinética , Modelos Químicos , Óxido de Zinco/químicaRESUMO
The temperature-dependence emission spectra of lower and upper part of Eu(3+) -doped LiNbO3 crystals from 77 to 600 K were investigated under the excitation of a 488 nm light. The results show that, for upper part of crystal, the fluorescence intensity of Eu3+ ions increases with the temperature increase, however, for lower part, the intensity first increases with the temperature increase, and then decreases obviously with temperature increase. The variations of fluorescence intensity for lower and upper part of crystals are explained. The emission intensity of Eu3+ ions in LN is the total effect of thermally excited emission, phonon-assistant absorption and temperature-quenching effect.
