The gene encoding thermostable lactate dehydrogenase (Tm-LDH) was cloned into the plasmid pHsh from Thermotoga maritima, and expressed in Escherichia coli JM 109. The recombinant protein was purified to homogeneity by a simple step, heat treatment. The recombinant enzyme had a molecular mass of 33 kDa. The optimal temperature and pH of Tm-LDH were observed 95 degrees C and 7.0. The purified enzyme had a half-life of 2 h at 90 degrees C, and exhibited better stability over a pH range from 5.5 to 8.0. The K(m) and V(max) values were 1.7 mmol/L, 3.8 x 10(4) U/mg of protein for pyruvate, and 7.2 mmol/L and 1.1 x 10(5) U/mg for NADH, respectively. The expression of Tm-LDH in T7 system could not obtain high efficiency, but it has been soluble over-expression in pHsh system and reached 340 mg/L. The superior stability and productivity of Tm-LDH will lay the foundation of its industrial-scale fermentation and application in the NAD regeneration.
Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; metabolism ; L-Lactate Dehydrogenase ; biosynthesis ; Molecular Weight ; Recombinant Proteins ; biosynthesis ; Temperature ; Thermotoga maritima ; enzymology

AIM:To investigate the role of anti-miR-21 oligonucleotide ( AMO) in the anti-leukemic activity of decitabine (DCA) in vitro.METHODS:AMO and scramble oligonucleotide (SCR) were constructed and transfected into HL-60 cells.The miR-21 expression was analyzed by real-time PCR to identify the transfection efficiency .The cells were treated with DCA at gradient concentrations (0.5, 2.0 and 4.0 μmol/L) for 48 h.The mRNA expression of human period circadian protein 3 (hPer3) was detected by real-time PCR.The early apoptotic rates were determined by flow cy-tometry with Annexin V/PI staining.Mean fluorescence intensities ( MFI) of CD117 and CD11b were also measured by flow cytometry.RESULTS:The miR-21 relative expression level in AMO group was significantly lower than that in blank group and SCR group (P<0.01).IC50 of DCA in AMO group was significantly lower than that in blank group and SCR group (P<0.01).With the same concentration of DCA, the early apoptotic rate, the mRNA expression of hPer3 and the MFI of CD11b in AMO group were significantly higher than those in blank group and SCR group (P<0.01).The MFI of CD117 in AMO group were significantly lower than those in blank group and SCR group ( P<0.01 ) .CONCLUSION:Activation of hPer3 expression plays an important role in enhanced anti-leukemic activity of decitabine by AMO in vitro.

Objective:To analyze the treatment outcomes and prognoses of children with head and neck non-parameningeal rhabdomyosarcoma (HNnPM RMS).Methods:A retrospective analysis was performed on the clinical data of children with HNnPM RMS admitted to Beijing Children′s Hospital from September 2012 to September 2022. The clinical features, comprehensive treatment modes and prognoses of the patients were analyzed. The overall survival rate (OS) and event free survival rate (EFS) were calculated using the Kaplan-Meier method, and univariate analysis was performed using the Log-rank test.Results:A total of 70 children were included in this study, 38 males and 32 females, with a median age of 47 months (2-210 months). Pathological subtypes including the embryonal in 27 cases, the alveolar in 36 cases and the spindle cell and sclerosing in 7 cases. Thirty children (83.3%) with alveolar type were positive for FOXO1 gene fusion. All 70 children underwent chemotherapy, including 38 with neoadjuvant chemotherapy and 32 with adjuvant chemotherapy. Sixty of 70 children underwent surgery, of whom, 10 underwent two or more surgeries. There were 63 children underwent radiotherapy, including 54 with intensity-modulated radiation therapy, 4 with particle implantation and 5 with proton therapy. The median follow-up was 45 (5-113) months, the 5-year OS was 73.2%, and the 5-year EFS was 57.7%. Univariate analysis showed lymph node metastasis ( χ2=5.022, P=0.025), distant metastasis ( χ2=8.258, P=0.004), and high Intergroup Rhabdomyosarcoma Study (IRS) group ( χ2=9.859, P=0.029) as risk factors for poor prognosis. Before June 2016, the 5-year OS based on BCH-RMS-2006 scheme was 63.6%, and after 2016, the 5-year OS based on CCCG-RMS-2016 scheme was 79.6%. Conclusion:Multidisciplinary combined standardized treatment can offer good treatment outcome and prognosis for children with HNnPM RMS. Local control is a key to the efficacy of comprehensive treatment.