S-methyl-L-cysteine sulfoxide (SMCO) is a non-protein sulfur-containing amino acid with a variety of functions. There are few reports on the enzymes catalyzing the biosynthesis of SMCO from S-methyl-L-cysteine (SMC). In this study, the flavin-containing monooxygenase gene derived from Schizosaccharomyces pombe (spfmo) was heterologously expressed in Escherichia coli BL21(DE3) and the enzymatic properties of the expressed protein were analyzed. The optimum catalytic conditions of the recombinant SpFMO were 30 ℃ and pH 8.0, under which the enzyme activity reached 72.77 U/g. An appropriate amount of Mg²⁺ improved the enzyme activity. The enzyme kinetic analysis showed that the K_m and k_cat/K_m of SpFMO on the substrate SMC were 23.89 μmol/L and 61.71 L/(min·mmol), respectively. Under the optimal reaction conditions, the yield of SMCO synthesized from SMC catalyzed by SpFMO was 12.31% within 9 h. This study provides reference for the enzymatic synthesis of SMCO.
Schizosaccharomyces/genetics* ; Escherichia coli/metabolism* ; Recombinant Proteins/metabolism* ; Cysteine/biosynthesis* ; Mixed Function Oxygenases/metabolism* ; Schizosaccharomyces pombe Proteins/metabolism* ; Oxygenases/metabolism* ; Kinetics

BACKGROUND: A large number of experiments in vivo and in vitro have shown that mesenchymal stem cells may obviously inhibit the lymphocytes and other immunocytes.OBJECTIVE: To investigate the effect of human umbilical cord mesenchymal stem cell transplantation on the immune function and prognosis of patients suffering decompensated liver cirrhosisn due to hepatitis B.METHODS: 118 patients with decompensated cirrhosis due to hepatitis B were randomly divided into control group (n=59) and observation group (n=59). The two groups all received normal medical treatment, and in addition, the observation group also received human umbilical cord mesenchymal stem cell transplantation. (4.0-4.5)×108 stem cells were transplanted twice by intervention via proper hepatic artery (10 mL) and intravenous infusion (10 mL) within 1 week after admission. The levels of serum interleukin-6, tumor necrosis factor-α, interleukin-10, transforming growth factor-β and the percentage of lymphocyte subsets in the peripheral blood were determined in the two groups before and 1, 4 weeks after treatment. The model for end-stage liver disease (MELD) score and Child-Pugh score of 118 patients after treatment for 12 weeks were observed and recorded, and liver failure, complications and survival during follow-up period in the two groups were observed. RESULTS AND CONCLUSION: After treatment for 1 and 4 weeks, the levels of serum interleukin-6 and tumor necrosis factor-α in the observation group were significantly lower than those in the control group (P < 0.05 or P <0.001), but the levels of serum interleukin-10 and transforming growth factor-β in the observation group were significantly higher than those in the control group (P < 0.05 or P < 0.001). After treatment for 1 week, the percentagesof CD3+CD4+T cell and CD4+CD25+Treg cells in the observation group were significantly higher than those in the control group (P < 0.001), but the percentages of CD3+CD8+ T cells and CD3-CD19+ B cells were significantly lower than those in the control group (P < 0.05 or P < 0.001). After treatment for 4 weeks, the percentages of CD3+ T cell ,CD3+CD4+ T cells and CD4+CD25+ Treg cells in the observation group were significantly higher than those in the control group (P < 0.05 or P < 0.001), but the percentages of CD3+CD8+ T cell and CD3-CD19+ B cells were significantly lower than those in the control group (P < 0.05 or P < 0.001). After treatment for 12 weeks, the MELD and Child-Pugh scores in the observation group were significantly lower than those in the control group (P < 0.05).During the follow-up period, none of the cases in the observation group developed liver failure, but five cases in the control group did. In addition, the incidence of complications and cumulative mortality in the observation group were significantly lower than those in the control group (P < 0.05). These results show that the human umbilical cord mesenchymal stem cell transplantation may alleviate liver inflammation and improve liver function, and then may reduce the incidence of hepatic failure and mortality for patients with decompensated cirrhosis due to hepatitis B.

Objective To investigate the molecular mechanisms of glial cell derived neurotrophic factor in promoting proliferation of spermatogonial stem cell.Methods RNAi expression vectors,targeted at GDNF,were constructed and transfected into SSCs from 5 to 7 days old mice.The SSCs with highest effectiveness of GDNF interfere was set as study group.And the SSCs without GDNF interfere was considered as control group.The ELISA method was used to compare the proliferative rate between study group and control group.Flow cytometry,RT-PCR were used to detect the expression of GDNF,RTKs,Fyn and FAK's mRNA,and the apoptosis of SSCs.Results From 1 to 4 days after transinfection,the absorbable A value in study group was 0.45 ± 0.02,0.68 ± 0.03,1.12 ± 0.03,2.24 ± 0.04,respectively.Meanwhile,the same item in control group was 0.46 ± 0.03、0.73 ± 0.02、1.32 ± 0.05、1.15 ± 0.06,respectively (P ＜ 0.05).There were significant different between experiment groups (25.43 ± 1.91) ％ and control group (5.61 ± 0.16)％ in the apoptosis rates of SSCs (P ＜ 0.05).Significant differences were noted between experimental group and control group(P ＜ 0.05).The mRNA expression rates of GDNF was (12.32 ± 1.22) ％ in study group and (54.25 ± 1.34)％ in control group (P ＜0.01).The mRNA expression rates of RTKs and Fyn and FAK in study group and control group were (16.24 ± 1.35)％ vs (45.35 ± 1.37)％,(18.32 ±1.34)％ vs (38.37 ± 1.55)％,(20.04 ± 1.65)％ vs (43.27 ± 1.28)％,respectively (P ＜0.05).Conclusions The glial cell line derived neurotrophic factor was important in course of SSCs' proliferation,which may up-regulating the expression of RTKs,Fyn and FAK.

BACKGROUND:Previous studies have shown that monocytes can transdifferentiate into vascular endothelial cells under the induction of various factors including vascular endothelial growth factor(VEGF).It remains poorly understood whether monocytes can be induced to transdifferentiate into lymphatic endothelial cells in vitro.OBJECTIVE:To explore the possibility of the transdifferentiation of monocytes into lymphatic endothelial cells under inflammatory condition.METHODS:Fresh monocytes from peripheral blood were collected by Ficoll density gradient centrifugation and cultured in an endothelial cell medium,followed by incubation in fibronectin-plated well or treated with tumor necrosis factor a for 24 hours,respectively.The expression of specific markers of lymphatic endothelial cells,such as LYVE-1,Podoplanin,Porx-1 and VEGF receptor 3(VEGFR-3),as well as the endothelial cells markers,such as vWF,endothelial nitric oxide synthase(eNOS)and VEGFR-2,were detected by RT-PCR and immunochemical methods.RESULTS AND CONCLUSION:Prior to induction,monocytes were positive to LYVE-1,but negative for Podoplanin,Porx-1,and VEGFR-3,vWF,eNOS,as well as VEGFR-2.Following induction,the cultured mononcytes were positive for Podoplanin,Prox-1 and VEGFR-3,but remained negative for vWF,eNOS and VEGFR-2.It suggested that monocytes can be induced to express the markers of lymphatic endothelial cells stimulated by fibronectin or tumor necrosis factor a.