Objective:To screen and isolate an eco-friendly,u thermophilic and potent L-asparaginase producing bacterium,with novel immunological properties that may obviates hypersensitivity reactions.Methods:In the present stud...Objective:To screen and isolate an eco-friendly,u thermophilic and potent L-asparaginase producing bacterium,with novel immunological properties that may obviates hypersensitivity reactions.Methods:In the present study baclerial strain isolated for extracellular L-asparaginase production from hotspring,identified by morphological,biochemical and physiological tests followed by t6S rDNA technology and the L-asparaginase production ability was tested by both semi quantitative and quantitative enzymatic assay.Result:The bacterial strain was identified as Bacillus sublilis strain hswx88(GenBank Accession Number JQ237656.1).The extracellular enzyme yielding capacity isolate Bacillus subtilis strain hswx88(23.8 IU/mL)was found to be 1.7 and 14.5 limes higher than the reference organism Pectobacterium carotovorum MTCC 1428(14.2 IU/mL)and Bacillus sp.BCCS 034(1.64 IU/mL).Conclusion:The isolate is eco-friendly and useful to produce bulk quantity of extracellular,thermophilic L-asparaginase for the treatment of various tumor cases and for preparation of acrylamide free fry food preparation.展开更多
Objective:To isolate marine bacteria,statistically optimize them for maximum asparaginase production.Methods:In the present study,statistically based experimental designs were applied to maximize the production of L-a...Objective:To isolate marine bacteria,statistically optimize them for maximum asparaginase production.Methods:In the present study,statistically based experimental designs were applied to maximize the production of L-asparaginase from bacterial strain of Bacillus cereus（B.cereus） MAB5（HQ675025） isolated and identified by 16S rDNA sequencing from mangroves rhizosphere sediment.Results:Plackett-Barman design was used to identify the interactive effect of the eight variables viz.yeast extract,soyabean meal,glucose,magnesium sulphate,KH<sub>2</sub>PO<sub>4</sub>,wood chips,aspargine and sodium chloride.All the variables are denoted as numerical factors and investigated at two widely spaced intervals designated as -1（low level） and +1（high level）. The effect of individual parameters on L-asparaginase production was calculated.Soyabean meal,aspargine,wood chips and sodium chloride were found to be the significant among eight variables.The maximum amount of L-asparaginase produced（51.54 IU/mL） from the optimized medium containing soyabean meal（6.282 8 g/L）,aspargine（5.5 g/L）,wood chips（1.383 8 g/L） and NaCl（4.535 4 g/L）.Conclusions:The study revealed that,it is useful to produce the maximum amount of L-asparaginase from B.cereus MAB5 for the treatment of various infections and diseases.展开更多
This study investigated the intracellular localization of asparagine synthetase （ASNS） in the relation with chemoresistance in leukemia. pIRES-GFP-ASNS-Flag/Neo expression vector was transiently tansfected into SK-N...This study investigated the intracellular localization of asparagine synthetase （ASNS） in the relation with chemoresistance in leukemia. pIRES-GFP-ASNS-Flag/Neo expression vector was transiently tansfected into SK-N-MC cells and 297T cells respectively. Immunofluorescence and Western blot analysis were performed for cellular localization of ASNS respectively. U937 cells were treated with L-asparaginase for 48 h and examined for endogenous ASNS expression on plasma membrane by immunofluorescence staining. Immunofluorescence staining showed that the transiently expressed ASNS was partly localized on transfected-SK-N-MC cell surface. Moreover, Western blotting exhibited that ASNS expressed both in cytosol and on plasma membrane of transfected-293T cells. Immunofluorescence staining with anti-ASNS-specific monoclonal antibody revealed that endogenous ASNS was localized on the plasma membrane of U937 cells, except for its distribution in the cytosol. In addition, ASNS exhibited a higher expression on plasma membrane after treatment with L-asparaginase as compared with the untreated cells. It was concluded that the subcellular translocation of ASNS may play an important role in L-asparaginase resistance in leukemia cells.展开更多
Objective: To improve the efficacy of refractory midfacial peripheral T-cell non-Hodgkin’s lymphoma (MPTC-NHL) with L-asparaginase (L-ASP) based salvage chemotherapy. Methods: 21 patients with refractory MPTC-NHL wer...Objective: To improve the efficacy of refractory midfacial peripheral T-cell non-Hodgkin’s lymphoma (MPTC-NHL) with L-asparaginase (L-ASP) based salvage chemotherapy. Methods: 21 patients with refractory MPTC-NHL were analyzed. 11patients (L-ASP group) received L-asparaginase based salvage chemotherapy consisting of L-asparaginase, vincristine and dexame-thosone. 10 patients (control group) received salvage combination chemotherapy without L-asparaginase. Results: Complete remission rates were 45.6% for L-ASP group and 0.0% for control group (p<0.05). Overall response rates (CR+PR) were 63.6% for L-ASP group and 10.0% for control group, respectively (p<0.05). 2-year survival rates were 45.5% for L-ASP group and 0.0% for control group (p<0.05). The major adverse effects of L-ASP were leukopenia, elevation of serum bilirubin and hyperglycemia. Conclusion: The preliminary clinical study shows that the L-ASP based salvage chemotherapy may improve the response rate and 2-year survival rate of the patients with refractory MPTC-NHL. It is necessary to continue the study further.展开更多
The present study describes the production optimization of recombinant L-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Escherichia coli BL21 (DE3) at batch and fed batch bioreactor level. Production of re...The present study describes the production optimization of recombinant L-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Escherichia coli BL21 (DE3) at batch and fed batch bioreactor level. Production of recombinant L-asparaginase II in batch and fed batch mode was found to be 1.34 and 5.38 folds higher, respectively as compared to shake flask culture. SDS-PAGE and native PAGE of the purified enzyme revealed that molecular mass of the subunits and native enzyme are ~37.5 kDa and ~150 kDa, respectively. Optimum range of pH and temperature for hydrolysis of L-asparagine were found to be 7.5 - 8.5 and 47°C - 52°C, respectively. The recombinant enzyme is very specific for its natural substrate, L-asparagine. The activity of recombinant L-asparaginase II is improved by mono cations and diverse effectors including Na+, K+, L-cystine, L-histidine, glutathione and 2-mercaptoethanol whereas, it is moderately inhibited by different divalent cations and thiol group blocking reagent. The kinetic parameters Km, Vmax, kcat and Km/Kcat of purified recombinant L-asparaginase II were determined. The purified L-asparaginase II possesses no partial glutaminase activity, which is prerequisite to reduce the possibility of side effects during the course of anti-cancer therapy.展开更多
An important factor in the emergence and progre sion of osteosarcoma(OS)is the dysregulated expression of microRNAs(miRNAs).Transcription factor 7-like 1(TCF7LI),a member of the T cell factor/lymphoid enhancer factor(...An important factor in the emergence and progre sion of osteosarcoma(OS)is the dysregulated expression of microRNAs(miRNAs).Transcription factor 7-like 1(TCF7LI),a member of the T cell factor/lymphoid enhancer factor(TCF/LEF)transcription factor family,interacts with the Wnt signaling pathway regulator β-catenin and acts as a DNA-specific binding protein.This study sought to elucidate the impact of the interaction between miR 3293p and TCF7L1 on.the growth and apoptosis of OS and analyze the regulatory expression relationship between miRNA and mRNA in osteosarcoma cells using a variety of approaches.MiR329-3p was significantly downregulated,while TCF7L1 was considerably up-regulated in all examined OS cell lines.Additionally,a clinical comparison study was performed using the TCGA database.Subsequently,the regulatory relationship between miR-329-3p and TCF7L1 on the proliferation and apoptosis of OS cells was verified through in vitro and in vivo experiments.When miR 329-3p was transfected into the OS cell line,the expression of TCF7L1 decreased,the proliferation of OS cells was inhibited,the cytoskeleton disintegrated,and the nucleus condensed to fom apoptotic bodies.The expression of proteins that indicate apoptosis increased simultaneously.The cell cycle was arrested in the G0/G1 phase,and the G1/S transition was blocked.The introduction of miR 3293p also inhibited downstream Cyclin D1 of the Wnt pathway.Xenograf experiments indicated that the overexpression of miR-329-3p signi ficanly inhibited the growth of OS xenografts in nude mice,and the expression of TCF7L1 and C-Myc in tumor tssues decreased.MiR 329-3p was significantly reduced in OS cells and played a suppressive role in tumorigenesis and proliferation by targeting TCF7L1 both in vitro and in vivo.Osteosarcoma cell cycle arrest and pathway inhibition were observed upon the regulation of TCF7LI by miR 3293p.Summarizing these results,it can be inferred that miR.3293p exerts anticancer efects in osteosarcoma by inhibiting TCF7L1.展开更多
基金Support by the All India Council for Technical Education.New Delhi,India through"Research Promotion Scheme"(AICTE Letter-F.No.8023/RID/RPS-17/(POLICYIV)/(GOVT.)/2011-12.Dt:09/07/2012,Veh.No.:340)
文摘Objective:To screen and isolate an eco-friendly,u thermophilic and potent L-asparaginase producing bacterium,with novel immunological properties that may obviates hypersensitivity reactions.Methods:In the present study baclerial strain isolated for extracellular L-asparaginase production from hotspring,identified by morphological,biochemical and physiological tests followed by t6S rDNA technology and the L-asparaginase production ability was tested by both semi quantitative and quantitative enzymatic assay.Result:The bacterial strain was identified as Bacillus sublilis strain hswx88(GenBank Accession Number JQ237656.1).The extracellular enzyme yielding capacity isolate Bacillus subtilis strain hswx88(23.8 IU/mL)was found to be 1.7 and 14.5 limes higher than the reference organism Pectobacterium carotovorum MTCC 1428(14.2 IU/mL)and Bacillus sp.BCCS 034(1.64 IU/mL).Conclusion:The isolate is eco-friendly and useful to produce bulk quantity of extracellular,thermophilic L-asparaginase for the treatment of various tumor cases and for preparation of acrylamide free fry food preparation.
文摘Objective:To isolate marine bacteria,statistically optimize them for maximum asparaginase production.Methods:In the present study,statistically based experimental designs were applied to maximize the production of L-asparaginase from bacterial strain of Bacillus cereus（B.cereus） MAB5（HQ675025） isolated and identified by 16S rDNA sequencing from mangroves rhizosphere sediment.Results:Plackett-Barman design was used to identify the interactive effect of the eight variables viz.yeast extract,soyabean meal,glucose,magnesium sulphate,KH<sub>2</sub>PO<sub>4</sub>,wood chips,aspargine and sodium chloride.All the variables are denoted as numerical factors and investigated at two widely spaced intervals designated as -1（low level） and +1（high level）. The effect of individual parameters on L-asparaginase production was calculated.Soyabean meal,aspargine,wood chips and sodium chloride were found to be the significant among eight variables.The maximum amount of L-asparaginase produced（51.54 IU/mL） from the optimized medium containing soyabean meal（6.282 8 g/L）,aspargine（5.5 g/L）,wood chips（1.383 8 g/L） and NaCl（4.535 4 g/L）.Conclusions:The study revealed that,it is useful to produce the maximum amount of L-asparaginase from B.cereus MAB5 for the treatment of various infections and diseases.
基金supported by grants from the National Natural Science Foundation of China (Nos. 30672271 and 81000227)
文摘This study investigated the intracellular localization of asparagine synthetase （ASNS） in the relation with chemoresistance in leukemia. pIRES-GFP-ASNS-Flag/Neo expression vector was transiently tansfected into SK-N-MC cells and 297T cells respectively. Immunofluorescence and Western blot analysis were performed for cellular localization of ASNS respectively. U937 cells were treated with L-asparaginase for 48 h and examined for endogenous ASNS expression on plasma membrane by immunofluorescence staining. Immunofluorescence staining showed that the transiently expressed ASNS was partly localized on transfected-SK-N-MC cell surface. Moreover, Western blotting exhibited that ASNS expressed both in cytosol and on plasma membrane of transfected-293T cells. Immunofluorescence staining with anti-ASNS-specific monoclonal antibody revealed that endogenous ASNS was localized on the plasma membrane of U937 cells, except for its distribution in the cytosol. In addition, ASNS exhibited a higher expression on plasma membrane after treatment with L-asparaginase as compared with the untreated cells. It was concluded that the subcellular translocation of ASNS may play an important role in L-asparaginase resistance in leukemia cells.
文摘Objective: To improve the efficacy of refractory midfacial peripheral T-cell non-Hodgkin’s lymphoma (MPTC-NHL) with L-asparaginase (L-ASP) based salvage chemotherapy. Methods: 21 patients with refractory MPTC-NHL were analyzed. 11patients (L-ASP group) received L-asparaginase based salvage chemotherapy consisting of L-asparaginase, vincristine and dexame-thosone. 10 patients (control group) received salvage combination chemotherapy without L-asparaginase. Results: Complete remission rates were 45.6% for L-ASP group and 0.0% for control group (p<0.05). Overall response rates (CR+PR) were 63.6% for L-ASP group and 10.0% for control group, respectively (p<0.05). 2-year survival rates were 45.5% for L-ASP group and 0.0% for control group (p<0.05). The major adverse effects of L-ASP were leukopenia, elevation of serum bilirubin and hyperglycemia. Conclusion: The preliminary clinical study shows that the L-ASP based salvage chemotherapy may improve the response rate and 2-year survival rate of the patients with refractory MPTC-NHL. It is necessary to continue the study further.
文摘The present study describes the production optimization of recombinant L-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Escherichia coli BL21 (DE3) at batch and fed batch bioreactor level. Production of recombinant L-asparaginase II in batch and fed batch mode was found to be 1.34 and 5.38 folds higher, respectively as compared to shake flask culture. SDS-PAGE and native PAGE of the purified enzyme revealed that molecular mass of the subunits and native enzyme are ~37.5 kDa and ~150 kDa, respectively. Optimum range of pH and temperature for hydrolysis of L-asparagine were found to be 7.5 - 8.5 and 47°C - 52°C, respectively. The recombinant enzyme is very specific for its natural substrate, L-asparagine. The activity of recombinant L-asparaginase II is improved by mono cations and diverse effectors including Na+, K+, L-cystine, L-histidine, glutathione and 2-mercaptoethanol whereas, it is moderately inhibited by different divalent cations and thiol group blocking reagent. The kinetic parameters Km, Vmax, kcat and Km/Kcat of purified recombinant L-asparaginase II were determined. The purified L-asparaginase II possesses no partial glutaminase activity, which is prerequisite to reduce the possibility of side effects during the course of anti-cancer therapy.
基金The Fund of National Cancer Center Research and Development(26-A-4),The Grants-in-Aid for Scientific Research(Grant Nos.15K10451,16K10866 and 16K20063)from Japan Society for the Promotion of Science.
文摘An important factor in the emergence and progre sion of osteosarcoma(OS)is the dysregulated expression of microRNAs(miRNAs).Transcription factor 7-like 1(TCF7LI),a member of the T cell factor/lymphoid enhancer factor(TCF/LEF)transcription factor family,interacts with the Wnt signaling pathway regulator β-catenin and acts as a DNA-specific binding protein.This study sought to elucidate the impact of the interaction between miR 3293p and TCF7L1 on.the growth and apoptosis of OS and analyze the regulatory expression relationship between miRNA and mRNA in osteosarcoma cells using a variety of approaches.MiR329-3p was significantly downregulated,while TCF7L1 was considerably up-regulated in all examined OS cell lines.Additionally,a clinical comparison study was performed using the TCGA database.Subsequently,the regulatory relationship between miR-329-3p and TCF7L1 on the proliferation and apoptosis of OS cells was verified through in vitro and in vivo experiments.When miR 329-3p was transfected into the OS cell line,the expression of TCF7L1 decreased,the proliferation of OS cells was inhibited,the cytoskeleton disintegrated,and the nucleus condensed to fom apoptotic bodies.The expression of proteins that indicate apoptosis increased simultaneously.The cell cycle was arrested in the G0/G1 phase,and the G1/S transition was blocked.The introduction of miR 3293p also inhibited downstream Cyclin D1 of the Wnt pathway.Xenograf experiments indicated that the overexpression of miR-329-3p signi ficanly inhibited the growth of OS xenografts in nude mice,and the expression of TCF7L1 and C-Myc in tumor tssues decreased.MiR 329-3p was significantly reduced in OS cells and played a suppressive role in tumorigenesis and proliferation by targeting TCF7L1 both in vitro and in vivo.Osteosarcoma cell cycle arrest and pathway inhibition were observed upon the regulation of TCF7LI by miR 3293p.Summarizing these results,it can be inferred that miR.3293p exerts anticancer efects in osteosarcoma by inhibiting TCF7L1.
