DPPD (Rv0061) is a difficult to express protein of Mycobacterium tuberculosis that elicits strong and specific delayed type hypersensitiv-ity reactions in humans infected with M. tuber-culosis. Therefore DPPD is a mol...DPPD (Rv0061) is a difficult to express protein of Mycobacterium tuberculosis that elicits strong and specific delayed type hypersensitiv-ity reactions in humans infected with M. tuber-culosis. Therefore DPPD is a molecule that can improve the specificity of the tuberculin skin test, which is widely used as an aid for the di-agnosis of tuberculosis. However, a pitfall of our initial studies was that the DPPD molecule used to perform the skin tests was engineered as fu-sion molecule with another Mycobacterium protein. This approach was used because no expression of DPPD could be achieved either as a single molecule or as a fusion protein using a variety of commercially available expression systems. Here, we report the production and purification of rDPPD using a synthetic gene engineered to contain E. coli codon bias. The gene was cloned into pET14b expression vector, which was subsequently used to transform Rosetta 2(DE3)pLysS or BL-21(DE3)pLysS host cells. The recombinant protein was over-ex- pressed after induction with IPTG and its puri-fication was easily achieved at levels of 5 – 10 mg/l of bacterial broth cultures. The purified protein was confirmed to be DPPD by Mass Spectroscopy sequencing analysis. Moreover, purified rDPPD stimulated peripheral blood mononuclear cells of PPD positive blood do-nors to produce high levels of IFN-γ, thus con-firming that this molecule is biologically active. Because of the DPPD gene is restricted to the tuberculosis-complex organisms of Mycobacte-rium genus, this highly purified molecule should be useful for the identification of indi-viduals sensitized with tubercle展开更多
Background:The increasing occurrence of diabetes mellitus(DM)noted worldwide has considerably elicited concern in the recent past.DM is associated with elevated vascular complications,morbidity,mortality,and poor qual...Background:The increasing occurrence of diabetes mellitus(DM)noted worldwide has considerably elicited concern in the recent past.DM is associated with elevated vascular complications,morbidity,mortality,and poor quality of life.In this context,mesenchymal stem cells(MSCs)have shown significant therapeutic potentialities in managing and curing type 1 DM owing to their self-renewable,immunosuppressive,and differentiation capacities.We investigated the potential action of N,N′-diphenyl-1,4-phenylenediamine(DPPD),a well-known synthetic antioxidant to enhance the therapeutic ability of the adipose-derived stem cells(AD-MSCs)in alleviating kidney and liver complications in diabetic rats.Methods:Over the four weeks of experiments,albino male rats(n=36)were split into six test groups:control,DPPD(250 mg/kg,i.p.),STZ-diabetic(D),D+DPPD,D+AD-MSCs(1×10^(6)cell/rat,i.v.),and D+AD-MSCs+DPPD treated groups.Results:Significant declines in the renal and hepatic oxidative stress markers(MDA,ROS,and AGEs)were observed coupled with a significant elevation in many antioxidant marker levels(GSH,SOD,CAT,GPx,HO-1,and TAC)in the diabetic rats treated with either DPPD or AD-MSCs or their co-administered injection compared to the diabetic untreated rats.This was suggested to be the leading cause of amelioration of the kidney functions(as measured by urea,uric acid,and creatine levels)and liver functions(as evidenced by the levels of AST,ALT,ALP,bilirubin,total proteins,albumin,and globulins).Conclusion:DPPD and AD-MSCs co-administration showed superior results in terms of the enhancement of the relative hepato-renal function,indicating the beneficial role of DPPD supplementation in increasing the therapeutic potential of AD-MSCs.展开更多
文摘DPPD (Rv0061) is a difficult to express protein of Mycobacterium tuberculosis that elicits strong and specific delayed type hypersensitiv-ity reactions in humans infected with M. tuber-culosis. Therefore DPPD is a molecule that can improve the specificity of the tuberculin skin test, which is widely used as an aid for the di-agnosis of tuberculosis. However, a pitfall of our initial studies was that the DPPD molecule used to perform the skin tests was engineered as fu-sion molecule with another Mycobacterium protein. This approach was used because no expression of DPPD could be achieved either as a single molecule or as a fusion protein using a variety of commercially available expression systems. Here, we report the production and purification of rDPPD using a synthetic gene engineered to contain E. coli codon bias. The gene was cloned into pET14b expression vector, which was subsequently used to transform Rosetta 2(DE3)pLysS or BL-21(DE3)pLysS host cells. The recombinant protein was over-ex- pressed after induction with IPTG and its puri-fication was easily achieved at levels of 5 – 10 mg/l of bacterial broth cultures. The purified protein was confirmed to be DPPD by Mass Spectroscopy sequencing analysis. Moreover, purified rDPPD stimulated peripheral blood mononuclear cells of PPD positive blood do-nors to produce high levels of IFN-γ, thus con-firming that this molecule is biologically active. Because of the DPPD gene is restricted to the tuberculosis-complex organisms of Mycobacte-rium genus, this highly purified molecule should be useful for the identification of indi-viduals sensitized with tubercle
基金the Deanship of Scientific Research,Vice Presidency for Graduate Studies and Scientific Research at King Faisal University,Saudi Arabia,for financial support under the annual funding track(Grant 3730).
文摘Background:The increasing occurrence of diabetes mellitus(DM)noted worldwide has considerably elicited concern in the recent past.DM is associated with elevated vascular complications,morbidity,mortality,and poor quality of life.In this context,mesenchymal stem cells(MSCs)have shown significant therapeutic potentialities in managing and curing type 1 DM owing to their self-renewable,immunosuppressive,and differentiation capacities.We investigated the potential action of N,N′-diphenyl-1,4-phenylenediamine(DPPD),a well-known synthetic antioxidant to enhance the therapeutic ability of the adipose-derived stem cells(AD-MSCs)in alleviating kidney and liver complications in diabetic rats.Methods:Over the four weeks of experiments,albino male rats(n=36)were split into six test groups:control,DPPD(250 mg/kg,i.p.),STZ-diabetic(D),D+DPPD,D+AD-MSCs(1×10^(6)cell/rat,i.v.),and D+AD-MSCs+DPPD treated groups.Results:Significant declines in the renal and hepatic oxidative stress markers(MDA,ROS,and AGEs)were observed coupled with a significant elevation in many antioxidant marker levels(GSH,SOD,CAT,GPx,HO-1,and TAC)in the diabetic rats treated with either DPPD or AD-MSCs or their co-administered injection compared to the diabetic untreated rats.This was suggested to be the leading cause of amelioration of the kidney functions(as measured by urea,uric acid,and creatine levels)and liver functions(as evidenced by the levels of AST,ALT,ALP,bilirubin,total proteins,albumin,and globulins).Conclusion:DPPD and AD-MSCs co-administration showed superior results in terms of the enhancement of the relative hepato-renal function,indicating the beneficial role of DPPD supplementation in increasing the therapeutic potential of AD-MSCs.
