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31.
Cloning, expression, purification, and characterization of the acid alpha-mannosidase from Trypanosoma cruzi 总被引:1,自引:0,他引:1
Vandersall-Nairn AS; Merkle RK; O'Brien K; Oeltmann TN; Moremen KW 《Glycobiology》1998,8(12):1183-1194
The acid alpha-mannosidase of Trypanosoma cruzi is a broad-specificity
hydrolase involved in the catabolism of glycoconjugates, presumably in the
digestive vacuole. We have cloned the alpha-mannosidase gene from a T.cruzi
epimastigote genomic library. The alpha-mannosidase gene was determined to
be single copy by Southern analysis, and similar sequences were not
detected in genomic digests of either Trypanosoma brucei or Leishmania
donovani. The coding region was subcloned into the Pichia pastoris
expression vector pPICZ, and alpha-mannosidase activity was detected in the
medium of induced cultures. The recombinant alpha- mannosidase demonstrated
a pH optimum, inhibition by swainsonine, Km, and substrate specificity
consistent with the characteristics of the alpha-mannosidase previously
purified from T.cruzi epimastigotes. The recombinant enzyme was purified
103-fold from the culture medium of Pichia pastoris and had a native
molecular mass of 359 kDa by gel filtration. A combination of SDS-PAGE,
deglycosylation with endo H, and NH2-terminal sequencing indicates that the
enzyme is originally synthesized as a homodimeric polypeptide that is
subsequently cleaved to form a heterotetramer composed of 57 and 46 kDa
subunits. A polyclonal antibody raised to the recombinant enzyme was shown
to immunoprecipitate the alpha-mannosidase from T.cruzi cell extracts and
will be used in future immunolocalization studies.
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32.
Autologous disc cell implantation, growth factors and gene therapy appear to be promising therapies for disc regeneration. Unfortunately, the replicative lifespan and growth kinetics of human nucleus pulposus (NP) cells related to host age are unclear. We investigated the potential relations among age, replicative lifespan and growth rate of NP cells, and determined the age range that is suitable for cell-based biological therapies for degenerative disc diseases. We used NP tissues classified by decade into five age groups: 30s, 40s, 50s, 60s and 70s. The mean cumulative population doubling level (PDL) and population doubling rate (PDR) of NP cells were assessed by decade. We also investigated correlations between cumulative PDL and age, and between PDR and age. The mean cumulative PDL and PDR decreased significantly in patients in their 60s. The mean cumulative PDL and PDR in the younger groups (30s, 40s and 50s) were significantly higher than those in the older groups (60s and 70s). There also were significant negative correlations between cumulative PDL and age, and between PDR and age. We found that the replicative lifespan and growth rate of human NP cells decreased with age. The replicative potential of NP cells decreased significantly in patients 60 years old and older. Young individuals less than 60 years old may be suitable candidates for NP cell-based biological therapies for treating degenerative disc diseases. 相似文献