ABSTRACT
l-Glutamate decarboxylase (GAD; EC 4.1.1.15) is the rate-limiting enzyme involved in the synthesis of gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain. Imbalance in the conversion of glutamate to GABA has been implicated in a host of human diseases. Studies on the structure, function, and therapeutic use of GAD have been precluded by insufficient quantities of purified active enzyme. Here we report a novel methodology for the expression and large-scale production of enzymatically active, pure, recombinant human GAD65 and GAD67. This method circumvents the sequestering of expressed protein into insoluble inclusion bodies and reduces production of truncated proteins. The availability of sufficient quantities of purified HGAD65 and HGAD67 has allowed for the production of specific polyclonal antibodies that discriminate between the two isoforms. This methodology, in addition to providing key human brain enzymes, may be generally applicable to other systems.
Subject(s)
Brain/enzymology , Glutamate Decarboxylase/biosynthesis , Isoenzymes/biosynthesis , Cloning, Molecular , Escherichia coli , Glutamate Decarboxylase/genetics , Glutamate Decarboxylase/isolation & purification , Humans , Immunoblotting , Isoenzymes/genetics , Isoenzymes/isolation & purification , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Synaptosomes/enzymologyABSTRACT
We have tested the axial and torsional stability of femoral components after revision arthroplasty in a cadaver model, using impacted morsellised cancellous graft and cement. Each one of six matched pairs of fresh frozen human femora had either a primary or a revision prosthesis cemented in place. For the 'revision' experiments, all cancellous bone was removed from the proximal femur which was then over-reamed to create a smooth-walled cortical shell. An MTS servohydraulic test frame was used to apply axial and torsional loads to each specimen through the prosthetic femoral heads with the femur submerged in isotonic saline solution at 37 degrees C. The mean subsidence was 0.27 +/- 0.17 mm for the primary and 0.52 +/- 0.30 mm for the revision groups. The difference was statistically significant (p < 0.025), but the mean subsidence was <1 mm in both groups. The mean maximum torque before failure was 42.9 +/- 26.9 N-m for the primary and 34.8 +/- 20.7 N-m for the revision groups. This difference was not statistically significant (p > 0.015). Based on our results we suggest that revision of the femoral component using morsellised cancellous graft followed by cementing with a collarless prosthesis with a polished tapered stem restores the integrity of the proximal femur and provides immediate stability of the implant.
