ABSTRACT
OBJECTIVES: The objective of this study was to report outcome and postoperative complications following stabilisation of humeral condylar fractures in skeletally immature French bulldogs with a transcondylar screw combined with locking or hybrid locking plates. MATERIALS AND METHODS: Medical records from one referral hospital were reviewed to identify skeletally immature French bulldogs with humeral condylar fractures treated with a transcondylar screw and epicondylar locking or hybrid locking plates crossing the distal humeral physis. RESULTS: Forty-five fractures in 41 different dogs with a mean age of 4 months (range 3.5 to 5) were identified. Six cases had complications: two (4.4%) minor and four (8.9%) major. Short-term clinical outcome was excellent in 35 (77.8%), good in nine (20%) and poor in one (2.2%) case. Forty-one of 45 fractures reached radiographic union without further surgical intervention; the remaining four cases reached union following revision surgery. Long-term owner assessed outcome by telephone interview was graded as excellent in all available cases (26 of 41 dogs). CLINICAL SIGNIFICANCE: This study suggests that the stabilisation of humeral condylar fractures in skeletally immature French bulldogs with combinations of a transcondylar screw and epicondylar locking or hybrid locking plates crossing the distal humeral physis was safe and led comparable outcomes and complication rates to previous reports.
Subject(s)
Dog Diseases , Humeral Fractures , Animals , Bone Plates/veterinary , Bone Screws/veterinary , Dog Diseases/diagnostic imaging , Dog Diseases/surgery , Dogs , Fracture Fixation, Internal/veterinary , Humeral Fractures/diagnostic imaging , Humeral Fractures/surgery , Humeral Fractures/veterinary , Humerus , Treatment OutcomeABSTRACT
Matrix metalloproteinases are inhibited by a growing family of specific tissue inhibitors, TIMPs. The cDNA of the third member of the family, TIMP-3, was obtained by using a reverse transcription-polymerase chain reaction (RT-PCR) to amplify the corresponding mRNA from human placenta. Cloning and expression of the TIMP-3 were performed in Escherichia coli as a fusion protein with a 36 amino acid N-tail containing a His cluster. In the host vector system, rhTIMP-3 was stored intracellularly in its denatured, insoluble form in inclusion bodies. Slow dilution of denaturing and reducing agents, from rhTIMP-3 His bound to a metal affinity solid phase, was followed by partial acid removal of the N-tail, which leaves a residue of four amino acids. Circular dichroism, fluorescence and second-derivative UV spectroscopic analyses supported correct refolding of the recombinant and zymography showed inhibition of both MMP-2 and MMP-9 gelatinolytic activities. The role of the C-terminus, which has closer homology with TIMP-2 than TIMP-1, was also investigated: a C-truncated mutant, similarly cloned and expressed in E. coli, shows complete lack of inhibitory activity on MMP-9, still retaining some on MMP-2. The described protein engineering shows high yield of active inhibitor, unglycosylated as in the native form.
Subject(s)
Protease Inhibitors/metabolism , Protein Biosynthesis , Protein Folding , Amino Acid Sequence , DNA, Complementary , Escherichia coli , Gelatinases/antagonists & inhibitors , Humans , Matrix Metalloproteinase 2 , Matrix Metalloproteinase 9 , Matrix Metalloproteinase Inhibitors , Metalloendopeptidases/antagonists & inhibitors , Molecular Sequence Data , Protease Inhibitors/chemistry , Proteins/chemistry , Proteins/genetics , Proteins/metabolism , Recombinant Fusion Proteins , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , Tissue Inhibitor of Metalloproteinase-3 , Tumor Cells, CulturedABSTRACT
The in vitro and in vivo actions of ciliary neurotrophic factor (CNTF) suggest that endogenous CNTF plays a role in nervous system development and maintenance. CNTF produces most, possibly all, of its effects by binding to a protein referred to as CNTF receptor alpha (CNTFRalpha). Information on CNTFRalpha tissue expression and dynamics would be advanced by the availability of reagents suitable for studying the subcellular localization and trafficking of CNTFRalpha. This paper describes the genetic construction, synthesis, purification and properties of a chimeric protein in which a highly fluorescent form of the green fluorescent protein (GFP) has been fused to human CNTF. The fusion protein, termed GFP-CNTF, was expressed in Escherichia coli. Histidine tagging of GFP-CNTF permitted ready purification by means of immobilized Ni(II) chromatography. Under non-reducing conditions GFP-CNTF migrated on SDS-PAGE with an apparent molecular mass of 50 kDa, although under reducing conditions it behaved electrophoretically as a 67 kDa species. Despite these discrepancies, the molecular mass of GFP-CNTF determined by mass spectrometry (54755) agreed well with its deduced relative molecular mass of 54536. Importantly, the absorbance profile of the GFP chromophore in GFP-CNTF was not modified by the presence of the CNTF domain. Moreover, the fluorescence emission spectrum of GFP-CNTF overlapped that of GFP, showing neither a change in absorbance shift nor a difference in the fluorescence quantum yield. Circular dichroism spectroscopy confirmed that the CNTF and GFP domains of GFP-CNTF folded independently of each other. GFP-tagged CNTF was equipotent to human CNTF in supporting the survival of cultured embryonic chicken sensory and ciliary ganglion neurons. GFP-CNTF, but not GFP, bound to immobilized CNTFRalpha and was displaced by an excess of human CNTF. GFP-CNTF specifically labeled the Purkinje cell layer in cerebellar slices from adult rat. This report is the first to describe a GFP chimera with a neurotrophic factor as the fusion partner. GFP-CNTF should provide a valuable tool for elucidating the role of CNTFRalpha in nervous system function.
Subject(s)
Luminescent Proteins/genetics , Nerve Growth Factors/genetics , Nerve Tissue Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Ciliary Neurotrophic Factor , Green Fluorescent Proteins , Humans , Indicators and Reagents , Luminescent Proteins/chemistry , Molecular Sequence Data , Nerve Growth Factors/chemistry , Nerve Growth Factors/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Purkinje Cells/metabolism , Rats , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, Ciliary Neurotrophic Factor , Receptors, Nerve Growth Factor/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Spectrophotometry, AtomicABSTRACT
A clone positive for D-carbamoylase activity (2.7 kb HindIII-BamHI DNA fragment) was obtained by screening a genomic library of Agrobacterium radiobacter in Escherichia coli. This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da. The D-carbamoylase gene, named cauA, was placed under the control of T7 RNA-dependent promoter and expressed in E. coli BL21(DE3). After induction with isopropyl-thio-beta-D-galactopyranoside, the synthesis of D-carbamoylase in E. coli reached about 40% of the total protein. The expressed protein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa and showed an enhanced stability with respect to that of the wild-type enzyme derived from A. radiobacter. Site-directed mutagenesis experiments allowed us to establish that a Pro14-->Leu14 exchange leads to an inactive enzyme species, while a Cys279-->Ser279 exchange did not impair the functional properties of the enzyme.
