[Application of the method of anatomic coracoclavicular ligament reconstruction for distal clavicle fractures].

Objective: To assess the clinical results of the method of anatomic coracoclavicular ligament reconstruction for distal clavicle fractures. Methods: From August 2013 to January 2015, the super image system was used to measure the CT data of 16 patients suffering distal clavicle fractures before operation in Department of Orthopaedics , the First Affiliated Hospital of Nanjing Medical Univerisity. The fractures' morphological features and acromioclavicular dislocation degree were assessed. By referring to the data collected by the my research group on Chinese people's coracoclavicular ligament, the injuries of the coracoclavicular ligament were estimated, which was then to verify the actual injuries detected during operation. Coracoclavicular ligament reconstruction was performed on patients and screws or suture anchors fixing small bone blocks was used as an adjuvant therapy. Clinical and radiological follow-up was at 1, 3, 6 and 12 months after the procedure. The clinical outcomes were assessed pre- and postoperatively with Constant Scores. Anteroposterior radiographs for the bilateral acromioclavicular joints were obtained immediately after surgery and every follow-up.To compare the reduction maintenance, coracoclavicular distances of the injured shoulders were measured in preoperative and postoperative standard radiographs. Results: All patients received satisfactory fracture and acromioclavicular joint reduction. The average follow-up period was (12.6±3.9) months (ranging from 6 to 22 months). Fractures healed six months after the operation. The coracoclavicular distances increased from (7.8±1.4)mm at one month follow-up to (7.9±1.2)mm at the final follow-up (P>0.05), which could be considered as no difference statistically. The constant score significantly increased from (49.1±4.4) at one month follow-up to (93.8±2.1) at the final evaluation (P<0.001). Obvious loss of acromioclavicular joint reduction was not observed after the operation. Coracoid process and calvicle fractures did not appear. Fractures healed well and shoulder joints also functioned well. Patients' lives and work went back to normal. Conclusion: Accompanied by ligament injuries, distal clavicle fractures is different from middle fractures. Coracoclavicular ligament injury is a major cause of the acromioclavicular joint instability, and the focus of the surgery. Therefore, the application of the double-bundle coracoclavicular ligament reconstruction is a feasible method to treat distal clavicle fractures, displaying satisfying clinical results.

Optimization of adenovirus-mediated endothelial nitric oxide synthase delivery in rat hindlimb ischemia.

Adenovirus-mediated overexpression of endothelial nitric oxide synthase (eNOS) induces collateral artery development and substantially increases blood flow after induction of experimental acute hindlimb ischemia. However, the optimal technique of gene delivery for this or any other form of gene therapy in limb ischemia is still unknown. The purpose of this study was to determine the effect of the two most commonly used techniques, intra-arterial and intramuscular injection, on blood flow recovery, collateral artery development, and preservation of muscle mass. We compared intra-arterial injection under vascular isolation, intra-arterial injection under transient vascular occlusion, and intramuscular injection of phosphate buffered saline (PBS) or adenovirus encoding either the eNOS (AdeNOS) or LacZ (AdlacZ) gene after induction of acute hindlimb ischemia. Delivery of AdeNOS by both intra-arterial injection techniques increased eNOS activity (22.30 versus 10.56, P<0.01), blood flow (0.90+/-0.02 versus 0.69+/-0.07, P<0.001) and collateral artery development (17.56484 versus 13.74259, P<0.05) more than by intramuscular delivery. Intra-arterial injection under transient vascular occlusion led to better preservation of muscle mass, muscle architecture, and clinical ischemic index, but led to greater transgene expression in distant organs and contralateral limb muscles. Intra-arterial injection of AdeNOS under transient vascular occlusion is the optimal technique to reverse severe hindlimb ischemia in the rat. This is the first systematic comparison of different delivery techniques used in gene therapy of experimental hindlimb ischemia.

Two-cistron system overexpression of chloroplast glyceraldehyde-3-phosphate dehydrogenase subunit B and B-derivatives from spinach in Escherichia coli.

A gene coding for the subunit B (GapB) of chloroplast glyceraldehyde-3-phosphate dehydrogenase from spinach and its two derivatives (GapBc) lacking the GapB-specific C-terminal extension have been cloned by RT-PCR. These three genes have been overexpressed with full activity in Escherichia coli when a two-cistron expression system controlled by an inducible promoter P(trc) is used. With a suitable base composition of the first cistron, the expression level of GapB and the derivatives GapBc are expressed up to 15-20% of the total cell protein and around 20 mg of recombinant GapBcs with full activity are purified from 1 liter of cultured bacteria. The specific activity of the two derivatives GapBc (40-60 u/mg) is similar to that of GapA (50-70 u/mg) and lower than that of reported GapBc derivative (E. Baalmann, R. Scheibe, R. Cerff, and W. Martin, 1996, Plant Mol. Biol. 32, 505-513).

Overexpression in Escherichia coli and characterization of the chloroplast fructose-1,6-bisphosphatase from wheat.

An important Calvin cycle enzyme, chloroplast fructose-1, 6-bisphosphatase (FBPase) from wheat, has been cloned and expressed up to 15% of the total cell protein using a pPLc expression vector in Escherichia coli by replacing the codons in the 5'-terminal encoding sequence with optimal and A/T-rich ones. The overexpressed wheat FBPase is soluble, fully active, and heat stable. It can be purified by chromatography in turn on DEAE-Sepharose and Sephacryl S-200, and around 15 mg of purified enzymes (>95%) is obtained from 1 liter of cultured bacteria. Its special activity is 8.8 u/mg, K(cat) is 22.9/S, K(m) is 121 microM, and V(max) is 128 micromol/min. mg. The recombinant FBPase can be activated by DTT, Na(+), or low concentrations of Li(+), Ca(2+), Zn(2+), GuHCl, and urea, while it can be inhibited by K(+) or NH(+)(4).

Overexpression in Escherichia coli and characterization of the chloroplast triosephosphate isomerase from spinach.

An important Calvin cycle enzyme, chloroplast triosephosphate isomerase (cpTPI) from spinach, has been cloned and expressed in up to 15% of the total cell protein using the P(L) expression vector in Escherichia coli. An even higher level expression, up to 36% of the total protein, was achieved by replacing the nucleotide sequence between the ribosomal binding site and the initial codon, ATG, with an AT-rich sequence. Computer modeling revealed that the moderate change in the standard free energy (5'-DeltaG degrees ) of mRNA secondary structure in the translation initial region might be the major factor which led to the later high-level expression. The overexpressed spinach cpTPI was soluble and fully active and was able to be purified beyond 95% purity by DEAE-Sepharose and Sephadex G-75, and around 55 mg of purified enzymes was obtained from 1 liter of cultured bacteria. With d-glyceraldehyde 3-phosphate as substrate, K(m (D-3-P)) is 0. 68 mM, V(max (G-3-P)) is 3.16 x 10(4) micromol/min. mg, and K(cat (G-3-P)) is 4.51 x 10(3)/s; with dihydroxyacetone phosphate as substrate, the corresponding values are 7.27 mM, 1.04 x 10(3) micromol/min. mg, and 1.16 x 10(2)/s, respectively.