Strain NCIMB 10467, a lignin degrader, was reclassified as genus Burkholderia according to its 16S rDNA sequence. It seems that the metabolism of protocatechuate by this strain is diverse under the induction of various substrates. A 9505-bp DNA fragment extending from α conserved region of the gene, which encodes β subunit of orthocleavage protocatechuate 3, 4-dioxygenase(P34D; EC 1.13.11.3),was obtained by genome walking. Sequence analysis revealed two deduced open reading frames, pcaG and pcaH, encoding the α and β subunits of P34D respectively in this fragment. The P34D activity could be detected when pcaGH were expressed in E. Coli and the disruption of pcaH in strain NCIMB 10467 has lead to loss of its ability to catabolize protocatechuate. It was proved that the cloned pcaGH were encoding a functional protocatechuate 3, 4-dioxygenase which was necessary for the protocatechuate metabolism in this strain.

Objective By using a newly developed animal model of limited macular translocation (LMT) in rabbit eyes, to explore the complications,indications and predictability of LMT. Methods LMT models were performed in 15 rabbit eyes, and were divided into 4.5 mm group and 3.5 mm group by the length of scleral shortening.The effect of retinal translocation (distance and direction) and its complications were analyzed. In a separated group, postoperative corneal astigmatism was studied after release of scleral shortening suture 1 month after LMT. In order to study the impact of neuroretina retinal pigment epithelium (RPE) adhesion on LMT, retinas and choroids in 4 pigmented rabbit eyes were pretreated with photocoagulation. Results Success rate of LMT was 86 7%. In these cases, retinal translocations were achieved [(distance of 610 to 2 690 ?m, [AKx-]?s =(1 395?636) ?m; translocation angle ? of 10 38?, ?s =(22.7?8.3) ?]. The difference in scleral shortening between 4.5 mm and 3.5 mm groups did not significantly influence the final translocation. Major complications of LMT included intraocular bleeding, retinal break, and corneal astigmatism. Release of scleral shortening suture reduced corneal astigmatism and made it more regular, meanwhile, retinal translocation did not regress. The neuroretina RPE adhesion induced by retinal photocoagulation made neuroretinal hydrodissection difficult, and led to retinal break readily. . The difference in scleral shortening between 4.5 mm and 3.5 mm groups did not significantly influence the final translocation. Major complications of LMT included intraocular bleeding, retinal break, and corneal astigmatism. Release of scleral shortening suture reduced corneal astigmatism and made it more regular, meanwhile, retinal translocation did not regress. The neuroretina RPE adhesion induced by retinal photocoagulation made neuroretinal hydrodissection difficult, and led to retinal break readily. Conclusion Sufficient LMT models in rabbit eye were obtained with few postoperative complications. Direction of retinal translocation is an important factor influencing the effect of LMT, except for distance. Release of scleral shortening suture can effectively reduce corneal astigmatism without causing regression of retinal translocation. For the patients with a history of macular or para macular photocoagulation, LMT should be performed carefully.

[Objective]To evaluate the posterior approach treatment of old traumatic lumbar spinal stenosis,lumbar kyphosis,intervertebral space stenosis using vertebral plate decompression,nucleus pulposus removal,intervertebral fusion and CAPSTONE cage insertion and internal fixation with pedicle screw and rod,TLIF.[Method]Twenty patients with low lumbar spinal canal and intervertebral space stenosis,lumbar kyphosis caused by trauma were enrolled in this study.There were 13 males and 7 females,with an average age of 31.4 years (range 22-49).Four had lesions of L3、4,10 of L4、5 and 6 of L5S1.One patient was treated at 4 years after injury,3 within 9-15 months,6 within 3-6 months and 10 within 1-3 months after injury.All patients were treated with vertebral plate decompression,some serious patients underwent laminectomy decompression,nucleus pulposus removal,intervertebral fusion,CAPSTONE cage insertion and internal fixation with pedicle screw and rod,TLIF.One week after surgery,patients could wear thoracic waist-iliac orthosis and take out-of-bed activity.[Result]All the patients lumbodynia,lower limb pain,muscle strength and hypoesthesia improved within one week after operation.X-rays showed that both the intervertebral space and the physiological curvature improved to some degrees.Twenty patients were followed up(ranged,1 to 3 years)and the clinical outcomes were good.The average Franke1 grade was 1.4.Re-X-rays showed that interbody fusion was achieved in all 20 patients.No loss was found about the altitude of the intervertebral space and the physiological curvature.[Conclusion]For patients with old traumatic lumbar spinal stenosis,the recovery of body altitude is almost impossible.This method is simple and has less complications.It can be widely used in the clinical practice.

Objective To study the effects of ischemic postconditioning and ischemic preconditioning on cerebral ischemia-reperfusion injury following middle cerebral artery occlusion in rats.Methods A reversible focal cerebral ischemia-reperfusion injury was modeled using middle cerebral artery occlusion. Thirty male Sprague-Dawley rats were randomized into three groups (n = 10 in each group) : a cerebral ischemia-reperfusion group, an ischemic postconditioning group and an ischemic preconditioning group. The impairment of neurological function was scored and the infarct volume, the activity of superoxide dismutase and malondiadehyde (MDA) content were measured after the operation.Results In the ischemic postconditioning and preconditioning groups the neurological function was better and the infarction volume was significantly smaller compared with the model group. In the preconditioning group both infarction volume and neurological function were significantly better than in the postconditioning group. In the brain tissues of the preconditioning and postconditioning groups MDA content was lower, while the activity of superoxide dismutase was significantly higher than in the model group.Conclusions lschemic postconditioning can attenuate pathological injury induced by focal cerebral ischemia and repedusion. The neuroprotective effect induced by ischemic preconditioning is stronger than that induced by ischemic postconditioning.

AIM:To investigate the effect of small interfering RNA ( siRNA)-mediated ABCE1 knockdown on the survival, cell cycle and invasion of human bladder cancer cell line T24.METHODS:The siRNA against ABCE1 was constructed and transfected into the T24 cells with LipofectamineTM 2000.The expression of ABCE1 was detected by RT-PCR and Western blot.Flow cytometry was used to detect the cell cycle.The effects of ABCE1 gene silencing on prolifera-tion, migration and invasion of T24 cells were evaluated by CCK-8 assay, wound-healing assay and Transwell invasion as-say, respectively.RESULTS: Compared with control group and blank group, the mRNA and protein levels of ABCE1 were significantly decreased in experimental group after transfected with ABCE1 siRNA.The cell cycle was arrested at G0/G1 phase and the cell number in S phase was decreased in the T24 cells.Compared with control group and blank group, the proliferation of T24 cells in experimental group was inhibited significantly, and the migration and invasion abilities of T24 cells in experimental group decreased significantly.CONCLUSION:Knockdown of ABCE1 gene may decrease migration, invasion and proliferation abilities in T24 cells.

OBJECTIVE:To isolate and purify three principal degraded impurities of mitiglinide calcium (impurity A,B,C) and identify their structures,establish HPLC method for content determination of impurity A,B,C. METHODS:Mitiglinide calci-um was used as raw material and reacted with acid;3 impurities were then separated by HPLC and their structures were elucidated by IR,MS,1H NMR,13C NMR,LC-ESI-MS and ORD. 3 impurities of 3 batches of mitiglinide calcium were determined,and the determination was performed on Agilent Extend-C18 column with mobile phase consisted of 0.01 mol/L sodium acetate solution-ace-tonitrile-triethylamine(60:40:0.1,pH=3.0)at the flow rate of 1.0 ml/min. The detection wavelength was set at 210 nm and sam-ple size was 20 μl. The response tests of 3 impurities and mitiglinide calcium were conducted. RESULTS:After treated with acid, impurity A,B,C had been obtained,and their purity were 99.05%,98.87%,99.98%,respectively after isolation and purifica-tion;after identifying the structure, 3 impurities were S-2-bezylsuccinic acid, S-2-bezylsuccinic acid-4-methyl ester, methyl (2S)-2-benzyl-3-(cis-hexahydroisoindolin-2-ylcarbonyl) propionate;methodological study of content determination of impurities were all up to the requirement. The linear range of impurity A,B,C were 0.387 5-3.875,0.395-3.95 and 0.392 5-3.925 μg/ml(all r were 1.000 0). The response value of impurity A,B,C and mitiglinide calcium were 2.316 1,2.636 1,2.617 8 and 2.620 4,re-spectively. CONCLUSIONS:The structures of 3 principal degraded impurities of mitiglinide calcium have been identified and con-firmed;the content of them can be determined by HPLC main component self-comparison method.

Objective To investigate the strategies of surgical approaches,indications and surgical techniques of local resection for mid-lower rectal tumors and pelvic floor neoplasia.Methods Clinical data of 122 patients underwent local resection for mid-lower rectal tumors pelvic floor neoplasia between July 2004 and July 2008 were analyzed retrospectively.Results Transanal,transsacral,transsphincteric local resection was respectively performed in 45,and 32,and 45 patients.Pathological examination proved that benign tumors were account for 81 cases,pelvic floor neoplasia 16 cases,malignant tumors 25 cases.The masses were 5.6 cm(0 to 12 cm) apart from the anal border,and the mean tumors' diameter was 4.2 cm (0.5 to 11 cm).No case was diagnosed with positive margins upon final pathology of resected specimens.The rate of postoperative complications of transanal,transsacral,transsphincteric approaches was 8.9％ (4/45),18.8％ (6/32),20.0％ (9/45),respectively.The recurrence of transanal,transsacral,transsphincteric approaches was 6.7％ (3/45),9.4％ (3/32),4.4％ (2/45),respectively.Conclusions The three approaches for patients suffering from mid-lower rectal tumors and pelvic floor neoplasia have respectively advantages.Transsphincteric approach is the most useful methods,but with more postoperative complications,so it need more surgical techniques.

In order to explore the role of acetylcholine in the pathogenesis of Parkinson's disease (PD), the changes in the concentration of acetylcholine (Ach) in the striatum, the apoptosis of substantia nigra cells, the ultrastructure and the changes of Nissl cells in rats during the morbidity of PD, and the corresponding behaviors in rats with PD were observed. Rat PD model was established by using the modified Thomas method. Eighty-one rats were randomly divided into normal control, sham operation and PD groups and their behavior features were observed at post-operative day (POD) 7, 14 and 21 as three subgroups (n=9 each). The concentration of Ach in the striatum was determined by using high-performance liquid chromatography. The apoptosis of substantia nigra cells was assayed by using TUNEL method. The ultrastructural changes in the substantia nigra were observed under the electron microscopy, and the survival of neurons in the substantia nigra area was examined by using Nissl staining. In PD group at POD 7 to 21, the damage in the substantia nigra area was gradually aggravated, the concentration of Ach, apoptosis rate and turns of rotation were gradually increased, and the number of Nissl cells was gradually reduced over the time as compared with the normal control and sham operation groups (all P<0.05). It was concluded that there exist dynamic changes in Ach concentration, ethology and apoptosis of the substantia nigra cells during the morbidity of PD, suggesting the contribution of apoptosis to the morbidity of PD, and critical role of Ach in the pathogenesis of PD.

OBJECTIVE To investigate the prevalence of DHA AmpC ?-lactamases mediated by plasmid in Klebsiella pneumoniae in China.METHODS Antimicrobial susceptibility test was conducted by the methods of double agar dilution and ESBLs confirmatory in K-B method according to the criteria of guidelines of CLSI.AmpC ?-lactamases were detected on the basis that AmpC ?-lactamases could be inhibited by 3-aminophenylboronic acid(APB).Gene chip technology and PCR were used to detect ESBLs and AmpC gene.RESULTS Among total 34 isolates of K.pneumoniae 32(94.1%) produced AmpC ?-lactamases and ESBLs.The most common(38.3%) were types DHA and TEM and SHV.MIC50 and MIC90 of all strains to all tested antimicrobial agents were lower than 34 strains tested 0.25?g/ml and 0.5?g/ml.Fourteen strains AmpC and ESBLs were conjugated successfully.CONCLUSIONS DHA AmpC ?-lactamases mediated by plasmid are the most common in K.pneumoniae in General Hospital of PLA of China.The most common(38.3%) are types DHA and TEM and SHV.Fourteen(41.2%) strains can be spreaded by plasmid.

Aim To clone and express the 1.2kb cDNA fragment (1/753-2 934bp) of human integrin α 4 subunit. Methods The 1.2 kb cDNA fragment of human integrin α 4 subunit was amplified from HL-60 total RNA by RT-PCR, then it was subcloned into expression vector pGEX-3X and induced with IPTG. Results The 1.2 kb cDNA fragment of human integrin α 4 subunit was cloned. The sequencing indicated that there was only one missense mutation (Arg→ Gln) among the fragment, and this mutation won't affect antigenicity after analysed by GOLDKEY. Then the 1.2 kb cDNA was subcloned into expression vector pGEX-3X. The α 4 fragment was highexpressed in E.coli after induced with IPTG. Conclusion The 1.2kb cDNA fragment of α 4 subunit was obtained, and it was highexpressed in E.coli, it might be important for study on the function of α 4 integrins.