To achieve higher level expression of Interferon α2b (IFN-α2b) in methylotrophic yeast (Pichia pastoris), a cDNA fragment coding for the mature IFN-α2b was designed and synthesized based on the synonymous codon bias of P. pastoris and optimized G+C content. The synthetic IFN-α2b was inserted into the secreted expression vector pPICZαA, and then integrated into P. pastoris GS115 genome by electroporation. Multi-copy integrants in the Mut+ recombinant P. pastoris strain were screened by high concentrations of Zeocin. 120 hours culturing allowed expression of the IFN-α2b transformant up to 810 mg/L as detected by SDS-PAGE and quantitative methods. In addition, Western blot analysis showed that the recombinant proteins had immunogenicity. The significant antiviral activity of the recombinant IFN-α2b protein was verified by WISH/ VSV system, which was 3.3×105 IU/mL.

Objective To investigate the usefulness of different-b-value diffusion weighted imaging (DWI) in patients with early cerebral infarction and obtain the optimal b value of early cerebral infarction.Methods DWI at b-value of 1,000,2,000,and 3,000 s/mm2 was performed for 40 patients within 72 h after the onset of stroke using a GE Signa HDx 3.0T MRI scanner.Post-processing was done by the DWI specific software Function Tool to gain signal intensity and mean apparent diffusion coefficient in the lesions center and the contralateral normal brain tissue,respectively.The sensitivity of conventional MRI and different-b-value DWI was calculated in the diagnosis of early cerebral infarction.Results In 40 patients with early cerebral infarction,the sensitivity of b-values of 1,000,2,000,and 3,000 s/mm2 (DWIb=1 000,DWIb=2 000,DWIb=3 000) and conventional MRI in the diagnosis of early cerebral infarction were 100％,97.5％,97.5％,72.5％,respectively.With b value increased,signal intensity and ADC value declined.Under the condition of different b values,signal intensity and ADC value were statistically significant in region of interest (P＜0.05).Conclusion DWI is superior to conventional MRI scan in monitoring early cerebral infarction.With the increase of b value,the sensitivity is the higher to hyperacute cerebral infarction,the signal contrast is obvious,while signal to noise ratio of the image reduces.It is suggested that brain tissue contrast and the sensitivity to the new cerebral infarction is more predictable on b value =1,000 DWI than on the other DWIs.

OBJECTIVETo investigate the expression of angiotensin converting enzyme (ACE) and ACE2 Gene in lung of paraquat poisoning rats and the protection of sodium dimercaptopropane sulfonate (Na-DMPS).

METHODSOne hundred SD male rats were randomly equally divided into 4 groups:normal control group (10 rats), drug control group (40 rats), paraquat poisoning group (40 rats) and drug intervention group(40 rats). The paraquat poisoning and drug intervention group rats were injected intraperitoneally by paraquat (20 mg/kg). The rats in drug intervention group rats were protected by intraperitoneal injection with Na-DMPS (200 mg/kg) 15 min before exposure of paraquat. Behavioral changes of the rats and histological changes of lung tissues under light microscope were observed. And the expression of ACE and ACE2 mRNA in lung tissues of rats both in paraquat poisoned group and drug intervention group were measured by RT-PCR at different time of 6 h, 24 h, 3 and 7 d after poisoning.

RESULTSThe poisoning symptoms of shortness of breath, cramps appeared and deteriorated progressively in rats after paraquat exposure and the protection of NA-DMPS could delay and reduce these symptoms significantly. Histological appearance of disorganization of pulmonary capillary and alveolus, exudation in alveolar space, pulmonary edema, severe bleeding, and inflammatory cells infiltration were obvious in lungs of rats after paraquat poisoning, whereas the histological changes were extenuated by protection of NA-DMPS. As compared with normal control group (NC group), the expressions of ACE, ACE2 mRNA in lung tissue decreased, and the lowest level of ACE mRNA expressions appeared at 24 h (0.457 +/- 0.262), on 3 d (0.385 +/- 0.179) after Paraquat exposure (P < 0.05), while lowest level of ACE2 mRNA expressions appeared on 3 d (0.415 +/- 0.247), 7 d (0.365 +/- 0.215) (P < 0.05). As compared with paraquat poisoned group, the expressions of ACE mRNA in lung tissue of rats in NA-DMPS protected group increased significantly at 24 h (0.739 +/- 0.558) and 3 d (0.749 +/- 0.414) (P < 0.05), while the expressions of ACE2 mRNA increased markedly on 3 d (0.584 +/- 0.345) and 7 d (0.493 +/- 0.292) (P < 0.05). But the expression of ACEmRNA and ACE2 mRNA in lungs had no statistical significance between normal control group and drug intervention group (P > 0.05).

CONCLUSIONThe expressions of ACE and ACE2 mRNA in lung tissue of the rats with paraquat poisoning are decreased. Na-DMPS can effectively improve the balance of RAS in local lung tissue and reduce the pathological changes of lung tissue, delay the poisoning symptoms and show protective effects for acute lung injury induced by paraquat.

Animals ; Lung ; drug effects ; enzymology ; Male ; Paraquat ; poisoning ; Peptidyl-Dipeptidase A ; biosynthesis ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Unithiol ; pharmacology

Xanthomonas campestris pv. campestris (Xcc), the pathogenic agent of black rot disease in cruciferous plants, produces large amount of extracellular polysaccharide (EPS), which has found wide applications in industry. For the great commercial value of the xanthan gum, many of the genes involved in EPS biosynthesis have been cloned and the mechanism of EPS biosynthesis also has been studied. In order to clone genes involved in EPS biosynthesis, Xcc wild-type strain 8004 was mutagenized with transposon Tn5 gusA5, and a number of EPS-defective mutants were isolated in our previous work. The Tn5 gusA5 inserted sites of these mutants were located by using thermal asymmetric interlaced PCR, and results showed that two EPS-defective mutants were insertion mutants of the gene wxcA which involved in lipopolysaccharide (LPS) biosynthesis. The gene wxcA involved in lipopolysaccharide biosynthesis but dose not extracellular polysaccharide in others' report. wxcA::Tn5 gusA5 mutant 021C12, the polar mutant, was complemented with recombinant plasmid pLATC8570 harboring an intact wxcA gene in this work, but the yield of EPS of the wxcA::Tn5 gusA5 mutant was not restored. In order to identify the function of wxcA gene of Xcc 8004 strain, the gene wxcA was deleted by gene replacement strategy, and the no-polar mutant of wxcA was obtained. DeltawxcA mutant strain, named Xcc 8570, was confirmed by using both PCR and southern analysis. Beside the LPS biosynthesis of deltawxcA mutant was affected, The EPS yield of deltawxcA mutant strain reduced by 50% as compared with the wild-type strain 8004. DeltawxcA mutant could be complemented in trans with the intact wxcA gene, and the EPS yield of the mutant was restored. The combined data showed that wxcA gene not only involved in LPS biosynthesis but also EPS yield in Xcc 8004 strain.
Cell Proliferation ; Genes, Bacterial ; physiology ; Lipopolysaccharides ; biosynthesis ; Mutation ; Polysaccharides, Bacterial ; biosynthesis ; Xanthomonas campestris ; genetics

OBJECTIVEto study the oxidative stress of rats with acute paraquat poisoning and the intervention of Sodium Dimercaptopropane Sulfonate (NA-DMPS).

METHODSEighty male SD rats were randomizedly divided into: the normal control group (n=8), NA-DMPS control group (n=8), the PQ group (n=32, the rats were intraperitoneally injected with 1% PQ solution at the dosage of 20 mg/kg) and the NA-DMPS protected group (n=32). The rats in the groups of normal and NA-DMPS control were sacrificed 1d after administration of NS or NA-DMPS. And the rats in the PQ group and the NA-DMPS protected group were sacrificed at 6h, 1, 3, 7d after poisoning. Samples of serum, bronchoalveolar lavage fluid (BALF) and lung tissue were gathered. The MDA and CAT in serum, BALF and lung homogenate, the glutathione (GSH) in serum and BALF were measured. And the expression of Nuclear factor E2-related factor 2 (Nrf2) mRNA in lung was tested with RT-PCR.

RESULTSCompared with the normal control group, the activities of MDA and CAT in serum, BALF and lung homogenate are higher in both groups of PQ and NA-DMPS protected. And compared with the PQ group, the activities of MDA in serum, BALF and lung homogenate of the NA-DMPS protected group decreased significantly at 6h, 1d after poisoning, whereas the activities of CAT are higher at 6h, 1, 3d in serum and 1, 3d in BALF and lung homogenate (P<0.05 or P<0.001). The serum GSH at 6h, 3d of the NA-DMPS protected group [(730.07 +/- 16.23), (793.66 +/- 7.40)] were higher than those in the PQ group. And the BALF GSH at 1, 3d of the NA-DMPS protected group [(609.75 +/- 6.74), (631.83 +/- 12.03)] were also markedly higher than the PQ group (P<0.05 or P<0.001). The expression of NRF2 mRNA of the lung at 1, 3, 7d in the PQ group [(0.71 +/- 0.061), (1.023 +/- 0.158), (0.969 +/- 0.046)] and the NA-DMPS protected group [(1.005 +/- 0.06), (1.464 +/- 0.166), (1.066 +/- 0.191)] were significantly higher than those in the control groups. Compared with the PQ group, the expression of NRF2 mRNA of the lung increased markedly in the NA-DMPS protected group at 1, 3d (P<0.01).

CONCLUSIONNa-DMPS decreases the activity of MDA and increases the activity of CAT, GSH and the expression of Nrf2 mRNA. NA-DMPS can protected rats from PQ intoxication by improving the balance of redox reaction.

Acute Disease ; Animals ; Male ; Oxidative Stress ; drug effects ; Paraquat ; poisoning ; Rats ; Rats, Sprague-Dawley ; Unithiol ; pharmacology

The fine structure of enoxaparin sodium samples with different degree of 1,6-anhydro derivatives were analyzed with polyacrylamide gel electrophoresis, high performance liquid chromatography, ultraviolet spectroscopy, infrared spectroscopy and nuclear magnetic resonance spectroscopy. A further study of anticoagulation activity of enoxaparins was performed, including those on their inhibition activities of coagulation factor Xa (FXa) and thrombin (FIIa). The results showed that the anti-FXa and -FIIa activities of enoxaparins with different degree of 1,6-anhydro derivatives (20.0%-39.7%) with similar structure characteristics, had decreasing tendency when the degree of 1,6-anhydro derivatives increased. Especially, the anti-FXa activity was sensitive to the change of the degree of 1,6-anhydro derivatives.
Anticoagulants ; chemistry ; Enoxaparin ; chemistry ; Factor Xa Inhibitors ; chemistry ; Thrombin ; antagonists & inhibitors

OBJECTIVEBiomechanical properties between retrograde nail and condylar buttress plate for supracondylar femoral fracture were tested and compared. The functional operative results were also followed and analyzed.

METHODS(1) Six fresh adult moist cadaveric femoral specimens were used to create femoral supracondylar fracture models. Six specimens were divided into two groups. Group 1 was fixed with retrograde locking intramedullary nail and group 2 with condylar buttress plate. All specimens undergone biomechanical test and the data were analyzed using SPSS software. (2) From August 1995 to December 2000, 36 cases (37 limbs) of femoral supracondylar fracture were included. The follow-up period ranged from 2.1 years to 7.5 years.

RESULTS(1) Biomechanical tests showed: The stiffness of the condylar buttress plate in resisting axial compression force was smaller than that of the retrograde locking intramedullary nail. condylar buttress plate showed larger stiffness in resisting bending force than the retrograde locking intramedullary nail. (2) Clinical outcomes: According to Neer rating system, the functional scores were excellent in 26 cases, good in 8, fair in 1 and poor in 2. The excellent-good rate was 91.9%. The excellent-good rate of plating cases was 94.7% and retrograde nail was 93.3%. ROM of the knee joint ranges from 60 degrees to 135 degrees , with an average of 110 degrees.

CONCLUSIONThe excellent or good outcomes of femoral supracondylar fracture could be obtained through the operative treatment. The fixation of the retrograde locking and condylar buttress plate have their own advantages in biomechanical properties.

Adolescent ; Adult ; Aged ; Biomechanical Phenomena ; Bone Plates ; Bone Screws ; Female ; Femoral Fractures ; surgery ; Follow-Up Studies ; Fracture Fixation, Internal ; instrumentation ; methods ; Fracture Fixation, Intramedullary ; instrumentation ; methods ; Humans ; Male ; Middle Aged ; Treatment Outcome

Objective To report the outcome of emergency repair of severe complex defect in forearm by transplantation of free flap and simultaneous functional reconstruction.Methods From Mar.1994 to Aug.2003,4 cases with severe complex defect in forearm was repaired by transplantation of free skin flap, free skin flap combined with fibula flap,or fibula osteocutaneous flap in emergency.Simultaneously the flexion and extension function were repaired by muscle transfer and/or tendon grafting,tenonectomy.Results All the cases were successful.Follow-up period ranged from 1 to 3 years postoperatively.The blood-supply,tex- ture and elasticity of transferred flaps were excellent with good bone healing.Opposition of thumb with four fin- gers was good.Sensory recovery of the hand was satisfactory.Conclusion Transplantation of free flap com- bined with simultaneous functional reconstruction is an ideal method in emergency repair of severe complex de- fect in forearm.

In this study,we aimed to study the pattern visual evoked potentials (P-VEPs) in two eyes with varying visual acuity in one eye and to provide an objective estimation of visual acuity by comparing P-VEPs in one and two eyes.Thirty subjects were chosen,who had one eye with an acuity of 5.0,4.85,4.6,4.0,or scieropia and obstructed vision and the other eye with an acuity of 5.0,respectively.P-VEPs were detected under the large grating stimuli at 3×4 spatial frequency,moderate grating stimuli (12× 16 spatial frequency) and small grating stimuli (48×64 spatial frequency).Under large grating stimuli,there was no significant difference in P100 peak latency between the groups,nor was there a significant difference between the amplitude of two eyes and the amplitude of one normal-vision eye.Under moderate and small grating stimuli,there was a significant difference in P100 peak latency between the group with both eyes having an acuity of 5.0 and the group with visual acuity below 4.0 in one eye.There was a significant difference in P100 amplitude between the group with visual acuity of 5.0 in both eyes and the group with one normal-vision eye.There was no significant difference in the amplitude of two eyes and the amplitude of one normal-vision eye between any other two groups.In forensic identification,characteristics and variability of P-VEPs in one and two eyes can be used to identify malingering or decline in visual acuity.

OBJECTIVETo observe the expression and effect of thrombomodulin (TM) mRNA and endothelial protein C receptor (EPCR) mRNA in lung tissue of acute paraquat poisoned rats, and intervention of sodium dimercaptopropane sulfonate (Na-DMPS).

METHODSEighty male SD rats were randomizedly divided into four groups: the normal control group (n=8), the Na-DMPS control group (n=8, administered with 200 mg/kg Na-DMPS intraperitoneally), the PQ group (n=32, administered with 20 mg/kg 1% PQ intraperitoneally), the NA-DMPS protected group (n=32, administered with 200 mg/kg Na-DMPS intraperitoneally before with 20 mg/kg 1% PQ). The expression of TM mRNA and EPCR mRNA in the PQ group and the Na-DMPS protected group was evaluated at the six hour, on the first, third and seventh day.

RESULTSThe expression of TM mRNA and EPCR mRNA in lung tissue of poisoned rats, was significantly increased and reached the peak at the six hour, was decreased slowly on the first day, and returned to normal level on the seventh day. In the Na-DMPS protected group, at the six hour and on the first day, the expression of TM mRNA (1.071 +/- 0.097, 1.055 +/- 0.051) was less than that in the PQ group (P<0.05 or P<0.01). EPCR mRNA (0.678 +/- 0.005), (0.650 +/- 0.007) at the six hour and on the first day in the Na-DMPS protected group was less than that in the PQ group (P<0.05 or P<0.01).

CONCLUSIONThe expression of TM mRNA and EPCR mRNA of rats after PQ intoxication is increased, and can significantly be decreased after administered with Na-DMPS.

Animals ; Antigens, CD ; genetics ; metabolism ; Disease Models, Animal ; Lung ; metabolism ; pathology ; Male ; Paraquat ; poisoning ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptors, Cell Surface ; genetics ; metabolism ; Thrombomodulin ; genetics ; metabolism ; Unithiol ; therapeutic use