To obtain a number of extracellular penicillinase,the gene(penp)encoding penicillinse of Bacillus cereus ATCC10987 was cloned into expression vector in Bacillus subtilis,transformed into Bacillus subtilis DB104 deficient in two proteases.When recombinant bacteria was cultured in LB medium for 24 hours,the result of SDS-PAGE showed that the molecular weight of the protein was 28kDa and the penicillinase activity reached 339U/ml.By screening seven different fermentation media,the result showed that 4# medium is favorable to producing penicillinase by the recombinant cells than the others,with the yield of the enzyme being 1580U/ml.When the recombinant cells was cultured in 7 liter fermentor for 24h,the penicillinase activity reached 1255.8U/ml.

OBJECTIVE: To observe the effects of intermedin preconditioning on hypoxic injury in rat's cardiac myocytes and to provide the hypothetical mechanism of sudden cardiac death in the field of forensic pathology. METHODS: The H9c2 cultured rat cardiac myocytes were randomly divided into control group, hypoxia group and IMD group. The myocardial cell viability, cellular ultrastructure, intracellular calcium concentration and apoptosis rate were determined by MTT assay, transmission electron microscopy, laser scanning confocal microscope and flow cytometry, respectively. RESULTS: Compared with the control group, cell viability obviously decreased with inner ultrastructure injury in the hypoxia group (P<0.05), while cell viability significantly increased in the IMD group by reducing the hypoxia injury of cardiac myocytes (P<0.05). Compared with the control group, [Ca2+]i (fluorescence intensity) and apoptosis rate significantly increased in the hypoxia group, but decreased in the IMD group (P<0.05). CONCLUSION IMD increases the cell survival rate and decreases the cell apoptosis inhibited by intracellular calcium overload from hypoxia. This finding may reveal the mechanism of protective effects of myocardial hypoxia, and provide a scientific basis for the identification sudden cardiac death.
Animals ; Apoptosis ; Calcium ; Cell Hypoxia ; Cell Survival ; Hypoxia ; Myocardial Ischemia ; Myocardium/cytology* ; Myocytes, Cardiac/physiology* ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To investigate the relation between injury time and the expression of cytochrome c oxidase subunit VIc (COX6C) mRNA in skeletal muscle of rat after contusion. METHODS: A total of fifty-four SD rats were divided into the control group and the contusion groups (0.5, 1, 6, 12, 18, 24, 30, and 36 h after contusion), randomly. The contusion model was established by free fall drop of gravity hammer. At corresponding time point after contusion, the regular histology was examined and expression level of COX6C mRNA was tested by real-time PCR after extraction of total RNA from the tissues. RESULTS: The main pathological features of 6 h after injury included edema and hemorrhage in myocytes with no inflammatory cells found. After 6 hours, the findings included myocyte degeneration and necrosis, inflammatory cells infiltration, and fibrous connective tissue proliferation in the contused zone. The expression level of COX6C mRNA was higher than that of the control group within 6 h after contusion. The expression level was lower than that of the control group from 6-36 h after contusion. CONCLUSION The level of COX6C mRNA expresses in a regular way after contusion. It may be useful for estimating wound age in combination with the results of pathological features.
Animals ; Contusions/metabolism* ; Electron Transport Complex IV/metabolism* ; Muscle, Skeletal/metabolism* ; RNA ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Time Factors

OBJECTIVE: In order to understand which kind of function genes play an important role for estimating wound age, the variation of difference genes' mRNA expression were compared after injury. METHODS: The mRNA expression levels of seven candidate genes (ICAM-1, NF-κB, MX2, MT1, MT2, sTnI, and Cox6c) were analyzed in contused rat skeletal muscle at different time points using real-time fluorescent quantitative PCR (RT-qPCR). The raw Ct values were normalized relative to that of RPL32 mRNA, and converted to standard Ct values. At each time point after injury, the standard deviations (SD) of the standard Ct values were calculated by SPSS. RESULTS: The expression trends of the seven genes were all found to be related to wound age, but there were lower variation coefficients and greater reliability of s TnI and Cox6c when compared with other genes. CONCLUSION The genes encoding structural proteins or proteins that perform basic functions can be suitable for wound age estimation.
Animals ; Contusions/genetics* ; Forensic Pathology ; Gene Expression Profiling ; Intercellular Adhesion Molecule-1 ; Muscle, Skeletal/metabolism* ; NF-kappa B ; Proteins ; RNA, Messenger/metabolism* ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Regression Analysis ; Reproducibility of Results ; Time Factors ; Wound Healing/genetics*

OBJECTIVEIn order to fully understand hepatitis c virus (HCV) genotype 3b, 1a, 2b and 6a infection in China, We built HCV 5'-noncoding region (5'-NCR) of different genotypes and subtypes.

METHODSThe classification HCV into variable genotypes (subtypes) was carried on by programs A, B and C A. Using a combination of three restriction endonuclease BHH' (BsrB I, Hae II, Hinf I) digestions at the same time. The distinct genotypes were classified into 5 groups: genotype 1 (1a, 1b), 6a, 2 (2a, 2b), genotype 3 (3a, 3b), genotype4 (4a). B. With regard to genotype 1, we could distinguish subtype 1a from 1b using BstU I digestion. C. Using restriction endonuclease Hae III, genotype 2a, 2b, 3b, 4a, 6a are differentiated respectively.

RESULTS(1) HCV genotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 6a are fully discriminated by comparison with the genotypes regular samples. (2) Of the 93 patients, HCV genotype distribution in China was 66.67% for 1b, 18.28% for 2a, 3.23% for 1b/2b, 3b, 2b respectively. 2.15% for 2a/2b, 1b/2a respectively. 1.08% for 1a.

CONCLUSIONThis research indicated that adoption of HCV 5'-NCR A B C restriction endonuclease digestions techniques, might be sensitive and efficient to detect HCV and discriminate HCV genotype (subtypes) 1a to 6a.

5' Untranslated Regions ; chemistry ; DNA Restriction Enzymes ; Genotype ; Hepacivirus ; classification ; genetics ; RNA, Viral ; analysis

OBJECTIVETo study the clinical results of posterior pedicle screw fixation, transpedicular bone grafting and vertebral canaloplasty with ilium autografting in treating serious burst thoracolumbar fracture.

METHODSFrom March 2004 to March 2008,10 patients with serious burst thoracolumbar fracture, including 7 males and 3 females with age for 24-58 years (mean 41 years)were treated by posterior pedicle screw fixation, transpedicular bone grafting and total laminectomy with preservation of spinal process and vertebral canaloplasty with ilium autografting. The operative effects were assessed according to Frankel classification and radiologic results.

RESULTSAll patients were followed up from 1 to 4 years. There was no loosening or broken in instrumentation. The anterior edge height of the fractured vertebrae body was restored from (21.00 +/- 12.00)% to (95.00 +/- 4.20)%, and the posterior edge height of the fractured vertebrae body was restored from (70.00 +/- 15.00)% to (96.00 +/- 3.20)% postoperatively, which both demonstrated improvement compared with preoperative instance (P < 0.01). The Cobb angle was restored from (32.80 +/- 8.20) degrees to (4.20 +/- 1.60) degrees which also demonstrated improvement compared with the preoperative Cobb angle (P < 0.01). At least one grade recovery was observed in all cases except one patient with preoperative Frankel A degree. The result of Denis classification, P1, had 4 cases, P2 had 4, P3 had 1, P4 had 1.

CONCLUSIONPosterior pedicle screw fixation, transpedicular bone grafting and vertebral canaloplasty can obtain satisfactory results treating serious burst thoracolumbar fractures. It is a feasible method with advantages of simple operation, good efficacy, preservation of structure of posterior column which should be applied clinically.

Adult ; Bone Screws ; Bone Transplantation ; Female ; Fracture Fixation, Internal ; Humans ; Lumbar Vertebrae ; injuries ; surgery ; Male ; Middle Aged ; Radiography ; Spinal Fractures ; diagnostic imaging ; surgery ; Thoracic Vertebrae ; injuries ; surgery ; Young Adult

This study examined the effect of Notch-1 signaling on malignant behaviors of breast cancer cells by regulating breast cancer stem cells (BCSCs). BCSCs were enriched by using serum-free medium and knocked out of Notch-1 by using a lentiviral vector. Real-time polymerase chain reaction (RT-PCR) and Western blotting were used to detect the Notch-1 expression levels in breast cancer cell lines and BCSCs, and flow cytometry to detect the proportion of BCSCs in BCSC spheres. The BCSC self-renewal, migration, invasion, and tumorigenicity were examined by the tumor microsphere-forming assay and transwell assay and after xenotransplantation. The results showed that the Notch-1 silencing reduced the number of BCSC spheres, the proportion of BCSCs, and the number of cells penetrating through the transwell membrane. It also decreased the size of tumors that were implanted in the nude mice. These results suggest that Notch-1 signaling is intimately linked to the behaviors of BCSCs. Blocking Notch-1 signaling can inhibit the malignant behaviors of BCSCs, which may provide a promising therapeutical approach for breast cancer.

Objective To detect the expression of nuclear factor erythroid 2-related factor 2(Nrf2)to explore its change rule and effects in the repair process of skeletal muscle. Methods An injury model of skeletal muscle was established by injection of cardiotoxin into the gastrocnemius muscle of SD male rats. The muscle samples were taken from control group and injury group at each time point(1 h, 4 h, 8 h, 12 h, 16 h, 1 d, 3 d, 5 d, 7 d, 9 d, 13 d, 17 d, 21 d). The morphological changes of skeletal muscle in repair process were observed by H&E staining, and the content of reactive oxygen species(ROS)was detected. The expression level of Nrf2 protein was investigated by Western blotting and immunohisto-chemistry staining. Results After the skeletal muscle injury, the injury region was infiltrated with inflam-matory cells which constituted mainly by lymphocyte neutrophils and mononuclear cells. There were large amounts of immature muscle cells appeared at the 5th day and the muscle repair was completed at the 21th day. Comparing with control group, the ROS content decreased in injury group at the 12th hour(P<0.05), while Nrf2 expression increased at the 16th hour(P<0.05). Moreover, the positive rate of Nrf2 increased after injury, and peaked at 16 h-1 d, then decreased gradually and returned to the level of control group(P<0.05). Conclusion The Nrf2 protein involves in the regulation of the repair of skeletal muscle injury. The positive rate of Nrf2 shows a time-dependent expression, which can be used to esti-mate the wound age of skeletal muscle.

OBJECTIVE: To investigate the expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in contused skin and muscle of rats and the relationship between the ICAM-1 expression and the wound age. METHODS: The samples were taken at 0.5, 1, 6, 12, 18, 24 and 30 h after contusion of rats, respectively. Total RNA was extracted both from the skin and muscle samples of each group and reverse transcription polymerase chain reaction (RT-PCR) was conducted to synthesize the 1st strand cDNA. The amount of ICAM-1 mRNA in each sample was quantified using rp132 intrinsic fluorescent assay and compared by the 2 (-Delta Delta Ct) method with that from control samples. RESULTS: After contusion the expression of ICAM-1 mRNA in skin increased rapidly and peaked at 0.5 h, at 24 h degraded to the amount that was seven times as much as the control group, then rised again. The expression of ICAM-1 mRNA in muscle increased significantly within 0.5 h and peaked at 6 h, reached the minimum at 18 h, then increased again. CONCLUSION It is suggested that ICAM-1 mRNA analysis may be useful for estimation of early wound age because of its time-related expression after contusion in skin and muscle.
Animals ; Contusions/metabolism* ; Female ; Forensic Medicine ; Intercellular Adhesion Molecule-1/metabolism* ; Male ; Muscle, Skeletal/metabolism* ; RNA, Messenger/metabolism* ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Skin/metabolism* ; Time Factors

OBJECTIVE: To investigate the effect and potential mechanism of intermedin (IMD) in acute cardiac ischemic injury and to provide a new approach for exploring mechanism of sudden cardiac death. METHODS: Seventy-two healthy male rats were randomly divided into 3 groups: control, ischemic and the IMD-treated group. The activity of lactate dehydrogenase (LDH), malondialdehyde (MDA) and superoxide dismutase (SOD) in heart blood were tested by enzyme chemistry method. The mRNA changes of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs) in cardiac were measured by real-time PCR analysis. Myocardial cyclic adenosine monophosphate (cAMP) content was determined by enzyme linked immunosorbent assay (ELISA). Apoptosis related factors Bcl-2 and Bax were detected by immunohistochemistry. RESULTS: Comparing with the control group, LDH and MDA activity of ischemic group in heart blood increased and SOD activity decreased. The concentration of cAMP increased in ventricular muscle, Bcl-2 and Bax proteins expression ratio level decreased. The intravenation of IMD decreased the level of increased activity of LDH and MDA, and lessened the level of decreased activity of SOD. The mRNA expression of CRLR and RAMPs obviously increased in ventricular muscle. CONCLUSION The protective effect of IMD against myocardial ischemic injury could be caused by decreasing the oxidative stress of ischemia and inhibiting the myocardial apoptosis.
Adrenomedullin/pharmacology* ; Animals ; Apoptosis/drug effects* ; Calcitonin Receptor-Like Protein/metabolism* ; Cardiotonic Agents/pharmacology* ; Cyclic AMP/metabolism* ; Disease Models, Animal ; L-Lactate Dehydrogenase/metabolism* ; Male ; Malondialdehyde/metabolism* ; Myocardial Ischemia/pathology* ; Myocardium/pathology* ; Neuropeptides/pharmacology* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; RNA, Messenger/metabolism* ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Receptor Activity-Modifying Proteins/metabolism* ; Superoxide Dismutase/metabolism*