Glucose was transported by the large number of hexose transporters in yeast cells. There were 18 hexose transporter genes had been identified in Saccharomyces cerevisiae. However,as an excellent expression system,there was no information of these genes had been reported in Pichia pastoris. Based on high homologous recombination efficiency in yeast,we chose G418 resistance for screening,200 bp were cloned from the up and down sequences of HXT1 ORF respectively,then ligated to the 5′ and 3′ end of G418 resis-tance gene for recombination. After electroporation of GS115 spheroplast and screened through different G418 concentration plates,finally we obtained one HXT1 gene deletion mutant named GS115?HXT1. The growth rate and glucose consumption of this mutant were both lower than the wide type.

The purpose of this study was to determine the combined effect of transmyocardial laser revascularization (TMLR) and the implantation of endothelial progenitor cells (EPCs) on cardiac function of ischemic hearts in canines. The left anterior descending artery (LAD) was occluded to establish the canine model of acute myocardial infarct (AMI). Four weeks later, the animals were randomly divided into four groups: TMLR group, in which transmyocardial laser-induced channels were established at the ischemic region; EPCs+TMLR group, in which EPCs were locally transplanted into laser-induced channels at the ischemic region; EPCs group, in which the EPCs were injected into the ischemic region; control group, in which the AMI animals received neither TMLR nor EPCs. The peripheral blood (50 mL) was sampled in all groups. Mononuclear cells from the peripheral blood were separated and cultured to obtain spindle-shaped attaching (AT) cells in vitro. AT cells were labeled with 1, 1'-dioctadecyl-1 to 3,3, 3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) before injecting into the laser-induced channels or ischemic region. Four weeks after the first operation, TMLR was performed in the TMLR group and EPCs+TMLR group, and at the same time, the EPCs originating from the AT cells were mixed with calcium alginate (CA). Then the EPCs-CA composites were implanted into myocardial channels induced by laser in the EPCs+TMLR group, and into the myocardial infarct area in the EPCs group. All dogs underwent echocardiography at second month after LAD occlusion. Finally the samples of myocardium around the LAD were subjected to histochemical and immunohistologic examinations. The results showed there was no significant difference in the diameter of left atrium and ventricle before treatment among all groups (P>0.05). Eight weeks after modeling, the regional contractility in the LAD territory in the EPCs+TMLR group was increased as compared with control group and TMLR group, but there was no significant difference between control group and TMLR group. Neoangiogenesis was observed in the EPCs+TMLR group, and the fibrosis was seen in the TMLR group. There was no significant difference in neoangiogenesis around the channels induced by laser among EPCs+TMLR, EPCs and TMLR groups. It was concluded that TMLR combined with EPCs could improve the regional and global cardiac function in AMI, and augment neovascularizaiton in channels of ischemic myocardium induced by laser.

OBJECTIVE: To discuss the relation between degree of body impairment and that of thoracolumbar spinal injuries resulting from road traffic accidents, and sum up the experiences in body impairment assessment and its regularity. METHODS: For comprehensive body impairment assessment, 477 cases of thoracolumbar spinal injuries in road accidents have been sorted out, reassessed and rediagnosed. In addition, analyses have been undertaken about their treatment, the assessment of the degree of their thoracolumbar dysfunction,nerve dysfunction and the relations between injuries and sequelaes. RESULTS: The analyses show that the degree of thoracolumbar dysfunction and that of the post-injury nerve dysfunction don't necessarily depend on the quantity and degree of spinal injuries. However, the position suffering from the thoracolumbar spinal injuries has an immense impact on the thoracolumbar dysfunction, and the nerve impairment result mainly from the T1-1L spinal injuries. The research also shows that there has been a high misdiagnosis rate in hospital about the spinal injuries. CONCLUSION In body impairment assessment, the cause and effect relations between the injury and degree of injury extent should be analyzed, the injury extent should be employed as principal evidence, and the degree of spinal dysfunction should be taken into greater consideration.
Accidents, Traffic ; Adolescent ; Adult ; Age Distribution ; Aged ; Aged, 80 and over ; Disability Evaluation ; Female ; Humans ; Injury Severity Score ; Lumbar Vertebrae/physiology* ; Male ; Middle Aged ; Nervous System/physiopathology* ; Retrospective Studies ; Spinal Injuries/physiopathology* ; Thoracic Vertebrae/physiology* ; Young Adult

OBJECTIVETo construct a system of I-SceI and induce a site-specific DNA double-strand break (DSB) in the genome of HepG2 for using this system in future exploration of the potential mechanisms of HBV integration by DSB repair.

METHODSThe eukaryotic expression plasmid pEGFP2 was constructed and transfected into human hepatoma cell line HepG2. The positive neomycin-resistant transfected cell clones were generated by G418 selection. Then the positive cells containing an 18-bp I-SceI endonuclease site were transfected transiently with pCMV(3NLS) I-SceI, an I-SceI expression plasmid. At 24 h post-transfection with pCMV (3NLS) I-SceI, gamma-H2AX, as an early cellular marker of DSB, was detected using immunocytochemistry and Western blot analysis.

RESULTSRestriction analysis and DNA sequencing verified that the plasmid pEGFP2 was successfully constructed. gamma-H2AX increased significantly in cells transfected with the I-SceI system.

CONCLUSIONSGenomic DSB can be induced into HepG2 by introducing an I-SceI system. The cell model could provide us with a practical tool for further study to see if DSB is a potential target for HBV integration.

Carcinoma, Hepatocellular ; genetics ; DNA Breaks, Double-Stranded ; DNA Repair ; Flap Endonucleases ; genetics ; Hep G2 Cells ; Humans ; Liver Neoplasms ; genetics ; Plasmids

The purpose of this study was to determine the combined effect of transmyocardial laser revascularization (TMLR) and the implantation of endothelial progenitor cells (EPCs) on cardiac function of ischemic hearts in canines. The left anterior descending artery (LAD) was occluded to establish the canine model of acute myocardial infarct (AMI). Four weeks later, the animals were randomly divided into four groups: TMLR group, in which transmyocardial laser-induced channels were established at the ischemic region; EPCs+TMLR group, in which EPCs were locally transplanted into laser-induced channels at the ischemic region; EPCs group, in which the EPCs were injected into the ischemic region; control group, in which the AMI animals received neither TMLR nor EPCs. The peripheral blood (50 mL) was sampled in all groups. Mononuclear cells from the peripheral blood were separated and cultured to obtain spindle-shaped attaching (AT) cells in vitro. AT cells were labeled with 1, 1'-dioctadecyl-1 to 3,3, 3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) before injecting into the laser-induced channels or ischemic region. Four weeks after the first operation, TMLR was performed in the TMLR group and EPCs+TMLR group, and at the same time, the EPCs originating from the AT cells were mixed with calcium alginate (CA). Then the EPCs-CA composites were implanted into myocardial channels induced by laser in the EPCs+TMLR group, and into the myocardial infarct area in the EPCs group. All dogs underwent echocardiography at second month after LAD occlusion. Finally the samples of myocardium around the LAD were subjected to histochemical and immunohistologic examinations. The results showed there was no significant difference in the diameter of left atrium and ventricle before treatment among all groups (P>0.05). Eight weeks after modeling, the regional contractility in the LAD territory in the EPCs+TMLR group was increased as compared with control group and TMLR group, but there was no significant difference between control group and TMLR group. Neoangiogenesis was observed in the EPCs+TMLR group, and the fibrosis was seen in the TMLR group. There was no significant difference in neoangiogenesis around the channels induced by laser among EPCs+TMLR, EPCs and TMLR groups. It was concluded that TMLR combined with EPCs could improve the regional and global cardiac function in AMI, and augment neovascularizaiton in channels of ischemic myocardium induced by laser.
Animals ; Coronary Circulation ; Coronary Vessels ; pathology ; surgery ; Dogs ; Humans ; Muscle Contraction ; physiology ; Myocardial Ischemia ; pathology ; therapy ; Myocardium ; pathology ; Neovascularization, Physiologic ; physiology ; Stem Cell Transplantation ; methods ; Stem Cells ; Transmyocardial Laser Revascularization ; methods

OBJECTIVETo observe the effect of intracoronary transfer of autologous HO-1 overexpressed MSCs in porcine model of myocardial ischemia (1 h)/reperfusion.

METHODSApoptosis was assayed and cytokine concentrations in supernatant were measured in cells exposed to hypoxia-reoxygen in vitro. In vivo, Chinese male mini-pigs were allocated to the following treatment groups: control group (saline), MSCs group (MSCs), MSCs transfected with pcDNA3.1-nHO-1 (HO-1-MSCs). 1 x 10(7) of autologous stem cells or identical volume of saline was injected intracoronary into porcine hearts 1 h after ischemia. MRI assay and postmortem analysis were assessed 3 months after stem cell transplantation.

RESULTSIn vitro, cell apoptosis rate post hypoxia-reoxygen was significantly reduced in HO-1-MSCs group (30.30% +/- 7.64%) compared with that in MSCs group (56.93% +/- 4.68%, P < 0.001) and LacZ-MSCs group (55.88% +/- 4.38%, P < 0.001), VEGF was also significantly upregulated in HO-1-MSCs group [(768.44 +/- 78.38) pg/ml] compared with that in MSCs group [(555.27 +/- 67.67) pg/ml, P < 0.001] and LacZ-MSCs group [(522.97 +/- 71.45) pg/ml, P < 0.001]. In vivo, cardiac function was significantly improved in both MSCs transplantation groups compared to saline group (all P < 0.05 vs.saline) and the left ventricular ejection fraction was significantly higher in HO-1-MSCs group compared with that in MSCs group at 3 months after transplantation (53.50% +/- 2.09% vs. 49.54% +/- 2.74%, P = 0.017), capillary density in the peri-infarct area was also significantly higher in HO-1-MSC group than that in MSCs group [(14.59 +/- 2.39)/HPF vs. (11.78 +/- 2.48)/HPF, P = 0.033].

CONCLUSIONSEfficacy of HO-1 overexpressed MSCs on improving cardiac function and promoting angiogenesis was greater than those by MSCs in this porcine ischemia/reperfusion model.

Animals ; Apoptosis ; Cells, Cultured ; Genetic Vectors ; Heme Oxygenase-1 ; genetics ; Male ; Mesenchymal Stem Cell Transplantation ; Myocardial Infarction ; therapy ; Myocardial Ischemia ; therapy ; Swine ; Swine, Miniature ; Transfection

BACKGROUNDMesenchymal stem cells (MSCs) transplantation may partially restore heart function in the treatment of acute myocardial infarction (AMI). The aim of this study was to explore the beneficial effects of MSCs modified with heme xygenase-1 (HO-1) on post-infarct swine hearts to determine whether the induction of therapeutic angiogenesis is modified by the angiogenic cytokines released from the implanted cells.

METHODSIn vitro, MSCs were divided into four groups: (1) non-transfected MSCs (MSCs group), (2) MSCs transfected with the pcDNA3.1-Lacz plasmid (Lacz-MSCs group), (3) MSCs transfected with pcDNA3.1-hHO-1 (HO-1-MSCs group), and (4) MSCs transfected with pcDNA3.1-hHO-1 and pretreatment with an HO inhibitor, tin protoporphyrin (SnPP) (HO-1-MSCs + SnPP group). Cells were cultured in an airtight incubation bottle for 24 hours, in which the oxygen concentration was maintained at < 1%, followed by 12 hours of reoxygenation. After hypoxia/reoxygen treatment, ELISA was used to measure transforming growth factor (TGF-β) and fibroblast growth factor (FGF-2) in the supernatant. In vivo, 28 Chinese mini-pigs were randomly allocated to the following treatment groups: (1) control group (saline), (2) Lacz-MSCs group, (3) HO-1-MSCs group, and (4) HO-1-MSCs + SnPP group. About 1 × 10(7) of autologous stem cells or an identical volume of saline was injected intracoronary into porcine hearts 1 hour after MI. Magnetic resonance imaging (MRI) assay and postmortem analysis were assessed four weeks after stem cell transplantation.

RESULTSPost hypoxia/reoxygenation in vitro, TGF-β in the supernatant was significantly increased in the HO-1-MSCs ((874.88 ± 68.23) pg/ml) compared with Lacz-MSCs ((687.81 ± 57.64) pg/ml, P < 0.001). FGF-2 was also significantly increased in the HO-1-MSCs ((1106.48 ± 107.06) pg/ml) compared with the Lacz-MSCs ((853.85 ± 74.44) pg/ml, P < 0.001). In vivo, at four weeks after transplantation, HO-1 gene transfer increased the capillary density in the peri-infarct area compared with the Lacz-MSCs group (14.24 ± 1.66/HPFs vs. 11.51 ± 1.34/HPFs, P < 0.001). Arteriolar density was also significantly higher in HO-1-MSCs group than in the Lacz-MSCs group (7.86 ± 2.00/HPFs vs. 6.45 ± 1.74/HPFs, P = 0.001). At the same time, the cardiac function was significantly improved in the HO-1-MSCs group compared with the Lacz-MSCs group ((53.17 ± 3.55)% vs. (48.82 ± 2.98)%, P < 0.05). However, all these effects were significantly abrogated by SnPP.

CONCLUSIONMSCs provided a beneficial effect on cardiac function after ischemia/reperfusion by the induction of therapeutic angiogenesis, and this effect was amplified by HO-1 overexpression.

Animals ; Blotting, Western ; Cell Differentiation ; genetics ; physiology ; Heme Oxygenase-1 ; genetics ; metabolism ; Magnetic Resonance Imaging ; Mesenchymal Stromal Cells ; cytology ; enzymology ; metabolism ; Myocardial Reperfusion Injury ; enzymology ; metabolism ; Swine ; Swine, Miniature

OBJECTIVETo investigate the effect of muscular rehabilitation on recovery after sagittal split ramus osteotomy.

METHODSTwenty-seven patients undergoing sagittal split ramus osteotomy received passive motion training and muscle endurance training after surgery using the new-developed mouth-opening trainer. The passive motion training began 2-3 days after the removal of inter-maxillary elastraction. The instrument was inserted into the inter-maxillary space for 5 min per time, 3-4 times per day. When the mouth opening reached a range of 20-25 mm, the muscle endurance training initiated. The results of the training were compared with another 20 cases who received the same surgery.

RESULTSThe mouth opening of the patients who received muscular rehabilitation achieved the pre-surgical level 4 weeks after surgery [(34.72 ± 3.75) mm], the bite force reached the pre-surgical level 8 weeks after surgery [(231.08 ± 62.52) N], but in the 20 cases without training the mouth opening [(28.42 ± 4.11) mm] in 4 weeks and the bite force in 8 weeks [(210.90 ± 73.40) N] were lower after surgery than before surgery.

CONCLUSIONSThe systematic and standard muscle function training, accompanied with the use of the new-developed mouth-opening trainer could improve the functional recovery of the patients who receive sagittal split ramus osteotomies.

Bite Force ; Humans ; Mandible ; physiopathology ; Maxilla ; Osteotomy, Sagittal Split Ramus ; rehabilitation ; Range of Motion, Articular

AIM:To investigate the therapeutic effect of Qingqianliu ( QQL) antidiabetic prescription , contai-ning Cyclocarya paliurus, on type 2 diabetic rats.METHODS: Ten rats were randomly chosen as normal control group , and other rats were used to establish diabetic rat models by high-fat diet feeding plus streptocin intraperitoneal injection . Successfully modeling rats were randomly divided into high (300 mg· kg -1· d-1), medium (150 mg· kg-1· d-1) and low (75 mg· kg-1· d-1) doses of QQL treatment groups, and model control group (10 rats in each group).The rats re-ceived daily treatment for 6 weeks.Meanwhile, the therapeutic effect of QQL on these type 2 diabetic rats were evaluated via the body weight , the levels of serum glucose , insulin and glycosylated hemoglobin , the glucose tolerance , the pathologi-cal changes of pancreatic islands , antioxidative indexes and inflammaory factors .RESULTS:Compared with model control group, the body weight, serum insulin, glucose tolerance, serum SOD and serum GSH were increased , the serum glucose, glycosylated hemoglobin , MDA, IL-1βand TNF-αwere decreased , and the pathological changes of pancreatic islands were improved in type 2 diabetic rats with QQL treatment at high and low doses (P<0.05 or P<0.01).CONCLUSION:The QQL reduces the blood glucose , improves the glucose tolerance , and attenuates the damage of pancreatic islands .Its me-chanism may be related to antioxidative and anti-inflammatory effects .

OBJECTIVETo screen and identify differentially expressed proteins between adult female and male worms of Schistosoma japonicum(S.japonicum).

METHODSTwo rabbits infected with the cercaria were perfused with saline in carotid, and approximately two hundred adult female and two hundred male worms of S.japonicum were collected. Approximately 300 microg soluble and hydrophobic proteins of adult female and male worms of S.japonicum were extracted and then the proteins were separated by two-dimensional gel electrophoresis respectively. The analysis using ImageMaster Platinum 2D 5.0 resulted in differentially expressed proteins between adult female and male worms, which were subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry sequencing of tryptic peptides.

RESULTSThere were (255 +/- 10) and (224 +/- 12) spots detected for soluble proteins and (200 +/- 11) and (132 +/- 8) spots for hydrophobic proteins from adult female and male worms respectively. Six differential proteins were identified, five up-regulated proteins in female worms were thioredoxin, putative ferritin-1 heavy chain, chain B in solution structure of the human ubiquitin-conjugating-enzyme-like protein Mms2-Ubiquitin Complex, heat shock protein 10, cytoplasmic fatty acid binding protein variant H; while only one up-regulated proteins in male worms was identified as 48 kDa histamine receptor subunit peptide 4.

CONCLUSIONSeveral differentially expressed proteins between female and male worms of S. japonicum were recognized through screening and identifying differential proteins between female and male worms of S.japonicum.

Animals ; Electrophoresis, Gel, Two-Dimensional ; Female ; Helminth Proteins ; isolation & purification ; Male ; Mass Spectrometry ; Proteome ; isolation & purification ; Rabbits ; Schistosoma japonicum ; chemistry