Objective To study the enzymatic progress of amyloid precursor protein (APP), and construct the recombinant eukaryotic expression plasmid encoding the double mutations of APP and 2 different types of fluorescent protein. Methods The last 300 bases of APP named as C99 containing APP717 mutation was amplified by polymerase chain reaction (PCR) with the template pcDNA3.0-APP.The cyan and yellow fluorescence sequences named as CFP and YFP respectively were obtained by PCR with the template of digestion of the plasmid pcDNA3.0-CFP-CaM-YFP.The 54 bases in the middle of APP named as 54 bp containingSwedish mutation were synthesized. The 4 fragments, CFP, YFP, C99 and 54 bp were inserted into the vector pcDNA3.0.By genetic engineering the recombinant plasmid pcDNA3.0-CFP-54bp-YFP-C99 and pcDNA3.0-CFP-54bp-YFP were constructed and finally identified by enzyme digestion, PCR and sequencing. Then the 2 constructs were transfected into SH-SY5Y cells respectively. The expression of the fusion gene was examined by multi-photon con-focal microscopy and the fluorescence resonance energy transfer (FRET) signal was collected. The amyloid beta (Aβ)deposition was detected by immunocytochemistry. Results (1) DNA sequencing showed that the sequence of the construct was correct.(2)The cyan and yellow fluorescence could be seen by the multi-photon con-focal fluorescence microscopy. The fusion gene could be correctly expressed.(3)FRET was present in the cells expressing CFP-54bp-YFP,but absent in the cells expressing CFP-54bp-YFP-C99.(4)A13 labeled by YFP was detected by multi-photon con-focal microscope and deposited in the cytoplasm, membrane and in the intercellular space.(5)It was shown that by immunocytochemistry, the CFP-54bp-YFP-C99 expressionproduct could generate Aβ. The Aβ deposition was detected in the cell membrane, cytoplasma and intercellular space. Conclusions (1) The fusion protein could generate Aβ peptide by β and γ proteolytic processing.(2)Aβ may be generated in the conrse of APP transportation from cell plasma to cell membrane.(3)C99 is very important for the correct cleavage of APP. Our test data strongly suggest that C99 may function as the signal-like peptide. It may guide and direct the APP to the right location for the cleavage.(4)Before the Aβ deposition is formed outside of the cell, Aβ intracellular accumulation results in the change of cell shape.

Objective To established a diagnostic methods to idenfity Niemann-Pick disease type C (NPC) in China. Methods Two patients aged 5 and 20 years respectively who presented progressive neurologic regression and splenomegaly were subjected to filipin staining of cultured skin fibroblasts and genetic analyses of NPC1 gene. Results Although there were differences in onset ages and clinical presentations, filipin staining of the cultured skin fibroblasts confirmed the clinical diagnosis of NPC, showing an intense punctate pattern of fluorescence concentrating around the nuclei, consistent with the accumulation of unesterified cholesterol in NPC cells. Genetic sequence analysis further verified the results of filipin staining. The case 1 was compound heterozygous for M1142T(3425T＞C),R1186H(3557T＞C)and case 2 for Q88H(264G＞T),469_470insGT.The latter 2 mutations were novel, and the possibility of polymorphisms was not supposed by analyzing 134 DNA samples obtained form normal controls. Conclusions Filipin stainning of the cultured skin fibroblasts is a reliable method to clinically diagnose NPC with a sensitivity. Genetic diagnose should be performed where genetic analysis is allowed.

OBJECTIVE:To screen formulation of Insulin cream and to investigate the effects of insulin cream on wound healing in rabbits.METHODS:The best formulation of insulin cream was selected from 8 kinds of formulations with appearance and stability as index.Rabbit trauma model were induced and divided into matrix group,blank group,Insulin cream groups(high-dose,medium-dose and low-dose).Wound healing rate and healing time were compared among those groups.RESULTS:The fifth formulation possessed good appearance and stability,which included albolene,cetanol,glycerin monostearate,glycerol,etc.Compared with matrix group,the wound healing rates were obviously increased at different time points in Insulin cream groups(P

Objective To analyse the operation technique and therapeutic effect of "inserting" uretero-intestinal anastomosis in orthotopic bladder substitution.Methods Thirty-eight patients undergoing orthotopic bladder substitution operations were followed up,and the way of uretero-intestinal anastomosis in all Datients was the "inserting"uretero-intestinal anastomosis.The therapeutic effect was observed by radiation,cystoscopy,pathologic biopsy and blood test.Results The average follow-up time was(3 1.65±14.14)montll8.and the stricture rate was 4%(3/75),but no vesicoureteric reflux was found.The rate of leakage was 0.Nipples were formed at the site of anastomosis under the view of cystoscope,and among the 7 patients whose nipples were taken to be examined by histology,2 cases were intestinal epithelium which were taken at the base of nipple8.while the others were transitional epithelium which were taken at the top of nipples.The renal function of all patients was normal (Cr 54-135 μmol/L,BUN 3.2-9.4 mmol/L).Conclusion "Inserting"uretem-intestinal anastomosis is an ideal antireflux uretero-intestinal anastomosis method.

Objective To evaluate the efficacy of mitomycin (MMC) intravesical chemotherapy for superficial bladder carcinoma by in vitro chemosensitivity using histoculture drug response assay (HDRA).Methods Forty-one cases of superficial bladder transitional cell carcinoma(TCC) were obtained,including 30 males and 11 females with mean age of 55 years.Of them,10 cases were Ta and 31 were T1 according to TNM stage system (UICC 2002) while 9 cases were G1,22 were G2 and 10 were G3 (WHO1973).All cases had no chemotherapy history before operation and were operated to retain bladder.Tumor specimens were cultured by three-dimensional histoculture.HDRA with im-proved MTT assay was utilized for chemosenstivity test of MMC with 1 g/L concentration and 2 hours exposure.Growth inhibition rate (GI) exceeding 70% was defined as high-sensitive while less than 50% GI was defined as insensitive,others were moderate-sensitive.All cases were performed standard intravesical chemotherapy with MMC 40 mg plus 40 mt saline.Every case was followed up every 3 months.The patients were followed up for 5 years or untill recurrence.Results The MMC chem-osensitivity was different among 41 patients.Thirteen cases were insensitive including 1 of Ta,12 of T1 (P=0.009) and 1 of G1,7 of G2,5 of G3(P=0.101).Total recurrence rate was 39%(16/41) af-ter 3 to 5 years follow-up.There were 1 of Ta,15 of T1 (P=0.059) and 10 of G2 6 of G3 (P=0.016).Insensitive group recurrence rate was 77% (10/13) while sensitive group was 21% (6/28,P= 0.004).Patients in sensitive group showed a longer median time(49.2 months) than patients in insen-sitive group (16.5 months,P<0.001) according to Kaplan-Meier analysis with Log-rank test.The MMC chemosensitivity was independent prognostic factor examed by Cox regression analysis (P= 0.008).There was a 78% correlation rate of chemosensitivity by HDRA to clinical effect of MMC in-travesical chemotherapy.Conclusion HDRA could evaluate MMC intravesical chemotherapy for su-perficial bladder TCC,improve therapeutic effect and lower tumor recurrence rate.

Objective To evaluate the internal reliability of Psychosocial Function Evaluation for mental disabled people in day care unit. Methods 16 participants were evaluated twice by the same rater (social worker) in the unit at the Permanent stage and the Steering stage. Results Cronbach's α was greater than 0.7 in the both evaluation. The Spearman correlation coefficient between each dimension and total score were between 0.502 and 0.869. Conclusion The internal reliability of Psychosocial Function Evaluation is satisfactory for mental disabilities.

Cysticercosis is caused by the metacestode form of Taenia solium-Cysticercus cellulosae and it causes great economic losses and threatens the people's health. There are some problems on how to control cysticercosis, in order to resolve the key problem that the native antigen to diagnose and prevent cysticercosis is very limited and is not satisfied, Pichia pastoris Expression System was used to express recombinant P2 protein. The interested P2 gene was got by digesting the pGEM - P2 vector using restriction endonuclease, then it was inserted into the secretory pPIC9K Pichia pastoris expression vector and transformed into E. coli. Positive recombinant plasmids were selected sequenced and named pPIC9K-P2 and it was linearized by Sal I and Bgl II, then the linear DNA transfored into Pichia pastoris GS115 by electroporation. The recombinant expression vector pPIC9K - P2 integrated into GS115 via homologous recombination between the transforming DNA and regions of homology within the genome. The transformants were screened for multicopy recombinants using G418 and were distinguished for Mut phenotypes by MD and MM. Two different phenotypes were generated-HIS+ MUT+ (Methanol utilization plus) and HIS+ MUT(S) (Methanol utilization slow). PCR analysis of the multicopy recombinants indicated that the P2 gene was integrated within the genome of pichia Pastoris. The multicopy recombinants were named GS115/pPIC9K - P2HIS+ MUT+ and GS115/pPIC9K-P2HIS+ MUT(S), both HIS+ MUT+ and HIS+ MUT(S) were induced with methanol. The results of SDS-PAGE and Western blot demonstrated that the culture supernatant of the induced Pichia pastoris contained P2 protein which was accumulated up to 33 % of total proteins in the culture supernant and its molecular weight is 12.6kD. The results of the clinical study indicated that the expression P2 protein could be used to diagnose human cysticercosis and swine cysticercosis as diagnosis antigen.
Animals ; Blotting, Western ; Cysticercosis ; diagnosis ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Genome, Fungal ; genetics ; Helminth Proteins ; genetics ; metabolism ; Humans ; Phosphoproteins ; genetics ; metabolism ; Pichia ; Polymerase Chain Reaction ; Recombinant Proteins ; genetics ; metabolism ; Swine ; Taenia ; metabolism

OBJECTIVETo optimize the adsorption condition of cation-exchange chromatographic media Streamline SP for separation and purification of anti-HBsAg Fab fragment from E. coli.

METHODSThe adsorption of the target protein for separation and purification by the cation-exchange chromatographic media Streamline SP was tested using test tube method in balanced buffer solution with different pH values and ion concentrations. The adsorption effect was then verified by cation-exchange chromatography using 1-ml Streamline SP prepacked column and 28-ml Streamline SP self-assembly column.

RESULTSAccording to the experiment results of test tube method, the loading buffer with pH of 4.4 and ionic concentration of 100 to 600 mmol/L could achieve optimal target protein adsorption effect by cation-exchange chromatographic media Streamline SP, as verified by cation-exchange chromatography with 1-ml SP prepacked column and 28-ml Streamline SP self-assembly column.

CONCLUSIONThe optimal condition of cation-exchange chromatography selected by test tube method can be applied for separation and purification of anti-HBsAg Fab fragment from E. coli.

Adsorption ; Cation Exchange Resins ; Chromatography, Ion Exchange ; methods ; Escherichia coli ; genetics ; metabolism ; Hepatitis B Antibodies ; isolation & purification ; metabolism ; Hepatitis B Surface Antigens ; immunology ; Humans ; Immunoglobulin Fab Fragments ; isolation & purification ; metabolism

OBJECTIVETo investigate the clinical significance of erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) in the treatment of spinal tuberculosis.

METHODSSixty-seven patients (41 males and 26 females, ranging in age from 23 to 61 years) with active spinal tuberculosis in our hospital (from Mar. 2004 to Mar. 2007) were included in this study. The tuberculosis focus were located either in cervical spine, thoracic spine or in lumbar spine. After 4 to 6 weeks anti-tuberculosis chemotherapy, all the patients underwent one-stage operation (focus debridment) and auto-bone graft combined with internal fixation. Blood test for ESR and CRP were carried out at different times before and after operation.

RESULTSThe average ESR was (79.4 +/- 35.6) mm/h, and the average CRP was (44.3 +/- 17.5) mg/L before chemotherapy, indicating active tuberculosis focus. After 4 to 6 weeks chemotherapy, the average ESR was (45.3 +/- 21.0) mm/h,and the average CRP was (26.7 +/- 11.8) mg/L, the differences were statistically (P < 0.05), and the clinical symptoms of spinal tuberculosis relieved in all patients. Four weeks after operation, the average ESR dropped to (42.8 +/- 16.5)mm/h, the average CRP dropped to (23.8 +/- 10.0) mg/L statistically (P < 0.05). Eight weeks after operation, the average value of ESR and CRP were at normal level in 47 cases, indicating inactive tuberculosis focus. Focus healing was achieved in 65 patients after short-term chemotherapy.

CONCLUSIONThe level of ESR and CRP are high in active spinal tuberculosis and low when focus controlled. ESR and CRP are reliable parameters in evaluation the treatment and prognosis of spinal tuberculosis.

Adult ; Blood Sedimentation ; C-Reactive Protein ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Perioperative Care ; Tuberculosis, Spinal ; blood ; diagnosis ; drug therapy ; surgery ; Young Adult

OBJECTIVETo investigate the effect of Ganoderma lucidum polysaccharides (GLPs) on hemodynamic and antioxidation in the T2DM rats.

METHODSD rats were fed high-fat diet for 4 weeks and then were injected STZ (30 mg x kg(-1)) to induce the type 2 diabetes mellitus (T2DM). Once the T2DM models were set successfully, rats were randomized into six groups: normal group (NG), group of diabetes mellitus (DMG), groups of low dosage (GLPs-LG), middle dosage (GLPs-MG), high dosage (GLPs-HG) and berberine (BerG). They received GLPs with different dosages (200, 400, 800 mg x kg(-1)) and berberine (30 mg x kg(-1)) continually for 16 weeks. At 16th weekend, the following indices of rats were measured respectively: blood glucose, hemodynamic including LVSP, LVEDP, dp/dt(max) and -dp/dt(max) and the contents of NO, SOD, MDA, GSH-Px, CAT in cardiac tissue. Besides, myocardial ultrastructure was observed by electron microscope.

RESULTBoth the middle dosage and the high dosage of GLPs could low blood glucose effectively, and they could reduce LVEP but increase -dp/dt(max). Meanwhile, they could activate GSH-Px, CAT, SOD, NO, but reduce MDA in cardiac tissue and improve the myocardial ultrastructure. Compared to the DM group, the middle dosage, high dosage of GLPs and berberine showed significant improvement. Compared to the berberine group, the middle dosage showed the same effect, but the high dosage was more effective than berberine.

CONCLUSIONThere is a confirmed action of GLPs in improving the hemodynamic and antioxidation in cardiac tissue of T2DM rats.

Animals ; Antioxidants ; metabolism ; Catalase ; metabolism ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; Diabetes Mellitus, Type 2 ; drug therapy ; metabolism ; Female ; Glutathione ; metabolism ; Hemodynamics ; drug effects ; Hypoglycemic Agents ; chemistry ; therapeutic use ; Male ; Malondialdehyde ; metabolism ; Microscopy, Electron, Transmission ; Myocardium ; metabolism ; ultrastructure ; Nitric Oxide ; metabolism ; Polysaccharides ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Reishi ; chemistry ; Superoxide Dismutase ; metabolism