In order to study the structure-function relationship in the protein which is incorporated with p-nitro-L-phenylalanine,the method of MD(Molecular Dynamics) simulation was established and successfully used in the analysis of protein which contains p-nitro-L-phenylalanine.The force field of CHARMM can only stimulate protein with natural amino acid in NAMD.Compared with phenylalanine,p-nitro-L-phenylalanine just has one more group of nitro.If the parameter of group of nitro was defined,the protein containing p-nitro-L-phenylalanine can be simulated.CGenFF-paramchem was used to calculate the energy and topological structure of p-nitro-L-phenylalanine' s new bonds (r),angles (θ),dihendrals (φ) and improper angle (ψ).And then the new defined parameter and topology information was input into the related parameter files and topology files in CHARMM.On the basis of correct parameter,NAMD can successfully simulate the modified BAFF(B lymphocyte stimulator) which contains p-nitro-L-phenylalanine.The changes in structure indicated that there might be new B cell epitopes.The temperature distribution of each frame in the process of dynamics stimulation was in accord with normal distribution,which proved the defined force field parameters was feasible.The RMSD of whole protein solution systemis 2.5.Calculate each resides' RMSF in BAFF,the RMSF of p-nitro-L-phenylalanine's residue is 3.7,which is obviously higher than that of the other residues in β-pleated sheet,and close to the loop rings,indicate that there might be variation in the area of p-nitro-L-phenylalanine residue and might produce new comformational epitopes.The results of MD stimulation will guide the immunogenicity experiments of p-nitro-L-phenylalanine modified proteins.

Purpose The research was conducted to evaluate the stability of N-terminal site-specific PEGylated uricase. Methods The enzyme activity is used as an index to evaluate the thermal stability (at 4-80 ℃) , the pH stability, the ability of avoiding the trypsin digestion and half-life in vivo of PEG-uricase, then it is compared with uricase. Results PEGylated uricase is thermally more stable than uricase between 4 ℃ and 60 ℃, but at 70 ℃, the enzyme activity of both PEG-uricase and uricase decreases sharply. In the test of pH stability, the curve of uricase shows an obvious decrease of enzyme activity at 5 .2-6.0 and 9.2-10.0. The behavior of PEG-uricase indicates that the destabilizing was prevented effectively by PEGylation. The test of anti-trypsin digestion suggests that PEG-uricase retains 70% of its enzyme activity,but uricase only 20% ,200 minutes after being reserved in trypsin solution. Stability in vivo indicates that the half-life of PEG-uricase is 1 530 min and uricase, only 45 min. Conclusion PEGylated uricase has improved thermal stability, the pH stability, ability of protecting from trypsin digestion and stability in vivo.

Autoimmune Diseases ; diagnosis ; immunology ; pathology ; surgery ; Humans ; Immunoglobulin G ; blood ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Pancreatectomy ; Pancreatitis ; diagnosis ; immunology ; pathology ; surgery

Ubiquitous chromatin opening element ( UCOE ) , composed of the promoters of human housekeeping genes, prevents transgene from silencing and produces consistent, stable and high-level gene expression irrespec-tive of the chromosomal integration site. The research studied the influence of different UCOE element parts on antibody expression in CHO cells. UCOE 1. 5 kb from chromobox homolog 3 ( CBX3 ) , UCOE 2. 5 kb from the heterogeneous ribonucleoprotein A2/B1 ( HNRPA2 B1 ) and the whole UCOE 4. 0 kb were inserted into the anti-body light and heavy chain vectors, respectively, and transfected into CHO cells using antibiotics-Zeocin and Blasticidin for pressure selection. Four groups of monoclonal cells were harvested and antibody expression of each group was detected. The monoclonal cells with UCOE 1. 5 kb and UCOE 2. 5 kb increased 1. 5 to 2-fold in the level of antibody expression, wheareas, monoclonal cells with UCOE 4. 0 kb increased 3 to 4-fold. The enhance-ment of two housekeeping promoter genes on antibody expression could stack up.

To optimize Chinese hamster ovary (CHO)expression system and establish a process of screening CHO cell lines with high productivity;neomycin-phosphotransferase (NPT)expressed by the resistance marker gene on the expression vector was mutated with amino acid D at 261 changed to G.After selection by culturing with G418;the survival rate of CHO cells bearing mutant-NPT was significantly lower than that of the cells bear-ing wide type NPT.An enhanced green fluorescent protein (EGFP)was genetically linked to the N terminus of the IgG1 Fc fragment part to generate an EGFP-Fc fusion protein regarded as a report gene;which verified that the resistance of mutant-NPT to G418 was weakened.By comparing fluorescence assay of EGFP intensity in stable transfections after selection with the same concentration of G418 for 3 weeks;mutant-selected pools expressed more exogenous protein than the WT-selected pools.Therefore;the ratio of high producers in a transfected cell population greatly increased.

Objective To explore the antibacterial activity of garlic solution against Campylobacter jejuni,and to provide theoriti-cal basis for further utilization of garlic.Methods Mashed garlic and extracted garlic solution.Antimicrobial susceptibility was de-termined by usig K-B disk diffusion test and broth dilution method.Results The minimal inhibitory concentration(MIC)of garlic solution against campylobacter jejuni was 40%.It showed positive correlation between bacteriostatic effect and concentration of gar-lic solution.Conclusion Garlic solution has strong bacteriostasis function to campylobacter jejuni.

Objective To explore the rationality of pancreaticojejunostomy and double purse embedded pancreaticogastrostomy in pancreaticoduodenectomy through comparing the short-term effects between pancreaticojejunostomy and double purse embedded pancreaticogastrostomy in pancreaticoduodenectomy.Methods A retrospective review was performed for 38 patients who were underwent pancreaticoduodenectomy at the second affiliated hospital of Kunming Medical University from January 2010 to December 2012.Results All 38 pancreaticoduodenectomy were performed successfully.The pancreatic fistula (0％),operationtime [(327.50±32.56) min],postoperative hospital days [(10.31 ±2.44) d] were found to be significantly lower in double purse embedded pancreaticogastrostomy group than those of pancreaticojejunostomy group [3/12,(425.83 ± 33.77) min,(14.33 ± 4.14) d],which has statistical significance(P＜0.05).Although the incidence of bile leakage,postoperative abdominal bleeding,delayed gastric emptying,infection of incision,infection of lung,intraoperative amount of bleeding are also lower than that of pancreaticojejunostomy group,which has no statistical significance.Conclusions Double purse embedded pancreaticogastrostomy is a digestive tract reconstruction which is safer and more reasonable than pancreaticojejunostomy.The former one is operated simply and the operation time is shorter.There are great advantages in the prevention of complications such as pancreatic fistula after pancreaticoduodenectomy.

Anti-Bacterial Agents ; therapeutic use ; Antigens, CD20 ; metabolism ; CD3 Complex ; metabolism ; CD4 Antigens ; metabolism ; Fever ; drug therapy ; virology ; Herpesvirus 6, Human ; isolation & purification ; Humans ; Lymph Nodes ; pathology ; virology ; Lymphadenitis ; drug therapy ; metabolism ; pathology ; virology ; Male ; Middle Aged ; Roseolovirus Infections ; drug therapy ; metabolism ; virology

In order to identify Peucedani Radix, Peucedani Decursivi Radix and their adulterants, the internal transcribed spacer 2 (ITS2) regions of Peucedani Radix, Peucedani Decursivi Radix and their adulterants were amplified and bidirectionally sequenced based on the Principles for Molecular Identification of Traditional Chinese Materia Medica Using DNA Barcoding, which has been promulgated by Chinese Pharmacopoeia Commission. Sequences were analyzed and assembled by Codon Code Aligner V3. 7.1. The relevant data were analyzed by MEGA 5. 0. Species identification analyses were performed by using the nearest distance methods and neighbor-joining (NJ) methods. The result showed that the ITS2 sequence lengths of Peucedani Radix were 229-230 bp and the average intra-specific genetic distances were 0.005. The ITS2 sequence lengths of Peucedani Decursivi Radix were 227 bp and the sequences contained no variation site. The average inter-specific K2P genetic distance of Peucedani Radix, Peucedani Decursivi Radix and their adulterants species were 0.044 and 0.065 respectively. The minimum inter-specific divergence is larger than the maximum intra-specific divergence of Peucedani Decursivi Radix. The nearest distance methods and NJ trees results indicated that Peucedani Radix, Peucedani Decursivi Radix and their adulterants species could be identification clearly. The ITS2 regions can stably and accurately distinguish Peucedani Radix, Peucedani Decursivi Radix and their adulterants.
Apiaceae ; classification ; genetics ; DNA Barcoding, Taxonomic ; methods ; DNA, Ribosomal Spacer ; Drug Contamination

In order to verify whether p-nitrophenylalanine-containing BAFF vaccine can be used as a candidate molecule for the treatment of autoimmune diseases with BAFF over-expression,a soluble mutant of B cell activating factor belonging to the TNF Family (smBAFF) and its pNO2Phe mutant(pNO2Phe65smBAFF),which site specific incorporated pNO2Phe at position 65 of smBAFF,were expressed and purified.In order to evaluate the feasibility of using pNO2Phe65 smBAFF to treat BAFF-over-expressed autoimmune diseases,we investigate its Lymphocyte-stimulating capacity,immunogenicity and inhibitory effect of serum on biological activity of natural BAFF.The pharmacological activity of pNO2Phe65 smBAFF was evaluated using a cGVHD(graft-versus-host disease) induced SLE mouse model.Results indicated that pNO2Phe65 smBAFF,could bind to mouse lymphocytes but could not promote the proliferation of mouse lymphocytes.Moreover,the incorporation of pNO2Phe significantly increased the immunogenicity and induced cross-antibody,which can inhibit the biological activity of natural BAFF.In cGVHD induced SLE mouse model,pNO2Phe65 smBAFF can significantly reduce the symptoms of the disease and play a therapeutic role.Therefore,pNO2Phe65 smBAFF can be used as a candidate molecule for the treatment of autoimmune diseases with BAFF over-expression.