Acupuncture Analgesia ; Animals ; Central Nervous System ; metabolism ; Electroacupuncture ; Nitric Oxide ; metabolism ; Pain Threshold ; physiology ; Rats ; Rats, Wistar

Objective To investigate the efficacy of combined monitoring of motor evoked potentials with transcranial electrical stimulation (TES-MEP),somatosensory evoked potentials (SEP) and spontaneous electromyo-graphy (s-EMG) in tuberculosis surgery involving the thoracic,lumbar and sacral vertebrae.Methods Twenty-seven patients with tuberculosis of the thoracic vertebrae (T2-L2) received intra-operative SEP and TES-MEP monito-ring.Combined SEP,TES-MEP and spontaneous EMG monitoring were employed in 11 patients with tuberculosis of the lumbar or/and sacral vertebrae (L3-S1).SEP and TES-MEP were used to precisely observe the status of the sen-sory and motor pathways; s-EMG responses were used to more accurately localize nerve root irritation.ResuIts (1) SEP monitoring was successful in all of the operations.TES-MEPs were successfully monitored in 35 of them (92.1％).Combined motor and sensory monitoring was successfully achieved in 35 cases (92.1％).Abnormal SEPs were observed in 3 cases (7.9％),while abnormal MEPs were observed in 11 cases (28.9％).Abnormality in both the SEP and TES-MEP occurred in 2 cases (5.3％).There were 9 cases (23.7％) where the SEPs were nor-mal and the TES-MEPs were abnormal.In only 1 case (2.6％) was the SEP normal and the MEP abnormal.The false negative rate was 0％ with combined SEP and TES-MEP monitoring,while the false positive rate was 5.3％.There were 2 cases complicated by post-operative neurological deficits.(2) Spontaneous EMG monitoring can accu-rately determine the functioning of lumbar nerve roots during lumbar or lumbosacral tuberculosis surgery.Among 5 cases where EMG responses were observed,4 cases occurred during the spinal canal and nerve root decompression,1 case occurred in the orthopedic reset phase.Conclusions (1) During tuberculosis surgery involving thoracic,lumbar or sacral vertebrae,combined monitoring of SEPs and TES-MEPs can reflect the physiological and pathological condition of the spinal cord after ruling out interfering factors.This can improve monitoring and help assure the safety of lumbar surgery.(2) Intra-operative s-EMG monitoring can accurately reveal nerve root function in real time,help-ing to avert nerve root injury in lumbar and lumbosacral tuberculosis surgery.

A fusion protein of glucagons-like peptide-1 and human serum albumin (GLP-1/HSA) was expressed and secreted into the fermentation broth with recombinant Pichia pastoris. The productivity of expressed GLP-1/HSA could reach 63.6mg/L in 10L fermentor. After concentrated with hollow-fiber ultrafiltration membrane, GLP-1/HSA was purified from fermentation broth by hydrophobic chromatography, negative ion exchange chromatography and gel filtration chromatography in turn. The HPLC analysis showed that the purified GLP-1/HSA had an overall purity of 95.8%. Furthermore, the analysis of in vivo activity indicated that GLP-1/HSA had the bioactivity of native GLP-1, and could significantly reduce blood glucose level 4h after intraperitoneal administration. It was concluded that a great deal of GLP-1/HSA with higher purity could be harvested by Pichia pastoris expression system and the established purification methods. Preliminary studies show a new potential for developing the long-acting GLP-1 analogs for clinical applications.

Pseudomonas sp. TS1138 isolated from soil samples was able to form L-cysteine from DL-2-Amino-△2-Thia-zoline-4-Carboxylic Acid (DL-ATC) after cultured 16 hours . The optimum carbon and nitrogen soruces of strain growth and enzyme formation are glucose and urea. This enzyme was induced by DL-ATC. The product was identified to be L-Cysteine based on thin layer chromatography, optical rotation and HPLC studies.

The L-ATC hydrolase and L-SCC amidohydrolase which convert L-ATC to L-cysteine in Pseudomonas sp.TS-1138 are purified about 83.9 and 90.3 fold by salting-out method, Sephadex G-75 gel chromatography, DEAE-cellulose 52 ion exchange and Sephadex G-100 gel chromatography, etc. The purified enzyemes are both demonstrated by SDS-PAGE to be a homogeneous protein. Their molecular weight are about 37.5kD and42.8kDa respectively. The optimum reaction temperature are both 35℃, and the optimum pH are 7.0 and 8.0 respectively. The Km of the two enzymes are 0.67 mmol/L and 0.15 mmol/L, and the Vmax are 0.39?10 -3mmol/L?min and 0.42?10 -3mmol/L?min respectively.

The L-cysteine desulfhydrase gene (cd) of Pseudomonas sp.TS1138 was amplified by PCR,and the amplified gene was recombined in the cloning vector pBluescript SKII.The 1.2kb DNA fragment containing cd was sequenced,and its homology with other desulfhydrases was blast; then the cd was cloned into the expression vector pET-21a(+), and afterward expressed by IPTG inducement.The expression protein was purified by Ni-NTA His-Bind Resin.Then the expression protein was identified by the method of activity staining of desulfhydrase, and the characterization of L-cysteine desulfhydrase and the critical role it played in the L-cysteine biosynthetic pathway were discussed.

A phage display single-chain variable fragment (scFv) library against Bursaphelenchus xylophilus cellulase (BXC) was constructed and used to screen the specific antibodies binding to BXC. The total RNA was extracted from fresh spleens of BALB/C mice immunized with BXC. Gene fragments encoding VH and VL were amplified by RT-PCR and assembled into a single chain by overlapping PCR with a linker DNA encoding the peptide (Gly4Ser)3. The recombinant fragments were cloned into the phagemids (pCANTABSE) and electroporated into E. coli TG1. The recombinant phagemids were rescued by reinfection of helper phage M13K07. The repertoire of the phage display antibody was about 5 x 10(4). The specific antibodies against BXC were obtained after five rounds of affinity selection. The positive phage clone was used to infect E. coli HB2151. SDS-PAGE and western blot analysis showed that the soluble scFv antibodies expressed bound specifically to BXC. The studies laid foundation for quarantine and pathological study of Bursaphelenchus xylophilu.
Animals ; Cellulase ; genetics ; immunology ; Cloning, Molecular ; Electroporation ; Escherichia coli ; genetics ; metabolism ; Female ; Helminth Proteins ; genetics ; immunology ; Immunoglobulin Variable Region ; genetics ; Mice ; Mice, Inbred BALB C ; Nematoda ; enzymology ; Peptide Library ; Pinus ; parasitology ; Plant Diseases ; parasitology ; Recombinant Proteins ; biosynthesis ; genetics

The clinical application of Chinese patent medicines has suffered sever problems and required guidelines for clinical practices. Currently, the expert consensus method is more suitable for formulating clinical practice guidelines of Chinese patent medicines than the evidence-based method. However, there remain problems in the application of the expert consensus method. This study proposed a derivative expert consensus method--a method for formulating clinical practice guidelines of common Chinese patent medicines based on clinical practices, and introduced the method in terms of research thought, methodology and implementation procedure.
Evidence-Based Medicine ; standards ; Humans ; Nonprescription Drugs ; standards

An about 700 bp DNA fragment was amplified from genome DNA of S. aureus TSTw by PCR. This fragment was cloned into pGEM-7Zf(+) and the recombinant plasmid was transformed into E. coli DH5 alpha. The sequencing result of the recombinant plasmid demonstrated that it contains seb gene with 717 bp (without signal encoding region of 81 bp) which has the same nucleotide sequence as described in literature. The seb gene was cloned into expression vector 7ZTS and was transformed into E. coli JM109 (DE3). The expression level of SEB was as high as 33.3% of the cell total proteins.
Cloning, Molecular ; Enterotoxins ; biosynthesis ; genetics ; Escherichia coli ; genetics ; Genetic Engineering ; Recombinant Proteins ; biosynthesis

The rare codons of a fragment in staphylococcal enterotoxin A gene were turned into the most high usage frequency codons in E. coli by overlap PCR technique. Genes of sea and seam were cloned into 7ZTS expression vector and transformed into JM109(DE3), respectively. The result shows that expression level of sea gene was very low, but the expression level of seam was as high as 15% of total cell proteins. The expression product shows activity of antitumor in vivo.
Animals ; Base Sequence ; Codon ; genetics ; Electrophoresis, Polyacrylamide Gel ; Enterotoxins ; genetics ; metabolism ; pharmacology ; Escherichia coli ; genetics ; Gene Expression Regulation, Bacterial ; Male ; Mice ; Molecular Sequence Data ; Neoplasms, Experimental ; drug therapy ; pathology ; Point Mutation ; Recombinant Proteins ; genetics ; metabolism ; pharmacology