Acupuncture Points ; Diagnosis, Differential ; Humans ; Medicine, Chinese Traditional

Objective:To express human interferon-?2b gene and to explore the feasibility of expressing human gene in plant cells.Methods:The hIFN-?2b coding sequence was amplified by PCR with specific primers and plasmid pBV889 was used as a template,subcloned into middle vector pMD18-T and binary vector pBI121 to obtain plant expression vector pBIFN. The pBIFN was transformed into Agrobacterium tumefaciens strain LBA4404. Then hIFN-?2b gene was introduced into Ginseng callus cells via Agrobacterium-mediated transformation. The positive cells were screened by G418. The transgenic Ginseng calli were confirmed by PCR,RT-PCR,Western blot and WISH/VSV system.Results:Stable integration of the hIFN-?2b gene in the Ginseng callus cells′ genome was confirmed by PCR analysis. RT-PCR analysis showed that there were transcription products. Western blot implied that the given protein was hIFN-?2b. WISH/VSV system assay showed that the expressed hIFN-?2b possessed relatively lower bioactivity.Conclusion:HIFN-?2b has been expressed in transgenic Ginseng calli, which facilitates further investigation of improving the curative effect of orally administered hIFN-?2b.

Objective: To construct subtracted cDNA libraries of stomach tumors and normal stomach tissue using suppression subtractive hybridization(SSH).Methods: cDNA Library subtraction was performed using the protocol described in the Clontech PCR-Select cDNA Subtraction Kit. cDNA was synthesized from 2 μg of poly A+RNA from the tumor and normal tissues using AMV reverse transcriptase. The tester and driver cDNAs were digested with RsaⅠ, a four-base-cutting restriction enzyme that yields blunt ends. The tester cDNA was then subdivided into two portions, and each was ligated with different cDNA adaptor. Two hybridizations were performed. In the first, an excess of driver was added to each sample of tester. Hybridization kinetics led to equalization and enrichment of differentially expressed sequences. During the second hybridization, the two primary hybridization samples were mixed together without denaturing and thus the templates were generated from differentially expressed sequences for PCR amplification. Using suppression PCR, only differentially expressed sequences were amplified exponentially and after second PCR amplification the background was reduced and differentially expressed sequences were further enriched. The cDNAs were then directly inserted into a T/A cloning vector to generate a stomach tumor-specific subtracted cDNA library. Results: The amplified library contained 800 positive clones. Plasmid inserts were PCR amplified and showed 250－700 bp inserts. Conclusions: The successfully constructed subtracted cDNA library of gastrocarcinoma and normal tissue enables us to compare two populations of mRNA and obtain clones of genes that expressed in one population but not in the other.The characterization of these genes will allow them to be exploited for their diagnostic and therapeutic potentials.

OBJECTIVETo study the blood enriching effects of Paeoniae Radix Rubra and Paeoniae Radix Alba, paeoniflorin and albiflorin on mouse model of blood deficiency caused by γ-ray radiation.

METHODBuild mouse model of blood deficiency induced by γ-ray radiation. Paeoniae Radix Rubra and Paeoniae Radix Alba were given during modeling. The amount of WBC was detected af- ter the treatment. Based on the result of WBC and paeoniflorin content, albiflorin content in Paeoniae Radix Rubra and Paeoniae Radix Alba, the same model and the same method were used to comparatively study the effect of blood enriching of paeoniflorin and albiflorin.

RESULTOn the 7th day, the amount of WBC in model mice treated with 2 g x kg(-1) Paeoniae Radix Alba and 2 g x kg(-1) Paeoniae Radix Rubra significantly increased compared with that of model group (P < 0.05). In another experiment with the same model, the amount of WBC in model mice treated with 120 mg x kg(-1) paeoflorin and 120 mg x kg(-1) albiflorin significantly increased (P < 0.05) compared with that of model group on the 7th day. On the 10th day, the amount of WBC in rats treated with 120 mg x kg(-1) paeoflorin increased significantly (P < 0.05) compared with that of model group. Compared with the same dose of paeoniflorin, the amount of WBC in mice treated with albiflorin had no significant difference.

CONCLUSIONAll Paeoniae Radix Alba, Paeoniae Radix Rubra, paeoniflorin and al- biflorin can raise the amount of WBC and have the effect of enriching blood induced by radiation, while paeoniflorin and albiflorin have a similar result in this model. The result indicated that both paeoniflorin and albiflorin are effective constituents in Paeoniae Radix Alba, and paeoniflorin work as the common effective constituent in both Paeoniae Radix Rubra and Paeoniae Radix Alba.

Animals ; Bridged-Ring Compounds ; pharmacology ; Gamma Rays ; adverse effects ; Glucosides ; pharmacology ; Leukocyte Count ; Leukocytes ; cytology ; drug effects ; radiation effects ; Male ; Mice ; Monoterpenes ; pharmacology ; Rats

In order to obtain active recombinant PNGase F in Escherichia coli, a prokaryotic expression vector pET28a/PNGase F was constructed. Amplification of PNGase F was obtained using PCR technique employing suitable primers designed according to the PNGase F gene sequence from Flavobacterium nmeningosepticum. The expression of PNGase F gene in LB medium or M9 medium led to the formation of inclusion body and soluble protein, respectively. The refolding of the denatured inclusion body was successful by gradual dilution. Further purification of the refolded protein and soluble crude extract from M9 medium with Ni2+ -NTA argarose resulted a 90% purified PNGase F. The purified protein catalyzed the complete and intact cleavage of N-linked oligosaccharides from various glycoproteins. The efficiency of this cleavage was affected by the substrate status in the reaction system. In summary, we have developed an enzyme production system where PNGase F was over-expressed in recombinant E. coli. This system can provide more than 15 mg/L purified active PNGase F. This purified active PNGase F can be used as tools in analyzing the oligosaccharide structure.
Bacterial Proteins ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Flavobacterium ; enzymology ; genetics ; Glycosylation ; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism

AIM: To investigate the affected proteins by tumor necrosis factor (TNF)-? in endothelial cells, and further explore the potential molecular mechanism of TNF-? on endothelial cells. METHODS: Nitric oxide (NO) production in the cultured human umbilical vein endothelial cells (HUVECs) was measured by a NO assay kit. Proteomic alterations were analyzed using two-dimensional electrophoresis, and peptide mass fingerprinting with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. RESULTS: NO production in HUVECs decreased significantly after TNF-? treatement. Proteomics analysis showed 21 protein spots were changed including 9 spots that were increased and 11 spots that were decreased after TNF-? stimulation, and 1 spot was only detected in TNF-? activated cell gels. CONCLUSIONS: The decreased expression of ecNOS by TNF-? might result in decrease in NO production. Up-regulated MAP/ERK kinase 3 expression might imply that TNF-? activates the expression of adhesion molecules. Cytoskeletal protein actin is also involved in TNF-? injuried HUVECs. Proteomic analysis can find some clues for identifying new potential target of TNF-?. [

Objective To investigate the clinical value for treatment of diabetic foot with PTA and PTA combined cinepazide maleate.Methods In 24 cases of diabetic associated vascular disease of lower limb,12 cases were treated with PTA and other 12 cases were treated with PTA combined einepazide maleate,We analysed and compared clinical effects before and after the procedure,together with 3 months follow up.Results In patients treated with PTA,the clinical symptom scores of posttreatment and follow-up decreased;ABI and TcPO_2 increased significantly.The clinical symptom score and ABI of follow-up remained,stable,but TcPO_2 decreased significantly.Control angiography showed improvement in degree of vascular stenosis and peripheral staining of 11 patients after treatment.The vascular patency remained in 12 patients and the peripheral staining decreased in 7 patients on follow-up.In patients treated with VIA combined cinepazide maleate,the clinical symptom score,ABI and TcPO_2 after treatment and on follow-up showed no signifcant changes compared with those in patients treated by PTA.F,Control angiography showed that the degree of vascular stenosis and peripheral staining were improved in 12 patients after treatment.The vascular pateney was maintained and peripheral staining was improved on follow-up.Before and after treatment,there were no significant differences in clinical symptom score.ABI and TcPO_2 between patients treated with PTA and PTA combined cinepazide maleate,however,there were significant differences in clinical symptom score and TcPO_2 on follow-up.Conclusion PTA can significantly improve clinical symptom of diabetic foot and the application of cinepazide maleate is a benefitial and necessary supplement.PTA combined cinepazide maleate can be taken as one of the conventional treatment plans for diabetic foot.(J Intervent Radiol,2007,16:811-815)

Permeabilized cells containing trehalose synthase from Meiothermus sp.CBS-01 was immobilized by the integration of entrapment in alginate beads with cross-linking and electrostatic self-assembly coating technique.Coating on alginate beads with diazo-resin and poly(styrene sulfonate) can protect the beads from degradation in phosphate buffer,whereas cross-linking with carbodiimide improves the thermostability of trehalose synthase entrapped in alginate beads.By co-immobilization of permeabilized cells using entrapment-cross-linking- coating method,the enzyme recovery was 32%,and the optimum temperature of the enzyme was still 60℃,but the optimum pH was shifted from 6.5 to 7.In batch manner,a maximum conversion yield of 60% trehalose from maltose could be reached by the immobilized cells.Within 4 times repeated use of the immobilized cells,and each time was operated at 50℃ for 24h,more than 50% conversion yield of trehalose could be maintained with less than 20% loss of the enzyme activity.

Objective To evaluate the results of NiTi shape memory alloy four-corner arthrodesis concentrator (NTMA-FCAC) for carpal collapse. Methods We reviewed retrospectively 13 patients who underwent scaphoid excision with four-corner arthrodesis using NTMA-FCAC for carpal collapse from August 2006 to June 2009. There were eight males and five females, with an average age of 38 years (range, 23-61years). The cause of carpal collapse was SNAC in 7 cases, perilunate dislocations in five and SLAC in one.The injury mechanisms included traffic accidents (5 cases), falling from a height (4 cases), crashes (3 cases)and sprain (1 case). Objective measurements included grip strength and range of the wrist. Radiographs were performed in all patients. A visual analogue scale (VAS) was used to assess wrist pain. The results were evaluated according to the Krimmer wrist scores. Results The mean follow-up time was 26.5 months (range,6-36 months). Clinical evaluation yielded the mean grip strength of (32.49±6.21) kg (80.8% of opposite side).The mean range of the wrist reached over 53% of the healthy side. Non-union and wound infection were not seen. The mean VAS scores had improved from 4.46±1.27 preoperatively to 1.31 ±0.95 postoperatively. The mean pain scores under stress had improved from 7.00±1.41 preoperatively to 2.62±1.26 postoperatively.There were remarkable differences between them. The mean Krimmer wrist score was 79. Conclusion Four-corner arthrodesis using NTMA-FCAC is an effective method for carpal collapse, preserving a majority of wrist function.

Objective To investigate the relationship between killer cell immunoglobulin-like receptors(KIR)and HLA by distribution of KIR gene in leukemia patients and their siblings who have same HLA-A/B/DR typing.Methods KIR genotypes were detected by PCR-SSP on 78 patients and their siblings who have same HLA-A/B/DR typing.Results There were 48.72%in 78 patients who had same KIR genotypes with their siblings while the 44.87%patients had different KIR genotypes with their siblings.There was no difference in frequency between patients and their siblings(P＞0.05).There were no differences in frequency among chronic myelocytic leukemia(CML),non acute lymphoblagtic leukemia(NALL)and acute lymphoblagtie leukemia(ALL)but the frequency of KIR2DS4 in CML was higher than others.Condusion The KIR gene and HLAⅠ antigen are heredity independently and relatively stable.The factor of disease has little effect on KIR gene.