Insect antifreeze protein (AFP) has high antifreeze activity. Antifreeze proteins can be used in cryopreservation of biological tissues and cells. We expressed an antifreeze protein from the desert beetle Microdera punctipennis in yeast and determined the function of the protein at low temperatures. Yeast expression vector, pPIC9K-Mpafp698, was constructed and transformed into Pichia pastoris GS115. The expression of MpAFP698 was induced by methanol, and identified by tricine SDS-PAGE and Western blotting. Mpafp698 gene was inserted into the genome of the host yeast strain GS115, and correctly expressed. Hardly any yeast's own protein was secreted into the media. Cryoprotective experiments showed that MpAFP698 can significantly protect mouse liver as well as other mouse organs from cold damage compared with those in the control of Bovine serum albumin (BSA) addition. Besides, the hemolysis of blood cells protected by MpAFP698 at 4 degrees C was reduced and the survival rate of SF9 cells protected by MpAFP698 after freezing and thawing was increased compared to those of the control with BSA addition. Our results showed that MpAFP698 can be expressed in yeast, which allows a convenient purification of the MpAFP protein that has the cryoprotective effect.
Animals ; Antifreeze Proteins ; biosynthesis ; Blotting, Western ; Cold Temperature ; Coleoptera ; Cryoprotective Agents ; chemistry ; Electrophoresis, Polyacrylamide Gel ; Freezing ; Insect Proteins ; biosynthesis ; Mice ; Pichia ; metabolism ; Sf9 Cells

objective To evaluate endoscopic surgery for high risk patients over 70 years old with severe acute cholangitis. Methods From October 1991 to October 2003, 84 high risk patients over 70 years old suffering from severe acute cholangitis were included and divided into laparotomy surgery (ST) and endoscopic surgery (ET) group. Results 80 patients were cured. The care rate was 81.2% in ST group and 91.6% in ET group. The mortality rate was lower in ET compared with ST (9.1% vs 18.8%). The rate of complication was reduced from 37.5% in ST group to 13.9% in ET group. The average duration of drainage was reduced from 49.4 days in ST group to 18.6 days in ET group. The total treatment days were shortened from 46.2 days to 22.8 days comparing ST with ET. Conclusions Endoscopic surgery should be the first choice for the senile and high risk patients with severe acute cholangitis. Endoscopic surgery was convenient, with mininal injury, safe and effective, and period of treatment was shortened, especially for elderly or critically ill patients, as well as those with MODS or previous billiary operations.

Objective To investigate T cell-mediated anti-HBV effects of dendritic cells (DCs) sensitized by HBsAg in patients with HBV infection in vitro. Methods DCs isolated from peripheral blood mononuclear cells of HBV patients were cultured and proliferated in vitro, and then were stimulated with pure HBsAg before DCs maturation, and co-cultured with auto-T lymphocytes after DCs maturation. The T lymphocytes were then harvested 5 days later and added into the supernatant of 2.2.15 cell cultures. The supernatants were collected on 1st, 3rd, 5th and 7th day, respectively. ELISA was employed to detect HBeAg and the secretion of HBsAg, as well as to measure the concentration of IFN-? and IL-12 in culture supernatents. Results The proliferation and cytotoxicity of auto-T cells were markedly enhanced in the antigen stimulated DCs group compared with that of DCs without loaded antigen and only T lymphocyte group (P

Objective To study the effects ofYiqi Huoxue Jiedu Recipe on immunomodulatory of patients with advanced ovarian cancer.Methods It was performed in a single-arm, open-label study with 40 patients with advanced epithelial ovarian cancer. All patients were treated withYiqi Huoxue Jiedu Recipe lasting for 4 weeks (200 mL, twice a day). The main endpoint was the quantities of CD4+CD25+ cells, CD8+CD28- regulatory T cells and CD8+CD28+ cytotoxic T cells measured by flow cytometry, and the accessory endpoints were variations of symptoms which included pain, fatigue, and numbness etc.Results The quantities of CD4+CD25+ and CD8+CD28- cells significantly decreased compared with those of pre-treatment (P<0.05); the quantity of CD8+CD28+ cell increased significantly (P<0.05). Except for pain, significant improvement showed in fatigue, tinnitus, numbness, poor appetite, abdominal discomfort, insomnia, diarrhea and abnormal urinate (P<0.05).ConclusionYiqi Huoxue JieduRecipe is proved to be effective in regulating immunosuppression advanced ovarian cancer and improving patients’ life quality.

This study was aimed to discuss possible problems in the data collection process of traditional Chinese medicine (TCM) clinical medicine and to propose appropriate solutions. Based on analysis of clinical data features and data collection demands among 20 TCM clinical research bases, reference were made on key technologies of the Electronic Medical Records' integration and sharing, to propose the solution of TCM clinical data collection. The re-sults showed that proposing the metadata and data entities synchronized intersecting strategy, analyzing key points on this strategy, and proposing appropriate solutions, found the technical processes of the TCM clinical data collec-tion. It was concluded that the analysis and solution on key points of TCM clinical data collection provided the basis and the reference to data collection issues of TCM clinical research base.

To investigate the effects of electro-acupuncture at Shuigou (DU26) on latency and amplitude of motor evoked potential (MEP) in rats after cerebral infarction.

Objective To evaluate the endoscopic managements of acute cholangitis of severe type (ACST) accompanied with multiple organ dysfunction syndrome (MODS). Methods A total of 122 ACST patients accompanied with MODS from January 2000 to October 2008 underwent endoscopic treatment in two time periods. In critical phase, emergent endoscopic retrograde cholangiopancreatography (ERCP) plus en-doscopic naso-biliary drainage (ENBD) were performed to correct critical situation of the patients. After sta-bilization, endoscopic sphincterotomy (EST) plus stone removal, EST plus stent placement, or laparoscopy was performed according to the causes of ACST. Results Emergent endoscopic managements succeeded in all patients of critical phase. At third day post-operation, a reduction in white blood cell count, serum total bilirubin, body temperature, and rate of patients with shock, mental symptoms and purulent bile juice was a-chieved. Recovery rate of dysfunction organs was 60.2% at one week after emergent procedure, and 82. 6% at 2 weeks post-operation. Selective EST plus stone removal was performed in 36 patients with a success rate n one session at 91.7%. Laparoscopic cholecystectomy was performed in 85 patients with a success rate of 95.3%. Stent was placed in 16 patients with an effective rate of 81.3% at 3 months post the procedure. No severe complication or death occurred during the whole therapeutic course. The 6-month survival rate of 10 cancer cases was 70%. Conclusion Therapeutic ERCP plus ENBD is the first choice for acute severe cholangitis accompanied with MODS, while EST plus biliary lithotomy, or EST plus stent placement, or com-bined laparoscopy are ideal methods for subsequent treatment.

Protocarcinogenic gene Her-2/neu was closely related to the development of gastric cancer. Herceptin is an antibody according to Her-2/neu and is used for treating breast cancer. Topo Ⅱ α is the important Nuclear enzymes needed by DNA duplicate, and is closely related to the proliferation of malignant tumors, so it is the important target enzymes of cancer chemotherapy. Her-2/neu and Topo Ⅱ α are located in the same chromosomes, there are correlation between the two genes' proliferation or loss. We reviewed the expression of Her-2/neu and Topo Ⅱ α in the stomach and the new progress of the correlation between the two genes home and abroad in this paper, which provides new methods for the prevention and treatment of gastric cancer.

The paper introduces the status of TCM symptoms information application,constructs the symptoms knowledge classification system by taking the TCM Diagnostics as the knowledge source,constructs the symptoms knowledge representation model based on the classification and coding technologies,and draws a conclusion that the data analysis result is consistent with the actual clinical situation based on collecting symptom data in the Electronic Medical Records (EMR) and correlation analysis using this model.

Objective To explore the mechanism of combined treatment with methotrexate (MTX) and Ga-Al-As laser irradiation for rheumatoid arthritis (RA) and to assess the effectiveness of Ga-Al-As laser therapy for RA. Methods Twenty-two patients with RA were randomly and evenly divided into two groups: the treatment group treated with Ga-Al-As laser irradiation combined with MTX and the control group treated with MTX only. Ten age-matched normal subjects were observed as normal controls. The amount of CD4 + CD25 + regulatory T cells in peripheral blood (PB) of the normal controls and that in the PB and synovial fluid (SF) of the 22 patients before and after therapy were counted by flow cytometry. Meanwhile, the amount of prostaglandin E2 (PGE2) in synovial fluid of the patients was measured before and after treatment by enzyme-linked immunosorbent assay(ELISA). Results After combined treatment the clinical symptoms of the patients were improved significantly, and the amount of PGE2in SF decreased significantly. The count of CD4 + CD25 + regulatory T cells in PB of RA patients was ( 3.84 ±3.20) % , compared to ( 10.05 ± 7.04) % in healthy individuals. The count of CD4 + CD25 + regulatory T cells in SF of RA patients was ( 14.89 ± 12.30) % , much higher than that in PB. The count of CD4 + CD25 + regulatory T cells in SF decreased significantly in treatment group compared to control group (P ＜0.05). Conclusion Ga-Al-As laser irradiation eombined with MTX can effectively improve the clinical symptoms of RA patients. It may be related to the decrease of amount of PGE2 and count of CD4 + CD25 + regulatory T cell in PB and SF.