There are still many problems at liquid state culture especially large-scale culture for Helicobacter pylori at present. Using analytical technique of single factor test statistics, a suitable medium and shaking culture conditions for Helicobacter pylori have been selected. The experimental results showed: under the optimal shaking culture conditions Hp could be growed well enough in Brucella broth with ?-CD. The thalli weight reached 0.6g/100mL.

This study was aimed to investigate the expression level of Wnt5a gene in some hematologic diseases and leukemic cell lines so as to provide a basis for further research of Wnt5a role and its mechanism in hematologic malignancies. The mononuclear cells of peripheral blood and bone marrow were isolated by human lymphocytic isolation solution. The expression of Wnt5a gene in specimen of 31 cases and three leukemic cell lines (Jurkat, K562, HL-60) were detected by RT-PCR. The results showed that in four out of five AML cases, negative or weak positive expressions were observed and negative expressions were observed also in K562 and HL-60 cells. Only in one AML case with complete remission and Jurkat cells the strong positive expressions were observed. The negative expression was observed in all six CML cases. In three out of four ALL cases, the expression was positive or weak positive and one negative. The expressions in two CLL cases were negative. Out of two MM cases, the expression in one was weak positive and in other was negative. Out of three lymphoma cases, the expression in one case was weak positive and in other two cases were negative. There were positive or weak positive expressions in two cases of AA, two cases of IDA, three cases of ITP, one cases of PV and ET cases. It is concluded that there have obvious down-regulated or lost expression of Wnt5a gene in 31 cases of hematologic disease and myelocytic leukemic cell lines except ALL samples. Nevertheless there have general positive expression of Wnt5a in cases of non-malignant hematologic diseases. These results suggest that the genesis of myelocytic leukemia is related to the down-regulated expression of Wnt5a.
Adolescent ; Adult ; Aged ; Child ; Down-Regulation ; Gene Expression Regulation, Leukemic ; Hematologic Neoplasms ; genetics ; metabolism ; Humans ; Middle Aged ; Proto-Oncogene Proteins ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Cells, Cultured ; Wnt Proteins ; metabolism ; Wnt-5a Protein ; Young Adult

Heat-labile enterotoxin (LT) from Escherichia coli is a bacterial protein toxin with an AB5 hexamer structure. LT is a powerful mucosal adjuvant when co-administered with soluble antigens. However, its use in mucosal immunity is inconvenient because of its low yield and depolymerization during long-term storage under normal condition. In this study, we report an efficient expression system and optimized purification and storage strategy of LT. A gene encoding LT was cloned into the vector pET11c and transformed in E. coli BL21(DE3). By growing this strain on modified M9-CAA medium, LT was expressed efficiently. About 46mg/L LT could be purified from the supernatant of bacteria lysate. Using D(+)-Immobilized galactose column, LT could be purified at a wide pH range with various elution buffers. The optimized elution buffers are TEAN (pH 7.3) containing 0.3mol/L galactose and carbonate buffer (pH 10.4) containing 0.3mol/L galactose. After dried by freeze and placed in 4 degrees C, LT dissolved in TEAN (pH 7.3) and carbonate buffer (pH 10.4) were assayed by HPLC. The results indicated that the integrity of AB5 hexamer was kept well. LT could undergo long-term storage under this condition. This was proved to be an optimized strategy of LT storage. The results of GM1 binding assay and toxicity assay showed that the purified recombinant LT has normal biological character.
Animals ; Bacterial Toxins ; genetics ; isolation & purification ; metabolism ; pharmacology ; CHO Cells ; Cell Shape ; drug effects ; Chromatography, High Pressure Liquid ; Cricetinae ; Cricetulus ; Electrophoresis, Polyacrylamide Gel ; Enterotoxins ; genetics ; isolation & purification ; metabolism ; pharmacology ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; isolation & purification ; metabolism ; pharmacology ; Female ; Genetic Vectors ; genetics ; Mice ; Mice, Inbred C57BL ; Toxicity Tests

In eukaryotes protein phosphorytion is a key event. By reversible protein phosphorylation eukaryotes control many cellular processes including signal transduction, gene expression, the cell cycle etc. Phosphoproteomics involves identification of phosphoproteins and phosphopeptides, localization of the exact residues that are phosphorylated and quantitation of phosphorylation. Because protein phosphorylation is a dynamic process, and it is present at low abundance within cells, and the phosphorylated sites on proteins might vary, and mass spectrometry (MS) signals from phosphopeptides are usually suppressed etc., so phosphoprotein analysis have more difficulties than nonphosphoprotein. In this article, we outline several analysis techniques for separation, identification and quantitation of phosphorylated proteins and peptides, and discuss the progress in these techniques. At present, MS is still an essential core identification technology for phosphoproteomic studies, To search better enrichment strategies are the main challenges in this rapidly evolving field. A major goal of quantitative proteomics is precise quantification and identification of proteins in complex mixtures. A common method for quantitative proteome analysis is the stable isotope labeling method. Today there is no single method that supersedes all others techniques for Phosphoproteomic studies. With continued development of sample preparation techniques and instrumentation, it should be possible to perform a global analysis of protein phosphorylation.
Animals ; Humans ; Mass Spectrometry ; Phosphoproteins ; analysis ; Phosphorylation ; Proteomics ; methods

OBJECTIVETo study the pathogenetic role of tissue factor (TF) in endothelial-injury in GVHD.

METHODSGene and protein expressions of TF in the organs of allogenic hematopoietic stem cell transplantation (allo-HSCT) and autologous HSCT (auto-HSCT) mice were determined by real-time PCR and Western blot. The effect of allogeneic T lymphocytes on the expression of TF and other cytokines and activation of MAPKs in human umbilical vein endothelial cells (HUVECs) was detected by flow cytometry, real-time PCR or Western blot. The influence of TF antibodies (SB203580 and SP600125) on allogeneic T lymphocytes-induced cytokines expression was also tested.

RESULTS(1) TF gene and protein expression in the liver, skin, small intestine and stomach of allo-HSCT mice was significantly elevated about 15.1+/-2.1, 5.5+/-1.4, 9.7+/-2.3, 14.2+/-2.9 folds and 13.5+/-2.7, 6.2+/-0.9, 7.9+/-1.6, 15.3+/-3.2 folds respectively compared with that of auto-HSCT mice. (2) Allogeneic CD4+ CD8+ T lymphocytes significantly enhanced TF, VCAM-1, TNF-alpha, IFN-gamma and IL-6 expression in TNF-alpha prestimulated HUVECs. (3) Allogeneic T lymphocytes enhanced p38MAPK and JNK phosphorylation in HUVECs, but did not affect ERK phosphorylation. p38 MAPK JNK inhibitors SB203580 and SP600125 reduced allogeneic T lymphocytes-induced TF expression in HUVECs. (4) SB203580 and SP600125 down-regulated allogeneic T lymphocytes-induced VCAM-1, TNF-alpha, IFN-gamma, IL-6 expression in HUVECs.

CONCLUSIONTF mediates vascular endothelial-injury and activation in GVHD via phosphorylation of p38MAPK and JNK.

Animals ; Anthracenes ; pharmacology ; Cells, Cultured ; Disease Models, Animal ; Endothelial Cells ; metabolism ; Endothelium ; pathology ; Endothelium, Vascular ; cytology ; Graft vs Host Disease ; metabolism ; pathology ; Hematopoietic Stem Cell Transplantation ; Humans ; Imidazoles ; pharmacology ; Interferon-gamma ; metabolism ; Interleukin-6 ; metabolism ; Mice ; Mitogen-Activated Protein Kinases ; metabolism ; Pyridines ; pharmacology ; T-Lymphocytes ; Thromboplastin ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

Objective To explore the applicability of Singapore semi-quantitative risk assessment mode and Romania risk assessment model in occupational health risk assessment. Methods We employed two risk assessment models to evaluate the risk of key positions in papermaking factories, electroplating factories, and chemical factories. Then we compared the results with occupational exposure limits, classification of occupational hazards and literature reports. Results The results of Singapore model showed that the total risk ratio was 0.40 ±0.16. The risk levels of papermaking factories, electroplating factories, and chemical factories were low-medium, negligible-low and low-very high, respectively. The risk ratio of three industries were 0.42±0.04, 0.31±0.10 and 0.62±0.15. The results of Romania model showed that the total risk ratio was 0.43 ±0.11. The risk levels of papermaking factories, electroplating factories, and chemical factories were respectively low-high, minimal-low and low-very high. The risk ratio of three industries was 0.46±0.13, 0.38±0.08 and 0.52±0.11, respectively. The risk levels of electroplating factories were higher than papermaking factories and chemical factories (P<0.05) . There was no significant difference between risk levels of papermaking factories and chemical factories (P>0.05) . There was significant difference between the occupational health risk levels assessed by the two models (P >0.05) . Conclusion These findings suggest that Singapore semi-quantitative risk assessment model and Romania risk assessment model both can be applied for the occupational health risk assessment of different workplaces, such as papermaking factories,electroplating factories, and chemical factories. The risk assessment results of the two methods are basically identical.

OBJECTIVETo investigate the level and distribution characteristics of anemia of the minority ethnic group children in Yunnan.

METHODThe cases with anemia were surveyed from 13 336 samples of 15 minority ethnic groups and Han children in Yunnan by Taking the method of random cluster sampling.

RESULTThe prevalence of anemia among the children under 7 years of age of 15 ethnic groups of minority in Yunnan was 13.6%. There are differences among the different ethnic groups (χ(2) = 716.33, P < 0.01), the highest was 26.6% in Jingpo, the lowest was 3.5% in Bai. There were differences among the different regions, the prevalence of anemia was high in the border regions City, the highest was 23.8% in Dehong; the prevalence of anemia was low in inland cities, the lowest was 2.7% in Fugong, and was higher in border areas. The prevalence of anemia was higher in boys (13.6%) than in girls (12.1%). There were differences among the different age in the different ethnic groups (6 months to 1 years old: χ(2) = 70.52, P < 0.01; 1 - 2 years old:χ(2) = 185.86, P < 0.01; 2 - 5 years old: χ(2) = 296.12, P < 0.01; 5 - 6 years old:χ(2) = 107.11, P < 0.01; 6 - 7 years old:χ(2) = 185.02, P < 0.01), the highest was 59.0% of Deang in 1 to 2 years old children. The trend of change was that the highest prevalence was seen in 6 months to 1 year old children, the prevalence gradually declined among older children, but rose again in children 6 years of age or older.

CONCLUSIONThe prevalence of anemia was 13.6% among the children of 15 ethnic minority under 7 years of age in Yunnan. There were differences among different ethnic groups of minority in different prefectures. There were differences among different ethnic groups of different age groups, but it was highest in 6 months to 1 year old children, it declined among older children, and rose in children 6 years of age or older. The prevalence of anemia was related to the ethnic and geographic factors.

Age Distribution ; Altitude ; Anemia ; diagnosis ; epidemiology ; ethnology ; Biomarkers ; analysis ; Child ; Child, Preschool ; China ; epidemiology ; ethnology ; Ethnic Groups ; Female ; Hemoglobins ; analysis ; Humans ; Infant ; Infant, Newborn ; Male ; Minority Groups ; statistics & numerical data ; Prevalence ; Sampling Studies ; Sex Distribution

OBJECTIVETo express a multi-copy specific epitope recombinant protein of herpes simplex virus type 1 (HSV-1) with immuno-reactivity.

METHODSMulti-copy genes with a specific epitope of HSV-1-glycoprotein G 112-127 were constructed by DNA recombination and cloned in E. coli JM109 pGEM-5Zf. The positive recombinants were determined by SDS-PAGE and Western blotting.

RESULTSThe recombinants with 4, 8, 16 and 32 copies of gG 112-127 were obtained. The 8-copy recombinant was expressed by 17.5%, mainly as inclusion body. And it reacted with antiserum HSV-1, but not with antiserum HSV-2.

CONCLUSIONThe HSV-1-gG112-127 recombinant could be used to distinguish HSV-1 and HSV-2 in ELISA.

Antigen-Antibody Reactions ; DNA, Recombinant ; Epitopes ; biosynthesis ; immunology ; Herpesvirus 1, Human ; immunology ; Herpesvirus 2, Human ; immunology ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; Viral Envelope Proteins ; biosynthesis ; immunology

OBJECTIVE: To investigate the protective effect of isoflurane delayed preconditioning on myocardial ischemia reperfusion injury and the potential mechanism in rabbits. METHODS: Thirty New Zealand male white rabbits were randomly assigned to 3 groups: Control group; I/R group; and 2.0% isoflurane group. Isoflurane group was exposed to 2.0% isoflurane-100% oxygen for 2 hours. Control group and I/R group were exposed to 100% oxygen for 2 hours and served as untreated controls. Twenty-four hours later I/R group and isoflurane group underwent 40 minutes of coronary occlusion followed by 2 hours of reperfusion. Blood samples were taken from the arterial line at 20 minutes before the occlusion(T1), 20 minutes after the occlusion(T2), 40 minutes after the occlusion(T3), 1 hours after the reperfusion(T4), and 2 hours after the reperfusion(T5) to determine the plasma level of TNF-alpha. At the end of the reperfusion, infarct size and area at risk were defined by Evans and TTC staining. The heart was harvested and levels of the p38MAPK activity were determined by Western blot, and ultrastructures were observed under the electron microscope. RESULTS: The p38MAPK activity of isoflurane group was significantly lower than that of I/R group (P<0.05). Isoflurane significantly (P<0.05) reduced the infarct size(19.7%+/-2.8% in isoflurane group) of the left ventricular area at risk as compared with the controls (37.8%+/-1.7% in I/R group).The injury of I/R group was worse than that of isoflurane group under the light microscope. Isoflurane group had a lower level of TNF-alpha than I/R group. CONCLUSION Isoflurane can inhibit p38MAPK activity during myocardial ischemia reperfusion and modulate the cytokine expression, which may be one of the molecular mechanisms of isoflurane delayed preconditioning on cardioprotection.
Animals ; Ischemic Preconditioning, Myocardial ; methods ; Isoflurane ; pharmacology ; Male ; Myocardial Reperfusion Injury ; pathology ; prevention & control ; Myocardium ; ultrastructure ; Rabbits ; Random Allocation ; Tumor Necrosis Factor-alpha ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVE: To investigate the effects of ulinastatin (UTI) on cerebral inflammatory response during cardiopulmonary bypass (CPB). METHODS: Twenty-four NYHA II-III patients (13 males and 11 females) aged 23-45 years, undergoing elective cardiac valve replacement under hypothermic CPB were randomly divided into 2 groups: ulinastatin group (Group U, n=12) and control group (Group C, n=12). In group U, UTI (1.2 x 10(4) U/kg) was given intravenously after the induction of anesthesia, 0.6 x 10(4) U/kg UTI was added to the priming solution, and 0.6 x 10(4) U/kg UTI was given about 5 min before the aortic decamping. In Group C, normal saline was given instead of UTI. Internal jugular vein was cannulated and the catheter was advanced retrogradely till jugular bulb. Blood samples were taken simultaneously from artery and jugular bulb after induction of anesthesia (T1), 60 min (T2) and 6 h (T3) after discontinuation of CPB for determination of TNFalpha, IL-6, IL-8 and IL-10. The juguloarterial gradients of these cytokines (deltaTNFalpha, deltaIL-6, deltaIL-8, and deltaIL-10) were calculated. RESULTS: In Group C, arterial levels of TNFalpha, IL-6, IL-8, IL-10 at T2 and T3, deltaTNFalpha, deltaIL-8 and deltaIL-10 at T2, deltaTNFalpha, deltaIL-6 and deltaIL-10 at T3 significantly increased (P < 0.01). deltaIL-8 increased at T3 (P < 0.05). In Group U, arterial levels of IL-6, IL-8, IL-10 at T2, arterial levels of IL-6, IL-8,IL-L-10 and deltaTNFalpha, deltaIL-8 at T3 significantly increased (P < 0.01). Arterial levels of TNFalpha at T2 and T3, deltaTNFalpha, deltaIL-10 at T2, deltaIL-6 at T3 increased (P < 0.05). Arterial levels of TNFalpha, IL-6 and deltaTNFalpha, deltaIL-8 at T2, arterial levels of TNFalpha and deltaIL-6 at T3 in Group U were lower than those in Group C (P < 0.05). Arterial levels of IL-6 at T3, IL-8 at T2 and T3 in Group U were significantly lower than those in Group C (P < 0.01). Arterial levels of IL-10 and deltaIL-10 at T3 in Group U were higher than those in Group C (P < 0.05). CONCLUSION Systemic and cerebral activation of inflammatory response during CPB can be alleviated by ulinastatin.
Adult ; Cardiopulmonary Bypass ; adverse effects ; Encephalitis ; etiology ; metabolism ; prevention & control ; Female ; Glycoproteins ; therapeutic use ; Heart Valve Prosthesis Implantation ; Humans ; Interleukin-10 ; blood ; Interleukin-6 ; blood ; Interleukin-8 ; blood ; Male ; Middle Aged ; Trypsin Inhibitors ; therapeutic use ; Tumor Necrosis Factor-alpha ; metabolism