Research on novel pullulanase has major significance on the domestic industrialization of pullulanase and the breakdown of foreign monopoly. A thermophilic bacteria LM 18-11 producing thermostable pullulanase was isolated from Lunma hot springs of Yunnan province. It was identified as Anoxybacillus sp. by 16S rDNA phylogenetic analysis. Full-length pullulanase gene was cloned from Anoxybacillus sp. LM18-11. The optimum temperature of the pullulanase was between 55 and 60 degrees C with a half-life as long as 48 h at 60 degrees C; and its optimum pH was between 5.6 and 6.4. V(max) and K(m) of the pullulanase was measured as 750 U/mg and 1.47 mg/mL, which is the highest specific activity reported so far. The pullulanase crystals structure showed a typical alpha-amylase family structure. The N-terminal has a special substrate binding domain. Activity and substrate binding were decreased when the domain was deleted, the V(max) and K(m) were 324 U/mg and 1.95 mg/mL, respectively. The pullulanase was highly heterologous expressed in Bacillus subtilis by P43 promoter. The extracellular enzyme activity was 42 U/mL, which increased more than 40 times compared to the initial strain. This pullulanase has good application prospects.
Anoxybacillus ; classification ; enzymology ; China ; Glycoside Hydrolases ; metabolism ; Hydrogen-Ion Concentration ; Phylogeny ; RNA, Ribosomal, 16S ; genetics ; Temperature

Adolescent ; Body Height ; Body Weight ; Child ; Female ; Growth and Development ; Humans ; Male ; Papilloma ; surgery ; Thyroid Neoplasms ; surgery ; Thyroidectomy ; adverse effects

Objective To observe the effects of T-lymphocyte function subsets,IL-4 and IFN-γ cell factor in different dose and stage after irradiation.Methoda The C57BL/6j mice were divided into shammed irradiation group and model groups.The radiation hurt model was induced by 60Co gamma rays(0.7,1.4,2.8 and 5.6 Gy).The changes of T-lymphocyte subsets CD3,CD4,CD8,IL-4 and IFN-γ in spleen cells were analyzed by flow cytometry in acute injury stage and recovery stage after irradiation.Results The lymphocyte subsets CD3 +,CD4+ and CD8 + decreased after irradiation,which were related to the irradiation dose.At 1 day after irradiation,the decreasing level of IFN-γ was higher than that of IL-4.When irradiation dose was over 2.8 Gy,IL-4 / IFN-γ showed a markedly increased compared with control group.At 25 days after irradiation,CD3 +,CD4+,CD8 +,CD4 +/CD8 +,IL-4 and IFN-γ recovered obviously,but they did not recover to the normal level of shammed irradiation group.Conclusions The depression of mouse immune function induced by γ-irradiation might be caused by changes of CD3,CD4,CD8 and CD4/CD8 ratio,especially the imbalance of IL4 and IFN-γ.

Objective To compare the changes in nitric oxide (NO), total ability of anti-oxidize antioxidase, superoxide dismutase (SOD), glutathion peroxidase (GSH-PX), catalase (CAT) and maleic dialdehyde(MDA) in rats after single and accumulative 60Co γ-irradiation . Methods 48 rats were randomly divided into two groups of single and accumulative irradiation. Each group was irradiated by 60Co gamma-rays. The total irradiation doses were 0.4, 0.8, 1.6, 3.2 and 6.4 Gy, respectively in each group. The NO, total ability of anti-oxidize antioxidaze, SOD, GSH-PX, CAT and MDA in serum were measured at day 1 after last irradiation. Results Compared with the single irradiation group, the NO (52.6-117.9 μmol/ml), total ability of anti-oxidize antioxidaze (3.3-26.2 U/ml), the antioxidase activity of the SOD (26.3-167.5 U/ml), GSH-PX (740.8-2462.4 U/ml), CAT (3.3-29.4 U/ml) and the content of MDA(29.3-155.1 nmol/ml) of mt serum in accumulative irradiation group were increased after irradiation, which was related with the accumulative irradiation dose. For instance, total ability of anti-oxidize antioxidase (26.2 U/ml), antioxidase activity of the SOD (167.5 U/ml) and CAT (29.4 U/ml) in 0.4 Gy group of accumulative irradiation were significandy increased when compared with those of control group. However, the content of MDA in accumulative irradiation group was obviously higher than that in single radiation group when the irradiation doses delivered over 3.2 Gy, which might be correlated with higher antioxidase activity. Conclusions Low dose of accumulative gamma-rays irradiation can induce the stimulative effect of antioxidase activity. However, higher dose of accumulative gamma-rays irradiation can damage the activity of antioxidase.

Adenocarcinoma ; blood ; diagnosis ; pathology ; Biopsy, Needle ; Humans ; Male ; Neoplasm Invasiveness ; Neoplasm Staging ; methods ; Prostate-Specific Antigen ; blood ; Prostatic Intraepithelial Neoplasia ; blood ; diagnosis ; pathology ; Prostatic Neoplasms ; blood ; diagnosis ; pathology

Arthrography ; Bone Neoplasms ; diagnosis ; diagnostic imaging ; pathology ; Bone and Bones ; diagnostic imaging ; pathology ; Diagnosis, Differential ; Diagnostic Errors ; Humans ; Immunohistochemistry ; Joint Diseases ; diagnosis ; diagnostic imaging ; pathology ; Joints ; diagnostic imaging ; pathology ; Radionuclide Imaging

Objective To develop a high-resolution melting ( HRM ) assay for rapidly screening Gilbert syndrome ( GS) and Crigler-Najjar syndrome ( CNS) associated with UGT1A1 defects.Method Methodology was developed .Then, we applied the established method to analyze 61 clinical samples from neonatal patients with severe unexplained unconjugated hyperbilirubinemia .Neonates with known risk factors for developing hyperbilirubinemia , such as ABO hemolysis, G6PD deficiency, sepsis, hypoxic ischemic encephalopathy were excluded .Five pairs of PCR primers were designed to detect the five common mutations (G211A, C686A, C1091T, C1352T and T1456G) in Asia population.PCR and HRM Assay conditions were optimized.UGT1A1 genotyping in clinical samples was performed by using the established HRM analysis , and all results were subsequently confirmed by direct DNA sequencing .Results The mutants were readily differentiated by using HRM analysis .In this study, 42 neonates were identified with UGT1A1 mutation, and 4 different known variants were detected .Conclusion HRM analysis in this study was economical, convenient, rapid, effective for screening UGT1A1 gene mutations, which can serve as an reliable method for the clinical diagnosis of GS and CNS and the large-scale molecular epidemiological research of UGT1A1 gene-related diseases.

Stroke is an important public health problem both in China and worldwide.Stroke genetics research has made great progress in recent years, especially the genome-wide association study (GWAS) and the emergence of epigenetics, has brought a breakthrough in this field. They studied the pathogenesis of stroke from the genetic level and the environmental factor levels. Although there are still many problems to be solved, the prospect of stroke genetics is bright.

Objective To evaluated the role of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in the diagnosis of thoracic tuberculosis.Methods The study was retrospective,from September 2009 to September 2012,38 patients who underwent EBUS-TBNA were finally diagnosed of thoracic tuberculosis,with enlarged hilar or mediastinal Iymph nodes on chest enhanced computed tomography(≥ 1.0 cm).Patients in whom EBUS TBNA was nondiagnostic subsequently underwent surgical biopsy.All the patients had a minimum of 6 months clinical and radiologic follow-up.Results EBUS-TBNA was performed on a total of 88 lymph node stations in 38 patients.Of the enlarged lymph nodes,60(68.18％) were located in the mediastinal region and the remaining 28 (31.82 ％) around the hilum or interlobar area.Of the 38 patients,EBUS-TBNA achieved definitive diagnosis in 34 patients(89.47％).EBUS was well tolerated by all of the patients with no complications.Conclusion EBUS-TBNA is a safe procedure with a high yield for the diagnoses of thoracic tuberculosis.

The present study was designed to test whether Rho-kinase (ROCK) specific inhibitor fasudil (HA-1077) could contribute to migration and vasculogenesis of endothelial cells differentiated from rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. rBMSCs were separated by gradient centrifugation on lymphocytes separation medium from bone marrow of Sprague-Dawley rats, and were cultured, purified and expanded in vitro. Cells of passage 2 to 3 were induced to differentiate into endothelial lineage cells by HG-DMEM plus EGM-2. These cells were identified as endothelial cells with positive factor VIII and Ulex europaeus agglutinin-1 expressions and DiI-Ac-LDL uptake. HA-1077 and VEGF synergistically promoted cell migration, especially in response to transwell chamber assay. When the cells were cultured on ECMatrix™, they showed cellular protrusions and/or cords of aligned cells resembling primitive capillary-like structures at 8 to 12 h of incubation. HA-1077 promoted cell migration and formation of capillary-like tubes. The length of the total capillary tubes was longer than that in the control group (P<0.05). When the cells were exposed to a combination of VEGF and HA-1077, the number of the capillary-like networks and the stability of tube increased. The results obtained suggest that HA-1077 can promote migration and vasculogenesis of endothelial cells differentiated from rBMSCs in vitro.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cell Movement ; drug effects ; Endothelial Cells ; cytology ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Sprague-Dawley ; rho-Associated Kinases ; antagonists & inhibitors