Purification and biochemical characterization of a novel thermophilic exo-ß-1, 3-glucanase from the thermophile biomass-degrading fungus Thielavia terrestris Co3Bag1

Background: The aim of this work was to purify and characterize exo-ß-1,3-glucanase, namely, TtBgnA, from the thermophilic fungus Thielavia terrestris Co3Bag1 and to identify the purified enzyme. Results: The thermophilic biomass-degrading fungus T. terrestris Co3Bag1 displayed ß-1,3-glucanase activity when grown on 1% glucose. An exo-ß-1,3-glucanase, with an estimated molecular mass of 129 kDa, named TtBgnA, was purified from culture filtrates from T. terrestris Co3Bag1. The enzyme exhibited optimum activity at pH 6.0 and 70°C and half-lives (t1/2) of 54 and 37 min at 50 and 60°C, respectively. Substrate specificity analysis showed that laminarin was the best substrate studied for TtBgnA. When laminarin was used as the substrate, the apparent KM and Vmax values were determined to be 2.2 mg mL-1 and 10.8 U/mg, respectively. Analysis of hydrolysis products by thin-layer chromatography (TLC) revealed that TtBgnA displays an exo mode of action. Additionally, the enzyme was partially sequenced by tandem mass spectrometry (MS/MS), and the results suggested that TtBgnA from T. terrestris Co3Bag1 could be classified as a member of the GH-31 family. Conclusions: This report thus describes the purification and characterization of TtBgnA, a novel exo-ß-1,3-glucanase of the GH-31 family from the thermophilic fungus T. terrestris Co3Bag1. Based on the biochemical properties displayed by TtBgnA, the enzyme could be considered as a candidate for potential biotechnological applications.

Effects of inhibiting of BAG-1 gene expression by RNA interference on prolifer-ation and apoptosis of cutaneous squamous cell carcinoma / 中国免疫学杂志

Objective:To investigate the effect of inhibiting of BAG-1 gene expression on proliferation and apoptosis of cutaneous squamous cell carcinoma.Methods:siRNA of BAG-1 was transfected in cutaneous squamous cell carcinoma A431 by Lipo-fectamineTM2000,cells were transfected for 48 h,the BAG-1 mRNA expression was detected by RT-PCR,BAG-1 protein expression was detected by Western blot;CCK8 and flow cytometry were used to detect the cell proliferation and apoptosis;Western blot was used to detect the expression of Bax,β-catenin and Survivin protein;IL-6 and VEGF content were detected by ELISA kit.Results:Compared with control group and negative control group,transfection of BAG-1 with siRNA could significantly inhibit the expression of BAG-1 at the transcriptional and translational levels;compared with the control group,the cells proliferation in BAG-1-siRNA group significantly decreased,apoptosis rate was significantly increased,Bax protein expression was up-regulated,expression of β-catenin,Survivin protein were down regulated,the contents of IL-6 and VEGF were decreased significantly(P<0.05).Conclusion:Silencing of BAG-1 expression can reduce the proliferation of cutaneous squamous cell carcinoma,promote apoptosis,up regulate Bax expression and down regulate the expression of Wnt/β-catenin signaling pathway,and reduce the secretion of IL-6 and VEGF factors.

The expression and clinical significance of PTEN,P27 and BAG-1 in endometrial carcinoma / 重庆医学

Objective To stduy the expression of PTEN ,P27 and BAG-1 in endometrial carcinoma ,and to analysis the clinical significance .Methods The expression of PTEN ,P27 and BAG-1 in 102 cases of endometrial carcinoma ,90 cases of endometrial hy-perplasia and 44 cases of normal endometrium tissue were detected by immunohistochemistry .Results PTEN and P27 in endome-trial carcinoma and hyperplasia tissue showed lower expression ,BAG-1 showed high expression in them .The expression of PTEN and BAG-1 were well correlated to histology degree and muscular infiltration .BAG-1 expression also had a well correlated to clinical stage .In this study ,we had not find a correlated between the expression of P27 and clinical and pathology characteristic of patien . PTEN expression had a Pearson relevance to P27 or BAG-1 expression ,but there was no correlation between P27 and BAG-1 .Con-clusion PTEN and P27 in endometrial carcinoma and hyperplasia tissue showed lower expression ,BAG-1 showed high expression in them .PTEN ,P27 and BAG-1 might play a role in the development of endometrial cancer .

BAG-1, EGFR, and PARP-1 expressions in breast cancer and their clinical significance / 中国肿瘤临床

To observe BAG-1, EGFR, and PARP-1 expressions in invasive breast cancer and its correlation with clini-cal pathological indicators, as well as to evaluate their clinical significance. Methods:The BAG-l, EGFR, and PARP-1 expressions in a tissue microarray of invasive breast cancer and peritumoral tissues were detected through immunohistochemical staining. The clinical and pathological significance of BAG-1, EGFR, and PARP-1 were evaluated. Results:The BAG-1, EGFR, and PARP-1 expression lev-els are higher in invasive breast cancer tissues than in peritumoral tissues (P<0.05). BAG-1 expression in invasive cancer tissues is not related to age, tumor site, lymph node metastases, and clinical TNM staging of patients, but is related to size, grade, ER, PR, and HER-2 expressions and molecular subtype (P<0.05). EGFR expression is related to size, clinical TNM staging, and molecular subtype (P<0.05). PARP-1 expression is related to grade, lymph node metastases, ER, and molecular subtype (P<0.05). BAG-1 expression is not significantly correlated with EGFR and PARP-1 in all cases, but BAG-1 and PARP-1 expressions are positively correlated in tri-ple-negative breast cancer tissues (P<0.05). Results of the univariate analysis revealed that the BAG-1 and PARP-1 expressions and the molecular subtypes are associated with the prognoses of breast cancer patients. Multivariate analysis revealed that BAG-1 and PARP-1 expressions are factors that are independent of the prognosis. Conclusion: BAG-1, EGFR, and PARP-1 overexpressions in human breast tissues suggest that BAG-1, EGFR, and PARP-1 are related to breast cancer development. BAG-1, EGFR, and PARP-1 are poten-tial biomarkers of breast cancer diagnosis and prognosis.

The Effect of Norcantharidin on Human Rectal Cancer Colo 320 Cells / 昆明医科大学学报

Objective To investigate the effect of norcantharidin on growth inhibition and induction of apoptosis of human rectal cancer Colo 320 cells. Methods Norcantharidin (NCTD) in different concentrations were added to rectal cancer Colo 320 cells. Morphological characteristics of apoptosis were observed using the light microscope and transmission electron microscope. The expressions of Bag-1 and Bcl-2 proteins were tested by Western blotting. The growth inhibition of Colo 320 cells on the cell cycle was observed by flow cytometry. Results The apoptosis morphological changes of Colo 320 cells were observed by the light microscope and transmission electron microscopy. Flow cytometry analysis showed that the cell count of G2/M phase in experimental group was higher than that in control group ( <0.05) but the cell counts of G0/G1 and S phases have decreased in experimental group after treatment with NCTD at the concentrations of 5μg/mL, 10μg/mL and 20 μg/mL, and presented dosage dependence relations. The expressions of Bag-1 and Bcl-2 proteins have decreased. Conclusion Norcantharidin has inhibitory effect on rectal cancer Colo 320 cells, and the effect may be related to the cell cycle arrest and apoptosis.

Real-Time RT-PCR on SAG1 and BAG1 Gene Expression during Stage Conversion in Immunosuppressed Mice Infected with Toxoplasma gondii Tehran Strain

Toxoplasmic encephalitis is caused by reactivation of bradyzoites to rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immunocompromised hosts. Diagnosis of this life-threatening disease is problematic, because it is difficult to discriminate between these 2 stages. Toxoplasma PCR assays using gDNA as a template have been unable to discriminate between an increase or decrease in SAG1 and BAG1 expression between the active tachyzoite stage and the latent bradyzoite stage. In the present study, real-time RT-PCR assay was used to detect the expression of bradyzoite (BAG1)- and tachyzoite-specific genes (SAG1) during bradyzoite/tachyzoite stage conversion in mice infected with T. gondii Tehran strain after dexamethasone sodium phosphate (DXM) administration. The conversion reaction was observed in the lungs and brain tissues of experimental mice, indicated by SAG1 expression at day 6 after DXM administration, and continued until day 14. Bradyzoites were also detected in both organs throughout the study; however, it decreased at day 14 significantly. It is suggested that during the reactivation period, bradyzoites not only escape from the cysts and reinvade neighboring cells as tachyzoites, but also converted to new bradyzoites. In summary, the real-time RT-PCR assay provided a reliable, fast, and quantitative way of detecting T. gondii reactivation in an animal model. Thus, this method may be useful for diagnosing stage conversion in clinical specimens of immunocompromised patients (HIV or transplant patients) for early identification of tachyzoite-bradyzoite stage conversion.

The role of BAG-1 in endoplasmic reticulum stress and the enhancement of cisplatin sensitivity in A549 lung cancer cells induced by tunicamycin / 肿瘤

Objective: To observe the induced effect of low dose of tunicamycin on endoplasmic reticulum stress (ERS) of A549 human lung adenocarcinoma cells in vitro and the change of Bcl-2 associated athanogene 1 (BAG-1) protein expression, as well as the changes of cisplatin (DDP) sensitivity and the BAG-1 protein expression. Methods: The expressions of GRP78 (a marker protein in ERS) and BAG-1 proteins in A549 cells treated with low dose of tunicamycin were detected by Western blotting. The change of half inhibitory concentration (IC50) of DDP for A549 cells after pretreatment with tunicamycin was measured by MTT method, and the DDP sensitivity was determined by flow cytometry (FCM). The Western blotting was carried out to detect the expressions of BAG-1 and procaspase-12 proteins in A549 cells induced by DDP after pretreatment with tunicamycin. Results: The ERS in A549 cells could be induced by pretreatment with 1.25 μg/mL tunicamycin for 8 h, which displayed that the expression levels of GRP78 and BAG-1 proteins were both up-regulated (P <0.05). The ERS could increase the DDP sensitivity in the A549 cells (P <0.05). Before ERS occurred, all of three doses of DDP (1.25, 2.5 and 5 μg/mL) could up-regulate the expression level of BAG-1 protein (P <0.05), and this effect was more obvious in 1.25 μg/mL DDP-treated A549 cells. When ERS occurred, the expression levels of BAG-1 protein in all of three doses of DDP (1.25, 2.5 and 5 μg/mL)-treated A549 cells were down-regulated in a dosedependent manner as compared with that in the A549 cells without DDP treatment (P <0.05). The DDP dose of 2.5 and 5 μg/mL could induce apoptosis by ERS pathway, and the expression levels of BAG-1 and procaspase-12 proteins were both down-regulated during this process (P <0.05). Conclusion: BAG-1 may be one of the important regulatory factors in both pathways of ERS- and cisplatin-induced apoptosis of lung cancer cells, and ERS-related apoptotic pathway may be one of the important pathways of cisplatininduced apoptosis of lung cancer cells. Copyright © 2012 by TUMOR.

Correlation between the expression of BAG-1, p53 and platinum-based chemotherapeutic sensitivity in NSCLC / 肿瘤研究与临床

Objective To detect the expression of BAG-1 and p53 in postoperative patients paraffin section of non-snmll cell lung cancer (NSCLC), and retrospectively analyze the relationships of clinical feature and platinum-based chemotherapeutic sensitivity in NSCLC, to further investigate the resistance mechanism of platinum. Methods The expression of BAG-1 and p53 was measured by immunohistochemistry in paraffin sections of 125 NSCLC postoperative patients. The existence and prognosis were analyzed after follow-up. Results BAG-1 and p53 showed high expression in NSCLC (positive rate 64.8 %, 46.7 %); BAG-1 positive or p53 negative expression had survival advantage(P<0.05); The group of BAG-1, p53 negative expression could benefit from platinum-based chemotherapy (P<0.05). Conclusion Expressions of BAG-1, p53 correlates with platinum-based chemotherapeutic sensitivity, and maybe become new types of clinical prognostic indicators and contribute to individualized treatment options for NSCLC.

Silencing BAO-1 gene by eukaryotic expression vector containing short hairpin RNA in mouse melanoma B16F10 cells / 中华皮肤科杂志

Objective To construct the eukaryotic expression plasmids of short hairpin RNA (shRNA) specific for mouse Bcl-2-assoeiated athanogene 1 (BAG-1) and to observe their inhibitory effects on the expression of BAG-1 gene in mouse melanoma B16FI0 ceils. Methods Plasmids named pRNAT-U6.1/Neo-BAG-1, were designed and constructed to target the mouse BAG-1 mRNA coding region. LipofectaminTM 2000 was used to transfect plasmids into BI6F10 cells. Negative plasmid-transfected and tmtransfected B16F10 cells served as negative and blank controls respectively. Forty-eight hours following transfection, G418 was used to select the resistant cells. The mRNA and protein expression of BAG-1 gene was measured by reverse transcription-PCR and Western blot respectively about 1 month after the transfection. Results The eukaryotic expression plasmids, pRNAT-U6.1/Neo-BAG-1, were constructed, and verified by restriction enzyme digestion and DNA sequencing. The transfection rate in B16F10 cells was 20% -30%. Compared with the blank control, the mRNA and protein expression of BAG-1 in BI6FI0 cells was significantly inhibited by BAG-1 shRNA (both P<0.05), and the inhibition rates were (77±4)% and (62 ±2)%, respectively. Conclusions These results indicate that the eukaryotic expression vectors containing shRNA against BAG-1 gene, pRNAT-U6.1/Neo-BAG-1, are successfully constructed, and can significantly inhibit the expression of BAG-1 gene in mouse melanoma B16F10 cells.

Expression of Heat Shock Protein 70 and Bag-1 Protein in Gastroenteric Cancer / 上海第二医科大学学报

Objective To observe the expression of bag-1 and heat shock protein (HSP) 70 in clinical malignant tumors and the correlation between them. Methods RT-PCR and Immunohistochemistry were employed to examine the expression of bag-1 and HSP70 in 69 cases with gastroenteric cancer (54 cancer tissues and 15 non-cancer tissues). Results The expression of Bag-1 was detected in 96.3% of the cases, much higher than that in the control group (P0.05), and no correlation was observed between histological grade and HSP 70 expression. Conclusion There exists definite relationship between the gastroenteric cancer and the expression of bag-1 and HSP70, and there are close correlation between the tumor progress and the expression of bag-1.

Expression and Significance of Apoptosis Related Gene BAG-1 in Digestive Tract Cancer by Tissue Microarray / 中国康复理论与实践

@#ObjectiveTo detect BAG1 expressions in digestive tract cancer by tissue microarray and to evaluate its clinical significance.MethodsTissue microarray of digestive tract cancer and normal tissues were analyzed by DAKO Envision system immunohistochemical staining for apoptosis related gene BAG-1 expression.ResultsThe positive rate of BAG-1 expression among esophagus cancer,gastric cancer and rectal cancer were higher than that of normal tissues respectively(P<0.01).ConclusionThere is an overexpression of BAG-1 in digestive tract cancer,which suggest that apoptosis related gene BAG-1 may be related to these cancer.

Expression and significance of anti-apoptotic gene Bag-1 and Bcl-2 in colorectal cancer / 中国现代普通外科进展

Objective:To investigate the expressions of Bag-1 and Bcl-2 in colorectal cancer and evaluate their clinical significance.Methods:Bag-1 and Bcl-2 expressions were studied by immunohistochemical Streptavidin-Biotin-Peroxidase Complex(SABC)method in 64 samples of colorectal cancer tissues,10 normal colorectal tissues.Results:Bag-1 was positively related to the tumor grade,distant metastasis,stages of Dukes and prognosis,but was not related to the pathological cell types,tumor diameter,depth of invasion and lymph node metastasis.Bcl-2 was not correlated to these clinicopathologic factors.There was a positive correlation between Bag-1 and Bcl-2(r=0.475).Conclusion:The over expression of Bag-1 and Bcl-2 protein in colorectal cancer can affect the generation of colorectal cancer by participating in the regulation of apoptosis.

Expression Pattern and Prognostic Correlation of BAG - 1 Protein in Breast Cancer / 대한암학회지

PURPOSE: The objective of this study was to understand the expression of BAG-1 in the human breast cancer. MATERIALS AND METHODS: We studied its expression in one hundred and thirteen patients diagnosed with breast cancer in Dong-A university hospital between 1992 and 1996 by performing immunohistochemical staining with BAG-1 monoclonal antibody. RESULTS: Of the 113 breast carcinoma examined, 62.0% were positive for BAG-1 cyto- plasmic expression, 28.0% were positive for nuclear BAG-1 expression and 9.7% were positive for both BAG-1 cytoplasmic and nuclear expression. The higher histologic grade was correlated with the higher cytoplasmic expression (p<0.05). Except for histologic grade, no correlation was observed between BAG-1 expression and conventional prognostic factors such as age, menopausal status, metastatic status of the axillary lymh nodes, cathepsin-D, p53, C-erbB-2, DNA ploidy, S-phase fraction, PCNA (proliferating cell nuclear antigen). CONCLUSION: The high histologic grade was found to correlate with positive BAG-1 cyto- plasmic staining which did not correlate with conventional prognostic factors. Our data indicate that furthermore investigation is warranted to define the role of BAG-1 as an meaningful prognostic factor in patients with newly diagnosed breast cancer.

Expressions of BAG-1 and NF-?B P65 in the mononuclearcell of multiple myeloma patients / 中国临床药理学与治疗学

AIM:To investigate the expressions of BAG-1 and NF-?B P65 in the multiple myeloma(MM)and approach the effects of BAG-1 and NF-?B P65 in the pathogenesis of MM and their clinical significance,which would be useful to find a theoretical basis for the treatment of MM.METHODS:The patients diagnosed MM in Yijishan hospital of wannan medical college from 2006.10 to 2008.10 as the experimental group,while the bone trauma patients selected as the control group.The mononuclearcells of bone marrow in the patients were extracted and the BAG-1and NF-?B P65 mRNA RT-PCR were detected.RESULTS:The expression of BAG-1 mRNA(71.4%)in the MM patients was higher than that in the control group(P=0.007).The expression of NF-?B P65 mRNA(75.0%)in the MM patients was higher than that in the control group(P=0.004).There was a positive correlation between the BAG-1 and NF-?B P65 mRNA.The tumor load was high in the MM patients with overexpression of BAG-1 and NF-?B P65,which was ralated to a number of clinical parameters indexes.BAG-1 and NF-?B P65 were positive expressed of MM tumor load,relatived with a number of clinical indicators.CONCLUSION:The expressions of BAG-1 and NF-?B P65 in MM are high,there are no difference between the experimental group and control group.BAG-1 and NF-?B P65 were related to a number of clinical indexes.BAG-1 and NF-?B P65 may be involved in the occurrence and the development of disease,and they can be used as one of the prognosis indexes.

The expression of anti-apoptosis gene bag-1 and its relation to the differentiation of intrahepatic cholangiocarcinoma / 中国普通外科杂志

Objective To investigate the expression of anti-apoptosis gene bag-1 and its relation to the differentiation of intrahepatic cholangiocarcinoma (ICC). Methods Immunohistochemical techniques were adopted to study the expression of bag-1 in ICC tissue ( n=48) and para-hepatocarcinoma bile duct ( control group, n=25). Results Expression of bag-1 in the ICC group was significantly higher than that in the control group. In the ICC group (P

Cloning and Expression of a Bradyzoite-Specific Gene of Toxoplasma gondii and Immunoreactive Analysis on the Recombinant Antigen / 中国寄生虫学与寄生虫病杂志

Objective To clone and express bradyzoite antigen 1(BAG1) gene of T. gondii,and analyze the immunoreactivity of the recombinant product. Methods The differentiation of T. gondii RH strain tachyzoites into bradyzoites was induced in vitro,and the coding sequence of BAG1 was amplified from bradyzoites by RT-PCR. The PCR product was analyzed by sequencing. The BAG1 coding sequence was further subcloned into the plasmid pET32a(+). The plasmid pET32a(+)-BAG1 was then transformed into BL21(DE3) to express after IPTG induction. The expression product was purified with Ni-NTA agarose and the purified BAG1 was further analyzed by Western blotting and ELISA. Results BAG1 cDNA was amplified from bradyzoites. After IPTG induction,BAG1 was expressed in a fusional form in E. coli. Western blotting showed that the purified recombinant protein could be specifically recognized by sera from mice chronically infected by T. gondii B36 strain. ELISA showed that the positive rate of T. gondii IgG antibodies of 350 human sera detected by the recombinant BAG1(17.4%) was higher than by recombinant SAG1 (12.6%)(P