Revealing protein-protein interactions at the transcriptome scale by sequencing

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人胱硫醚β-合酶及其截短型片段的表达、纯化和活性测定

将人胱硫醚β-合酶(CBS)基因克隆至质粒pGEX-4T-1中,获得的重组质粒pGEX-4T-1-CBS转入大肠杆菌E.coli Rosetta (DE3)菌株,构建了高效表达CBS的重组菌E.coli Rosetta (pGEX4T-1-CBS)。重组菌在0.1mmol/L的IPTG于30℃诱导16h,可溶性CBS表达量达到28mg/L培养基。将重组菌破碎后上清液经GSTrap Fast Flow亲和层析一步纯化得到CBS融合蛋白,在凝血酶柱上切割缓冲液中加入3%甘油和0.1%CHAPS可以有效抑制酶切后CBS聚沉,酶活性回收率为54.8%,蛋白质产率为15.2mg/L培养基,纯度达到95%,单位酶活为143U/mg,终浓度为1mmol/L的S-腺苷甲硫氨酸(AdoMet)可使CBS单位酶活提高5.1倍,达到735U/mg。同时构建了表达CBS_1-413(删除了CBS羧基端调控域138个氨基酸残基)的重组菌E.coli Rosetta (pETDuet-1-CBS_1-413),经过一步HisTrap Fast Flow亲和层析,酶活性回收率为74.3%,蛋白质产率为12.8mg/L培养基,纯度达到95%,单位酶活为965U/mg; 还表达和纯化了胱硫醚β-裂解酶(CBL),并在此基础上建立了一种新的CBL偶联的CBS酶活性测定方法。     

模拟胃酸条件下的幽门螺杆菌CAT特异性抗体活性研究

幽门螺杆菌是常见的感染性病原菌,人类多种疾病发生与此菌感染有关。预防和治疗菌体感染及引发的相关疾病仍是现代医学面临的课题。实验利用原核表达的幽门螺杆菌过氧化氢酶(1~380 aa)免疫家兔,获得效价为1∶6 000的特异性抗血清,经硫酸铵沉淀法得到初步纯化的抗体。在体外模拟胃酸环境下(pH3.4)将抗体进行水解。SDS-PAGE结果显示,抗体的重链能被水解。水解后的抗体产物经ELISA方法检测,仍然具有与抗原特异性结合的能力。实验结论证实,在体外环境下特异性幽门螺杆菌抗体保护作用不会被胃蛋白酶的水解而破坏,提示口服特异性抗体预防和治疗幽门螺杆菌感染可能是一条可行的途径。     

Hypoxia induces beta-amyloid in association with death of RGC-5 cells in culture

Beta-amyloid (Aβ) derived from amyloid precursor protein (APP) has been associated with retinal degeneration in Alzheimer’s disease (AD) and glaucoma. This study examined whether hypoxia exposure induces Aβ accumulation in RGC-5 cells. While levels of APP mRNA and protein significantly increased in the cells, elevated abundance of Aβ was also observed in cells and culture medium between 12 or 24 and 48 h after 5% O₂ hypoxia treatment. Additionally, there is a close relationship between induction of APP and Aβ and intracellular accumulation of ROS along with loss of mitochondrial membrane potential followed by the death of RGC-5 cells in culture under hypoxia. These results suggest a possible involvement of APP and Aβ in the death of RGCs challenged by hypoxia.     

Activation of ROCK by RhoA is regulated by cell adhesion, shape, and cytoskeletal tension

Adhesion to the extracellular matrix regulates numerous changes in the actin cytoskeleton by regulating the activity of the Rho family of small GTPases. Here, we report that adhesion and the associated changes in cell shape and cytoskeletal tension are all required for GTP-bound RhoA to activate its downstream effector, ROCK. Using an in vitro kinase assay for endogenous ROCK, we found that cells in suspension, attached on substrates coated with low density fibronectin, or on spreading-restrictive micropatterned islands all exhibited low ROCK activity and correspondingly low myosin light chain phosphorylation, in the face of high levels of GTP-bound RhoA. In contrast, allowing cells to spread against substrates rescued ROCK and myosin activity. Interestingly, inhibition of tension with cytochalasin D or blebbistatin also inhibited ROCK activity within 20 min. The abrogation of ROCK activity by cell detachment or inhibition of tension could not be rescued by constitutively active RhoA-V14. These results suggest the existence of a feedback loop between cytoskeletal tension, adhesion maturation, and ROCK signaling that likely contributes to numerous mechanochemical processes.     

DNA-binding activity of TNF-alpha inducing protein from Helicobacter pylori

Tumor necrosis factor-alpha (TNF-alpha) inducing protein (Tipalpha) is a carcinogenic factor secreted from Helicobacter pylori (H. pylori), mediated through both enhanced expression of TNF-alpha and chemokine genes and activation of nuclear factor-kappaB. Since Tipalpha enters gastric cancer cells, the Tipalpha binding molecules in the cells should be investigated. The direct DNA-binding activity of Tipalpha was observed by pull down assay using single- and double-stranded genomic DNA cellulose. The surface plasmon resonance assay, indicating an association between Tipalpha and DNA, revealed that the affinity of Tipalpha for (dGdC)10 is 2400 times stronger than that of del-Tipalpha, an inactive Tipalpha. This suggests a strong correlation between DNA-binding activity and carcinogenic activity of Tipalpha. And the DNA-binding activity of Tipalpha was first demonstrated with a molecule secreted from H. pylori.     

Structural and functional characterization of galactooligosaccharides in Nostoc commune: beta-D-galactofuranosyl-(1-->6)-[beta-D-galactofuranosyl-(1-->6)]2-beta-D-1,4-anhydrogalactitol and beta-(1-->6)-galactofuranosylated homologues

A new class of galactooligosaccharides has been identified from the terrestrial cyanobacterium Nostoc commune by MS and NMR techniques. These consist of beta-D-galactofuranosyl-(1-->6)-[beta-D-galactofuranosyl-(1-->6)]n-beta-d-1,4-anhydrogalactitols with n ranging from 2 to 8, corresponding to compounds designated 1 through 7. In total these saccharides amounted to approximately 0.35% of the dry thallus of N. commune, while in several other cyanobacteria they were not detected. Possibly they play some role in protection from damage by heat and desiccation as suggested by experiments with heterologous systems. For example, phosphoglucomutase (EC 2.7.5.1) from rabbit muscle was protected against heat inactivation by these oligosaccharides, and alpha-amylase (EC 3.2.1.1) from porcine pancreas by the oligosaccharides 6 and 7. The homologues of lower molecular mass, however, enhanced heat sensitivity of alpha-amylase. The viability of Escherichia coli was completely abolished by desiccation, whereas in the presence of 4 survival rates were approximately 50% of controls not subjected to desiccation. The newly identified saccharides are compared with known galactofuranose-based oligo- and polysaccharides and possible biological functions of them are discussed.     

DNA damage in peripheral blood lymphocytes in patients during combined chemotherapy for breast cancer

Combined chemotherapy is used for the treatment of a number of malignancies such as breast cancer. The target of these antineoplastic agents is nuclear DNA, although it is not restricted to malignant cells. The aim of the present study was to assess DNA damage in peripheral blood lymphocytes (PBLs) of breast cancer patients subjected to combined adjuvant chemotherapy (5-fluorouracil, epirubicin and cyclophosphamide, FEC), using a modified comet assay to detect DNA single-strand breaks (SSB) and double-strand breaks (DSB).

Forty-one female patients with advanced breast cancer before and after chemotherapy and 60 healthy females participated in the study. Alkaline and neutral comet assays were performed in PBLs according to a standard protocol, and DNA tail moment was measured by a computer-based image analysis system.

Breast cancer patients before treatment had higher increased background levels of SSB and DSB as compared to healthy women. During treatment, a significant increase in DNA damage was observed after the 2nd cycle, which persisted until the end of treatment. Eighty days after the end of treatment the percentage of PBLs with SSB and DSB remained elevated, but the magnitude of DNA damage (tail moment) returned to baseline levels. There was no correlation between PBL DNA damage and response to chemotherapy.

DNA-SSB and DSB in PBLs are present in cancer patients before treatment and increase significantly after combined chemotherapy. No correlation with response to adjuvant chemotherapy was found. Biomonitoring DNA damage in PBLs of cancer patients could help prevent secondary effects and the potential risks of developing secondary cancers.