A novel in vitro co-culture model to examine contact formation between astrocytic processes and cerebral vessels

Here, we developed a novel in vitro co-culture model, in which process-bearing astrocytes and isolated cerebral microvessels from mice were co-cultured. Astrocytes formed contacts with microvessels from both adult and neonatal mice. However, concentrated localization of the immunofluorescence signal for aquaporin-4 (AQP4) at contact sites between perivascular endfoot processes and blood vessels was only detected with neonatal mouse microvessels. Contact between astrocytic processes and microvessels was retained, whereas concentrated localization of AQP4 signal at contact sites was lost, by knockdown of dystroglycan or α-syntrophin, reflecting polarized localization of AQP4 at perivascular regions in the brain. Further, using our in vitro co-culture model, we found that astrocytes predominantly extend processes to pericytes located at the abluminal surface of microvessels, providing additional evidence that this model is representative of the in vivo situation. Altogether, we have developed a novel in vitro co-culture model that can reproduce aspects of the in vivo situation and is useful for assessing contact formation between astrocytes and blood vessels.     

灰飞虱内生病毒HiPV外壳蛋白VP1多克隆抗体的制备及在病毒检测中的应用

【目的】前期发现水稻条纹病毒(rice stripe virus, RSV)可与介体灰飞虱Laodelphax striatellus体内的HiPV病毒(Himetobi P virus, HiPV)互作。本研究旨在制备HiPV外壳蛋白VP1的多克隆抗体,并评估其在HiPV病毒检测中的可用性,以为深入研究HiPV-RSV和HiPV-灰飞虱的互作机制提供技术支持。【方法】以RT-PCR方法从灰飞虱成虫体内扩增HiPV主要外壳蛋白基因VP1,然后将VP1基因亚克隆至原核表达载体pET-32a中,构建表达载体pET-VP1。将重组质粒转化大肠杆菌Escherichia coli BL21 (DE3),经IPTG诱导、Ni²⁺-NTA亲和层析纯化,获得重组蛋白,免疫新西兰大白兔,制备抗体。【结果】从灰飞虱体内克隆到774 bp的HiPV外壳蛋白基因VP1,经原核表达、纯化,获得分子量约47.5 kD的融合蛋白,免疫新西兰大白兔后获得VP1多克隆抗体。该抗体间接ELISA效价达1∶819 200,与HiPV外壳蛋白VP1有特异性反应,而与灰飞虱蛋白无交叉反应。利用该多克隆抗体建立了检测单头灰飞虱成虫体内HiPV的Western blot和免疫捕获RT-PCR方法,检测结果显示HiPV在携带和不携带RSV的灰飞虱高亲和性群体内均广泛存在。【结论】利用制备的HiPV的VP1多克隆抗体可特异性检测灰飞虱体内HiPV。本研究为HiPV病毒的快速检测以及HiPV-RSV互作、HiPV-灰飞虱互作研究提供了技术支持。     

除草剂2,4-D特异性多克隆抗体的制备及鉴定

目的是制备特异性的2,4-D多克隆抗体.用活性酯和混合酸酐法将半抗原2,4-D分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)共价偶联;以2,4-D-BSA作免疫原免疫两只新西兰白兔;以2,4-D-OVA包被96孔酶标板,用间接酶联免疫吸附分析(ELISA)法检测抗血清效价和竞争性ELISA法检测2,4-D.结果显示2,4-D与BSA和OVA的偶联比率分别为32∶1和18∶1,抗血清效价＞6.4×105,在优化条件下2,4-D的最低检测限达37ng/mL.实验成功地制备了2,4-D特异性多克隆抗体,为其ELISA方法的建立提供了条件.     

Special Features of the DNA-Hydrolyzing Activity of the Antibodies in Systemic Lupus Erythematosus

Two types of IgG anti-DNA antibodies exhibiting DNA-hydrolyzing activity have been isolated from blood serum of patients with systemic lupus erythematosus. This DNase activity of antibodies differs from serum DNases by the non-processive mode, temperature resistance, pH optimum, and the rate of DNA hydrolysis. It is suggested that the anti-DNA antibody molecule possessing DNase activity contains two sites: one site determines specificity of antibody-DNA interaction, whereas the other is responsible for manifestation of the catalytic activity.     

Evidence that the TRP-1 protein is unlikely to account for store-operated Ca2+ inflow in Xenopus laevis oocytes

The role of the TRP-1 protein, an animal cell homologue of the Drosophila transient receptor potential Ca²⁺ channel, in store-operated Ca²⁺ inflow in Xenopus laevis oocytes was investigated. A strategy involving RT-PCR and 3 and 5 rapid amplification of cDNA ends (RACE) was used to confirm and extend previous knowledge of the nucleotide and predicted amino acid sequences of Xenopus TRP-1 (xTRP-1). The predicted amino acid sequence was used to prepare an anti-TRP-1 polyclonal antibody which detected the endogenous oocyte xTRP-1 protein and the human TRPC-1 protein expressed in Xenopus oocytes. Ca²⁺ inflow (measured using fura-2) initiated by 3-deoxy-3-fluoroinositol 1,4,5-trisphosphate (InsP₃F) or lysophosphatidic acid (LPA) was completely inhibited by low concentrations of lanthanides (IC₅₀ = 0.5 M), indicating that InsP₃F and LPA principally activate store-operated Ca²⁺ channels (SOCs). Antisense cRNA or antisense oligodeoxynucleotides, based on different regions of the xTRP-1 cDNA sequence, when injected into Xenopus oocytes, did not inhibit InsP₃F-, LPA- or thapsigargin-stimulated Ca²⁺ inflow. Oocytes expressing the hTRPC-1 protein, which is 96% similar to xTRP-1, exhibited no detectable enhancement of either basal or InsP₃F-stimulated Ca²⁺ inflow and only a very small enhancement of LPA-stimulated Ca²⁺ inflow compared with control oocytes. It is concluded that the endogenous xTRP-1 protein is unlikely to be responsible for Ca²⁺ inflow through the previously-characterised Ca²⁺-specific SOCs which are found in Xenopus oocytes. It is considered that xTRP-1 is likely to be a receptor-activated non-selective cation channel such as the channel activated by maitotoxin.     

Studies on a Nigerian isolate of banana streak badnavirus: I. Purification and enzyme linked immunosorbent assay

A Nigerian isolate of banana streak badnavirus (BSV) was purified and a polyclonal antiserum was produced in mice. The antiserum titre was between 1:10 000 and 1:40 000 in enzyme linked immunosorbent assay (ELISA), and showed a good specificity to BSV antigens. Comparative tests were carried out to determine the sensitivity and reliability of BSV antigen detection by double antibody sandwich (DAS)-ELISA, triple antibody sandwich (TAS)-ELISA, antigen coated plate (ACP)-ELISA, and protein-A coated antibody sandwich (PAS)-ELISA. TAS-ELISA using rabbit polyclonal antiserum to trap BSV and mouse polyclonal antiserum to detect the virus particles, was more sensitive than ACP-ELISA and PAS-ELISA and detected BSV in plant extracts from both symptomatic and some asymptomatic plants. However, immunosorbent electron microscopy detected more BSV-infected plants from asymptomatic plant samples than did TAS-ELISA. Results of this study showed that detection of BSV antigens in sap extracts by TAS-ELISA was most efficient with symptomatic tissues which occurred most frequently in the ‘cool rainy’ season. This suggests that for more reliable BSV-indexing of field samples, tissue sampling should be done during the rainy season when most BSV-infected plants express severe symptoms.     

Endothelial connexin32 enhances angiogenesis by positively regulating tube formation and cell migration

The gap junction proteins connexin32 (Cx32), Cx37, Cx40, and Cx43 are expressed in endothelial cells, and regulate vascular functions involving inflammation, vasculogenesis and vascular remodeling. Aberrant Cxs expression promotes the development of atherosclerosis which is modulated by angiogenesis; however the role played by endothelial Cxs in angiogenesis remains unclear. In this study, we determined the effects of endothelial Cxs, particularly Cx32, on angiogenesis. EA.hy926 cells that had been transfected to overexpress Cx32 significantly increased capillary length and the number on branches compared to Cx-transfectant cells over-expressing Cx37, Cx40, and Cx43 or mock-treated cells. Treatment via intracellular transfer of anti-Cx32 antibody suppressed tube formation of human umbilical vein endothelial cells (HUVECs) compared to controls. In vitro wound healing assays revealed that Cx32-transfectant cells significantly increased the repaired area while anti-Cx32 antibody-treated HUVECs reduced it. Ex vivo aorta ring assays and in vivo matrigel plaque assays showed that Cx32-deficient mice impaired both vascular sprouting from the aorta and cell migration into the implanted matrigel. Therefore endothelial Cx32 facilitates tube formation, wound healing, vascular sprouting, and cell migration. Our results suggest that endothelial Cx32 positively regulates angiogenesis by enhancing endothelial cell tube formation and cell migration.     

Isolation of a hemidesmosome-rich fraction from a human squamous cell carcinoma cell line

Hemidesmosomes are cell-to-matrix adhesion complexes anchoring keratinocytes to basement membranes. For the first time, we present a method to prepare a fraction from human cultured cells that are highly enriched in hemidesmosomal proteins. Using DJM-1 cells derived from human squamous cell carcinoma, accumulation of hemidesmosomes was observed when these cells were cultured for more than 10 days in a commercial serum-free medium without supplemental calcium. Electron microscopy demonstrated that numerous electron-dense adhesion structures were present along the basal cell membranes of DJM-1 cells cultured under the aforementioned conditions. After removing cellular materials using an ammonia solution, hemidesmosomal proteins and deposited extracellular matrix were collected and separated by electrophoresis. There were eight major polypeptides, which were determined to be plectin, BP230, BP180, integrin α6 and β4 subunits, and laminin-332 by immunoblotting and mass spectrometry. Therefore, we designated this preparation as a hemidesmosome-rich fraction. This fraction contained laminin-332 exclusively in its unprocessed form, which may account for the promotion of laminin deposition, and minimal amounts of Lutheran blood group protein, a nonhemidesmosomal transmembrane protein. This hemidesmosome-rich fraction would be useful not only for biological research on hemidesmosomes but also for developing a serum test for patients with blistering skin diseases.     

PHD finger protein 8蛋白多克隆抗体的制备、纯化与检测

PHD finger8(PHF8)蛋白是最新发现的一种带有PHD结构域和Jmjc结构域的蛋白。现有研究表明其可能在基因转录、组蛋白去甲基化等方面发挥重要作用。为研究其功能,本研究构建原核表达载体pET41b-PHF8(aa886-936),在大肠杆菌Escherichia coli BL21中诱导表达带有GST标签的PHF8(aa886-936)亲水片段融合蛋白,并纯化该片段作为抗原免疫家兔,再以CNBr活化Sepharose4B微珠纯化抗血清制备PHF8特异性多克隆抗体。Western blotting以及免疫荧光检测表明该抗体具有很好的特异性,同时免疫荧光染色的结果也表明PHF8蛋白定位于细胞核。     

肺炎链球菌假想蛋白SPD0873的表达纯化及保守性分析

目的通过构建原核表达载体,获得纯化的肺炎链球菌S．pn重组假想蛋白SPD0873,并制备多克隆抗体,进一步分析其在常见S．pn菌株中的保守性。方法分离培养D39型肺炎链球菌,获取其基因组DNA。利用PCR方法扩增去除信号肽的spd0873序列,采用基因体外重组法将spd0873序列克隆到原核表达载体pET-32（a）内,测序鉴定。将重组质粒转化到E．coli Rossetta（DE3）中,经IPTG诱导大量表达融合6个组氨酸标签的SPD0873重组蛋白,经Ni—NTA树脂纯化后,获得的重组蛋白用SDS—PAGE和Western印迹鉴定;将鉴定后纯化的蛋白免疫BALB／C小鼠制备多克隆抗体,并用间接ELISA检测多克隆抗体的效价,Western印迹方法分析多克隆抗体的特异性,同时,鉴定该蛋白在5种常见肺炎链球菌分离株的保守性。结果克隆的spd0873序列与GenBank中的数据相符,并实现了SPD0873蛋白高水平的可溶表达。纯化蛋白免疫BALB／C小鼠获得高滴度、高特异性的的多克隆抗体,Western印迹验证SPD0873蛋白在5株常见肺炎链球菌菌株中均有表达。结论成功制备了高滴度、高特异性的SPD0873蛋白多克隆抗体,同时,检测到SPD0873蛋白在5种常见的肺炎链球菌菌株中非常保守,为研究该蛋白的生物学功能及肺炎链球菌多肽联合疫苗的研制奠定了基础。