大鼠BDNF基因真核表达载体的构建

从23日龄大鼠脑组织中抽提总RNA,应用RT-PCR技术扩增脑源性神经营养因子(BDNF)基因片段,将此片段克隆入T载体,酶切反应鉴定。然后双酶切BDNF-T载体和空质粒pEGFP(N1),将所获目的片段和线性空载体用T4 DNA连接酶连接,构建真核表达载体pEGFP(N1)-BDNF,并进行酶切反应鉴定及DNA测序。序列测定的结果与GenBank比较,所克隆的BDNF基因从起始密码子ATG到终止密码子TAG全长共750 bp序列完全相同。结果表明成功构建真核表达载体pEGFP(N1)-BDNF,为进一步研究BDNF基因表达,神经系统疾病治疗和生物制药奠定基础。     

Expression of tissue factor in experimental bovine pneumonic pasteurellosis and endotoxemia

Tissue factor (TF), a cell surface-associated cofactor and activator of coagulation factor VII, has been implicated in the local and systemic activation of coagulation associated with sepsis. This study describes the pattern of TF expression in experimental bovine pneumonic pasteurellosis and endotoxemia. Immunohistochemical techniques were used to localize TF antigen in tissue sections. Tissue factor expression was not observed in tissues from control animals. In response to Pasteurella haemolytica challenge, TF was expressed within alveolar walls, by mononuclear inflammatory cells within alveoli, and in walls of arteries, arterioles, bronchi, and bronchioles. Tissue factor was not detected in unaffected lung, liver, spleen, lymph node or kidney tissue. Administration of Escherichia coli endotoxin intravenously resulted in tissue factor expression in lung, spleen, and lymph node tissue. Results of this study indicate that TF is expressed locally at sites of inflammation and systemically in endotoxemia. Therefore, TF may be involved in coagulation events associated with pneumonic pasteurellosis.     

N-acetylserotonin increases cell proliferation and differentiating neuroblasts with tertiary dendrites through upregulation of brain-derived neurotrophic factor in the mouse dentate gyrus

In this study, we investigated the effects of N-acetylserotonin (NAS) on cell proliferation and neuroblast differentiation in the mouse dentate gyrus using anti-Ki67 and anti-doublecortin (DCX) antibodies. Ki67 is expressed in the nucleus or on the surface of chromosomes during all of the active phases of the cell cycle, and DCX is expressed in neuronal precursor cells as well as in immature neurons. At 17 weeks of age, 20 mg/kg of NAS or the same volume of vehicle was intraperitoneally administered once a day for 3 weeks. The animals were sacrificed 2 hr after the last vehicle or NAS treatment. NAS treatment significantly increased the number of Ki67-positive nuclei and DCX-immunoreactive neuroblasts with well-developed dendrites (tertiary dendrites) compared to the vehicle-treated group. However, the number of DCX-immunoreactive neuroblasts without tertiary dendrites was not changed. The administration of NAS also significantly increased the protein levels of brain-derived neurotrophic factor (BDNF) in the dentate gyrus. This result suggests that NAS significantly promotes cell proliferation and the number of differentiating neuroblasts with tertiary dendrites through an increase in BDNF levels in the mouse dentate gyrus.     

Expression and characterization of a recombinant soluble form of bovine tumor necrosis factor receptor type I

A recombinant soluble bovine tumor necrosis factor receptor type I (sboTNF-RI) was expressed in the methylotrophic yeast Pichia pastoris and evaluated for its ability to inhibit bovine tumor necrosis factor alpha (TNF-alpha) cytotoxicity. A cDNA encoding the extracellular domain of bovine TNF-RI was placed under the control of the powerful and tightly regulated alcohol oxidase1 (AOX1) gene promoter of the pPICZa A vector and the resulting construct integrated into the 5' region of the alcohol oxidase genes of GS115 and KM71 strains of Pichia. Soluble bovine TNF-RI was secreted into the medium following induction of the AOX1 gene promoter with methanol, and purified to greater than 95% purity by ion-exchange chromatography. In in vitro assays, the purified recombinant sboTNF-RI will block the cytolytic activity of bovine TNF-alpha on WEHI 164 cells clone 13 by 50% when used at a concentration of 170 microg/ml, and by nearly 90% when used at a concentration of 310 microg/ml. Results of this study suggest that recombinant sboTNF-RI may have therapeutic value as a TNF inhibitor in cattle with coliform mastitis.     

Expression, purification, and in vitro biological activities of recombinant bovine granulocyte-colony stimulating factor

Neutrophils are essential components of the innate immune system and they play a critical role in the defense of host against bacterial and fungal infections. The colony stimulating factors are a class of glycoproteins that are required for proliferation, differentiation, and functional activation of hematopoietic progenitor cells. Granulocyte-colony stimulating factor (G-CSF) is a member of this regulatory family of cytokines that specifically stimulates proliferation and maturation of precursor cells in the bone marrow into fully differentiated and functional neutrophils. G-CSF also modulates the biological activities of mature neutrophils in circulation. A bovine G-CSF (bG-CSF) cDNA clone (previously isolated and sequenced in our laboratory) was expressed in Escherichia coli and the biological activities of the solubilized protein from purified inclusion bodies were examined. Flow cytometric analysis of membrane antigen density of neutrophils activated with bG-CSF revealed an upregulation in the expression of CD11a (>114%), CD11b (>148%), CD11c (>87%), and CD18 (>109%). Expression of L-selectin was decreased by more than 43%. There was no change, however, in the expression of CD14. These findings indicate that recombinant bG-CSF (rbG-CSF) expressed in E. coli is biologically active and exerts the same type of effects on neutrophils in vitro as those of human G-CSF (hG-CSF).     

Olfactory ganglioneuroblastoma in a dog: case report and literature review

A 7-y-old, intact male Alaskan Malamute was presented with a 3-mo history of stertor and epistaxis. Computed tomography of the skull revealed generalized loss of gas throughout both nasal passages with replacement by a soft tissue mass that traversed the cribriform plate. Histopathology revealed neoplastic neuroblast cells arranged in anastomosing cords, as well as separately located aggregates of ganglion cells. Both neoplastic cell populations demonstrated immunoreactivity to MAP-2, TuJ-1, and synaptophysin. Neuroblastic cells additionally exhibited punctate immunoreactivity to MCK and CK8/18. We document here both the positive neural immunohistochemical markers for this neoplasm, as well as propose possible histomorphologic variants.     

Electroacupuncture at the Zusanli and Baihui acupoints ameliorates type-2 diabetes-induced reductions in proliferating cells and differentiated neuroblasts in the hippocampal dentate gyrus with increasing brain-derived neurotrophic factor levels

In the current study, we investigated whether electroacupuncture (EA) can inhibit pathological reductions in neurogenesis. Zucker diabetic fatty (ZDF) rats at 7 weeks of age were anesthetized with zoletil, and sham-acupuncture or EA at the Zusanli (ST36) and Baihui (GV20) acupoints was administered once a day for 5 weeks. In the ZDF group that received sham-EA (ZDF-Sham group), the blood glucose level was significantly increased together with age as compared to the control littermates [Zucker lean control (ZLC) rat]. In contrast, proliferating cells and differentiated neuroblasts were significantly decreased in the ZDF-Sham group compared to the ZLC group. Although EA treatment decreased blood glucose levels, this was not statistically significant when compared to blood glucose levels changes in the ZDF-Sham group. However, proliferating cells and differentiated neuroblasts were significantly increased with EA in ZDF rats as compared to those in the ZDF-Sham group. Brain-derived neurotrophic factor (BDNF) levels were significantly decreased in hippocampal homogenates of ZDF-Sham group compared to those in the ZLC group. The EA treatment significantly increased the BDNF levels compared to those in the ZDF-Sham group, and BDNF levels in this group were similar to those in the ZLC group. These results suggest that EA at ST36 and GV20 can ameliorate the reductions in proliferating cells and differentiated neuroblasts in the dentate gyrus induced by type-2 diabetes without significantly reducing blood glucose levels with increasing BDNF levels.     

Expression of Kit, the receptor for stem cell factor, in bovine peripheral blood

The expression of Kit, the receptor for stem cell factor (SCF), on bovine peripheral blood cells (PBCs) was examined by using monoclonal antibodies against the bovine Kit protein. Flow cytometric analysis showed that approximately 1.5% of PBCs expressed Kit. In cytospin preparations, the morphology of most Kit+ PBCs was similar to that of large lymphocytes. Subsets of Kit+ PBCs coexpressed CD3, IgM, and/or CD11b but not CD14 or G1. SCF did not induce the proliferation of Kit+ PBCs in vitro. These results indicate that Kit is expressed on subsets of lymphocytes in bovine peripheral blood, but the ligand of Kit, SCF, does not directly induce the proliferation of this cell population.     

牛碱性成纤维细胞生长因子基因在大肠杆菌中的表达及其表达产物的生物学活性

采用巢式PCR方法克隆了牛18ku-bFGF基因完整的编码序列,并构建了原核表达载体pET-28a-bFGF,将其转化大肠杆菌BL21,在25℃低温条件下,用0.5 mmol/L IPTG诱导表达5 h,用Ni-NTA亲和纯化细胞裂解上清液,经Western-blotting检测,结果显示,在特定的诱导条件下,重组牛bFGF基因在大肠杆菌中获得了表达,并且主要以可溶性状态存在于细胞中。经检测,纯化后的重组蛋白能显著促进成纤维细胞的增殖（P〈0.05）,其活性与商品用重组人18ku-bFGF没有差异（P〉0.05）。表明,所获得的可溶性重组牛18ku-bFGF蛋白具有较高的生物学活性,可用于后续研究工作。     

Expression of a bioactive bovine interleukin-12 using baculovirus

Recombinant baculoviruses that express recombinant bovine interleukin-12 (rboIL-12) subunits, p35 and p40 subunits were constructed. A recombinant virus containing the p40 subunit gene expressed the p40 subunit as a 40kDa monomer and an 80kDa disulfide-linked homodimer in the infected insect cells and in the culture supernatant. The p35 subunit was expressed in a 30kDa monomer in the infected cells but not in the supernatant. Superinfection of both recombinant viruses into the cells in a spinner flask resulted in the formation of a 70kDa disulfide-bonded heterodimer detected in the supernatant by immunoblotting using anti-p40 and anti-p35 subunits antibodies. The superinfected culture supernatant showed induction of IFNgamma mRNA synthesis and IFNgamma production in bovine peripheral blood mononuclear cells. Thus, the bioactive rboIL-12 was produced in large scale using a baculovirus expression system.     

Quantitation of bovine macrophage colony-stimulating factor in bovine serum by ELISA

We established an enzyme-linked immunosorbent assay (ELISA) system for the quantitation of bovine macrophage colony-stimulating factor (M-CSF) and used it to measure the serum M-CSF levels in bovine fetuses and calves. The average serum M-CSF level was 2.7+/-1.5 ng/ml in 39 calves under 100 days old, and 1.8+/-0.8 ng/ml in 15 cattle between 101 and 418 days old. Fetal sera samples (n = 6) prepared from cattle between 150 and 280 days of gestational age had a higher average level of M-CSF (8.8+/-1.4 ng/ml). Alteration in serum M-CSF levels in each individual calf was also measured. The serum levels of M-CSF in calves at 0-1 day after birth ranged from 0.52 to 7.3 ng/ml. During the period 113-125 days after birth, serum levels were around 1.4+/-0.39 ng/ml. Although serum M-CSF levels generally decreased as the age of calves advanced, differences among individuals, especially among newborn calves, were observed.     

Expression of a 50 kDa putative receptor for bovine viral diarrhea virus in bovine fetal tissues.

The expression of a 50 kDa bovine viral diarrhea virus putative receptor in different bovine fetal tissues from 3-month old fetuses was studied. The receptor expression was examined by immunocytochemical staining and by immunoblotting using antiidiotypic probe (anti-D89). Intense specific staining in enterocytes of the small and large intestines, cortical tubular epithelial cells of kidneys, respiratory epithelial cells of the trachea and esophageal mucosal epithelial cells was observed, demonstrating the strong expression of bovine viral diarrhea virus receptor in the tissues. Weak staining was found in cerebellum, thymus, spleen, liver, cerebrum, and lung tissues; however, heart tissues were negative. Immunoblotting results correlated with the immunoperoxidase staining assays. Thus, the expression levels of the receptor are variable in different tissues. This pattern of expression may provide clues to the pathogenic potential of bovine viral diarrhea virus in the bovine fetus.