Isolation, cloning, sequencing of brain type aromatase and its expression in male and female Wrasse, Pseudolabrus sieboldi

Pseudolabrus sieboldi, wrasse being a diurnal spawner provides a good opportunity to study the endocrine mechanism of estrogen formation in brain and gonads. Moreover, an extremely large amount of E2 was produced in serum and testis of wrasse. It is assumed that the presence of E2 may play a major role in diurnal gametogenesis in male fish. In this study brain type aromatase have been isolated, cloned and sequenced from the brain of wrasse. Further, the expression pattern of brain type aromatase in gonads and adult tissue of male and female fish have been analyzed. In addition, the diurnal expression pattern of brain type aromatase in both male and female fish brain during spawning season have been analyzed. The P450arom (br) was isolated, cloned and sequenced from both male and female bamboleaf wrasse. The P450arom (br) gene (1877 sequenced nucleotide) contains an ORF of 1470 bp, a 5′-UTR of 18 bp and at least 407 bp in 3′-UTR. The amino acid sequence homology in the coding region of wrasse P450arom (br) is high compared to that of medaka, Oryzias latipes (80%), rainbow trout type 2, Oncorhynchu mykiss (78.2%), fugu, Takifugu ribripes (78%) rainbow trout type 1, (76%), goldfish, Carassius auratus (66.8%) and zebrafish, Danio rerio (66.2%). Expression study reveals that P450arom (br) mRNA were most abundant in brains of both male and female fish throughout the day during the spawning season. RT-PCR study revealed that P450arom (br) was expressed in skin, anal fin and tail fin of both male and female wrasse. P450arom (br) was not detected at any time of the spawning day in either ovary or testis of wrasse.     

人溶菌酶定点突变基因在毕赤酵母中的表达及活性分析

利用PCR法将人溶菌酶(h LYZ)成熟肽与载体信号肽连接处的氨基酸残基进行定点突变,突变基因亚克隆至酵母分泌型表达载体p PICZαA,重组载体线性化后通过电击转化法转化巴斯德毕赤酵母X-33,利用含Zeocine抗生素的YPDS平板筛选高拷贝转化子。转化子酵母经甲醇诱导后取培养基离心上清液进行SDS-PAGE和活性检测。结果显示,突变体h LYZ活力和表达量较野生型均有显著提高。表明定向改变h LYZ信号肽残基性质可以提高其表达量和分泌效率。     

天蚕素抗菌肽及其在动物生产中的应用

天蚕素抗菌肽具有提高动物生产性能、改善动物机体免疫力、维护动物肠道菌群平衡等多种生理功能。文章从天蚕素抗菌肽的来源、结构、作用机理、生物学功能及其在动物生产中的应用入手,并对其存在的问题和发展前景进行分析。     

Anti-Diabetic Effects of CTB-APSL Fusion Protein in Type 2 Diabetic Mice

To determine whether cholera toxin B subunit and active peptide from shark liver (CTB-APSL) fusion protein plays a role in treatment of type 2 diabetic mice, the CTB-APSL gene was cloned and expressed in silkworm (Bombyx mori) baculovirus expression vector system (BEVS), then the fusion protein was orally administrated at a dose of 100 mg/kg for five weeks in diabetic mice. The results demonstrated that the oral administration of CTB-APSL fusion protein can effectively reduce the levels of both fasting blood glucose (FBG) and glycosylated hemoglobin (GHb), promote insulin secretion and improve insulin resistance, significantly improve lipid metabolism, reduce triglycerides (TG), total cholesterol (TC) and low density lipoprotein (LDL) levels and increase high density lipoprotein (HDL) levels, as well as effectively improve the inflammatory response of type 2 diabetic mice through the reduction of the levels of inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Histopathology shows that the fusion protein can significantly repair damaged pancreatic tissue in type 2 diabetic mice, significantly improve hepatic steatosis and hepatic cell cloudy swelling, reduce the content of lipid droplets in type 2 diabetic mice, effectively inhibit renal interstitial inflammatory cells invasion and improve renal tubular epithelial cell nucleus pyknosis, thus providing an experimental basis for the development of a new type of oral therapy for type 2 diabetes.     

Antimicrobial Activity of Peptides Derived from Olive Flounder Lipopolysaccharide Binding Protein/Bactericidal Permeability-Increasing Protein (LBP/BPI)

We describe the antimicrobial function of peptides derived from the C-terminus of the olive flounder LBP BPI precursor protein. The investigated peptides, namely, ofLBP1N, ofLBP2A, ofLBP4N, ofLBP5A, and ofLBP6A, formed α-helical structures, showing significant antimicrobial activity against several Gram-negative bacteria, Gram-positive bacteria, and the yeast Candida albicans, but very limited hemolytic activities. The biological activities of these five analogs were evaluated against biomembranes or artificial membranes for the development of candidate therapeutic agents. Gel retardation studies revealed that peptides bound to DNA and inhibited migration on an agarose gel. In addition, we demonstrated that ofLBP6A inhibited polymerase chain reaction. These results suggested that the ofLBP-derived peptide bactericidal mechanism may be related to the interaction with intracellular components such as DNA or polymerase.     

Marine Antimicrobial Peptide TP4 Exerts Anticancer Effects on Human Synovial Sarcoma Cells via Calcium Overload,Reactive Oxygen Species Production and Mitochondrial Hyperpolarization

Synovial sarcoma is a rare but aggressive soft-tissue sarcoma associated with translocation t(X;18). Metastasis occurs in approximately 50% of all patients, and curative outcomes are difficult to achieve in this group. Since the efficacies of current therapeutic approaches for metastatic synovial sarcoma remain limited, new therapeutic agents are urgently needed. Tilapia piscidin 4 (TP4), a marine antimicrobial peptide, is known to exhibit multiple biological functions, including anti-bacterial, wound-healing, immunomodulatory, and anticancer activities. In the present study, we assessed the anticancer activity of TP4 in human synovial sarcoma cells and determined the underlying mechanisms. We first demonstrated that TP4 can induce necrotic cell death in human synovial sarcoma AsKa-SS and SW982 cells lines. In addition, we saw that TP4 initiates reactive oxygen species (ROS) production and downregulates antioxidant proteins, such as uncoupling protein-2, superoxide dismutase (SOD)-1, and SOD-2. Moreover, TP4-induced mitochondrial hyperpolarization is followed by elevation of mitochondrial ROS. Calcium overload is also triggered by TP4, and cell death can be attenuated by a necrosis inhibitor, ROS scavenger or calcium chelator. In our experiments, TP4 displayed strong anticancer activity in human synovial sarcoma cells by disrupting oxidative status, promoting mitochondrial hyperpolarization and causing calcium overload.     

Effects of ischemic postconditioning on neuronal Akt signaling pathways in hippocampus CA1 area after cerebral ischemia in tree shrews

AIM: To investigate the phosphatidylinositol 3-kinase/Akt (PI-3K/Akt) Ser-473/Thr-308/ phosphorylation (Akt /Akt ) and the intensity of the neurons in happocampus CA1 area under the conditions of thrombotic cerebral ischemia and postconditioning in tree shrews. METHODS: The thrombotic focal cerebral ischemia was induced by photochemical reaction in tree shrews. Two hundred and ten minutes after cerebral ischemia, ischemic postconditioning was established by repeated cliping of ipsilateral carotid. The distribution of Akt and Akt , and neuronal ultrastructure in hippocampus CA1 area were observed by the methods of electronic microscopy and immunohistochemistry. The phosphorylation intensity was measured by determining the optical gray value. RESULTS: The photochemical reaction induced cerebral ischemia and resulted in obvious lesions in hippocampus CA1 neurons. The damages of ultrastructure in the hippocampus were diminished by postconditioning. Correspondingly, in ischemia group, although the Akt showed positive during 72 h, the positive Akt was only observed at the time point of 4 h, and went negative at the time points of 24 h and 72 h. In postconditioning group, Akt at the time points of 4 h, 24 h and 72 h were positive,and Akt at the time points of 24 h and 72 h was also positive. CONCLUSION: Cerebral ischemia leads to neuron lesions in tree shrew hippocampus and the postconditioning decreases the damage. The Akt and Akt may play important roles in the protective mechanism.     

Early-onset type 1 diabetes causes brain injury and mitochondrial dysfunction

AIM:To explore the main risk factors of diabetic encephalopathy and to investigate the changes of mitochondria in early-onset type 1 diabetes. METHODS:Single dose of streptozotocin was injected through the femoral vein to establish type 1 diabetes model in rats. The levels of glucose, triglyceride and cholesterol in plasma, cerebrospinal fluid and cerebral cortex were measured. Oxidative and antioxidative status was evaluated by determining the levels of malondialdehyde and glutathione in the isolated mitochondria of cerebral cortex. Furthermore, the level of active AMP-activated protein kinase(AMPK) in the isolated mitochondria was detected by Western blotting. Degenerative neurons were identified by Fluo-Jade-C staining in serial brain sections. Autophagy-lysosome was observed under electron microscope. RESULTS:The main risk factor in the development of diabetic encephalopathy was hyperglucemia, which increased the Fluo-Jade-C positive neurons in the cerebral cortex of diabetic rats. The content of malondialdehyde was increased and glutathione was decreased in diabetic rats compared with the control animals. The activity of AMPK was lower in diabetic brain than that in normal brain. Aggregated autophagysome and mitochondria enveloped by autophagy-lysosome were observed in pyramidal cells of cerebral cortex in diabetic rats. CONCLUSION:Persistent hyperglycemia and mitochondrial dysfunction contribute to the diabetic encephalopathy at an early stage in type 1 diabetes, indicating that the mechanism may be partly related to the oxidative stress and activation of autophagy.     

Affecting factors of nylon monofilament induced focal cerebral ischemia in rats

AIM: To study factors affecting the stability of nylon monofilament for intraluminal middle cerebral artery occlusion in rats.METHODS:Successful rates and infarct volume of ischemic model were compared by polyvinyl alcohol (PVA) coated and silicon coated nylon monofilament intraluminal occlusion of middle cerebral artery under condition of ligation and without ligation of peterygopalatine artery.RESULTS:The successful rates were 60% and 53% in two groups under condition of ligation of peterygopalatine artery, and 20% and 27% in other two groups under condition of without ligation of peterygopalatine artery respectively. The intracranial length of nylon monofilament were about 7 mm in successful model and 4 mm in failure model. Animals in the PVA coated nylon monofilament group showed neurological dificit signs earlier, and had a significantly larger infarct volume at 12 hours of ischemia than those in the silicon coated group.CONCLUSIONS:The ligation of peterygopalatine artery is critical in the success of making this kind of ischemic model. The appropriate position of nylon monofilament entering crania and the good expansibility of PVA play an important role in occlusion of blood flow. Because of the relatively lower successful rate, a new substitute with unique quality, proper hardness and better elasticity needs to be explored in the future.     

Design and immune activity detection of long peptide vaccines targeting lung cancer antigen COX-2

AIM:To design long peptides based on cytotoxic T-lymphocytes (CTL) epitope prediction for lung cancer antigen cyclo-oxygenase-2 (COX-2) and to study the immune activity of the long peptides. METHODS:HLA-A2 epitopes from COX-2 protein were predicted by NetCTL 1.2, SYFPEITHI and IEDB. The CTL epitope-concentrated area was analyzed, and the appropriate length of long peptides were designed. In vitro activity experiments were used to verify the immune activity of the long peptides. ELISPOT assay and intracellular cytokine staining assay were used to investigate the ability of the peptide to induce specific restricted CTLs and release of interferon-γ (IFN-γ). The ability of the peptides to induce T-cell response was investigated by lactate dehydrogenase (LDH) and CFSE cytotoxicity assay in vitro. RESULTS:ELISPOT and intracellular cytokine staining assay showed P315-338 and P375-401 were able to induce specific CTLs and higher levels of IFN-γ release. The results of LDH and CFSE cytotoxicity assays showed the CTLs induced by P315-338 and P375-401 lysed the target cells. CONCLUSION:Two long peptides pointing to lung cancer antigen COX-2 are successfully identified, which could be used as immunotherapy vaccine in future.