鸡痘病毒282E4弱毒株TK基因限制性酶切图谱分析

以限制性内切酶ＢａｍＨＩ、Ｘｂａｌ、Ｃｌａｌ、Ｈ１ｎｄⅢ、Ｎｃａｌ对含有鸡痘病毒（ＦＰＶ）２８２Ｅ４弱毒株３．７ｋｂＨｉｎｄⅢＴＫ基因片段的重组质粒ｐＳＬ１进行单酶和它们之间双酶酶切。结果表明：３．７ｋｂＨｉｎｄⅢＴＫ基因片段上有２个Ｃｌａｌ切点，２个Ｘｂａｌ切点，１个Ｎｃａｌ切点，没有ＢａｍＨＩ切点。随后，用澳大利亚ＦＰＶ２．２ｋｂＨｉｎｄⅢ＋ＣｌａｌＴＫ基因作探针，对各种酶切片段进行Ｓｏｕｔｈｅｒｎ印迹杂交，进一步确定ＴＫ基因位于２．２ｋｂＨｉｎｄⅢ＋Ｃｌａｌ片段中。杂交结果与限制性酶切图谱分析结果相一致。该基因的限制性酶切图谱与澳大利亚ＦＰＶ疫苗株的基本相同。     

The response of phospholipase C/protein kinase C and adenylyl cyclase/protein kinase A pathways in porcine theca interna cells to opioid agonist FK 33-824

Opioids were found as factors affecting porcine ovarian steroidogenesis. The mechanism of opioid action, however, on porcine theca interna cells is completely unknown. Therefore, the present study was designed to investigate the possible involvement of two intracellular pathways, phospholipase C/protein kinase C and adenylyl cyclase/protein kinase A, in opioid signal transduction in porcine theca cells treated with mu opioid receptor agonist, FK 33-824. Incubation of the cells for 4 h with FK 33-824 at the dose 1 nM resulted in decreases in inositol phosphate accumulation as well as androstenedione (A(4)), testosterone (T), and estradiol (E(2)) secretions. Protein kinase C (PKC) inhibitors, staurosporine (1-100 nM), D-sphingosine (10-500 nM), and PKCi (100-2000 nM), both added alone and together with the opioid agonist, depressed release of the steroid hormones. PKC activator, phorbol ester (PMA, 1-100 nM), used alone was without effect on theca cell steroidogenesis, but added in combination with FK 33-824 abolished inhibitory influence of the opioid on A(4), T, and E(2) output. The steroid hormone secretion by PKC-deficient theca cells was inhibited by the opioid agonist. FK 33-824 also suppressed PKC activity reducing [(3)H]PDBu specific binding to theca cells, whereas ionomycin (a positive control) increased labeled phorbol ester binding to the cells. In the next experiment, cAMP release from theca cells during 2 and 4 h incubations with FK 33-824 (1-100 nM), naloxone (10 microM; opioid receptor antagonist), and LH (100 ng/mL; a positive control) was examined. FK 33-824 at the dose 1 nM inhibited cAMP secretion during 2 h incubation, but had no effect during longer incubation. LH in a manner independent on incubation time multiplied cAMP release. Protein kinase A inhibitor, PKAi (100-2000 nM), alone and in combination with FK 33-824 (1 nM), inhibited A(4), T, and E(2) secretions by theca cells. PKA activator, 8BrcAMP (10-1000 microM), stimulated the steroid hormone release, but this stimulatory effect was diminished in the presence of FK 33-824. The results allow to suggest that opioid peptides affect porcine theca cell steroidogenesis and their acute action on the cells is connected with the inhibition of phospholipase C/protein kinase C and adenylyl cyclase/protein kinase A signal transduction systems.     

A non-radiometric method for measuring serum thymidine kinase activity in malignant lymphoma in dogs

The aim of this study was to evaluate an enzyme-linked immunosorbent assay (ELISA), for determination of serum thymidine kinase 1 (sTK1) activity in dogs with malignant lymphoma (ML) and compare it with a thymidine kinase (TK) radioenzymatic assay (TK-REA). The TK-REA has recently been shown to be useful in determining the clinical stage and prognosis in canine ML. In addition, serum lactate dehydrogenase (LDH) was measured. Forty-five dogs were included in the study. Sixty serum samples from these dogs, stored in a tumour serum sample bank (stored at -20 degrees C), were analysed. Apart from 37 dogs with ML, four normal dogs as well as two dogs with mammary carcinomas, one dog with bladder carcinoma, and one dog with malignant fibrous histiocytoma were included. Staging of ML was based on the modified World Health Organization (WHO) staging system for canine ML. The diagnosis of all tumours was verified by histopathology. The TK activity (units per litre [U/L]) ranged from 1.0 to 607.9 in the TK-REA analysis and from 1.1 to 510 in the TK-ELISA (normal reference value <7U/L). The range for LDH was between 12 and 1194 U/L (normal reference value <228 U/L). There was a significant correlation between the TK-REA and the TK-ELISA. The correlation coefficient (CC) was 0.97 and the standard error of the estimate (SEE) was 3.7 U/L. There was no correlation between LDH and either the TK-REA or the TK-ELISA (CC=0.53 for both assays; SEE=26.7 and 12.7 U/L, respectively). Most of the variation in LDH was still within the normal reference range. The mean LDH in dogs with high-stage (stage IV+V) disease was 201.9 U/L. The corresponding values for the TK-REA and TK-ELISA were 109 and 109.9 U/L, respectively. The significant relation between the TK-REA and the TK-ELISA was confirmed by Bland-Altman analysis. The TK-ELISA assay, because of its relative simplicity, will permit measurement of TK in cases of ML in dogs to become a routine procedure.     

In vitro effect of recombinant human tumor necrosis factor-alpha on canine neutrophil apoptosis

Neutrophils (polymorphonuclear leukocytes: PMNs) are essential for the host defense against various infections and are often injurious to the host, causing inflammatory diseases where tumor necrosis factor-alpha (TNF-alpha) is suggested to play an important role. Since an effect of TNF-alpha on canine PMN apoptosis has not been studied, canine PMNs were stimulated with recombinant human (rh)TNF-alpha in the present study to investigate the effect of TNF-alpha on canine PMN apoptosis. PMN apoptosis and function to produce ROS were assessed by flow cytometry. Delayed apoptosis was observed in the PMNs treated with rhTNF-alpha at 100 ng/ml, accompanied by retention of capability to produce ROS. However, PMN apoptosis was accelerated by rhTNF-alpha combined with cycloheximide. Therefore, it is indicated that TNF-alpha is able to activate anti- and pro-apoptotic pathways in PMNs and that the inhibition of PMN apoptosis by TNF-alpha requires protein synthesis in the PMNs.     

双歧杆菌脂磷壁酸对胃癌SGC7901细胞SphK1表达的影响

SphK1 mRNA水平采用半定量RT-PCR检测;SphK1蛋白表达水平采用Western blot测定.研究双歧杆菌脂磷壁酸(LTA)对胃癌SGC7901细胞内鞘氨醇激酶1(SphK1)基因和蛋白表达的影响.结果表明,经200、500 μg· mL-1 LTA处理72 h后,SGC7901细胞内SphK1 mRNA...     

Effects of Astragalus polysaccharide on expression of HSP70, PKB and P53 in rat cerebral cortex with cerebral ischemia and reperfusion

AIM:To explore the molecular effects of Astragalus polysaccharide(AP) on improving nervous functions and preventing neuronal apoptosis in rat cerebral cortex with cerebral ischemia and reperfusion. METHODS:One hundred and twenty male Wister rats were randomly divided into sham operation group(SOG), model groups(MG-1 d, 3 d and 7 d), low-dose AP treatment groups(L-APTG-1 d, 3 d and 7 d), and high-dose AP treatment groups(H-APTG-1 d, 3 d and 7 d). The right middle cerebral artery of the rats in MG and AGTG was intercepted by operation to induce ischemic brain injury. The rats in L-APTG and H-APTG were treated with AP at the doses of 5 mg/kg and 15 mg/kg by intraperitoneal injection, respectively. On the 1st day, 3rd day and 7th day after operation, those animals were sacrificed to collect the brain specimens for the study after cerebral blood flow reperfusion and determination of neurological deficit scores. The structural changes of the neurons were observed under electron microscope. Apoptosis was analyzed by flow cytometry. The protein levels of heat-shock protein 70(HSP70), protein kinase B(PKB) and P53 in cerebral corical neurons were determined by immunohistochemical staining and Western blotting. RESULTS:The neurological deficit scores and the apoptotic rate of cerebral cortical neurons in H-APTG were significantly lower than those in MG and L-APTG(P<0.05). The structures of the neurons in H-APTG, such as ribosome endoplasmic reticulum, nucleolus, Golgi complex, mitochondria, etc, were better than those in MG and L-APTG. On the 1st day, 3rd day and 7th day, the protein levels of HSP70 and PKB in cerebral cortical neurons in H-APTG were significantly higher than those in L-APTG, which were significantly higher than those in MG(P<0.05). However, the P53 protein level in H-APTG was significantly lower than that in L-APTG, which was significantly lower than that in MG(P<0.05). CONCLUSION:AP improves nervous functions and inhibits neuronal apoptosis during ischemia and reperfusion. The molecular mechanisms are associated with variations of protein expression in cerebral cortical neurons, such as promotion of HSP70 and PKB and inhibition of P53.     

Effects of Dryk1A,ASF and alternative splicing of CaMKⅡδ on myocardial hypertrophy in renovascular hypertensive rats

AIM: To investigate the role of dual-specificity tyrosine phosporylation-regulated kinase 1A (Dyrk1A)-alternative splicing factor (ASF)-calcium/calmodulin-dependent protein kinase Ⅱδ (CaMK Ⅱδ) pathway in the progression of myocardial hypertrophy in renovascular hypertensive rats. METHODS: The renovascular hypertension was induced by two-kidney one-clip (2K1C) method. The changes of blood pressure and myocardial hypertrophy were measured. The techniques of RT-PCR and Western blotting were used to detect CaMKⅡδ alternative splicing and the protein expression of Dyrk1A and ASF, respectively. RESULTS: Eight weeks after operation, systolic blood pressure (SBP) and diastolic blood pressure (DBP) in 2K1C rats increased (P<0.05). The increases in left ventricular weight (LVW), the ratio of LVW to body weight (BW) and the area of myocardial cells indicated that the hypertensive rats developed significant cardiac hypertrophy. The protein expression of Dyrk1A and mRNA expression of CaMKⅡδA and δB were significantly increased, while the protein expression of ASF and mRNA expression of CaMKⅡδC were decreased compared with sham-operated control rats (P<0.05). Treatment with Dryk1A inhibitor epigallocatechin gallate (EGCG) or harmine effectively attenuated cardiac hypertrophy and reversed the changes in the protein expression of Dyrk1A, ASF and alternative splicing of CaMKⅡδ (all P<0.05). CONCLUSION: Dyrk1A-ASF-CaMKⅡδ pathway plays a role in the development of myocardial hypertrophy in renovascular hypertensive rats.     

植物甜菜碱醛脱氢酶的生物信息学分析

运用生物信息学的方法,对已在GenBank数据库中注册的甜菜、菠菜、山菠菜、玉米、辽宁碱蓬等植物甜菜碱醛脱氢酶(BADH)的氨基酸序列进行分析。结果表明:植物BADH属于稳定蛋白质,含有较丰富的丙氨酸和谷氨酸;不同植物BADH的氨基酸序列具有较高的同源性,含有与酶功能相关的十肽保守序列及半胱氨酸残基;不同植物的BADH具有不同的亚细胞定位;分子进化研究表明,BADH可作为植物遗传分化和分子进化研究的重要依据;氨基酸序列中不存在信号肽;分子中不存在跨膜结构域,可能受蛋白激酶的磷酸化;α?螺旋是多肽链中的主要结构元件;蛋白质保守区域包含典型的醛脱氢酶功能结构域。该研究结果为开展BADH的酶学特性及植物抗逆的分子机理研究提供理论参考。     

Up-regulation of Snail1 expression in renal tubular epithelial cells through Akt/GSK-3β pathway under high-glucose condition

AIM: To examine the effects of high glucose (HG) on the expression of Snail1 and protein kinase B (Akt)/glycogen synthase kinase 3β (GSK-3β) in primary renal tubular epithelial cells (RTECs). METHODS: The primary RTECs were randomly treated with normal glucose, high glucose or D-mannitol for 30 min~72 h. RT-PCR and Western blotting were used to observe the expression of Snail1, Akt and GSK-3β at mRNA and protein levels in these cells. The primary cultured RTECs were pretreated with LY294002 (a PI3K inhibitor, 25 μmol/L) to observe the specific inhibitory effects of phosphatidylinositol 3-kinase (PI3K) on HG-induced expression of Snail1 protein. RESULTS: Treatment of RTECs with HG resulted in increased mRNA and protein levels of Snail1, Akt1, and phosphorylation of Akt and GSK-3β. LY294002 blocked the HG-induced up-regulation of p-Akt, p-GSK-3β and Snail1 expression at protein level, but no effect of LY294002 was seen on the total protein expression of Akt1 and GSK-3β. HG did not affect the expression of GSK-3β at mRNA and protein levels. CONCLUSION: HG-induced up-regulation of Snail1 may be regulated by Akt/GSK-3β pathway in RTECs.     

Artesunate negatively controls expression of cyclin D1 and activity of AP-1 by inhibiting the activation of ERK1/2 protein in HSC-T6 cells

AIM: To investigate the effects of artesunate(Art) on the expression of ERK1/2, AP-1 and cyclin D1 in rat hepatic stellate cells (HSCs), and to elucidate the molecular mechanism of Art against hepatic fibrosis. METHODS: HSC-T6 cells were treated with platelet-derived growth factor BB(PDGF-BB) to induce cell proliferation. The cells were divided into control group, PDGF-BB group, PDGF-BB+Art groups (with 6.25 mg稬^-1, 25 mg稬^-1or 50 mg稬^-1 of Art) and PDGF-BB+PD98059 group. The level of collagen type I in the supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of ERK1/2 and cyclin D1 were measured by RT-PCR. The protein levels of p-ERK1/2 and cyclin D1 in HSC-T6 cells were detected by Western blotting. The activity of AP-1 was analyzed by electrophoretic mobility shift assay. RESULTS: The concentration of collagen type I was significantly higher in PDGF-BB group than that in control group (P<0.05), and decreased in PDGF-BB+Art group and PDGF-BB+PD98059 group in comparison with that in PDGF-BB group (P<0.05, P<0.01). The protein level of ERK1/2 in PDGF-BB+Art group (50 mg稬^-1) was lower than that in PDGF-BB group (P<0.05), and was even lower in PDGF-BB+PD98059 group (P<0.01). The mRNA expression of cyclin D1 in PDGF-BB+Art groups (25 mg稬^-1and 50 mg稬^-1) and PDGF-BB+PD98059 group were significantly lower than that in PDGF-BB group (P<0.05). The protein levels of p-ERK1/2 and cyclinD1 were the highest in PDGF-BB group, and significantly lower in PDGF-BB+Art groups (6.25 mg稬^-1, 25 mg稬^-1 and 50 mg稬^-1) and PDGF-BB+PD98059 group (P<0.05, P<0.01). The AP-1 binding activity in HSC-T6 cells was down-regulated by Art. CONCLUSION: Artesunate inhibits the proliferation of HSC-T6 cells in vitro by inhibiting the activation of ERK1/2, thus down-regulating the activity of AP-1 and expression of cyclin D1.