粟酒裂殖酵母N-糖酰胺酶在大肠杆菌中的表达、纯化及活性分析

根据GenBank中公布的粟酒裂殖酵母(Schizosaccharomyces pombe)N-糖酰胺酶(Png1p)cDNA序列, 设计并合成一对特异性引物, 利用RT-PCR技术从粟酒裂殖酵母中克隆出糖酰胺酶cDNA。将得到的基因克隆到表达载体pET-15b中。重组质粒转入大肠杆菌BL21(DE3)中, 经诱导表达和纯化提取后, 进行酶活测定。实验结果表明, 该酶的分子量约为39 kD, 纯化后的重组N-糖酰胺酶可以对变性处理的糖蛋白进行糖链的切除, 且这种作用需要还原剂DTT的辅助作用; N-糖酰胺酶只对错误折叠的糖蛋白有作用, 对天然的糖蛋白没有作用。等量粟酒裂殖酵母Png1p在不同温度、pH、DTT浓度和底物变性温度下对等量核糖核酸酶B(RNase B)的脱糖基化检测发现, 重组酶的最适反应温度30°C, 最适反应pH为7.0, 需要的最适DTT浓度为10 mmol/L, 底物在100°C处理10 min时酶的脱糖基化率最高。     

Identification of metabolic fluxes in hepatic cells from transient 13C-labeling experiments: Part I. Experimental observations

An experimental set-up for acquiring metabolite and transient (13)C-labeling data in mammalian cells is presented. An efficient sampling procedure was established for hepatic cells cultured in six-well plates as a monolayer attached to collagen, which allowed simultaneous quenching of metabolism and extraction of the intracellular intermediates of interest. Extracellular concentrations of glucose, amino acids, lactate, pyruvate, and urea were determined by GC-MS procedures and were used for estimation of metabolic uptake and excretion rates. Sensitive LC-MS and GC-MS methods were used to quantify the intracellular intermediates of tricarboxylic acid cycle, glycolysis, and pentose phosphate pathway and for the determination of isotopomer fractions of the respective metabolites. Mass isotopomer fractions were determined in a transient (13)C-labeling experiment using (13)C-labeled glucose as substrate. The absolute amounts of intracellular metabolites were obtained from a non-labeled experiment carried out in exactly the same way as the (13)C-labeling experiment, except that the media contained naturally labeled glucose only. Estimation of intracellular metabolic fluxes from the presented data is addressed in part II of this contribution.     

Cyclophilin A binds to AKT1 and facilitates the tumorigenicity of Epstein-Barr virus by mediating the activation of AKT/mTOR/NF-κB positive feedback loop

The AKT/mTOR and NF-κB signalings are crucial pathways activated in cancers including nasopharyngeal carcinoma (NPC), which is prevalent in southern China and closely related to Epstein-Barr virus (EBV) infection. How these master pathways are persistently activated in EBV-associated NPC remains to be investigated. Here we demonstrated that EBV-encoded latent membrane protein 1 (LMP1) promoted cyclophilin A (CYPA) expression through the activation of NF-κB. The depletion of CYPA suppressed cell proliferation and facilitated apoptosis. CYPA was able to bind to AKT1, thus activating AKT/mTOR/NF-κB signaling cascade. Moreover, the use of mTOR inhibitor, rapamycin, subverted the activation of the positive feedback loop, NF-κB/CYPA/AKT/mTOR. It is reasonable that LMP1 expression derived from initial viral infection is enough to assure the constant potentiation of AKT/mTOR and NF-κB signalings. This may partly explain the fact that EBV serves as a tumor-promoting factor with minimal expression of the viral oncoprotein LMP1 in malignancies. Our findings provide new insight into the understanding of causative role of EBV in tumorigenicity during latent infection.     

The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

In mouse intestine, caveolae and caveolin‐1 (Cav‐1) are present in smooth muscle (responsible for executing contractions) and in interstitial cells of Cajal (ICC; responsible for pacing contractions). We found that a number of calcium handling/dependent molecules are associated with caveolae, including L‐type Ca²⁺ channels, Na⁺‐Ca²⁺ exchanger type 1 (NCX1), plasma membrane Ca²⁺ pumps and neural nitric oxide synthase (nNOS), and that caveolae are close to the peripheral endo‐sarcoplasmic reticulum (ER‐SR). Also we found that this assemblage may account for recycling of calcium from caveolar domains to SR through L‐type Ca ⁺ channels to sustain pacing and contractions. Here we test this hypothesis further comparing pacing and contractions under various conditions in longitudinal muscle of Cav‐1 knockout mice (lacking caveolae) and in their genetic controls. We used a procedure in which pacing frequencies (indicative of functioning of ICC) and contraction amplitudes (indicative of functioning of smooth muscle) were studied in calcium‐free media with 100 mM ethylene glycol tetra‐acetic acid (EGTA). The absence of caveolae in ICC inhibited the ability of ICC to maintain frequencies of contraction in the calcium‐free medium by reducing recycling of calcium from caveolar plasma membrane to SR when the calcium stores were initially full. This recycling to ICC involved primarily L‐type Ca²⁺ channels; i.e. pacing frequencies were enhanced by opening and inhibited by closing these channels. However, when these stores were depleted by block of the sarco/endoplasmic reticulum Ca²⁺‐ATPase (SERCA) pump or calcium release was activated by carbachol, the absence of Cav‐1 or caveolae had little or no effect. The absence of caveolae had little impact on contraction amplitudes, indicative of recycling of calcium to SR in smooth muscle. However, the absence of caveolae slowed the rate of loss of calcium from SR under some conditions in both ICC and smooth muscle, which may reflect the loss of proximity to store operated Ca channels. We found evidence that these channels were associated with Cav‐1. These changes were all consistent with the hypothesis that a reduction of the extracellular calcium associated with caveolae in ICC of the myenteric plexus, the state of L‐type Ca²⁺ channels or an increase in the distance between caveolae and SR affected calcium handling.     

多聚赖氨酸改性壳聚糖对神经细胞的作用

壳聚糖是一种具有优良的生物相容性的生物可降解材料。研究几种与壳聚糖相关的材料对神经细胞生长的促进作用。实验方法以在材料上培养神经细胞为主。选用胎鼠大脑皮层神经元和神经胶质瘤细胞9L。另外 ,本实验还使用ELISA法测量细胞外基质粘附分子在材料上的吸附量 ,并测量各材料的接触角以研究细胞在材料上的吸附和铺展。结果发现壳聚糖对神经细胞的生长有良好的促进作用 ,而壳聚糖表面涂敷多聚赖氨酸和壳聚糖与多聚赖氨酸混合材料是比壳聚糖更好的促神经细胞生长的生物材料 ,都是很有应用前景的神经修复材料。     

菜心溶菌酶的提纯及酶学性质

菜心（ＢｒａｓｓｉｃａｃａｍｐｅｓｔｒｉｓＬ．ｓｓｐ．ｃｈｉｎｅｎｓｉｓｖａｒ．ｕｔｉｌｉｓ）叶子高速捣碎后,滤液经酸碱处理,硫酸铵分步沉淀,凝胶柱层析等步骤分离纯化溶菌酶,酶比活力达３４１４．６Ｕ／ｍｇ,纯化倍数为１９７．４。菜心溶菌酶在较宽的温度或ｐＨ值范围均有活性,最适温度为６０℃,最适ｐＨ值为５．８,底物Ｋｍ值为８７μｇ／ｍＬ。该酶对热和酸碱的稳定性较高,巯基和酪氨酸残基不是该酶活性中心的必需基团。     

In vitro enhancing effects of thymic dendritic cells on apoptotic cell death of thymocytes

Using terminal deoxynucleotide transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay and propidium iodide-DNA staining flow cytometry assay, the effects of mouse thymic dendritic cells (MTSC4) on the process of programmed cell death of thymocytesin vitro were investigated. It was noticed that thymocytes bound to MTSC4 used in this study. That the percentages of apoptotic nuclei of the bound thymocytes on MTSC4 were much higher than those of medium-cultured thymocytes, while the bound thymocytes on mouse thymic epithelial cell (MTEC1) showed much lower percentages of apoptosis. FACS analysis quantitatively confirmed the observation. Phenotype analysis showed that MTSC4 induced the deletion of CD4 + CD8 + cells and CD4 + CD8-.cells in 18 h of coculture. The results suggest that the negative selection of medullary thymocytes may be achieved by thymic dendritic cells through their enhancing effects on apoptosis. Project supported by the National Natural Science Foundation of China (Grant No.39670685) and FokYin Tung Education Foundation.     

Localization of vasopressin,serotonin and angiotensin II in endothelial cells of the renal and mesenteric arteries of the rat

Summary The localization of vasopressin, serotonin and angiotensin II in the endothelial cells of renal and mesenteric arteries was investigated using the pre-embedding peroxidase-antiperoxidase technique for electron microscopy. Vasopressin-and serotonin-positive endothelial cells were present in both renal and mesenteric arteries while angiotensin II-positive cells were observed in the mesenteric artery exclusively. Both arteries showed less than 10% immunoreactive cells. The lack of angiotensin II in the endothelial cells of the renal artery suggests that there may be subtle physiological differences between the renal and mesenteric arteries with respect to the local control of blood flow.     

Sertoli and Leydig cell numbers and gonadotropin receptors in rat testis from birth to puberty

Summary In testes of rats from 2 to 60 days of age, we examined the number of Sertoli cells (SC) and Leydig cells (LC) as well as the binding of radioiodinated gonadotropins to frozen sections and homogenates. The number of SC per testis increased only during the first 2 postnatal weeks, whereas that of LC was stable up to days 7–10 and increased thereafter. The uptake of ¹²⁵I-labelled human follicle-stimulating hormone (¹²⁵I-FSH) to frozen sections was confined to sex cords or seminiferous tubules, while that of ¹²⁵I-labelled human choriogonadotropin (¹²⁵I-hCG) matched the distribution of LC in the interstitium. High affinity receptors for FSH and hCG were found in homogenates at all stages studied. The number of FSH receptors per testis increased steadily, whereas that of hCG receptors was low until days 7–10 and rose afterwards. Thus, SC in rat testis appear to proliferate in the presence of fetal LC during the first 2 postnatal weeks and to differentiate concomitantly with the emergence of the adult LC generation after day 10. The complement of FSH receptors in SC remains constant as they proliferate and increases after day 21 as they differentiate. The hCG receptor number is relatively fixed in each LC generation, being higher in adult compared to fetal LC.     

Atrophy of compartments of rat lymph nodes related to an entry of lethally altered lymphocytes

Summary This paper reports the occurrence of an accumulation of lethally altered lymphocytes in the subcapsular sinus of a compartment or compartments of some lymph nodes, an unusual feature best developed in nodes of the mesenteric site in aging athymic animals. Many of these cells are rod-like. In other compartments, similar lymphocytes occurred at various depths in the nodal parenchyma. This was accompanied by the disappearance of a compartment's populations of normal lymphoid cells. The observations reveal that lymphocytes, altered in a tissue, may reach the subcapsular sinus of the draining node compartment and migrate into its parenchyma which then undergoes atrophy. The likely involvement of mast cells is discussed.This work was funded by the Medical Research Council of Canada.