硫酸化对金顶侧耳多糖构象及生物活性的影响

从金顶侧耳子实体中分离纯化一半乳甘露聚糖ＰＣ－３。该多糖在水中为无规线团构象,与Ｃｏｎ Ａ可相互结合并产生沉淀,对柯萨奇病毒ＣＢ５有一定的抑制作用,ＰＣ－３经硫酸化修饰后,由于同性电荷的排斥作用,使糖链的无规线团扩展呈伸展状态,局部可能形成螺旋。硫酸化的ＰＣ－３与Ｃｏｎ Ａ不能结合成多糖－蛋白复合物,但显著地提高了抗病毒ＣＢ５的活性。     

化学修饰对金顶侧耳多糖抗病毒（CB_5）活性的影响

从金顶侧耳子实体中分离纯化一半乳甘露聚糖ＰＣ－３,其分子量约为６７ｋＤ,一级结构为α（１→６）糖苷键相连的Ｇａｌ构成分子的主链,部份残基Ｃ_２上带有分支,分支结构为α（１－２）Ｍａｎ－α（１－２）Ｍａｎ。按高碘酸氧化、部份酸水解,甲基化、硫酸化程序对ＰＣ－３的结构进行化学修饰;并分别将ＰＣ－３及其衍生物与柯萨奇病毒Ｂ_５进行体外实验,结果表明,ＰＣ－３经硫酸化后,显著地提高了抗病毒活性;而高碘酸氧化,甲基化,部份酸水解产物则降低了抗ＣＢ_５的活性。     

化学修饰对金顶侧耳多糖抗病毒（CB5）活性的影响

从金侧耳子实体中分离纯化一半乳甘露聚糖ＰＣ－３,其分子量约为６７ＫＤ,一级结构ａ（１→６）糖苷键相连的Ｇａｌ构成分子的主链,部份残基Ｃ２上带有分支,分支结构为ａ（１－２）Ｍａｎ－ａ（１－２）Ｍａｎ。按高碘酸氧化、部份酸水解、甲基化、硫酸程序对ＰＣ－３的结构进行化学修饰;并分别将ＰＣ－３及其衍生物与柯萨厅病毒Ｂ５进行体外实验,结果表明,ＰＣ－３经硫酸化后,显著地提高了抗病毒活性;而高碘酸氧化,甲基化     

金顶侧耳多糖PC—4的结构确定与抗肿瘤活性的研究

３％氯乙酸浸提过的金顶侧耳子实体中分离纯化另一水溶性多糖ＰＣ－４。该多糖分子量经为１８９ｋＤ。纸层析与气相层析分析表明其为单一聚糖。经高磺酸氧化,Ｓｍｉｔｈ降解,甲基化,层析,气质联机分析,核磁其振（Ｈ－ＮＭＲ,１３Ｃ－ＮＭＲ）谱及红外光谱测定等,可确定Ｃ－４的主链结构由β－（１→３）糖苷键相连的葡萄糖构成,部份残基Ｃ６,上带有分支。约每５个糖残基有两个侧链,侧链仅为１个葡萄糖残基。     

金顶侧耳胞内多糖生物活性研究

对金顶侧耳Pleurotus citrinopileatus胞内多糖的热水浸提工艺进行研究,并研究了该多糖的抑菌活性以及对超氧阴离子自由基和亚硝基的清除作用。结果表明,金顶侧耳胞内多糖的最佳提取工艺为:提取温度60℃,液料比100:1,时间4h,此条件下多糖提取率可达17.65%。该多糖对埃希氏大肠杆菌和金黄色葡萄球菌均有抑制作用且对超氧阴离子自由基和亚硝基有较强的清除作用,说明该多糖具有明显的生物活性。     

金顶侧耳次级代谢产物及抗氧化活性研究(英文)

采用硅胶柱色谱、ODS柱色谱、Sephadex LH-20柱色谱、HPLC等分离方法,对金顶侧耳大米发酵乙酸乙酯提取物进行分离;通过HR-ESI-MS、1H-NMR和13C-NMR等光谱学方法对其结构进行鉴定。并检测铁氰化钾还原能力、1,1-二苯基-2-苦基苯肼(DPPH)自由基清除能力和亚铁离子螯合能力。从金顶侧耳大米发酵粗提物中分离鉴定了吲哚甲醛、苯肽等6个化合物,吲哚甲醛为首次从该菌中分离得到,活性测试表明金顶侧耳粗提取物具有一定的还原能力,其大米发酵产物有可能开发成为一种新型功能性食品。     

金顶侧耳的深层液体培养及相关检测*

研究表明,金顶侧耳深层培养的适宜碳源为葡萄糖和玉米粉,适宜氮源为酵母膏和豆饼粉,经0~48h的适应期, 48~96h的增殖生长期后进入稳定生长期,120h生物量可达30g/100ml(鲜重)以上。金顶侧耳营养液多糖含量>6.0mg/ml,氨基酸含量≥790 mg/ml; 经动物实验证明,其具有显著的抗疲劳作用及提高机体免疫力作用;重金属含量及微生物指标符合国家标准。     

丙酮提取金顶侧耳发酵液多糖的优化设计

以液体深层发酵法培养金顶侧耳,发酵液以丙酮为提取剂在不同的浓度﹑时间﹑温度条件下提取多糖,利用正交设计试验分析,得到了一种优化方案。试验结果表明:发酵液多糖提取最优方案是提取温度为70℃、丙酮浓度为75%、提取时间为1.5h。     

红栓菌多糖的分离纯化与分析

液体发酵得到的红栓菌（Ｐｙｃｎｏｐｏｒｕｓｃｉｎｎａｂａｒｉｕｓ）菌丝经沸水抽提,胰匀浆处理和Ｓｅｖａｇｅ法除蛋白,ＤＥＡＥＳｅｐｈａｄｅｘＡ－２５与ＳｅｐｈａｄｅｘＧ－１５０柱层析,乙醇沉淀得到二种多糖组分（简称ＰＣ＿１、ＰＣ＿２）。经聚丙烯酰胺电泳,冻融分析鉴定为均一体。ＰＣ＿１和ＰＣ＿２的,总糖含量分别为８１．５％和７７．３％,分子量分别为２３０００和１３０００．两者的红处光谱具多糖特征吸收峰,在紫外区无明显吸收现象,基本不含氮。ＰＣ＿１单糖组成为Ｄ－葡萄糖、Ｄ－半乳糖及Ｄ－甘露糖,ＰＣ＿２为Ｄ－半乳糖、Ｄ－甘露糖。     

侧耳属食用菌多糖的研究进展

侧耳多糖是从侧耳属食用菌子实体、菌丝体、发酵液或菌糠中提取的一种活性代谢产物。从侧耳多糖的提取方法、结构以及生理作用等方面进行了综述,并对其应用前景进行了展望,旨在进一步发掘和利用侧耳属食用菌的食用价值和药用价值,扩大其在食品和医药领域的应用范围。     

Transport of methotrexate into PC-3 human prostate cancer cells

Recombinant BmRad51 and BmDmc1, silkworm homologs of the Escherichia coli RecA proteins catalyzing the homologous DNA pairing, were purified from E. coli cells carrying expression vectors. These possessed different enzymatic properties in the joint molecule formation between single-stranded circular DNA and homologous linear double-stranded DNA. The requirement of single-stranded circular DNA for the efficient reaction was twofold higher in BmRad51 than in BmDmc1. Although able to mediate the joint molecule formation independently, a complex of the two enzymes formed prior to single-stranded DNA binding was found to have augmented efficiency of the pairing reaction.     

3-D measurement of osmotic dehydration of isolated and adhered PC-3 cells

Cell dehydration during freezing results from an elevated concentration of electrolytes in the extracellular medium that is deeply involved in cellular injury. We undertook real-time threedimensional (3-D) observation of osmotic dehydration of cells, motivated by a comparison of cellular responses between isolated cells in suspension and cultured cells adhering to a surface since several studies have suggested a difference in freeze tolerance between cell suspensions and monolayers. A laser confocal scanner was used with a perfusion microscope to capture sectional images of chloromethylbenzamido (DiI)-stained PC-3 cells that were exposed to an increase in NaCl concentration from 0.15 to 0.5 M at 23 °C. Change in cell volume was determined from reconstructed 3-D images taken every 2.5 s. When cells were exposed to an elevated NaCl concentration, isolated cells contracted and markedly distorted from their original spherical shape. In contrast, adhered cells showed only a reduction in height and kept their basal area constant. Apparent membrane hydraulic conductivity did not vary considerably between isolated and adhered cells, suggesting a negligible effect of the cytoskeletal structure on the rate of water transport. The surface area that contributed to water transport in adhered PC-3 cells was nearly equal to or slightly smaller than that present in isolated cells. Therefore, the similarity in properties and dimensions between isolated and adhered cells indicate that there will be similar extents of dehydration, resulting in a similar degree of supercooling during freezing.     

Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells

Most clinically approved biomarkers of cancer are glycoproteins, and those residing on the cell surface are of particular interest in biotherapeutics. We report a method for selective labeling, affinity enrichment, and identification of cell-surface glycoproteins. PC-3 cells and primary human prostate cancer tissue were treated with peracetylated N-azidoacetylgalactosamine, resulting in metabolic labeling of cell surface glycans with the azidosugar. We used mass spectrometry to identify over 70 cell surface glycoproteins and biochemically validated CD146 and integrin beta-4, both of which are known to promote metastatic behavior. These results establish cell-surface glycoproteomics as an effective technique for discovery of cancer biomarkers.     

A third xylanase from Trichoderma reesei PC-3-7

A third xylanase (Xyn III) from Trichoderma reesei PC-3–7 was purified to electrophoretic homogeneity by gel filtration and ion-exchange chromatographies. The enzyme had a molecular mass of 32 kDa, and its isoelectric point was 9.1. The pH optimum of Xyn III was 6.0, similar to that of Xyn II, another basic xylanase of  T. reesei. The purified Xyn III showed high activity with birchwood xylan but no activity with cellulose and aryl glycoside. The hydrolysis of birchwood xylan by Xyn III produced mainly xylobiose, xylotriose and other xylooligosaccharides. The amino acid sequences of the N-terminus and internal peptides of Xyn III exhibited high homology with the family F xylanases, showing that they were distinct from those of Xyn I and Xyn II of  T. reesei, which belong to family G. These results reveal that Xyn III is a new specific endoxylanase, differing from Xyn I and Xyn II in  T. reesei. It is noteworthy that this novel xylanase was induced only by cellulosic substrates and l-sorbose but not by xylan and its derivarives. Furthermore,  T. reesei PC-3-7 produced Xyn III in quantity when grown on Avicel or lactose as a carbon source, while  T. reesei QM9414 produced little or no Xyn III. Received: 7 November 1997 / Received last revision: 2 February 1988 / Accepted: 23 February 1998     

Pro-angiogenic effect of PC-3 exosomes in endothelial cells in vitro

The progression to a castration-resistant prostate cancer can occur after treatment with androgen deprivation therapy, resulting in poor prognosis and ineffective therapy response. Hormone dependence transition has been associated with increased tumor vascularization. Considering that exosomes are important components in communication between tumor cells and the microenvironment, we examined the angiogenic potential of exosomes released from Pca cell lines with distinctive profiles of androgen response through exosomes isolation, microscopy and uptake, functional assays follow up by microarray, RT-qPCR and bioinformatics analysis. HUVEC cells treated with PC-3 exosomes (androgen independent) showed increased invasion and tube formation ability. In order to identify microRNAs (miRNAs) related to the angiogenic response, the characterization of exosomal miRNA profile was performed. As result we suggest that the miR-27a-3p could be involved in the pro-angiogenic effect of PC-3 exosomes.     

Molecular lipidomics of exosomes released by PC-3 prostate cancer cells

The molecular lipid composition of exosomes is largely unknown. In this study, sophisticated shotgun and targeted molecular lipidomic assays were performed for in-depth analysis of the lipidomes of the metastatic prostate cancer cell line, PC-3, and their released exosomes. This study, based in the quantification of approximately 280 molecular lipid species, provides the most extensive lipid analysis of cells and exosomes to date. Interestingly, major differences were found in the lipid composition of exosomes compared to parent cells. Exosomes show a remarkable enrichment of distinct lipids, demonstrating an extraordinary discrimination of lipids sorted into these microvesicles. In particular, exosomes are highly enriched in glycosphingolipids, sphingomyelin, cholesterol, and phosphatidylserine (mol% of total lipids). Furthermore, lipid species, even of classes not enriched in exosomes, were selectively included in exosomes. Finally, it was found that there is an 8.4-fold enrichment of lipids per mg of protein in exosomes. The detailed lipid composition provided in this study may be useful to understand the mechanism of exosome formation, release and function. Several of the lipids enriched in exosomes could potentially be used as cancer biomarkers.     

Neuroprotective potential of epigallo catechin-3-gallate in PC-12 cells

Oxidative stress is a major player in aging and neurodegenerative disorders. Macromolecular damage occurs as a result of oxidative stress that affects the mitochondria. Mitochondrial damage leads to cell death by apoptosis or necrosis. EGCG is a tea polyphenol that protects the cells against oxidative stress. Neuroprotective potential of EGCG was tested against H₂O₂ induced oxidative stress in PC-12 cells. PC-12 cells were grown in tissue culture flasks. Oxidative stress was induced by adding H₂O₂ to the cells. EGCG was also added and the cell death was assessed using MTT assay. Oxidative stress was assessed by protein carbonyl and thiol status. Mitochondrial membrane potential was studied using JC-1 staining. TNF-α levels were assessed using ELISA. H₂O₂ increased the protein carbonyl content and reduced the thiol status in the PC-12 cells. Cell death was increased in H₂O₂ treated cells as shown by MTT assay. Mitochondrial membrane potential was also decreased along with increase in TNF-α level in H₂O₂ treated cells. EGCG brought about an increase in the cellular thiol status and decreased the protein carbonyl content in the PC-12 cells. Cell death was attenuated by EGCG treatment along with an increase in mitochondrial membrane potential and decrease in TNF-α level. EGCG conferred its antioxidant potential to PC-12 cells as evident by decreased protein damage. Mitochondrial membrane potential was improved along with a decrement in the cell death in PC-12 cells. EGCG acts as a good neutraceutical antioxidant to render neuroprotectivity to PC-12 cells.     

Secretion of endogenous kallikreins 2 and 3 by androgen receptor-transfected PC-3 prostate cancer cells

Androgen independent PC-3 cells lack androgen receptor (AR) expression and do not produce kallikrein 2 (hK2) or 3 (prostate-specific antigen, PSA). In this paper, we examined the ability of androgens to stimulate PSA and hK2 production in AR transfected PC-3 cells (PC-3(AR)) and compared this to LNCaP cells. PSA and hK2 were measured in the culture medium and cell lysates using an ELISA-based immunofluorometric assay. Only androgens were able to induce PSA and hK2 secretion in PC-3(AR) cells in a dose- and time-dependent manner depending on the level of AR present. The level of androgen-induced PSA and hK2 secretion in PC-3(AR) cells was approximately 1.5 and 0.9% that induced in LNCaP cells, respectively. Insulin-like growth factor-I (IGF-I), which has been shown to activate AR in the absence of ligand, did not activate PSA secretion in the absence of androgen, but further increased the dihydrotestosterone-induced PSA secretion in PC-3(AR) cells. The lack of PSA and hK2 production in parental PC-3 cells is thus a result of their lack of AR expression. PSA and/or hK2 production in PC-3(AR) cells can thus serve as an endogenous reporter system to investigate AR action or to screen putative endocrine disrupters.     

Cellulase induction by lactose in Trichoderma reesei PC-3-7

In an attempt to clarify the function of lactose in cellulase induction, experiments were carried out on cellulase formation by lactose along with other sugars in a resting cell system of Trichoderma reesei PC-3-7, a hypercellulase-producing mutant. Although lactose alone induces little cellulase under the conditions used, a synergistic effect on cellulase formation was observed following the respective addition of sophorose, cellobiose or galactose to lactose. The lactose consumption was more rapid when these sugars were added than in their absence. Furthermore, following lactose addition 10 h after the beginning of cultivation in the presence of cellobiose, cellulase formation was initiated with only a little lag, and lactose consumption started immediately, being complete in 14 h. \-Galactosidase induction experiments suggested that the rapid consumption of lactose is possibly not dependent on lactose degradation by the enzyme. From these results, it is suggested that lactose may function as an inducer for cellulase formation if it is taken up in the mycelium of T. reesei PC-3-7, and that sophorose, cellobiose or galactose may induce a putative lactose permease. *** DIRECT SUPPORT *** AG903066 00005     

慢病毒介导的稳定表达LBH的前列腺癌细胞株的建立

目的:构建稳定表达LBH基因的人前列腺癌细胞株PC-3M-LBH,探讨LBH基因对PC-3M细胞增殖能力的影响。方法:构建表达LBH基因的重组慢病毒载体并制备出相应的慢病毒,感染低表达LBH基因的人前列腺癌PC-3M细胞后,经嘌呤霉素筛选获得细胞克隆;实时荧光定量PCR和蛋白印迹法(Western-Blot)分别检测细胞株中LBH的mRNA、蛋白表达水平;采用CCK-8法检测表达LBH基因后细胞增殖能力的改变。结果:成功构建了重组慢病毒表达质粒p Lenti-LBH并包装出了慢病毒,感染前列腺癌细胞后经嘌呤霉素筛选得到PC-3M-LBH细胞株;PC-3M-LBH细胞株中LBH基因的mRNA和蛋白表达显著上调;相对母细胞和NC对照组,PC-3M-LBH细胞在接种后第4天即出现明显的生长抑制,到第6天其生长抑制率达到19.7%。结论:构建的细胞株能稳定表达LBH基因,该基因的表达能显著抑制前列腺癌PC-3M细胞的体外增殖。