陶瓷膜过滤收获微藻的效能与膜污染特征

考察了平板陶瓷膜在次临界通量条件下多周期过滤收获小球藻细胞的效能以及跨膜压差、阻力构成、不可逆污染组分等膜污染特征。结果表明,次临界通量条件下,采用孔径100 nm的陶瓷膜多周期过滤能够有效收获普通小球藻。84%~98%的进水藻细胞以滤饼层方式被截留,通过物理清洗膜表面能够获得高浓度藻液,浓缩因子高于34。3个过滤周期内膜污染均呈现先缓慢增加后急剧升高的两阶段变化趋势。随过滤周期增加,膜污染急剧升高阶段显著加速,其主要原因是小球藻细胞及其EOM形成的协同污染效应以及水力不可逆污染的逐渐累积。水力不可逆污染组分的有机物分子量特征是100~1 000 Da的低分子酸以及1~10 kDa的蛋白类有机物,其中多种芳香族类蛋白和可溶性微生物产物是主要组分。     

陶瓷膜过滤收获微藻的效能与膜污染特征

考察了平板陶瓷膜在次临界通量条件下多周期过滤收获小球藻细胞的效能以及跨膜压差、阻力构成、不可逆污染组分等膜污染特征。结果表明,次临界通量条件下,采用孔径100 nm的陶瓷膜多周期过滤能够有效收获普通小球藻。84%⁹8%的进水藻细胞以滤饼层方式被截留,通过物理清洗膜表面能够获得高浓度藻液,浓缩因子高于34。3个过滤周期内膜污染均呈现先缓慢增加后急剧升高的两阶段变化趋势。随过滤周期增加,膜污染急剧升高阶段显著加速,其主要原因是小球藻细胞及其EOM形成的协同污染效应以及水力不可逆污染的逐渐累积。水力不可逆污染组分的有机物分子量特征是10098%的进水藻细胞以滤饼层方式被截留,通过物理清洗膜表面能够获得高浓度藻液,浓缩因子高于34。3个过滤周期内膜污染均呈现先缓慢增加后急剧升高的两阶段变化趋势。随过滤周期增加,膜污染急剧升高阶段显著加速,其主要原因是小球藻细胞及其EOM形成的协同污染效应以及水力不可逆污染的逐渐累积。水力不可逆污染组分的有机物分子量特征是100¹ 000 Da的低分子酸以及11 000 Da的低分子酸以及1¹0 kDa的蛋白类有机物,其中多种芳香族类蛋白和可溶性微生物产物是主要组分。     

旋毛虫两个发育时期丝氨酸蛋白酶的结构及功能的比较与分析

目的比较并分析旋毛虫成虫期及新生幼虫期丝氨酸蛋白酶基因和蛋白的结构及功能。方法应用NCBI、EMBL、MEGA、ExPasy、TMHMM、SignalP、PROSITE、Coils、SYFPEITHI等多种在线生物信息学网站和软件,对旋毛虫成虫期和新生幼虫期丝氨酸蛋白酶基因(Zh68和T668)的同源性、系统发育进化状况进行分析,并预测相应蛋白(SP1和SP2)的基本理化性质、跨膜区、motif、结构域、亚细胞定位、二级及三级结构、细胞抗原表位等。结果 Zh68和T668为旋毛虫特有基因,但核苷酸同源性仅为48%,SP1和SP2的氨基酸同源性为27.5%。SP1和SP2均为不稳定的亲水性蛋白,N-末端均有一个信号肽的分泌蛋白;二级及三级结构类似,并预测出两者最佳B及T细胞抗原表位,但SP2有一个跨膜螺旋结构,为膜蛋白。结论旋毛虫成虫期的SP1是非跨膜蛋白,可能参与虫体在肠道寄生及发育、繁殖;新生幼虫期的SP2是跨膜蛋白,可能参与虫体的入侵及迁移。     

大豆多肽对前列腺癌PC-3细胞增殖及凋亡的影响

目的探讨大豆多肽对前列腺癌PC-3细胞增殖及凋亡的影响,为临床应用大豆多肽治疗前列腺癌提供实验依据。方法用不同浓度的大豆多肽(5、10、15、20μmol/L)处理前列腺癌PC-3细胞不同时间(24、48、72 h),采用MTT法检测细胞的增殖活力,倒置显微镜观察细胞的形态,流式细胞术检测细胞凋亡率及细胞周期。结果大豆多肽可抑制前列腺癌PC-3细胞的增殖,将细胞周期阻滞在G2/M期,诱导细胞凋亡,中晚期细胞凋亡趋势明显,且呈明显的剂量与时间依赖性;随着大豆多肽浓度的增加,前列腺癌PC-3细胞密度逐渐降低,边缘趋于圆滑,细胞间隙逐渐增大,局部可见部分已固缩的细胞及死亡的细胞碎片。结论大豆多肽可抑制前列腺癌PC-3细胞增殖,诱导细胞凋亡,在临床治疗前列腺癌方面具有广阔的应用前景。     

特异性抗原致敏的DC-CIK细胞对肿瘤细胞的杀伤效应

目的检测特异性抗原致敏的DC-CIK细胞对恶性肿瘤细胞的杀伤效应。方法采用肿瘤抗原致敏的DC与CIK细胞共培养,分析其免疫表型,并用MTT染色法检测其对肿瘤细胞的杀伤力。结果所获得DC-CIK细胞的CD3异质性T细胞群,对肾透明细胞癌786-0和前列腺癌PC-3细胞的杀伤率分别为70.64%和65.65%。结论DC-CIK细胞对肾透明细胞癌786-0细胞和前列腺癌PC-3细胞具有较强的杀伤效应。     

白藜芦醇诱导人皮肤鳞状细胞癌凋亡的蛋白质组学研究

为了探讨白藜芦醇抗皮肤鳞状细胞癌生物效应和分子机制,采用MTT法测定细胞活性,细胞流式法检测细胞凋亡率;SILAC蛋白质组学方法鉴定白藜芦醇调控的蛋白质分子。白藜芦醇呈浓度依赖性抑制皮肤鳞状细胞癌细胞的活性,细胞的存活率从100%降为56.7%;50μg/mL白藜芦醇处理导致37.3%细胞发生凋亡;鉴定了11个受白藜芦醇调控的蛋白质分子,其中BAG1,PDCD11,BCLAF1,HSPA9,YWHAZ等5个细胞凋亡相关蛋白被发现受白藜芦醇调控。     

黑水虻幼虫蛋白水解物的制备及营养价值评价

采用碱提酸沉法提取黑水虻幼虫脱脂虫粉中的粗蛋白,采用醇洗法进一步纯化得到精制蛋白,再经过碱性蛋白酶水解和超滤,分别得到3 kDa、3～10 kDa、10 kDa等3个组分,通过测定各组分中氨基酸组成进行营养价值评价。结果表明,碱提酸沉法和醇洗法可进一步提高脱脂虫粉蛋白含量,最高可达到(82.91±0.18)%;黑水虻幼虫蛋白水解物及其3种超滤组分的必需氨基酸比值(EAAI)在1.12～1.32之间,其中3 kDa组分的EAAI最高(1.32),其蛋白质品质最高,其第一限制性氨基酸为苏氨酸,可作为开发昆虫蛋白产品的理想原料。     

免疫蛋白质组学技术筛选布鲁菌高免疫原性膜蛋白

目的建立布鲁菌免疫蛋白质组学方法,从羊布鲁菌膜蛋白中筛选免疫候选抗原。方法提取羊布鲁菌16M膜蛋白,进行双向电泳(2-DE)分离,分别用牛、羊布鲁菌感染的阳性血清进行Westernblot分析,筛选出的免疫显性蛋白点进行LC-MS/MS质谱鉴定,并进行生物信息学分析。结果筛选出56个免疫显性蛋白点,鉴定成功35个,其中有12个蛋白是能够被牛、羊2种血清共同识别的抗原。数据检索显示,这些蛋白主要涉及布鲁菌的能量代谢,蛋白质、氨基酸合成,脂肪酸代谢及糖和辅酶的合成等生物过程。结论利用免疫蛋白质组学技术成功筛选出羊布鲁菌高免疫原性膜蛋白,为布鲁菌亚单位疫苗的研制提供了大量的候选抗原。     

胶原三股螺旋重叠蛋白1的理化性质和分子结构分析

目的分析胶原三股螺旋重叠蛋白1(collagen triple helix repeat containing 1,CTHRC1)的理化性质和分子结构,为进一步研究CTHRC1的分子功能及促癌机制提供参考。方法利用NCBI、ExPASy、SignalP、TMHMM、PSORTⅡ、SOPMA、Conserved Domain、SWISS-MODEL、STRING等软件对CTHRC1蛋白进行系统分析。结果 CTHRC1为一种碱性不稳定的亲水性蛋白,有信号肽,无跨膜区,定位于细胞外的可能性最大;CTHRC1二级结构的主要形式是无规卷曲,含有1个Collagen结构域,属于Collagen超家族;经GO和KEGG通路分析,CTHRC1相互作用蛋白主要包括FZD、DVL及ROR2。结论 CTHRC1可能参与Wnt、Hippo等信号通路,为进一步研究其分子作用机制提供了参考。     

胜利馏分油族组分分离及磺化

通过柱层析法将胜利馏分油分离为4个族组分,采用紫外光谱、红外光谱表征各组分的结构,结果表明:族组分a是不同链长的烷烃类,族组分b、c是芳烃,族组分d是以3～4芳香片线性排列的芳烃。用1HNMR、元素分析法鉴定了族组分b、c磺酸盐的平均分子结构,推测出:族组分b分子中平均含有1～2个芳环,侧链平均含有15～16个C原子,族组分c分子中平均含有2～3个芳环,侧链平均含有19～21个C原子。用发烟硫酸进行磺化的优化条件为:组分b和组分c的磺化温度分别是40、50℃;馏分油的磺化温度可确定为45℃,优化的酸油质量比为1∶1。组分b、c及馏分油的磺化产物的表面张力相差不大,胜利石油磺酸盐(SLS)有良好的界面活性。3种磺化产物的HLB值介于11～14,具有优良的乳化性能。     

Apoptotic Effects of Dietary and Synthetic Sphingolipids in Androgen-Independent (PC-3) Prostate Cancer Cells

Stress-induced activation and metabolism of plasma membrane sphingolipids results in intracellular ceramide accumulation and has been shown to induce apoptosis in human prostate cancer cells. This effect has been observed using synthetic ceramide analogs, such as C6-ceramide; however, the effects of naturally-occurring sphingolipids, such as C18-ceramide and sphingomyelin (CerPCho), on apoptosis and prostate cancer cell proliferation have not been examined. The results of the present study demonstrate that natural (CerPCho, C18-ceramide) and synthetic (C6-ceramide) sphingolipids reduced PC-3 cell proliferation by 15 ± 1.8, 17 ± 2.5, and 46 ± 2.1%, respectively (P < 0.05). These reductions in proliferation were due, in part, to increased cellular apoptosis. Treatment of PC-3 cells with CerPCho and C18-ceramide significantly increased apoptosis by 3.0 ± 0.8 and 3.6 ± 0.6%, respectively, compared to the untreated control, while the synthetic C6-ceramide significantly increased apoptosis by 55.7 ± 0.4%. C6-ceramide-induced apoptosis was associated with cell cycle arrest in the G₂/M phase, decreased extracellular signal-regulated kinase (ERK1/2) signaling and activation of the cell cycle regulatory protein, retinoblastoma (pRb). Treatment of PC-3 cells with C18-ceramide and CerPCho did not alter cell cycle distribution, pRb or ERK1/2 activation. Taken together, these results suggest that natural and synthetic sphingolipids induce apoptosis in PC-3 cells via distinct signaling mechanisms and potencies.     

Betel Nut Arecoline Induces Different Phases of Growth Arrest between Normal and Cancerous Prostate Cells through the Reactive Oxygen Species Pathway

Prostate cancer (PCa) is a reproductive system cancer in elderly men. We investigated the effects of betel nut arecoline on the growth of normal and cancerous prostate cells. Normal RWPE-1 prostate epithelial cells, androgen-independent PC-3 PCa cells, and androgen-dependent LNCaP PCa cells were used. Arecoline inhibited their growth in dose- and time-dependent manners. Arecoline caused RWPE-1 and PC-3 cell cycle arrest in the G2/M phase and LNCaP cell arrest in the G0/G1 phase. In RWPE-1 cells, arecoline increased the expression of cyclin-dependent kinase (CDK)-1, p21, and cyclins B1 and D3, decreased the expression of CDK2, and had no effects on CDK4 and cyclin D1 expression. In PC-3 cells, arecoline decreased CDK1, CDK2, CDK4, p21, p27, and cyclin D1 and D3 protein expression and increased cyclin B1 protein expression. In LNCaP cells, arecoline decreased CDK2, CDK4, and cyclin D1 expression; increased p21, p27, and cyclin D3 expression; had no effects on CDK1 and cyclin B1 expression. The antioxidant N-acetylcysteine blocked the arecoline-induced increase in reactive oxygen species production, decreased cell viability, altered the cell cycle, and changed the cell cycle regulatory protein levels. Thus, arecoline oxidant exerts differential effects on the cell cycle through modulations of regulatory proteins.     

Induction of Endoplasmic Reticulum Stress-Mediated Apoptosis by Aminosteroid RM-581 Efficiently Blocks the Growth of PC-3 Cancer Cells and Tumors Resistant or Not to Docetaxel

Aminosteroid derivative RM-581 was previously identified as an endoplasmic-reticulum (ER) stress inducer with potent in vitro and in vivo anticancer activities. We report its evaluation in androgen-independent prostate cancer (PC-3) cells. RM-581 efficiently blocks PC-3 cell proliferation with stronger activity than that of a selection of known antineoplastic agents. This later also showed a synergistic effect with docetaxel, able to block the proliferation of docetaxel-resistant PC-3 cells and, contrary to docetaxel, did not induce cell resistance. RM-581 induced an increase in the expression level of ER stress-related markers of apoptosis, potentially triggered by the presence of RM-581 in the ER of PC-3 cells. These in vitro results were then successfully translated in vivo in a PC-3 xenograft tumor model in nude mice, showing superior blockade than that of docetaxel. RM-581 was also able to stop the progression of PC-3 cells when they had become resistant to docetaxel treatment. Concomitantly, we observed a decrease in gene markers of mevalonate and fatty acid pathways, and intratumoral levels of cholesterol by 19% and fatty acids by 22%. Overall, this work demonstrates the potential of an ER stress inducer as an anticancer agent for the treatment of prostate cancers that are refractory to commonly used chemotherapy treatments.     

Toxicity of Jegosaponins A and B from Styrax japonica Siebold et al. Zuccarini in Prostate Cancer Cells and Zebrafish Embryos Resulting from Increased Membrane Permeability

(1) Background: Screening of medicinal herbs is one of the most powerful approaches to identifying novel therapeutic molecules against many human diseases. To avoid potential harmful effects during medicinal use, toxicity testing is necessary in the early stages of drug discovery. The objective of this study was to identify the cytotoxic mechanisms of jegosaponin A and B from Styrax japonica Siebold et al. Zuccarini; (2) Methods: We screened Japanese medicinal herb extracts using PC-3 prostate cancer cells and found that a methanol extract isolated from the unripe fruit of Styrax japonica Siebold et al. Zuccarini (SJSZ) had an inhibitory effect on cell viability. We further performed fractionation assays with PC-3 cells and identified the bioactive compounds using LC/MS and NMR analysis. We clarified the toxic mechanisms of these compounds using PC-3 cells and zebrafish embryos; (3) Results: We identified two active molecules, jegosaponin A and jegosaponin B, in the inhibitory fractions of the methanol extract. These jegosaponins are toxic to zebrafish embryos during the early developmental stage. Jegosaponin A and B showed strong haemolytic activity in sheep defibrinated blood (EC₅₀ = 2.1 μM, and 20.2 μM, respectively) and increased the cell membrane permeability in PC-3 cells and zebrafish embryos, which were identified using a membrane non-permeable DRAQ7, a fluorescent nucleus staining dye; (4) We identified the cytotoxic compounds jegosaponin A and B from SJSZ, which we showed to exhibit cell membrane disruptive properties using cell- and zebrafish-based testing.     

Cell Adhesion Regulates Expression of the Androgen Receptor and Coregulators in Different Prostate Cancer Cells

Prostate cancer cells adhere to a tumor basement membrane, while secretory epithelial cells reside in a suprabasal cell compartment. Since tumor cells are derived from suprabasal epithelial cells, they experience de-novo substratum adhesion in the context of oncogenesis. We therefore analyzed whether cell-matrix adhesion could affect the protein expression and activity of the AR. In this study, AR protein expression declined upon suspension of BPH-1-AR cells, but not in PC-3-AR cells shown by Western blot. In a time course study, BPH-1 cell lost AR expression within 6 hours, and the synthetic androgen, R1881 reduced the loss of AR expression. We further explored the mechanism of AR loss in suspended BPH-1 cells. BPH-1-AR cells underwent apoptosis (anoikis) when suspended for 2 – 5 hours. Suspension did not induce significant apoptosis or decreasing of AR expression in PC-3 cells. Inhibition of apoptosis in suspended BPH-1-AR cells, either by expression of Bcl-2 or Bcl-xl or by treatment with Z-VAD, a caspase inhibitor, prevented loss of AR protein. In contrast, the calpain protease inhibitor, ALLN, accelerated the loss of AR protein expression. Additionally, cell-matrix adhesion changed the expression of coregulators of AR in the mRNA level of prostate cancer cells. Our results demonstrate that AR protein expression was reduced through activation of cell death pathways, and thus indirectly through cell suspension in BPH-AR cells. The activity of AR can also be regulated by adhesion in PC-3-AR and LNCaP cells through affecting the coregulators level.     

Pharmacological Effects of Guava (Psidium guajava L.) Seed Polysaccharides: GSF3 Inhibits PC-3 Prostate Cancer Cell Growth through Immunotherapy In Vitro

The inhibitory effects of purified fractions isolated from guava seed polysaccharides (GSPS) including guava seed polysaccharide fraction 1 (GSF1), GSF2, and GSF3 on prostate cancer cells remain unclear. To clarify the anti-prostate cancer potential, GSPS, GSF1, GSF2, and GSF3 were isolated using Sepharose 6B gel filtration chromatography to assay their inhibitory effects on prostate PC-3 cell growth with direct action or indirect immunotherapy using either splenocyte conditioned media (SCM) or macrophage conditioned media (MCM). Correlations between cytokine profiles in the conditioned media and pro-apoptotic gene expression levels in the corresponding treated PC-3 cells were analyzed. Results showed that GSPS, GSF1, GSF2, and GSF3, particularly GSF3, through either direct action or indirect treatments using SCM or MCM, significantly (p < 0.05) inhibited PC-3 cell growth. GSF3 direct treatments increased pro-apoptotic Bax/anti-apoptotic Bcl-2 mRNA expression ratios in corresponding treated PC-3 cells. Either SCM or MCM cultured with GSF3 increased Fas mRNA expression levels in corresponding treated PC-3 cells. Both Th2-polarized and anti-inflammatory cytokine IL-10 either secreted in SCM or MCM were positively correlated with Fas mRNA expression levels in corresponding treated PC-3 cells. Our results suggest that GSF3 is a potent biological response modifier to decrease PC-3 cell growth through inducing apoptosis.     

The diphtheria toxin transmembrane domain as a pH sensitive membrane anchor for human interleukin-2 and murine interleukin-3

We have constructed two fusion proteins T-hIL-2 and T-mIL-3 in which human interleukin-2 (hIL-2) or murine interleukin-3 (mIL-3) are fused to the C-terminus of the diphtheria toxin transmembrane domain (T domain). Two additional fusion proteins, T-(Gly4-Ser)2-hIL-2 and T- (Gly4-Ser)2-mIL-3, were derived by introduction of the (Gly4-Ser)2 spacer between the T domain and cytokine components. Recognition of the hIL-2 receptor or the mIL-3 receptor by the corresponding recombinant proteins was demonstrated by their capacity to stimulate cytokine- dependent cell lines. All proteins retained the capacity of the T domain to insert into phospholipid membranes at acidic pH. Finally, anchoring of both cytokines to the membrane of lipid vesicles or living cells was assessed by specific antibody recognition. Our results show that the T domain fused to the N-terminus of a given protein can function as a pH sensitive membrane anchor for that protein.      

Involvement of Intercellular Adhesion Molecule-1 Up-Regulation in Bradykinin Promotes Cell Motility in Human Prostate Cancers

Prostate cancer is the most commonly diagnosed malignancy in men and shows a predilection for metastasis to distant organs. Bradykinin (BK) is an inflammatory mediator and has recently been shown to mediate tumor growth and metastasis. The adhesion molecule intercellular adhesion molecule-1 (ICAM-1) plays a critical role during tumor metastasis. The aim of this study was to examine whether BK promotes prostate cancer cell migration via ICAM-1 expression. The motility of cancer cells was increased following BK treatment. Stimulation of prostate cancer cells with BK induced mRNA and protein expression of ICAM-1. Transfection of cells with ICAM-1 small interfering RNA reduced BK-increased cell migration. Pretreatment of prostate cancer cells with B2 receptor, phosphatidylinositol 3-kinase (PI3K), Akt, and activator protein 1 (AP-1) inhibitors or mutants abolished BK-promoted migration and ICAM-1 expression. In addition, treatment with a B2 receptor, PI3K, or Akt inhibitor also reduced BK-mediated AP-1 activation. Our results indicate that BK enhances the migration of prostate cancer cells by increasing ICAM-1 expression through a signal transduction pathway that involves the B2 receptor, PI3K, Akt, and AP-1. Thus, BK represents a promising new target for treating prostate cancer metastasis.     

Inhibition of Stromal PlGF Suppresses the Growth of Prostate Cancer Xenografts

The growth and vascularization of prostate cancer is dependent on interactions between cancer cells and supporting stromal cells. The primary stromal cell type found in prostate tumors is the carcinoma-associated fibroblast, which produces placental growth factor (PlGF). PlGF is a member of the vascular endothelial growth factor (VEGF) family of angiogenic molecules and PlGF mRNA levels increase after androgen deprivation therapy in prostate cancer. In this study, we show that PlGF has a direct dose-dependent proliferative effect on human PC-3 prostate cancer cells in vitro and fibroblast-derived PlGF increases PC-3 proliferation in co-culture. In xenograft tumor models, intratumoral administration of murine PlGF siRNA reduced stromal-derived PlGF expression, reduced tumor burden and decreased the number of Ki-67 positive proliferating cells associated with reduced vascular density. These data show that targeting stromal PlGF expression may represent a therapeutic target for the treatment of prostate cancer.