HSP70基因在人胃癌、宫颈癌中高表达和癌变关系的研究

采用人的HSP70基因探针P17与人的胃癌、宫颈癌的癌组织中提取的RNA进行northern杂交，结果均出现了比正常组织较强的杂交信号。表明HSP70基因在癌变组织中呈现高表达，与组织的癌变程度表现出了很强的相关性，为进一步了解细胞的癌变机理和对癌症的预防及基因治疗提供了可靠的实验依据。     

人甲胎蛋白 AFP 在大肠杆菌中表达和鉴定

本研究通过应用基因工程技术,重组构建编码人全长甲胎蛋白AFP基因的原核表达载体pET22b-AFP,并将其转化到大肠杆菌宿主菌BL21（DE3）中进行诱导表达目的蛋白AFP。采用PCR法扩增编码AFP蛋白的cDNA序列片段,并将其克隆到含有His-tag的pET22b原核表达载体上;重组质粒经双酶切及测序鉴定正确后转化到大肠杆菌BL21（DE3）中进行IPTG诱导表达;对诱导过程中不同浓度的IPTG和不同的温度进行优化;选定最佳的优化条件进行诱导表达目的蛋白;SDS-PAGE电泳和Western Blot法鉴定表达产物。结果表明,通过PCR扩增技术获得了编码AFP蛋白的基因片段;重组质粒经双酶切和基因测序等方法鉴定后,确认了重组质粒已经构建成功;表达产物通过利用SDS-PAGE和Western Blot法检测到在66 kDa附近出现条带,与预期值相符,表明重组AFP蛋白已成功表达。     

Identification, characterization, and physiological actions of factor H as an adrenomedullin binding protein present in human plasma

A recently discovered adrenomedullin binding protein has been characterized as complement factor H, an important regulator of the complement cascade. This review will describe the evidence that led to the identification of factor H as an adrenomedullin binding protein and will address the implications that such binding has in the radioimmunoassay of AM in plasma. We will also describe the possible physiological implications of AM binding: namely, factor H suppresses the antimicrobial activity of AM, enhances AM-mediated induction of cyclic-AMP in rat fibroblasts, and augments the AM-mediated growth of a human cancer cell line. These initial studies suggest that factor H may be an important factor in the regulation of AM physiology. The elucidation of the mechanisms that modulate AM activity will be necessary for the understanding of the role of AM in normal and pathological conditions.     

Upregulation of TLRs and IL-6 as a Marker in Human Colorectal Cancer

Toll-like receptors (TLRs) not only form an important part of the innate immune system but also serve to activate the adaptive immune system in response to cancer. Real-time PCR; immunohistochemical stain and Western blotting analyses were performed to clarify molecular alterations in colorectal cancer (CRC) patients. We identified Toll-like receptor 1 (TLR1), TLR2, TLR4 and TLR8 gene expression levels and downstream gene, i.e., interleukin-6 (IL-6), IL-8, interferon-α (IFN-α) and myeloid differentiation primary-response protein-88 (MyD88), expression levels in CRC patients and in cancer cell lines. CRC tissues have higher TLR1, TLR2, TLR4, TLR8, IL-6 and IL-8 gene expression levels than do the normal colon mucosa (p < 0.05). TLR2 expression varied in different cell types (mucosa and lymphocytes). There was no difference in the MyD88 and IFN-α gene expression levels between cancerous and normal colon mucosa. CRC patients had higher levels of IL-6 (p = 0.002) and IL-8 (p = 0.038) expression than healthy volunteers did; and higher IL-6 and IL-8 expression was also found to signify a higher risk of recurrence. CL075 (3M002) treatments can reduce the production of IL-8 in different cancer cell lines. The signaling pathway of TLRs in cancer tissue is different from that in normal cells; and is MyD88-independent. Higher expression levels of TLR1, TLR2, TLR 4 and TLR 8 mRNA were related to upregulation inflammatory cytokines IL-6 and IL-8 gene expression in tissue and to the upregulation of IL-6 in blood. The concentration of IL-6 in serum can be used as an indicator of the possibility of CRC recurrence. Treatment with 3M002 can reduce IL-6 production in vitro and may prevent CRC recurrence. Our findings provide evidence that TLR1, TLR2, TLR4 and TLR8 gene expression induce downstream IL-6 and IL-8 gene expression; detection of these expression levels can serve as a CRC marker.     

CLONING OF A YEAST GENE WHICH CAUSES PHENOLIC OFF-FLAVOURS IN BEER

A gene (POF1) has been cloned, which confers upon yeast (Saccharomyces cerevisiae) the ability to decarboxylate phenolic acids such as ferulic and trans-cinnamic acid. This property was previously shown to be a cause of phenolic off-flavour production in wort fermentations. The identity of the cloned gene was confirmed as POF1 by gene disruption techniques. Southern blotting of total genomic DNA revealed that sequences homologous to POF1 are conserved in Pof^? brewing strains of Sacch. cerevisiae. The transformation of a Pof^? lager strain with the cloned POF1 gene led to the production of an aroma characteristic of a phenolic off-flavour, when the transformed strain was used in wort fermentations. This latter observation suggests that the Pof^? phenotype of brewers' yeast is specifically due to the absence of a functional POF1 gene.     

Molecular characterisation and comparison of Lactococcal phages of Pakistan and Germany

Bacteriophages of Lactococcus lactis ssp. diacetylactis isolated in Pakistan and Germany were compared in respect of protein profile and restriction analysis. SDS‐PAGE profile analysis shows similarities amongst the local isolates that have different protein profile when compared with phages from other regions. No marked difference was observed in their DNA homology. The data suggested that the bacteriophages isolated from Pakistan are different from the bacteriophages that have already been characterised from the other parts of the world.     

A role of sea buckthorn on Alzheimer’s disease

Evidence suggests that diets rich in antioxidants reduce the risk factors of Alzheimer’s disease (AD). Hippophae rhamnoides, commonly known as sea buckthorn (SB), is rich in antioxidants which could have direct effects on amyloid-beta (Aβ) levels and consequently influence AD pathogenesis. In this study, sea buckthorn powder (SBP) was administered at varying concentrations (0.6, 0.9, 1.2, 1.5 and 1.8 µg mL⁻¹) to cell cultures (BE(2)-M17) with 20 mm Aβ for 72 h. MTS test indicated that SB significantly increased cell viability in Aβ-induced cells up to 95%. Results of Western blot showed maximum 38% inhibition of Aβ compared to the control (Aβ only). ELISA demonstrated significantly lower amyloid-β level (6672 pg mL⁻¹) than the control (10189 pg mL⁻¹). Images of AFM further confirmed the presence of low quantity of amyloid beta in SBP-treated cells. These findings suggest that SB warrants further investigation as potential therapeutic agent in the treatment of AD.     

不同热加工处理方式对中华绒螯蟹原肌球蛋白的消化稳定性和致敏性的影响

目的 探究不同热加工处理方式对中华绒螯蟹原肌球蛋白的消化稳定性和致敏性的影响。方法 以中华绒螯蟹（Eriocheir sinensis）为原料,采用4种不同热加工方式（蒸煮、超声波结合蒸煮、反压蒸煮、高温高压）对蟹肉进行处理,参照美国药典中模拟胃、肠液消化实验方法,对处理后蟹肉的TM粗蛋白进行体外消化,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（Sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS–PAGE）、免疫印迹（Western blotting）和抑制性酶联免疫吸附测定（Inhibition enzyme linked immunosorbent assay,Inhibition ELISA）等技术分析其消化稳定性及消化产物的致敏性。结果 结果显示,与未处理的样品相比,普通蒸煮处理后TM的消化稳定性及致敏性没有明显变化（P>0.05）;超声波结合蒸煮、反压蒸煮以及高温高压处理对TM降解具有明显的促进作用,TM及其消化产物的致敏性也显著降低（P<0.05）。结论 实验中反压蒸煮与高温高压处理对致敏性消减的效果较好。本实验为过敏原消减机制的研究和低致敏性水产品的开发提供了理论依据。     

p42/44 MAPK 激酶抑制剂增强二烯丙基二硫化物诱导CNE2 细胞凋亡

目的: 探讨二烯丙基二硫化物(DADS) 诱导CNE2 细胞凋亡及p42/44 MAPK 信号转导通路对此过程的作用。方法: DADS 处理CNE2 细胞24 h 后, 荧光显微镜下观察形态学变化及凋亡细胞计数, 四甲基偶氮唑盐微量酶反应比色法测定细胞活性, 流式细胞仪检测凋亡细胞, 蛋白质印迹法检测磷酸化p42/44MAPK 表达。结果: DADS(50 ～ 150 μmol·L^-1) 作用24 h 后, 诱导CNE2 细胞产生典型的凋亡细胞形态学变化(核浓染核碎裂), 流式细胞仪结果显示, 随着DADS 给药浓度增加, 细胞凋亡率呈浓度依赖性逐渐升高, 细胞周期中各期细胞所占百分率的变化无规律, DADS(50 ～ 150 μmol·L^-1 ) 浓度依赖性刺激磷酸化p42/44 MAPK 的表达, p42/44 MAPK 抑制剂U0126 显著增强DADS 致凋亡作用。结论: DADS 诱导CNE2 细胞凋亡时激活磷酸化p42/44 MAPK 表达, 磷酸化p42/44 MAPK 抑制剂能增强DADS 诱导CNE2 细胞凋亡效应。     

A Mouse-Based Method to Monitor Wheat Allergens in Novel Wheat Lines and Varieties Created by Crossbreeding: Proof-of-Concept Using Durum and A. tauschii Wheats

Wheat allergies are potentially life-threatening because of the high risk of anaphylaxis. Wheats belong to four genotypes represented in thousands of lines and varieties. Monitoring changes to wheat allergens is critical to prevent inadvertent ntroduction of hyper-allergenic varieties via breeding. However, validated methods for this purpose are unavailable at present. As a proof-of-concept study, we tested the hypothesis that salt-soluble wheat allergens in our mouse model will be identical to those reported for humans. Groups of Balb/cJ mice were rendered allergic to durum wheat salt-soluble protein extract (SSPE). Using blood from allergic mice, a mini hyper-IgE plasma bank was created and used in optimizing an IgE Western blotting (IEWB) to identify IgE binding allergens. The LC-MS/MS was used to sequence the allergenic bands. An ancient Aegilops tauschii wheat was grown in our greenhouse and extracted SSPE. Using the optimized IEWB method followed by sequencing, the cross-reacting allergens in A. tauschii wheat were identified. Database analysis showed all but 2 of the durum wheat allergens and all A. tauschii wheat allergens identified in this model had been reported as human allergens. Thus, this model may be used to identify and monitor potential changes to salt-soluble wheat allergens caused by breeding.