High-throughput screening of cellular redox sensors using modern redox proteomics approaches

Cancer cells are characterized by higher levels of intracellular reactive oxygen species (ROS) due to metabolic aberrations. ROS are widely accepted as second messengers triggering pivotal signaling pathways involved in the process of cell metabolism, cell cycle, apoptosis, and autophagy. However, the underlying cellular mechanisms remain largely unknown. Recently, accumulating evidence has demonstrated that ROS initiate redox signaling through direct oxidative modification of the cysteines of key redox-sensitive proteins (termed redox sensors). Uncovering the functional changes underlying redox regulation of redox sensors is urgently required, and the role of different redox sensors in distinct disease states still remains to be identified. To assist this, redox proteomics has been developed for the high-throughput screening of redox sensors, which will benefit the development of novel therapeutic strategies for cancer treatment. Highlighted here are recent advances in redox proteomics approaches and their applications in identifying redox sensors involved in tumor development.     

两种非放射性标记方法在染色体原位杂交中敏感性的比较

通过原位杂交比较了地高辛配基和生物素标记探针,检测染色体单拷贝基因的敏感性。结果表明：在打点检测条上地高辛配基可检出３０ｆｇ低限探针ＤＮＡ,生物素为１ｐｇ。经原位杂交地高辛配基可检测出单拷贝基因,生物素未成功。     

麦类作物原位杂交影响因素的研究

为了更好地利用原位杂交技术进行麦类作物外源染色体的检测和基因定位,对麦类作物原位杂交的影响因素进行了研究。（１）利用缺口转译法对克隆的ＤＮＡ 片段进行生物素标记,即节省时间,又可以获得较高的标记效率;对麦类作物的基因组ＤＮＡ,宜采用随机寡核苷酸引物标记法进行生物素标记,适当延长标记时间可以提高标记效率。（２）共变性法比较适宜麦类作物的原位杂交,分别变性法如掌握不当易使染色体产生膨胀现象,变性温度过高也会使黑麦（Ｓｅｃａｌｅ ｃｅｒｅａｌ）染色体的轮廓模糊不清。（３）应根据麦类作物亲本之间的亲缘关系决定封阻ＤＮＡ 的使用浓度,并利用生物素标记的簇毛麦（Ｈａｙｎａｌｄｉａ ｖｉｌｌｏｓａ）基因组ＤＮＡ 为探针,从普通小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ ）－簇毛麦双二倍体的染色体中识别了簇毛麦的染色体。（４）麦类作物的原位杂交受洗脱强度的影响很大,利用甲酰胺进行洗脱可以获得背景清晰的原位杂交带型。随着原位杂交技术分辨率的不断提高,该技术还将用于单拷贝基因的染色体定位     

Research into selective biomarkers of erythrocyte exposure to organophosphorus compounds

Flowcytometric procedures provide distinct advantages over the colorimetric methods currently in use to monitor erythrocytes for exposure of patients to organophosphorus (OP) pesticides and chemical warfare agents; therefore, they warrant exploration. Two types of fluorescent probes—one to detect the total acetylcholinesterase on erythrocytes (RBC-AChE) and the other to distinguish between the active and OP-inhibited RBC-AChE—have been explored. Our studies demonstrate that a fluorescently conjugated fasciculin can be used to monitor total, active, and OP-inhibited RBC-AChE. However, a fluorescently tagged potent inhibitor of AChE, TZ2PIQ-A6 with a K_d of 33 fM, did not distinguish between the active and OP-inhibited RBC-AChE, nor did three different biotinylated OP compounds. The biotin-fluorescent avidin approach is not a viable procedure for monitoring RBC-AChE. Western blot studies indicate that there are at least 20 serine hydrolases on the surface of red blood cells (RBCs). Plans currently under way for the development of more specific probes to distinguish between active and OP-inhibited RBC-AChE are discussed.     

Effects of a ruminally protected B-vitamin supplement on milk yield and composition of lactating dairy cows

It is not clear if B vitamins supplied to the small intestine of dairy cows from dietary and rumen microbial sources are provided in sufficient quantity to maximize animal performance. Our objective was to determine effects of adding a ruminally protected B vitamin blend supplement, containing biotin, folic acid, pantothenic acid and pyridoxine, to the diet of high producing dairy cows on their productivity. Two dairy facilities located in California (USA) were used, one with mid lactation Holstein cows (Experiment 1) and the other with early lactation Holstein cows (Experiment 2). In each Experiment, cows were randomly assigned to treatment in a 2 × 2 crossover design with 28 d (Experiment 1) or 35 d (Experiment 2) experimental periods. In Experiment 1, milk and milk fat yield were unaffected by treatment, although milk fat proportion was lower (37.1 versus 36.3 g/kg; P<0.01), but milk protein yield was higher (1.21 versus 1.24 kg/d; P=0.02) in cows fed B vitamins. In Experiment 2, milk (39.60 versus 40.46 kg/d; P=0.02), milk fat (1.40 versus 1.47 kg/d; P<0.01) and milk protein yield (1.10 versus 1.16 kg/d; P<0.01), as well as milk energy output (113.2 versus 117.8 MJ/d; P<0.01) were all higher with B vitamin feeding. Body condition score (BCS) increased more with B vitamin feeding in Experiment 2, but was unaffected in Experiment 1. Body locomotion score (BLS) increased with B vitamin feeding in both experiments (P=0.01 and < 0.01, respectively), possibly an indication of reduced locomotory ability. Overall, productivity of high producing lactating dairy cows responded positively to feeding a mixture of ruminally protected B vitamins, although differences in the extent of the positive responses between experiments perhaps suggests that early lactation cows, with lower DM intake to milk yield ratios, may be more responsive to ruminally protected B vitamins than mid lactation cows, with higher DM intake to milk yield ratios.     

Surface plasmon resonance-based highly sensitive immunosensing for brain natriuretic peptide using nanobeads for signal amplification

Site-specific metal-catalyzed oxidation (MCO) was applied to characterize the metal-binding site (MBS) of recombinant human prolactin (hPRL), which belongs to the hematopoietic cytokine family. Copper and ascorbate of various concentrations were used to initiate the oxidation of hPRL, and the oxidation-sensitive motifs were characterized and quantitated by mass spectrometry. Based on the results obtained with 10 microM Cu(2+) and 0.3-2.0mM ascorbate, we propose that the MBS in hPRL is composed of His27, His30, and His173. This result shows the similarity of hPRL to human growth hormone (hGH), a member of the same family as hPRL, where the MBS is composed of His18, His21, and Glu174.     

白色念珠菌摄取生物素的机制研究

本文进一步实验显示处于生物素饥饿状态的白色念珠菌摄取生物素具可饱合性和特异性。从饱合曲线测算出主动摄入生物素的半数最大初速率（１／２Ｖｍａｘ）所需生物素浓度,即Ｋｍ值为０７９±０１１（平均数±标准差,ｎ＝８）;叠氮钠（５ｍＭｏｌ／Ｌ）处理后,其Ｋｍ值增到２３９±０６４μｍＭｏｌ／Ｌ（平均数±标准差,ｎ＝３）。生物素之结构同源分子,如脱硫生物素、生物胞素、甲基酯生物素和对硝基苯酯生物素等竞争性抑制其主动摄取生物素,但是２亚氨基生物素却完全无抑制作用。在生物素摄入充足的白色念珠菌（在含２０ｎＭｏｌ／Ｌ生物素的培养基中生长）,生物素主要通过简单的弥散方式进入菌体内。由于血清中的生物素含量在ｐＭｏｌ／Ｌ到几个ｎＭｏｌ／Ｌ之间,白色念珠菌在体内可能通过主动摄取生物素机制满足其营养代谢需求。本研究为发展抗白色念珠菌新举措可能提供某种理论基础     

The C-terminal domain of biotin protein ligase from E. coli is required for catalytic activity

Biotin protein ligase of Escherichia coli, the BirA protein, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. BirA also functions to repress the biotin biosynthetic operon and synthesizes its own corepressor, biotinyl-5'-AMP, the catalytic intermediate in the biotinylation reaction. We have previously identified two charge substitution mutants in BCCP, E119K, and E147K that are poorly biotinylated by BirA. Here we used site-directed mutagenesis to investigate residues in BirA that may interact with E119 or E147 in BCCP. None of the complementary charge substitution mutations at selected residues in BirA restored activity to wild-type levels when assayed with our BCCP mutant substrates. However, a BirA variant, in which K277 of the C-terminal domain was substituted with Glu, had significantly higher activity with E119K BCCP than did wild-type BirA. No function has been identified previously for the BirA C-terminal domain, which is distinct from the central domain thought to contain the ATP binding site and is known to contain the biotin binding site. Kinetic analysis of several purified mutant enzymes indicated that a single amino acid substitution within the C-terminal domain (R317E) and located some distance from the presumptive ATP binding site resulted in a 25-fold decrease in the affinity for ATP. Our data indicate that the C-terminal domain of BirA is essential for the catalytic activity of the enzyme and contributes to the interaction with ATP and the protein substrate, the BCCP biotin domain.     

红外光谱法在基因探测中的应用研究

应用干涉型傅里叶变换红外分光光度计及聚乙烯膜作载体,得到了单链、双链DNA、生物素-dUTP及经过缺口翻译参入生物素的DNA探针的红外光谱,观察到Bio-dUTP分子内化学键振动产生的特征吸收谱带。利用差谱技术比较了DNA与生物素标记DNA的差异。Bio-dUTP的特征吸收探测灵敏度可达0.1ng。同时分析了硝酸纤维膜与genescreen的红外透过性质。     

Cytotoxic effects of a novel maleimide derivative on epithelial and tumor cells

Novel N-triazolyl maleimide derivatives were synthesized by azide–alkyne Huisgen cycloaddition (1,3-dipolar cycloaddition) and tested for cytotoxicity against a cell line derived from human melanomas SK-Mel-28 and SK-Mel-103, and human umbilical vein endothelial cell lines (HUVEC). The 4l was chose to be biologically tested due to incorporation of benzyl triazolic to the nitrogen of maleimide has not been tested before, and due the satisfactory yield. The analysis of cell metabolism, using the MTT method, showed that the compound 4l impaired cell metabolism in HUVEC only in high concentration (100 µM). A lower concentration of compound 4l, whether in association or not with paclitaxel, was required to cause toxicity in both SK-Mel-28 and SK-Mel-103 cells in comparison with HUVEC cells. Moreover, the ability of 4l to cause cell death was evaluated by flow cytometry, and the data obtained highlighted the apoptotic action of 4l and paclitaxel co-treatment on Sk-Mel-28 cells only, which corroborated the greater efficacy of maleimide compounds against cancer cells. Together, our data provide promising data on the selectivity of maleimide compounds to cancer cells, and suggest that novel maleimide-substituted compounds may be synthesized and tested on different cancer cell lines, as primary or co-adjuvant agents of cancer cell toxicity.