混合型应力强度因子的光弹性多参数测定

提高裂纹尖端应力强度因子值的计算精度,对于准确分析受力结构的起裂条件和破坏模式具有重要意义.本文采用3D打印技术获得了不含残余应力的平板模型,高精度打印预置裂纹避免了传统加工过程产生残余应力的缺点;综合考虑奇异场和非奇异场对裂纹尖端区域应力场的影响,引入远场边界控制的三个常数项应力,提出了光弹性多参数法;采用三点弯曲试验,运用最小二乘法计算了不同载荷下纯I型和I-Ⅱ混合型应力强度因子值,并与理论解对比分析.结果表明:对于纯I型应力强度因子,计算结果的平均误差为6.1%,对于I-Ⅱ混合型应力强度因子,计算结果的平均误差分别为6.4%和5.5%,多参数法与理论解相比较小的计算误差验证了该方法的可靠性和准确性,可为精确计算应力强度因子的光弹性实验研究提供借鉴.     

ParaMEME: a parallel implementation and a web interface for a DNA and protein motif discovery tool

Many advanced software tools fail to reach a wide audience because they require specialized hardware, installation expertise, or an abundance of CPU cycles. The worldwide web offers a new opportunity for distributing such systems. One such program, MEME, discovers repeated patterns, called motifs, in sets of DNA or protein sequences. This tool is now available to biologists over the worldwide web, using an asynchronous, single-program multiple-data version of the program called ParaMEME that runs on an Intel Paragon XP/S parallel computer at the San Diego Super-computer Center. ParaMEME scales gracefully to 64 nodes on the Paragon with efficiencies > 72% for large data sets. The worldwide web interface to ParaMEME accepts a set of sequences interactively from a user, submits the sequences to the Paragon for analysis, and e-mails the results back to the user. ParaMEME is available for free public use at http://@www.sdsc.edu/CompSci/Biomed/ MEME.     

Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification

Although mass spectrometric peptide mapping has become an established technique for the rapid identification of proteins isolated by polyacrylamide gel electrophoresis (PAGE), the results of the identification procedure can sometimes be ambiguous. Such ambiguities become increasingly prevalent for proteins isolated as mixtures or when only very small amounts of the proteins are isolated. The quality of the identification procedure can be improved by increasing the number of peptides that are extracted from the gel. Here we show that cysteine alkylation is required to ensure maximal coverage in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mapping of proteins isolated by PAGE. In the described procedure, alkylation was performed prior to electrophoresis to avoid the adventitious formation of acrylamide adducts during electrophoresis. In this way, homogeneous alkylation was obtained with three different alkylating reagents (4-vinylpyridine, iodoacetamide, acrylamide). Cysteine alkylation was also used as a tool for the identification of cysteine-containing peptides. Using a 1:1 mixture of unlabeled acrylamide and deuterium-labeled acrylamide ([2,3,3'-D3]acrylamide), the proteins of interest were alkylated prior to electrophoretic separation. Peptide mixtures produced by trypsin digestion of the resulting protein bands were analyzed by MALDI-TOF MS, and the cysteine content of the peptides was inferred from the isotopic distributions. The cysteine content information was readily obtained and used to improve the protein identification process.     

Concanavalin A- and wheat germ agglutinin-conjugated lectins as a tool for the identification of multiple N-glycosylation sites in heterologous protein expressed in yeast

We report here a methodology that allows the identification of glycosylation sites by a combination of protein enzymatic digestion, glycopeptide separation on a reverse-phase HPLC column, and further recognition in a dot-blot system using concanavalin A-horseradish peroxidase. Wheat germ agglutinin-horseradish peroxidase is used as the recognition system for peptides generated after proteolytic digestion of endoglycosidase H deglycosylated protein. Glycosylation sites were confirmed by automatic Edman degradation and fast atom bombardment mass spectrometry. This methodology was applied to a model glycoprotein, alpha-amylase from Bacillus licheniformis, which is unglycosylated in its natural host and appears highly glycosylated when expressed in the methylotrophic yeast Pichia pastoris.     

Capillary electrophoresis/electrospray ionization high mass accuracy time-of-flight mass spectrometry for protein identification using peptide mapping

Capillary electrophoresis/electrospray ionization (CE/ESI) high mass accuracy time-of-flight mass spectrometry was used for the first time to characterize small proteins using peptide mapping. To identify small proteins, the intact proteins were first analyzed to obtain their average molecular weights with errors less than 1 Da. On-line capillary electrophoresis mass spectrometry of the tryptic digests of these small proteins was then performed to obtain the accurate molecular weights of the peptides with accuracies of approximately 10 ppm. Next, this information was used for the identification of the proteins using a protein database. It was found that high mass accuracy is an effective tool in reducing the list of most-likely proteins generated by the database. In addition, on-line collision-induced dissociation of the completely or partially resolved capillary electrophoresis peaks of the protein digests was used to unambiguously identify the sequences of these peptides. Each CE/ESI-MS analysis used only 5 nL of sample containing approximately 120 fmol of each peptide in protein digests. The results indicate that the combination of capillary electrophoresis and high resolution, high mass accuracy time-of-flight mass spectrometry is a viable option for the identification of small proteins using peptide mapping.     

The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein

Mycobacterium marinum, like Mycobacterium tuberculosis, is a slow-growing pathogenic mycobacteria that is able to survive and replicate in macrophages. Using the promoter-capture vector pFPV27, we have constructed a library of 200-1000 bp fragments of M. marinum genomic DNA inserted upstream of a promoterless green fluorescent protein (GFP) gene. Only those plasmids that contain an active promoter will express GFP. Macrophages were infected with this fusion library, and phagosomes containing fluorescent bacteria were isolated. Promoter constructs that were more active intracellularly were isolated with a fluorescence-activated cell sorter, and inserts were partially sequenced. The promoter fusions expressed intracellularly exhibited homology to mycobacterial genes encoding, among others, membrane proteins and biosynthetic enzymes. Intracellular expression of GFP was 2-20 times that of the same clones grown in media. Several promoter constructs were transformed into Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis. These constructs were positive for GFP expression in all mycobacterial strains tested. Sorting fluorescent bacteria in phagosomes circumvents the problem of isolating a single clone from macrophages, which may contain a mixed bacterial population. This method has enabled us to isolate 12 M. marinum clones that contain promoter constructs differentially expressed in the macrophage.     

An on-line method for the measurement of total protein output in biological fluids and secretory tissues after stimulation of intrinsic nerves and identification of neurotransmitters using immunohistochemical techniques

Proteins are essential ingredients of life and thus it is essential to measure the level of proteins in biological fluids and tissue homogenates. Several methods have previously been described in the literature for the estimation of proteins using either certain dyes which bind to specific groups of polypeptide side chains producing a protein dye colour complex, methods involving copper binding to peptide bonds or application of an eosin B red dye. In this study, an on-line automated technique based on the Lowry method has been used to estimate total protein output from the isolated lacrimal segments. This method can also be used to estimate total protein from saliva, or any other biological fluid, tissue homogenates or secretory tissues. The on-line automated method for the estimation of total protein from secretory tissues and biological fluid was designed mainly to obtain a rapid, simple and consistent graphical interpretation of result within 40-50 min of starting the experiment. The original chart recording of the time-course response can also be used for publication purposes. With this method, it is possible to investigate the effect of electrical field stimulation on the intrinsic secretomotor nerves employing either wire or platinum electrodes embedded in the perfusing chamber. Moreover, the tissue can also be stimulated with different concentrations of either drugs, hormones or neurotransmitters for different time periods. This method can also be combined with morphology whereby the stimulated tissue can be processed for neuropeptide or neurotransmitter immunohistochemistry to determine which neurotransmitters or neuropeptides are involved in the physiological responses. The automated method is simple and rapid and moreover, it can estimate accurately and directly at physiological pH small amount (ng-microgram) of proteins in effluent samples depending on the sensitivity of the chart recorder. In this study, neuropeptides and neurotransmitters were used as secretagogues in addition to electrical field stimulation.