A comprehensive characterization of the T-cell antigen receptor complex composition by microcapillary liquid chromatography-tandem mass spectrometry

It has become apparent that many intracellular signaling processes involve the dynamic reorganization of cellular proteins into complex signaling assemblies that have a specific subunit composition, function, and subcellular location. Since the elements of such assemblies interact physically, multiprotein signaling complexes can be isolated and analyzed. Recent technical advances in highly sensitive protein identification by electrospray-tandem mass spectrometry have dramatically increased the sensitivity with which such analyses can be performed. The T-cell antigen receptor (TCR) is an oligomeric transmembrane protein complex that is essential to T-cell recognition and function. The extracellular protein domains are responsible for ligand binding while intracellular domains generate and transduce signals in response to specific receptor-ligand interactions. We used microbore capillary chromatography-tandem mass spectrometry to investigate the composition of the TCR protein complex isolated from resting and activated cells of the murine T-cell line CD11.3. We identified all the previously known subunits of the TCR/CD3 complex as well as proteins previously not known to associate with the TCR. The catalytic activities of some of these proteins could potentially be used to interfere pharmacologically with TCR signaling.     

Antagonists to human and mouse vascular endothelial growth factor receptor 2 generated by directed protein evolution in vitro

Using directed in vitro protein evolution, we generated proteins that bound and antagonized the function of vascular endothelial growth factor receptor 2 (VEGFR2). Binders to human VEGFR2 (KDR) with 10-200 nM affinities were selected by using mRNA display from a library (10(13) variants) based on the tenth human fibronectin type III domain (10Fn3) scaffold. Subsequently, a single KDR binding clone (K(d) = 11 nM) was subjected to affinity maturation. This yielded improved KDR binding molecules with affinities ranging from 0.06 to 2 nM. Molecules with dual binding specificities (human/mouse) were also isolated by using both KDR and Flk-1 (mouse VEGFR2) as targets in selection. Proteins encoded by the selected clones bound VEGFR2-expressing cells and inhibited their VEGF-dependent proliferation. Our results demonstrate the potential of these inhibitors in the development of anti-angiogenesis therapeutics.     

Alteration of the carbon and nitrogen stable isotope composition of beef by substitution of grass silage with maize silage

This study investigated the effect of substituting grass silage (C3 photosynthetic plant product) with maize silage (C4 photosynthetic plant product) on the natural abundance carbon (delta13C) and nitrogen (delta15N) stable isotope composition of bovine muscle tissue. Forty-five continental crossbred heifers were assigned to one of three diets consisting of 3 kg of a barley-based concentrate plus grass silage, maize silage or an equal mixture (dry matter basis) of grass silage and maize silage, fed ad libitum, for 167 days. Substitution resulted in less negative delta13C values (P<0.001) in lipid-free muscle and in lipid, and also a lower delta15N (P<0.001) in lipid-free muscle. Feeding of maize silage was clearly reflected in the delta13C of muscle, with each 10% difference in the dietary C4 carbon intake resulting in a 0.9 to 1.0 per thousand shift of delta13C in lipid-free muscle and a 1.0 to 1.2 per thousand in lipid. Minimum detectable mean differences (95% confidence, power 0.80, n=15) in this experiment were about 0.5 per thousand and 1.0 per thousand for delta13C of lipid-free muscle and lipid, respectively, and about 0.5 per thousand for delta15N of lipid-free muscle. The power analysis presented here is useful for estimating minimum isotopic differences that can be detected between any two groups of beef samples with a given number of replicates. It is concluded that carbon stable isotope ratio analysis of meat can be used to quantify C3/C4 dietary constituents in beef production.     

Alteration of biotite by suspended montmorillonite

Summary Alteration of biotite flakes which were placed in suspensions of Na⁺ — and Mg⁺⁺-montmorillonite for different contact periods at two different temperatures, were followed using X-ray and petrographic microscope.The biotite seemed to alter to a mineral of the vermiculite type. Na⁺-montmorillonite changed the biotite more than Mg⁺⁺-montmorillonite at 21°C. At 50°C, however, no changes of the biotite flakes could be observed using Na⁺-montmorillonite in contrast to the case when Mg⁺⁺-montmorillonite was used. A good correlation between X-ray measurements and optical observations was found.With 3 figures and 3 tables     

Alteration of polymorphic selectivity through different crystallization mechanisms occurring in the same crystallization solution

We report a case in which two different crystallization mechanisms occurring in the same crystallization experiment are found to yield different polymorphic outcomes. In particular, we focus on crystallization of glycine from neutral aqueous solution. Crystallization in the bulk solution gives only the metastable alpha-polymorph, as observed in previous studies, whereas crystallization by evaporation of a thin film of the solution on the walls of the crystallization vessel is found to give rise to the thermodynamically stable gamma-polymorph, and furthermore produces an uncharacteristic crystal morphology for this polymorph. A detailed set of control experiments are described that elucidate mechanistic details relating to the latter crystallization process. The fact that crystallization on the walls of a crystallization vessel can yield a different polymorphic outcome from crystallization in the bulk solution in the same experiment has potentially much wider significance with regard to other polymorphic systems.     

Alteration of Bacterial Cellulose Properties by Diacetylglycerol

Bacterial cellulose (BC) was altered by means of 0.5-2.5% w/v diacetylglycerol in acetone-water through impregnation process provided DGBC composites. Results from the scanning electron microscope images, revealed that diacetylglycerol filled the pores of BC, leads to the significant enhanced in hydrophilicity and caused a smoother BC morphology. The addition of diacetylglycerol into BC caused a slightly changed in crystallinity indexes and bring about the reduction in tensile strength and Young's modulus but increased in elongation at break and toughness. The significant reduction of tensile strength and Young's modulus was achieved for DGBC 2.5% as well as for elongation at break and hydrophylicity improvement. Through the impregnation method, diacetylglycerol serves as biodegrada–ble and safe plasticizer, resulted in less rigid and higher ductility DGBC composites.     

Alteration of yeast activity by gamma radiation

Yeast is an important component in microbe based industrial technologies. Due to the techno-economic reasons, the fermentation technique has acquired renewed interest. The effect of -radiation on the fermentation reaction has been investigated. The studies show that exposure of the fermentation mixture to -radiation at 5 kGy enhance alcohol production, whereas irradiation at higher doses, viz., 10 kGy and 25 kGy caused a considerable reduction in the alcohol yield. Therefore, low dose irradiation of fermentation mixtures can be applied for increasing the alcohol production by about 25%.     

Enhancing signals of microfluidic impedance cytometry through optimization of microelectrode array

Microfluidic impedance cytometry shows a great value in biomedical diagnosis. However, the crosstalk between neighboring microelectrodes strongly weakens the impedance signal. Hereby, we demonstrate a novel microfluidic impedance cytometer consisted of sensing electrodes and ground electrodes (GNDs). The simulation reveals a signal enhancement by more than five times with GNDs compared to that without ones. We also found that the linear correlation between the impedance at a high frequency and that at a low frequency varies as microparticle size changes, which can be used for microparticle classification. The study can help with microelectrode optimization and signal processing for microfluidic impedance analysis.     

Band-gap engineering of semiconductor nanowires through composition modulation

Alloyed ternary CdS(1-x)Se(x) nanowires were synthesized by template-assisted electrodeposition, in which the ratio of S to Se in the nanowires was controlled by adjusting the relative amounts of the starting materials. Higher-resolution transmission electron microscopy (HRTEM) and X-ray diffraction (XRD) showed that the alloyed ternary CdS(1-x)Se(x) nanowires are highly crystalline, and no phase-separated Cd was observed in these nanowires. Optical measurements indicated that the band-gap engineering can be realized in these CdS(1-x)Se(x) nanowires through modulating the composition of S and Se. With broadly tunable optical and electrical properties, these alloyed nanowires could be used in color-tuned nanolasers, biological labels, and nanoelectronics.     

Single-cell protein analysis of a single mouse embryo by two-dimensional capillary electrophoresis

The characterization of protein expression from a single-cell mouse embryo using two-dimensional capillary electrophoresis (2D-CE) is described. These zygotes were obtained from Hsf1 gene knockout mice. Single zygotes were lysed off-column and proteins were fluorescently labeled using the fluorogenic dye 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). After injection, analytes were separated first according to molecular weight using capillary sieving electrophoresis (CSE) and then by micellar electrokinetic capillary chromatography (MEKC) to obtain protein expression fingerprints. Analytes were detected in a sheath flow cuvette using laser-induced fluorescence. In a 1-h 2D-CE separation, over 100 components were resolved with a spot capacity of 380.     

Alteration of uracil-DNA glycosylase activity by uracil dimers in DNA

Abstract The formation of colonies in solid medium was used as a criterion of viability to determine the effect of ultraviolet radiation on Trichomonas vaginalis. Both viability (colony) counts and total cell (hemocytometer) counts were used to estimate physiological ages of cell populations to be irradiated. Washed-cell suspensions in 0.6% saline were exposed to far- (254 nm) and near-UV (300–400 nm) radiation and dose-response survival curves were constructed from colony counts. The effect of far-UV was found to be independent of growth phase with the D₀ for exponential, early stationary, and late stationary cells 2.6, 2.7, and 2.7 J/m², respectively. Survival to near-UV increased with the age of cells with the estimated D₅₀ being 216 J/m² for exponential cells, 1360 J/m² for early stationary cells, and 4200 J/m² for late stationary cells. Exponential cells of Trichomonas gallinae irradiated with near-UV had a D₅₀ of 340 J/m². T. vaginalis is highly sensitive to far-UV relative to protozoa. T. vaginalis and T. gallinae are highly sensitive to near-UV relative to other microorganisms.     

CD36 signaling inhibits the translation of heat shock protein 70 induced by oxidized low density lipoprotein through activation of peroxisome proliferators-activated receptor ��

Oxidized LDL (OxLDL), a causal factor in atherosclerosis, induces the expression of heat shock proteins (Hsp) in a variety of cells. In this study, we investigated the role of CD36, an OxLDL receptor, and peroxisome proliferator-activated receptor γ (PPARγ) in OxLDL-induced Hsp70 expression. Overexpression of dominant-negative forms of CD36 or knockdown of CD36 by siRNA transfection increased OxLDL-induced Hsp70 protein expression in human monocytic U937 cells, suggesting that CD36 signaling inhibits Hsp70 expression. Similar results were obtained by the inhibition of PPARγ activity or knockdown of PPARγ expression. In contrast, overexpression of CD36, which is induced by treatment of MCF-7 cells with troglitazone, decreased Hsp70 protein expression induced by OxLDL. Interestingly, activation of PPARγ through a synthetic ligand, ciglitazone or troglitazone, decreased the expression levels of Hsp70 protein in OxLDL-treated U937 cells. However, major changes in Hsp70 mRNA levels were not observed. Cycloheximide studies demonstrate that troglitazone attenuates Hsp70 translation but not Hsp70 protein stability. PPARγ siRNA transfection reversed the inhibitory effects of troglitazone on Hsp70 translation. These results suggest that CD36 signaling may inhibit stress-induced gene expression by suppressing translation via activation of PPARγ in monocytes. These findings reveal a new molecular basis for the anti-inflammatory effects of PPARγ.     

Analysis of internucleosomal DNA fragmentation in apoptotic thymocytes by dynamic sieving capillary electrophoresis

The analysis, by slab gel electrophoresis, of internucleosomal DNA cleavage or laddering, characteristic of apoptosis in many cell systems, is labour intensive, difficult to automate and best only semi-quantitative. In this report we show that CE, using dilute solutions of hydroxyethylcellulose as a replaceable sieving matrix, can be applied to the relatively rapid analysis of DNA laddering in whole digests of apoptotic rat thymocytes. Also, using the sensitivity of laser-induced fluorescence detection and the highly sensitive nucleic acid stain YO-PRO-1, the CE method reported here can use 1000–2000 fold fewer cells than needed for traditional slab gel methods.     

Classification of green coffee beans by differences in protein composition obtained by matrix-assisted laser desorption/ionization mass spectrometry

It is well known that proteins and peptides play an important role in the flavour of roasted coffee, but little is reported in the literature about their characterization. In view of the potential of matrix-assisted laser desorption/ionization mass spectrometry in the analysis of proteins in complex mixtures, two varieties of coffee green beans, Arabicas and Robustas, were analyzed by this technique, in order to obtain fingerprints of their native proteins. Differences were observed between Arabicas and Robustas green beans, and cluster analysis allows differentiation of samples of the same variety from different plantations.