Toward the Proteome of the Human Peripheral Blood Eosinophil

Eosinophils (EOSs) are granular leukocytes that have significant roles in many inflammatory and immunoregulatory responses, especially asthma and allergic diseases. We have undertaken a fairly comprehensive proteomic analysis of purified peripheral blood EOSs from normal human donors primarily employing 2‐DE with protein spot identification by MALDI‐MS. Protein subfractionation methods employed included IEF (Zoom^® Fractionator) and subcellular fractionation using differential protein solubilization. We have identified 3141 proteins, which had Mascot expectation scores of 10^?3 or less. Of these 426 were unique and non‐redundant of which 231 were novel proteins not previously reported to occur in EOSs. Ingenuity Pathway Analysis showed that some 70% of the non‐redundant proteins could be subdivided into categories that are clearly related to currently known EOS biological activities. Cytoskeletal and associated proteins predominated among the proteins identified. Extensive protein posttranslational modifications were evident, many of which have not been previously reported that reflected the dynamic character of the EOS. This data set of eosinophilic proteins will prove valuable in comparative studies of disease versus normal states and for studies of gender differences and polymorphic variation among individuals.     

Aberrant protein expression in plasma and kidney tissue during experimental obstructive nephropathy

Kidney failure is a major health problem worldwide. Patients with end-stage renal disease require intensive medical support by dialysis or kidney transplantation. Current methods for diagnosis of kidney disease are either invasive or insensitive, and renal function may decline by as much as 50% before it can be detected using current techniques. The goal of this study was, therefore, to identify biomarkers of kidney disease (associated with renal fibrosis) that can be used for the development of a non-invasive clinical test for early disease detection. We utilized two protein-profiling technologies (SELDI-TOF MS and 2-D) to screen the plasma and kidney proteome for aberrantly expressed proteins in an experimental mouse model of unilateral uretric obstruction, which mimics the pathology of human renal disease. Several differentially regulated proteins were detected at the plasma level of day-3-obstructed animals, which included serum amyloid A1, fibrinogen α, haptoglobin precursor protein, haptoglobin and major urinary proteins 11 and 8. Differentially expressed proteins detected at the tissue level included ras-like activator protein 2, haptoglobin precursor protein, malate dehydrogenase, α enolase and murine urinary protein (all p<0.05 versus controls). Immunohistochemistry was used to confirm the up-regulation of fibrinogen. Interestingly, these proteins are largely separated into four major classes: (i) acute-phase reactants (ii) cell-signaling molecules (iii) molecules involved in cell growth and metabolism and (iv) urinary proteins. These results provide new insights into the pathology of obstructive nephropathy and may facilitate the development of specific assay(s) to detect and monitor renal fibrosis.     

Construction of human monoclonal single-chain Fv antibodies against small-cell lung cancer by phage display libraries derived from cell-immunized SCID mice engrafted with human peripheral blood lymphocytes

In this study, we describe a phage display strategy to obtain human monoclonal single-chain Fv (scFv) antibodies binding target cancer cell surface proteins. By developing a cancer cell immunization protocol for SCID mice engrafted with human peripheral blood lymphocytes in combination with an antibody phage display method, we have isolated phage antibodies binding small-cell lung cancer cell line H889 by subtractive selection. One of the isolated scFv antibodies, 12EAb, recognized the E2 component of pyruvate dehydrogenase complex (PDC-E2) by immunoprecipitation according to MALDI-TOF MS analysis. Furthermore, we have confirmed the plasma membrane localization of PDC-E2 in small-cell lung cancer cells by immunocytochemistry and cell surface protein biotinylation, although PDC-E2 is usually located in the mitochondrial matrix. These results, including unique localization of identified antigens, were obtained by proteomic approaches. The present methods can be applied to generate human monoclonal scFv antibodies against tumor cells and to identify new molecular targets for immunotherapy and markers for diagnosis.     

Isoform-specific expression and characterization of 14-3-3 proteins in human glioma tissues discovered by stable isotope labeling with amino acids in cell culture-based proteomic analysis

Human 14-3-3 proteins have isoform-specific expression and functions in different kinds of normal or tumor cells and tissues. However, the expression profiling of 14-3-3 proteins and isoform-specific biological functions are unclear in human glioma so far. In our study, the expression levels and characterization of 14-3-3 isoforms in human glioma tissues were investigated by a sensitive, accurate stable isotope labeling with amino acids in cell culture-based quantitative proteomic strategy. As a result, except unexpressed 14-3-3σ, the other six isoforms, with different expression levels, were existed in glioma tissues and para-cancerous brain tissues (PBTs). 14-3-3β and η were upregulated, whereas 14-3-3ζ was downregulated in glioma tissues compared with that in PBTs. And the other three isoforms 14-3-3ε, θ, and γ had similar expression levels in human glioma tissues and PBTs. Western blot and immunohistochemistry analysis were both consistent with the quantitative proteomic data. The loss of expression of 14-3-3σ was further discovered due to DNA high methylation in its coding region in glioma by methylation-specific PCR analysis. These results indicated that the four isoforms, including 14-3-3β, η, ζ, and σ, may play important roles in tumorigenesis of human glioma, which is probably used as potential biomarkers for diagnosis and targets for treatment of human gliomas in future.     

Insights into virus capsid assembly from non-covalent mass spectrometry

The assembly of viral proteins into a range of macromolecular complexes of strictly defined architecture is one of Nature's wonders. Unraveling the details of these complex structures and the associated self-assembly pathways that lead to their efficient and precise construction will play an important role in the development of anti-viral therapeutics. It will also be important in bio-nanotechnology where there is a plethora of applications for such well-defined macromolecular complexes, including cell-specific drug delivery and as substrates for the formation of novel materials with unique electrical and magnetic properties. Mass spectrometry has the ability not only to measure masses accurately but also to provide vital details regarding the composition and stoichiometry of intact, non-covalently bound macromolecular complexes under near-physiological conditions. It is thus ideal for exploring the assembly and function of viruses. Over the past decade or so, significant advances have been made in this field, and these advances are summarized in this review, which covers the literature up to the end of 2007.     

The application of inductively coupled plasma mass spectrometry in clinical pharmacological oncology research

Metal-based anticancer agents are frequently used in the treatment of a wide variety of cancer types. The monitoring of these anticancer agents in biological samples is important to understand their pharmacokinetics, pharmacodynamics, and metabolism. In addition, determination of metals originating from anticancer agents is relevant to assess occupational exposure of health care personnel working with these drugs. The high sensitivity of inductively coupled plasma mass spectrometry (ICP-MS) has resulted in an increased popularity of this technique for the analysis of metal-based anticancer drugs. In addition to the quantitative analysis of the metal of interest in a sample, ICP-MS can be used as an ultrasensitive metal selective detector in combination with speciation techniques such as liquid chromatography. In the current review we provide a systematic survey of publications describing the analysis of platinum- and ruthenium-containing anticancer agents using ICP-MS, focused on the determination of total metal concentrations and on the speciation of metal compounds in biological fluids, DNA- and protein-adducts, and environmental samples. We conclude that ICP-MS is a powerful tool for the quantitative analysis of metal-based anticancer agents from multiple sample sources.     

A novel method for three-dimensional observation of the vascular networks in the whole mouse brain

A novel method for acquiring serial images suitable for three-dimensional reconstruction of vascular networks in the whole brain of mouse was developed. The brain infused with a White India ink-gelatin solution was fixed and embedded in paraffin containing Sudan Black B through xylene also containing Sudan Black B. Each sliced surface of the paraffin block was coated with liquid paraffin and its image was serially acquired. Coating with liquid paraffin extremely improved the quality of the image. The series of serial images was free of distortion and a three-dimensional image was reconstructed without the problem of the alignment and registration of adjacent images. The volume-rendered image indicated three-dimensional distribution of blood vessels in a whole brain. No ghost or shadow was observed on a volume-rendered image of the White India ink-gelatin infused brain. The z-axial resolution examined on the orthogonal sections reconstituted from serial images obtained at an interval of 5 mum showed no cross talk, indicating that the z-axial resolution was no larger than 5 mum. A proper understanding of the vascular system in a whole brain is indispensable to reveal the development of the vascular system in the brain of normal and genetically manipulated mouse and vascular alterations in pathological situation, such as stroke and neurodegenerative disease. Although simple and inexpensive, this method will provide fundamental information on the vascular system in a whole brain.     

New insights into the morphology of Trypanosoma cruzi reservosome

Reservosomes are late endosomes present only in members of the Schizotrypanum subgenus of the Trypanosoma genus and are defined as the site of storage of endocytosed macromolecules and lysosomal enzymes. They have been extensively described in Trypanosoma cruzi epimastigote: are bounded by a membrane unit, present an electron-dense protein matrix with electron-lucent lipid inclusions, being devoid of inner membranes. Here we performed a detailed ultrastructural analysis of these organelles using a variety of electron microscopy techniques, including ultrathin sectioning, uranyl acetate stained preparations, and freeze fracture, either in intact epimastigotes or in isolated reservosomes. New informations were obtained. First, both isolated and in situ reservosomes presented small profiles of inner membranes that are morphologically similar to the membrane surrounding the organelle. In uranyl acetate stained preparations, internal membrane profiles turned out to be longer than they appeared in ultrathin section images and traversed the organelle diameter. Internal vesicles were also found. Second, endocytosed cargo are not associated with internal vesicles and reach reservosomes on board of vesicles that fuse with the boundary membrane, delivering cargo directly into reservosome lumen. Third, electron-lucent bodies with saturated lipid core surrounded by a membrane monolayer and with unusual rectangular shape were also observed. Fourth, it was possible to demonstrate the presence of intramembranous particles on the E face of both internal vesicles and the surrounding membrane. Collectively, these results indicate that reservosomes have a complex internal structure, which may correlate with their multiple functions.     

基于长周期光纤光栅的生物传感器设计及仿真

利用生物活性物质高效、专一的催化特性,通过生化反应中生成的复合物对包层折射率和半径的直接调制,提出了基于长周期光纤光栅的、可用于蛋白质等生物物质鉴别与分析的传感方案,并用数值计算方法对检测中所生成复合物薄膜层的折射率和厚度变化对耦合波长偏移和衰减的影响进行了仿真研究,为新型高灵敏度生物传感器的研发提供了理论依据。     

Protien side-chain conformational entropy derived from fusion data-comparison with other empirical scales

The loss of conformational entropy of protein side-chains isa major effect in the energetics of folding. The simplest approachis to enumerate the number of freely rotatable bonds. Recently,two scales of side-chain conformational entropy have been proposedbased on the definition of entropy as the Boltzmann samplingover all accessible states (S –Rp_iInp_i, where p_i is theprobability of being in a rotameric state). In one scale, derivedonly for aliphatic and aromatic side-chains, the values of p_iwere obtained from Monte Carlo simulations. In the other scale,the observed frequencies of different rotameric states in adatabase of protein crystal structures yielded an estimate forp_i. Here an empirical estimation of the fusion entropy of theside-chains is used to derive a third scale. The fusion entropyis obtained as a sum of empirically derived contributions fromcomponent hydrocarbon and functional groups. There is a goodagreement between the fusion scale and the other two scales.This suggests that the magnitude of conformational entropy isbeing correctly established