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PubMed2005

Nanoparticle decorated surfaces with potential use in glycosylation analysis.

A majority of all biologically active proteins are glycosylated and various diseases have proven to correlate with alterations in protein glycosylation. Sensitive identification of different glycoprotein glycoforms is therefore of great diagnostic value. Here we describe a method with potential for glycoprotein profiling, based on lectins as capture probes immobilized on particulate substrates in the nm-range. The nanoparticles present high concentrations of attachment sites for specific ligands and cause minimal steric hindrance to binding. In the present model study the mannose-binding lectin ConA has been coupled to polystyrene nanoparticles via a poly(ethyleneoxide) linker which protects the protein conformation and activity and prevents unspecific protein adsorption. The ConA-coated particles are accommodated at different spots on the analytical surface via oligonucleotide linkage. This attachment, which relies on the hybridization of complementary oligonucleotides, allows firm fixation of the particles at specific positions. The ConA attached to the particles has retained conformation and activity and binds selectively to a series of different glycoproteins. The results indicate the potential for using a multi-lectin nanoparticle array in glycoprotein mapping.

Cell Fractionation↗

PubMed2005

Proteomic studies of Plasmodium knowlesi SICA variant antigens demonstrate their relationship with P. falciparum EMP1.

Malaria variant antigens are encoded by large multigene families and expressed on the surface of infected erythrocytes. The Plasmodium knowlesi Schizont-infected cell agglutination (SICA) antigens are encoded by the SICAvar multigene family, and the P. falciparum erythrocyte membrane protein-1 (PfEMP1) antigens are encoded by the var gene family. Although these variant antigens share many fundamental features, P. knowlesi and P. falciparum are phylogenetically distantly related, and so far a significant level of sequence identity has not been observed in alignments of either the SICAvar and var gene families or their encoded proteins. In support of their orthologous relationship, however, here we demonstrate through proteomic analysis that the P. knowlesi SICA variant antigens share significant common sequences with P. falciparum EMP1 molecules. As many as forty P. knowlesi SICA peptides show identity with a particular P. falciparum EMP1, mapping throughout all characterized domains, including the externally exposed cysteine-rich domains that are characteristic of both proteins. These findings provide further validation of the classical in vivo P. knowlesi-rhesus monkey model system for advancing our understanding of the immunobiology of antigenic variation and variant antigen gene expression in Plasmodium.

Animals↗

PubMed2002

Amelioration of ozone-induced lung injury by anti-tumor necrosis factor-alpha.

Ozone (O(3)) is a significant component of atmospheric air pollution and produces detrimental effects in the lung. Although the mechanism of O(3)-induced lung inflammation and injury is unclear, the increased release of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) by lung cells following O(3) exposure may shed some light on this subject. To investigate the role of TNF-alpha in the O(3)-induced pulmonary insult, we intraperitoneally injected rats with either rabbit preimmune serum or rabbit antirat TNF-alpha 1 h prior to O(3) exposure. Approximately 12 h after the end of O(3) exposure the animals were sacrificed, the lungs lavaged, and tissue samples collected for expression of cytokine genes relevant to inflammation. The bronchoalveolar lavage fluid (BALF) was analyzed for albumin as a marker of pulmonary epithelial permeability changes and for fibronectin for its role in lung injury and repair. The lavage cells were collected, counted, and identified to quantitate the inflammatory response. Ozone exposure resulted in a significant increase in BALF albumin and fibronectin as compared to air-exposed controls and a significant increase in BALF polymorphonuclear leukocytes (PMNs). Antibody treatment produced a significant decrease in BALF albumin and PMNs as compared to O(3)-exposed rats given preimmune serum. Antibody treatment did not affect the BALF fibronectin concentration or the total cell count in the BAL. Tissue analysis for gene arrays revealed an activation of IL-1alpha, IL-6, and IL-10 in animals exposed to O(3). The gene expression was downregulated in animals treated with anti-TNF-alpha antibody prior to O(3) exposure. The results suggest a central role for TNF-alpha in the mechanistic pathways critical to lung inflammation. The significance of TNF-alpha in the inflammation and epithelial injury produced by ozone exposure reflects its overall contribution through modulation of other cytokines.

Albumins↗

PubMed2006

Optical technologies for the read out and quality control of DNA and protein microarrays.

Microarray formats have become an important tool for parallel (or multiplexed) monitoring of biomolecular interactions. Surface-immobilized probes like oligonucleotides, cDNA, proteins, or antibodies can be used for the screening of their complementary targets, covering different applications like gene or protein expression profiling, analysis of point mutations, or immunodiagnostics. Numerous reviews have appeared on this topic in recent years, documenting the intriguing progress of these miniaturized assay formats. Most of them highlight all aspects of microarray preparation, surface chemistry, and patterning, and try to give a systematic survey of the different kinds of applications of this new technique. This review places the emphasis on optical technologies for microarray analysis. As the fluorescent read out of microarrays is dominating the field, this topic will be the focus of the review. Basic principles of labeling and signal amplification techniques will be introduced. Recent developments in total internal reflection fluorescence, resonance energy transfer assays, and time-resolved imaging are addressed, as well as non-fluorescent imaging methods. Finally, some label-free detection modes are discussed, such as surface plasmon microscopy or ellipsometry, since these are particularly interesting for microarray development and quality control purposes.

Animals↗

PubMed2006

Application of antibody array technology in the analysis of urinary cytokine profiles in patients with chronic kidney disease.

AIMS: Emerging evidence suggests that the urinary excretion of cytokines is associated with the progression of chronic kidney disease (CKD). However, detection of urinary cytokines in high throughput is still a problem in clinical practice. In this cross-sectional study, we applied a novel proteomic technology, antibody array, to analyze urinary cytokine profiles in patients with CKD. METHODS: A total of 10 subjects including 7 CKD patients and 3 normal controls were studied. These patients with CKD were divided into two groups according to the levels of estimated glomerular filtration rate (eGFR): group A (eGFR >or=80 ml/min/1.73 m(2), n = 3) and group B (eGFR <or=40 ml/min/1.73 m(2), n = 4). Urine samples taken from age and gender-matched healthy volunteers (n = 3) served as control. Differential excretion of urinary cytokines was determined by human cytokine antibody array (Raybiotech, Norcross, Ga., USA). A 2-fold change in spot intensity compared to controls was considered as significant. In order to check the reliability of the data obtained by antibody array, urinary monocyte chemoattractant protein (MCP) 1 and tumor necrosis factor (TNF) alpha were determined by using enzyme-linked immunosorbent assay. RESULTS: A total of 15 cytokines varied significantly in urinary samples obtained from patients with CKD compared with normal controls. It was shown that the levels of MCP-1, RANTES, tissue inhibitor of metalloproteinase (TIMP) 1, TNF-alpha, vascular endothelial growth factor (VEGF), E-selectin, Fas, intercellular adhesion molecule 1, interleukin 2, matrix metalloproteinase (MMP) 2 and transforming growth factor beta in patients with CKD at stage 1-2 (group A) were 2- to 5-fold higher than those in normal controls, while the excretion of MCP-1, RANTES, TIMP-1, TNF-alpha and VEGF was further increased in the patients with renal insufficiency (group B). However, a lower excretion of urinary vascular cell adhesion molecule 1 and platelet-derived growth factor was found in patients with CKD compared with normal controls. Impressively, the urinary MMP-9 excretion was 492-fold higher in group A and 198-fold higher in group B than that in normal controls. The levels of MCP-1 and TNF-alpha correlated well with the relative n-fold change seen in the antibody array experiments. The rank correlation coefficients (r values) were 0.976 (p < 0.001) and 0.939 (p < 0.001) for MCP-1and TNF-alpha, respectively. CONCLUSION: Antibody array could serve as a fast, high-throughput and sensitive tool for detecting the excretion of urinary cytokines. Our study is the first to provide an important information regarding the utility of antibody array in evaluating the role of cytokines in the initiation and progression of CKD.

Adult↗

PubMed2005

SELDI-TOF-MS: the proteomics and bioinformatics approaches in the diagnosis of breast cancer.

Breast cancer has never had any good serum tumor markers. Therefore, we developed and evaluated a proteomics approach to searching for new biomarkers and building diagnostic models. SELDI-TOF-MS ProteinChip was used to detect the serum protein patterns of 49 breast cancer patients, 51 patients with benign breast diseases, and 33 healthy women. The diagnostic models were developed and validated using bioinformatics tools such as artificial neural networks and discriminant analysis. In total, four models were built and their sensitivities and specificities were satisfactory. The abilities of these models to diagnose stage I breast cancer were not worse than for stages II-IV (P>0.05). Four candidate biomarkers of breast cancer were found. The high sensitivity and specificity achieved by this method show great potential for the early detection of breast cancer and facilitation of discovering new and improved biomarkers.

Adult↗

PubMed2005

Regulation of hepatocyte activator inhibitor-1 expression by androgen and oncogenic transformation in the prostate.

Hepatocyte activator inhibitor-1 (HAI-1) is a transmembrane serine protease inhibitor that regulates the conversion of latent to active hepatocyte growth factor (HGF). Studies supporting a role for the HGF pathway in prostate carcinogenesis prompted an analysis of HAI-1 expression in the prostate. Here we analyze the regulation of HAI-1 expression by androgen, oncogenic transformation, and cancer progression. Immunohistochemical analysis revealed that HAI-1 expression was restricted to prostate epithelium, where staining occurred primarily in basal and atrophic luminal epithelial cells. Compared to normal glands, HAI-1 expression was significantly increased in localized prostate cancer and was present in most prostate cancer metastases. HAI-1 protein expression levels were sensitive to androgen in normal epithelium but not in cancer. Although androgen did not increase HAI-1 protein expression levels in LNCaP cells, it decreased HAI-1 surface expression, consistent with previous data from our group (Martin DB, Gifford DR, Wright ME, Keller A, Yi E, Goodlett DR, Aebersold R, Nelson PS: Quantitative proteomic analysis of proteins released by neoplastic prostate epithelium. Cancer Res 2004, 64:347-355). HAI-1 overexpression in cancer was predictive of prostate-specific antigen recurrence (relative risk, 1.24). These results suggest that HAI-1 regulates the HGF Met axis on prostate epithelial cells and influences HGF mediated tumor invasion and metastasis.

Androgens↗

PubMed2006

Protein microarrays for the diagnosis of allergic diseases: state-of-the-art and future development.

In the emerging field of Proteomics, protein microarrays represent an elegant solution for the simultaneous analysis of the (i) abundance, (ii) function and (iii) interaction of proteins on a system-wide scale. The great power of microarray-based miniature solid-phase immunoassays lies in their potential to investigate in parallel large numbers of analyte pairs while employing only minute amounts of biological sample material (e.g., serum). This has inspired researchers to adopt this approach to the development of novel diagnostic tests with to date published examples in the fields of autoimmune diseases, allergy and cancer. Here, we discuss recent advancements in the development of protein microarrays for the profiling of IgE antibodies in the diagnosis of Type-1 related allergic diseases.

Allergens↗

PubMed2006

Inhibitor and protein microarrays for activity-based recognition of lipolytic enzymes.

Protein and small-molecule microarrays are useful tools for high-throughput analysis of DNA-protein, protein-protein, and protein-small molecule interactions. Here we report on novel microarrays for activity screening of lipases and esterases based on phosphonic acid ester inhibitors. These compounds are activity recognition probes (ARPs) and bind to active serine hydrolases in a stoichiometric and irreversible manner. Protein microarrays were generated by spotting six different lipolytic enzymes onto hydrogel-coated glass slides. The activity of immobilized enzymes was determined after treatment with fluorescently labeled ARPs. Alternatively, biotinylated ARPs were bound to streptavidin slides in order to identify their affinity for enzymes in solution. Both systems, the protein- and ARP microarrays proved to be useful and versatile tools for the rapid identification and characterization of novel and known lipolytic enzymes.

Enzyme Inhibitors↗

PubMed2005

Recent advances in blood-related proteomics.

Blood is divided in two compartments, namely, plasma and cells. The latter contain red blood cells, leukocytes, and platelets. From a descriptive medical discipline, hematology has evolved towards a pioneering discipline where molecular biology has permitted the development of prognostic and diagnostic indicators for disease. The recent advance in MS and protein separation now allows similar progress in the analysis of proteins. Proteomics offers great promise for the study of proteins in plasma/serum, indeed a number of proteomics databases for plasma/serum have been established. This is a very complex body fluid containing lipids, carbohydrates, amino acids, vitamins, nucleic acids, hormones, and proteins. About 1500 different proteins have recently been identified, and a number of potential new markers of diseases have been characterized. Here, examples of the enormous promise of plasma/serum proteomic analysis for diagnostic/prognostic markers and information on disease mechanism are given. Within the blood are also a large number of different blood cell types that potentially hold similar information. Proteomics of red blood cells, until now, has not improved our knowledge of these cells, in contrast to the major progresses achieved while studying platelets and leukocytes. In the future, proteomics will change several aspects of hematology.

Amyloid↗

PubMed2005

The importance of experimental design in proteomic mass spectrometry experiments: some cautionary tales.

Proteomic expression patterns derived from mass spectrometry have been put forward as potential biomarkers for the early diagnosis of cancer and other diseases. This approach has generated much excitement and has led to a large number of new experiments and vast amounts of new data. The data, derived at great expense, can have very little value if careful attention is not paid to the experimental design and analysis. Using examples from surface-enhanced laser desorption/ionisation time-of-flight (SELDI-TOF) and matrix-assisted laser desorption-ionisation/time-of-flight (MALDI-TOF) experiments, we describe several experimental design issues that can corrupt a dataset. Fortunately, the problems we identify can be avoided if attention is paid to potential sources of bias before the experiment is run. With an appropriate experimental design, proteomics technology can be a useful tool for discovering important information relating protein expression to disease.

Biomarkers, Tumor↗

PubMed2004

Down-regulated expression of cytokeratin 18 promotes progression of human breast cancer.

PURPOSE: Cytokeratins (CKs) have been recognized for >20 years as structural marker proteins specific for epithelial cells. Recent expression profiling analyses indicate, however, that CK down-regulation may occur in breast cancer. EXPERIMENTAL DESIGN: Here we evaluated the expression pattern of CK18 by immunohistochemical analysis of primary breast carcinomas (n = 1458) spotted on a high-density tissue microarray. The findings were correlated to histopathological risk factors and clinical outcome. RESULTS: Down-regulation of CK18 (as compared to normal breast tissue) was observed in 25.4% of the tumors with a lower rate in lobular carcinomas (17.0%) than in ductal carcinomas (25.4%) or other histological entities (32.5%). CK down-regulation was significantly correlated to advanced tumor stage and high grade but not to axillary lymph node status. Kaplan-Meier survival analysis revealed CK18 as a prognostic indicator of overall survival (P = 0.015) and cancer-specific survival (P = 0.005). CONCLUSIONS: Down-regulation of the luminal CK18 is not rare and a clinically relevant event in breast cancer. This finding has important implications for the use of CK18 as epithelial tumor marker. The correlations with clinical follow-up suggest that CK18 might suppress tumor progression.

Aged↗

PubMed2003

Novel human and mouse genes encoding a shank-interacting protein and its upregulation in gastric fundus of W/WV mouse.

BACKGROUND AND AIMS: A division of labor exists between different classes of interstitial cells of Cajal (ICC) in the gastrointestinal tract. In the stomach and small intestine, ICC at the level of the myenteric plexus (IC-MY) act as slow wave pacemaker cells, whereas intramuscular ICC (IC-IM) in the stomach act as intermediaries in enteric motor neurotransmission. The muscle layers of the gastric fundus do not have IC-MY, therefore electric slow waves are not generated. Intramuscular ICC are absent in the gastric fundus of W/WV mutant mice, and excitatory and inhibitory motor nerve responses are reduced in these tissues. The absence of IC-IM in W/WV mutants in the fundus provides a unique opportunity to study the molecular changes that are associated with the loss of these cells. METHODS: The tissue gene expression of wild-type and W/WV mice from gastric fundus was assayed using a murine microarray chip analysis displaying a total of 8734 elements. RESULTS: Twenty-one queries were differentially expressed in wild-type and W/WV mice. One candidate gene, encoding a novel protein homologous to rat Shank-interacting protein (Sharpin) was significantly upregulated in fed and starved W/WV mice. The full-length clone of the murine gene and its human counterpart were isolated and designated as Shank-interacting protein-like 1 (SIPL1). Human SIPL1 complementary DNA encodes a protein of 345 amino acids. This gene was localized to chromosome 8. SIPL1 was abundantly expressed in human stomach and small intestine, and scarcely expressed in cecum and rectum. CONCLUSIONS: Gene analysis showed that SIPL1 differentially express in the gastric fundus of normal and W/WV mice. The upregulation of SIPL1 in the fundus of W/WV mice, and expression in the upper gastrointestinal tract suggest that the SIPL1 gene could be associated with ICC function in mice and humans.

Amino Acid Sequence↗

PubMed2004

Method for producing coded micro-carrier and test method by using a novel type biochip.

This paper provides a method for producing a novel type coded micro-carrier. A simple and cost effective solution for bio-molecule applications was developed. Application relevant items such as manufacture process, biospecific interaction, and analysis method are discussed. For low cost fabrication, the use of LIGA-like process is suggested. LIGA-like process is used as a dry patterning process in which an intense beam of light from an excimer laser is used to pattern a material directly. This process has found extensive application in the microelectronics industry for patterning of polymer materials. The use of LIGA-like techniques offers two attractive features: first, we can cut the polymer into many tiny micro-carriers with micrometer precision. Second, LIGA-like process allows to encode with high precision spatial information onto the micro-carrier that can be used in the identification of the bio-molecule. This paper gives a description of the basic idea, describes the fabrication of the novel micro-carrier that we called "coded micro-carrier," and of the image processing algorithms used for the analysis of bio-molecules. This study also provides a test method for identifying a bio-molecule, which includes mixing several coded micro-carriers with the hybridized unknown bio-molecules; and identifying the codes on the micro-carrier via image recognition system. The numbers and types of the known micro-carrier can be flexibly adjusted according to the number of tested bio-molecules.

Animals↗

PubMed2006

ProMAT: protein microarray analysis tool.

SUMMARY: ProMAT is a software tool for statistically analyzing data from enzyme-linked immunosorbent assay microarray experiments. The software estimates standard curves, sample protein concentrations and their uncertainties for multiple assays. ProMAT generates a set of comprehensive figures for assessing results and diagnosing process quality. The tool is available for Windows or Mac, and is distributed as open-source Java and R code. AVAILABILITY: ProMAT is available at http://www.pnl.gov/statistics/ProMAT. ProMAT requires Java version 1.5.0 and R version 1.9.1 (or more recent versions). ProMAT requires either Windows XP or Mac OS 10.4 or newer versions.

Algorithms↗

PubMed2005

Integrative genomic and proteomic analysis of prostate cancer reveals signatures of metastatic progression.

Molecular profiling of cancer at the transcript level has become routine. Large-scale analysis of proteomic alterations during cancer progression has been a more daunting task. Here, we employed high-throughput immunoblotting in order to interrogate tissue extracts derived from prostate cancer. We identified 64 proteins that were altered in prostate cancer relative to benign prostate and 156 additional proteins that were altered in metastatic disease. An integrative analysis of this compendium of proteomic alterations and transcriptomic data was performed, revealing only 48%-64% concordance between protein and transcript levels. Importantly, differential proteomic alterations between metastatic and clinically localized prostate cancer that mapped concordantly to gene transcripts served as predictors of clinical outcome in prostate cancer as well as other solid tumors.

Disease Progression↗

PubMed2006

Teaching molecular genetics: Chapter 3--Proteomics in nephrology.

The novel discipline of proteomics has experienced a rapid growth in the recent past and has great potentials for the future. The study of proteins on a genomic scale enables a large number of proteins to be analysed simultaneously. Moreover, proteomic analysis reveals the presence of protein isoforms and post-translational modifications, both of which have the potential to regulate protein complex formation, activity and function. As such, the assessment of the proteome, unlike genomic analysis, provides a view of biological processes at their level of occurrence. The knowledge thus gained is important not only for a better understanding of renal physiology and pathophysiology, but also for the identification of disease markers and the development of new therapies. This review applies the science of proteomics to nephrology: our aim is to give an overview of the discipline, providing background information and outlining the scope, advantages and limitations of proteomics.

Biomarkers↗

PubMed2006

Tolerizing DNA vaccines for autoimmune arthritis.

Current therapies for rheumatoid arthritis (RA) and other autoimmune diseases non-specifically suppress immune function, and there is great need for fundamental approaches such as antigen-specific tolerizing therapy. In this paper we describe development of antigen-specific tolerizing DNA vaccines to treat collagen-induced arthritis (CIA) in mice, and use of protein microarrays to monitor response to therapy and to identify potential additional autoimmune targets for next generation vaccines. We demonstrate that tolerizing DNA vaccines encoding type II collagen (CII) reduced the incidence and severity of CIA. Atorvastatin, a statin drug found to reduce the severity of autoimmunity, potentiated the effect of DNA vaccines encoding CII. Analysis of cytokines produced by collagen-reactive T cells derived from mice receiving tolerizing DNA encoding CII, as compared to control vaccines, revealed reduced production of the pro-inflammatory cytokines IFN-gamma and TNF-alpha. Arthritis microarray analysis demonstrated reduced spreading of autoantibody responses in mice treated with DNA encoding CII. The development of tolerizing DNA vaccines, and the use of antibody profiling to guide design of and to monitor therapeutic responses to such vaccines, represents a promising approach for the treatment of RA and other autoimmune diseases.

Animals↗