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PubMed2002

High-throughput molecular profiling of high-grade astrocytomas: the utility of fluorescence in situ hybridization on tissue microarrays (TMA-FISH).

Due to recent biological and technical advances, the list of potentially useful candidate genes is rapidly expanding in the study of brain tumors. However, traditional methods of screening individual genes in individual samples are slow and tedious, often with consumption of precious resources after only a few experiments. This study evaluates the feasibility of high-throughput molecular analysis using fluorescence in situ hybridization (FISH) on glioma tissue microarrays (TMA). A single microarray paraffin block was constructed using 65 WHO grade III and IV astrocytomas, sampled in duplicate with 0.6-mm-diameter punch cores. FISH was used to detect common alterations, such as EGFR amplification, chromosome 7, 9, and 10 aneusomies and deletions of 1p, 19q, PTEN, DMBT1, and p16. Of 585 hybridization sets, 508 (87%) yielded interpretable data, with hybridization failure in 33 (5.5%) and dislodged tissue in 44 sets (7.5%), respectively. Glioblastomas harbored significantly more alterations than anaplastic astrocytomas, with the overall frequencies of alterations similar to those reported using other techniques. The overall concordance rate between paired tumor core samples was 93%. We conclude that TMA-FISH is an efficient and reliable method for detecting molecular alterations in high-grade astrocytomas.

Agglutinins↗
PubMed2002

The predicted candidates of Arabidopsis plastid inner envelope membrane proteins and their expression profiles.

Plastid envelope proteins from the Arabidopsis nuclear genome were predicted using computational methods. Selection criteria were: first, to find proteins with NH(2)-terminal plastid-targeting peptides from all annotated open reading frames from Arabidopsis; second, to search for proteins with membrane-spanning domains among the predicted plastidial-targeted proteins; and third, to subtract known thylakoid membrane proteins. Five hundred forty-one proteins were selected as potential candidates of the Arabidopsis plastid inner envelope membrane proteins (AtPEM candidates). Only 34% (183) of the AtPEM candidates could be assigned to putative functions based on sequence similarity to proteins of known function (compared with the 69% function assignment of the total predicted proteins in the genome). Of the 183 candidates with assigned functions, 40% were classified in the category of "transport facilitation," indicating that this collection is highly enriched in membrane transporters. Information on the predicted proteins, tissue expression data from expressed sequence tags and microarrays, and publicly available T-DNA insertion lines were collected. The data set complements proteomic-based efforts in the increased detection of integral membrane proteins, low-abundance proteins, or those not expressed in tissues selected for proteomic analysis. Digital northern analysis of expressed sequence tags suggested that the transcript levels of most AtPEM candidates were relatively constant among different tissues in contrast to stroma and the thylakoid proteins. However, both digital northern and microarray analyses identified a number of AtPEM candidates with tissue-specific expression patterns.

Arabidopsis↗
PubMed2003

Comprehensive proteomic profiling identifies serum proteomic signatures for detection of hepatocellular carcinoma and its subtypes.

BACKGROUND: Detection of hepatocellular carcinoma (HCC) in patients with chronic liver disease (CLD) is difficult. We investigated the use of comprehensive proteomic profiling of sera to differentiate HCC from CLD. METHODS: Proteomes in sera from 20 CLD patients with alpha-fetoprotein (AFP) <500 microg/L (control group) and 38 HCC patients (disease group) were profiled by anion-exchange fractionation (first dimension), two types (IMAC3 copper and WCX2) of ProteinChip Arrays (second dimension), and time-of-flight mass spectrometry (third dimension). Bioinformatic tests were used to identify tumor-specific proteomic features and to estimate the values of the tumor-specific proteomic features in the diagnosis of HCC. Cross-validation was performed, and we also validated the models with pooled sera from the control and disease groups, serum from a CLD patient with AFP >500 microg/L, and postoperative sera from two HCC patients. RESULTS: Among 2384 common serum proteomic features, 250 were significantly different between the HCC and CLD cases. Two-way hierarchical clustering differentiated HCC and CLD cases. Most HCC cases with advanced disease were clustered together and formed two subgroups that contained significantly more cases with lymph node invasion or distant metastasis. For differentiation of HCC and CLD by an artificial network (ANN), the area under the ROC curve was 0.91 (95% confidence interval, 0.82-1.01; P <0.0005) for all cases and 0.954 (95% confidence interval, 0.881-1.027; P <0.0005) for cases with nondiagnostic serum AFP (<500 microg/L). At a specificity of 90%, the sensitivity was 92%. Both cluster analysis and ANN correctly classified the pooled serum samples, the CLD serum sample with increased AFP, and the HCC patient in complete remission. CONCLUSION: Tumor-specific proteomic signatures may be useful for detection and classification of hepatocellular cancers.

Carcinoma, Hepatocellular↗
PubMed2003

Biopsy of a biopsy: validation of immunoprofiling in gastric cancer biopsy tissue microarrays.

AIMS: Tissue microarrays offer an efficient way of examining a large number of tumour cases on a single glass slide. A major concern, however, is tumour heterogeneity. Also, the use of tissue microarrays in biopsy material is unexplored. The purpose of the present study was to assess the possibility and validity of arraying three 0.6-mm cores per case in endoscopic gastric cancer biopsies for immunophenotyping. METHODS AND RESULTS: Thirty-eight cases were studied with immunohistochemical staining for p53, CD44v6 and vascular endothelial growth factor. Full tissue sections were compared with triple core-tissue microarrays. Thirty-six cases contained three cores with tumour, one case contained two cores with tumour and one case contained only a single core with viable tumour and was excluded. Three further cores had been lost from three separate cases on the sections for immunohistochemistry. kappa values for whole-sections versus tissue microarrays ranged between 0.77 and 0.94. p53 immunohistochemical staining (interpretation as + or -) yielded the best result with only 1/37 mismatches, whereas CD44v6 (graded both for intensity and extent) showed 3/37 mismatches. The small depth of tissue in cores from biopsies necessitates all cores being arrayed flush with the face of the recipient wax block for maximizing the number of sections available. Compared with the first section over 30 additional 4- micro m sections were available before the first case (with one core left) had to be excluded and 80 sections before half the tissue cores were lost. CONCLUSIONS: It is impracticable to array more than 120-150 cores per block. Tissue microarray with three cores per case is feasible and valid for studying biopsy material.

Adult↗
PubMed2002

Characterization of a human sphingosine-1-phosphate receptor gene (S1P5) and its differential expression in LGL leukemia.

Large granular lymphocyte (LGL) leukemia is a lymphoproliferative disorder often associated with autoimmune disease. A central feature of this disease is dysregulation of apoptosis. In order to identify differentially expressed genes in LGL leukemia, microarray analysis was performed. We found many differentially expressed genes including several expression sequence tags (ESTs). As a systematic study, we selected one up-regulated EST (GenBank Accession number N47089) and further investigated. An LGL leukemia library was screened using this EST as a probe and a full-length sequence for a novel gene was identified. The deduced amino acid sequence revealed that the novel gene encodes a G-protein-coupled receptor gene that exhibits 86% identity with rat sphingosine-1-phosphate receptor (edg-8/nrg-1). This gene is present in brain, spleen, and peripheral blood mononuclear cells (PBMC) and is overexpressed in leukemic LGL.

Amino Acid Sequence↗
PubMed2003

Tissue microarray analysis reveals prognostic significance of syndecan-1 expression in prostate cancer.

BACKGROUND: Tissue microarrays (TMA) have recently emerged as powerful tools to rapidly analyze the clinical significance of new molecular markers in human tumors. Here, we have tested several molecular markers on a prostate TMA containing 637 different specimens. METHODS: The specimens were from 551 patients with prostate cancer and long-term follow-up information on progression (median 5.3 years), tumor-specific and overall survival (median 5.9 years). Eighty-six specimens from benign prostatic hyperplasia were included as controls. Expression of Ki67, Bcl-2, p53, CD-10 (neutral endopeptidase), and syndecan-1 (CD-138) was analyzed by immunohistochemistry. RESULTS: Gleason grade and Ki67 Labeling Index (LI) were independent predictors of early recurrence and poor survival. Bcl-2 predicted early recurrence, whereas p53 was associated with poor survival. Syndecan-1 overexpression also predicted early recurrence and was significantly associated with tumor specific survival, high Gleason grade, Ki67 LI, and Bcl-2 overexpression. Neoadjuvant hormonal therapy was associated with overexpression of Bcl-2 and inhibition of Ki67 LI and CD-10, but did not affect the expression of the remaining markers. CONCLUSIONS: The results of this TMA study confirm a dominant prognostic significance of Gleason grading and Ki67 LI in prostate cancer, as compared to a less pronounced role of Bcl-2, and p53. We identified syndecan-1 as a new prognostic factor and provide evidence for an androgen-dependent regulation of CD-10 expression.

Aged↗
PubMed2026

Immunoproteomic Profiling of Autoantibodies and Antibodies against Infectious Agents in Autoimmune Diseases.

Prior research investigated limited antibody sets within individual autoimmune diseases. Using the Nucleic-Acid Programmable Protein Array platform, we measured antibodies against 280 human, 40 viral, and 15 bacterial antigens in serum from 237 patients with 8 autoimmune diseases, including autoimmune gastritis (AG), autoimmune thyroiditis (AT), celiac disease (CD), idiopathic inflammatory myopathies (IIM), type 1 diabetes mellitus (T1D), rheumatoid arthritis (RA), Sj&#xf6;gren's disease (SjD), and systemic lupus erythematosus (SLE), and 112 controls. Candidate antibodies were identified by combining Firth logistic regression and machine learning. We identified disease-specific antibodies, ranging from 3 in IIM to 13 in SLE for IgG and 1 in CD to 13 in AG for IgA. Additionally, 63 IgG and 44 IgA antibodies were shared across two or more diseases. Notably, two IgG autoantibodies overlapped in up to five diseases: directed against STNM4 (SLE, SjD, T1D, CD, and RA) and TRIM21 (SLE, SjD, IIM, CD, and RA); and three IgA antibodies in up to seven diseases: directed against H1N1 Influenza A virus NP (IIM, SjD, AG, T1D, CD, RA, and AT) and Coxsackievirus B3MK012537 and Enterovirus C PVgp1 (IIM, SjD, AG, T1D, CD, SLE, and RA). These findings underscore the potential of antibody profiling in autoimmune disease characterization and biomarker discovery.

Humans↗
PubMed2003

Approaches to proteomic analysis of human tumors.

The completion of the Human Genome Project and the successful use of high-throughput array formats for mRNA measurements has spurred a revival of interest in the field of proteomics. Clinical cancer specimens will be a critical component of proteomic studies of cancer, both for discovering new insights into TSG function, and for validation of discoveries that are made in the laboratory. Global protein analysis methods hold great potential for facilitating the discovery of novel tumor suppressor genes, as well as providing a better understanding of the biochemical role of these proteins.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2002

Design of buffer exchange surfaces and sensor chips for biosensor chip mass spectrometry.

The feasibility of buffer exchange in biosensor chip mass spectrometry, along with the construction of base sensor chips and use of alternative chip chemistries, is demonstrated in this work. Beta-2-microglobulin (beta2m) was used as an analyte and captured in the first flow cell (FC1) on the sensor chip surface by an immobilized anti-beta2m antibody. Low pH buffer was then used to elute the captured analyte from the flow cell and route it to a second flow cell (FC2) downstream that served as a cation exchanger that retains the analyte. Following additional washes in FC1, the analyte present in FC2 was either eluted with a higher pH buffer (to demonstrate the possibility of elution into a downstream trypsin flow cell), or it was subjected to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis to verify its presence in FC2. In a separate experiment, a gold-sputtered glass slide (base chip) was activated through a formation of 11-mercaptoundecanoic acid self-assembled monolayer and via reaction with 1,1"-carbonyldiimidazole. The activated chip was placed manually into the biosensor and two surfaces (flow cells) were derivatized with antibodies to beta2m and cystatin C (cysC). To evaluate the chip performance, diluted human urine aliquot was injected over the flow cells. Following the surface plasmon resonance analysis, the chip was MALDI-TOF MS analyzed, yielding signals from beta2m and cysC from their respective flow cells. Artifacts arising from the surface chemistries were not observed in the analysis.

Buffers↗
PubMed2003

Expression of mucins and cytokeratins in primary carcinomas of the digestive system.

To determine the most optimal treatment of cancer patients, it is fundamental to classify human carcinomas according to their primary anatomical site of origin. As for some patients, it is difficult to identify cancers occurring at obscure location and overlapping adjacent sites. The aim of this study is to partition the primary site of 486 patients in cancers of the digestive system by the expression pattern of the mucins and cytokeratins typifying each site. The expressions of MUC1, MUC2, MUC5AC, MUC6, CK7, CK8, CK13, CK14, CK18, CK19 and CK20 were evaluated immunohistochemically in 426 adenocarcinomas and 60 hepatocellular carcinomas using the tissue-array method. The finding of MUC series showed their characteristics in case of MUC2 in the appendix cancer and MUC1 and 5AC in pancreas cancer. As for CKs 7, 13, and 19, and 20 had a feature in cancers of common bile duct, liver, and appendix, respectively. We classified cancers in 11 sites by characteristic expression of antibodies. The sensitivity, specificity, positive predictive value, and diagnostic efficacy of significant antibodies were calculated with deducing the dichotomous tree made by SPSS 10.0. Six of 11 antibodies, CK 7, CK13, CK19, CK20, MUC1, and MUC5AC distinguished 6 groups from 11 sites. We also executed the clustering of cancers to investigate total relationship among cancers. They fell into three categories, which corresponded to embryologic origin. Unlike other sites, the small intestine and colorectum cancers expressed significantly different patterns to their sublocations. Mucins and CKs showed expression patterns to classify the primary sites of digestive cancers and may be helpful in predicting the primary sites of digestive cancers.

Adenocarcinoma↗
PubMed2003

Molecular characterization of oncostatin M-induced growth arrest of MCF-7 cells expressing a temperature-sensitive mutant of p53.

Our previous studies have shown that treatment of MCF-7 breast cancer cells with cytokine oncostatin M (OM) results in a growth arrest and a concurrent decrease in p53 expression. It remains to be determined whether these two important events are directly connected, as changes in p53 protein levels can lead to variable biological outcomes. In this study we have generated stable cell lines (MCF7-ptsp53) that express p53Val135 a p53 temperature-sensitive mutant. We demonstrate that overexpression of the wildtype (wt) p53 at permissive temperature in MCF7-ptsp53 cells leads to growth arrest at the G2-M phase of the cell cycle. Inhibition of endogenous p53 function with the expression of mutant p53 protein at non-permissive temperature did not affect the OM-induced G1 cell cycle arrest. Microarray studies were further carried out to identify p53- and OM-regulated genes that mediate the G2/M or G1 cell cycle arrest. We show that the expression of p21 was upregulated and expressions of cdc2, cyclin B2 and protein regulator of cytokinesis 1 (PRC1) were suppressed by overexpression of the wt p53 in MCF7-ptsp53 cells at the permissive temperature. In contrast, OM treatment caused coordinate changes of mRNA expression of several cell cycle components including c/EBPdelta, cdc20, and thymidine kinase 1 (TK1) that mainly affect G1-S phase transition. All together, our results suggest that the downregulation of p53 transcription may be involved in some other cellular changes induced by OM but it is not directly connected to the antiproliferative activity of OM per se.

Antineoplastic Agents↗
PubMed2003

Microfluidic peroxidase biochip for polyphenol synthesis.

An enzyme-containing microfluidic biochip has been developed for the oxidative polymerization of phenols. The biochip consists of a simple T-junction with two feed reservoirs 20 mm apart and a microreaction channel 30 mm long. The channel is 15 microm deep and 200 microm wide at the center, giving a reaction volume of 90 nL. The biochip was fabricated using conventional photolithographic methods on a glass substrate etched using a HF-based solution. Fluid transport was enabled using electroosmotic flow. Soybean peroxidase was used as the phenol oxidizing catalyst, and in the presence of p-cresol and H(2)O(2), essentially complete conversion of the H(2)O(2) (the limiting substrate) occurred in the microchannel at a flow rate of ca. 290 nL/min. Thus, peroxidase was found to be intrinsically active even upon dramatic scale-down as achieved in microfluidic reactors. These results were extended to a series of phenols, thereby demonstrating that the microfluidic peroxidase reactor may have application in high-throughput screening of phenolic polymerization reactions for use in phenolic resin synthesis. Finally, rapid growth of poly(p-cresol) on the walls of the microreaction channel could be performed in the presence of higher H(2)O(2) concentrations. This finding suggests that solution-phase peroxidase catalysis can be used in the controlled deposition of polymers on the walls of microreactors.

Bioreactors↗
PubMed2003

Multienzyme catalysis in microfluidic biochips.

The attachment of enzymes to glass microfluidic channels has been achieved using a highly reactive poly(maleic anhydride-alt-alpha-olefin) (PMA)-based coating that is supplied to the microchannel in a toluene solution. The PMA reacts with 3-aminopropyltriethoxysilane groups linked to the glass surface to form a matrix that enables additional maleic anhydride groups to react with free amino groups on enzymes to give a mixed covalent-noncovalent immobilization support. Using a simple T-channel microfluidic design, with reaction channel dimensions of 200 microm wide (at the center), 15 microm deep, and 30 mm long giving a reaction volume of 90 nL, soybean peroxidase (SBP) was attached at an amount up to 0.6 microg/channel. SBP-catalyzed oxidation of p-cresol was performed in aqueous buffer (with 20% [v/v], dimethylformamide) containing H(2)O(2), with microfluidic transport enabled by electroosmotic flow (EOF). Michaelis-Menten kinetics were obtained with K(m) and V(max) values of 0.98 mM and 0.21 micromol H(2)O(2) converted/mg SBP per minute, respectively. These values are nearly identical to nonimmobilized SBP kinetics in aqueous-DMF solutions in 20-microL volumes in 384-well plates and 5-mL reaction volumes in 20-mL scintillation vials. These results indicate that SBP displays intrinsically native activity even in the immobilized form at the microscale, and further attests to the mild immobilization conditions afforded by PMA. Bienzymic and trienzymic reactions were also performed in the microfluidic biochip. Specifically, a combined Candida antarctica lipase B-SBP bienzymic system was used to convert tolyl acetate into poly(p-cresol), and an invertase-glucose oxidase SBP trienzymic system was used to take sucrose and generate H(2)O(2) for SBP-catalyzed synthesis of poly(p-cresol).

Catalysis↗
PubMed2003

Protein expression profiling arrays: tools for the multiplexed high-throughput analysis of proteins.

The completion of the human genome sequence has led to a rapid increase in genetic information. The invention of DNA microarrays, which allow for the parallel measurement of thousands of genes on the level of mRNA, has enabled scientists to take a more global view of biological systems. Protein microarrays have a big potential to increase the throughput of proteomic research. Microarrays of antibodies can simultaneously measure the concentration of a multitude of target proteins in a very short period of time. The ability of protein microarrays to increase the quantity of data points in small biological samples on the protein level will have a major impact on basic biological research as well as on the discovery of new drug targets and diagnostic markers. This review highlights the current status of protein expression profiling arrays, their development, applications and limitations.

Journal Article↗
PubMed2002

Multi-analyte analysis system using an antibody-based biochip.

A multi-analyte detection system using a unique antibody (Ab) biochip is described. The Ab-based biochip, also referred to as the protein biochip, uses a sensor array based on a complementary metal oxide silicon (CMOS) integrated circuit. The Ab-biochip has a sampling platform of four-by-four microarrays of antibodies deposited onto a Nylon membrane substrate. The micro-arrayed antibodies can be interrogated simultaneously or sequentially using the biochip sensing array detector with the use of a diffractive optical element illuminating each antibody spot individually. The usefulness of the Ab biochip is illustrated by the measurements of immunoglobulin G (IgG) used as the model analyte system. The detection limit for Cy5-labeled IgG molecules was 13 pg.

Animals↗
PubMed2003

Comparison of the performance characteristics of poly(dimethylsiloxane) and Pyrex microchip electrophoresis devices for peptide separations.

A comparative study of electrophoretic separations of fluorescently labeled peptides and amino acids on poly(dimethylsiloxane) (PDMS) and Pyrex microchips is presented. The separation parameters for each microchip substrate were compared, including electroosmotic flow, plate numbers, resolution, and limits of detection. The effect of buffer composition on the separation was also investigated. Acceptable separations were obtained for most peptides with both substrates; however, PDMS chips exhibited much lower separation efficiencies and longer analysis times.

Amino Acids↗
PubMedDate not supplied

Analysis of several fluorescent detector molecules for protein microarray use.

The utility of several streptavidin-linked fluorescent detector molecules was evaluated on two protein microarray platforms. Tested detector molecules included: Alexa Fluor 546; R-phycoerythrin (RPE), orange fluospheres; Cy3-containing liposomes (Large Unilamellar Vesicles, LUV) labelled with Cy3; and an RPE-antibody complex. The two array architectures tested consisted of an array of murine Fc-biotin and an array of murine IgG (the murine IgG array was probed with a biotinylated rabbit anti-murine IgG). These platforms allowed for the direct comparison of detector utility by detector recognition of array-bound biotin. All of the fluorescent detectors examined demonstrated utility on each of the array platforms. For the Fc-biotin array, detector signal intensity (background adjusted) was as follows: RPE-antibody complex > fluospheres > RPE > liposomes > Alexa 546: for the IgG array: RPE/antibody complex > RPE > fluospheres > Alexa546 > liposomes. The RPE-antibody complex fluoresced 67% and 150% more intensely than the next closest detector molecule for the Fc-biotin and the murine IgG arrays, respectively. A marked increase in background fluorescence (as compared to RPE alone) did not accompany the increase in signal intensity gained through RPE-antibody complex use (a true increase in signal:noise ratio). These results suggest that the RPE-antibody complex is superior to other molecules for fluorescent detection of analytes on protein microarrays.

Animals↗