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PubMedDate not supplied

ProteinChip technology: a new and facile method for the identification and measurement of high-density lipoproteins apoA-I and apoA-II and their glycosylated products in patients with diabetes and cardiovascular disease.

This paper describes a ProteinChip technology for the identification and quantification of apolipoprotein profiles in crude biological samples. Expression levels of apoA-I and apoA-II and their glycosylated products were accomplished using single 1 microL plasma samples. In the present studies, strong anionic and weak cationic exchanger ProteinChips (SAX2 and WCX2 chip surfaces) were tested, and the WCX2 chip was found to be selective for specific apolipoproteins. Using the WCX2 chip and analysis via surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS), apoA-I and apoA-II were separated as sharp peaks at 28 and 17 kD and did not overlap with other serum protein peaks. Since these assays can be completed on a large number of clinical samples in approximately 1 h, further development of this technique will facilitate both epidemiological studies and therapeutic trials in assessing the role of the apolipoproteins and their glycosylated products in atherosclerosis.

Apolipoprotein A-I↗

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↗

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↗

PubMed2003

Proteomic analyses of arsenic-induced cell transformation with SELDI-TOF ProteinChip technology.

In this study, we demonstrated that low levels (1.5 microM) of arsenite induces B[a]P-treated lung cell transformation. We then used a proteomic approach to identify protein expression by ProteinChips, which could potentially be important for transformation induced by this toxic metal. Most of the protein peaks in cell extracts of all samples, including the control, B[a]P-treated, and B[a]P + As-treated cells are identical. However, surface-enhanced laser desorption/ionization time of flight (SELDI-TOF) analysis with Cu-ProteinChips and WCX-ProteinChips revealed several dramatically different protein peaks that appeared in lung cells after being transformed by a treatment of 1.5 microM arsenite for 12 weeks. SAX2 ProteinChip also identified a prominent protein peak that was preferentially expressed in control cells. Interestingly, by using a SAX2 chip, we were able to detect several protein peaks that increased their expression in lung epithelial cells (LEC) treated with only B[a]P. Identification and characterization of these proteins may reveal the molecular basis of As-induced cell transformation and provide insight into the mechanisms by which arsenic induces carcinogenesis.

Animals↗

PubMed2002

Parallel analysis with optically gated sample introduction on a multichannel microchip.

As an alternative to the T-type injection on microchips, optically gated sample introduction previously has been demonstrated to provide fast, serial, and reproducible injections on a single-channel microchip. Here, the ability to perform high throughput, multichannel analysis with optically gated sample introduction is described using a voice coil actuator. The microchip is fixed on a stage, which moves back and forth via the voice coil actuator, scanning two laser beams across the channels on the microchip. For parallel analysis on a multichannel microchip, both the gating beam and the probe beam are scanned at 10 Hz to perform multiple injections and parallel detection. Simultaneous, fast separations of 4-choloro-7-nitrobenzofurazan (NBD)-labeled amino acids are demonstrated in multiple channels on a microchip. Serial separations of different samples in multiple channels are also reported. Optically gated sample introduction on multiple, parallel channels shows the potential for high-speed, high-throughput separations that are easily automated by using a single electronic shutter.

4-Chloro-7-nitrobenzofurazan↗

PubMed2003

A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard.

The comparison of two-dimensional (2-D) gel images from different samples is an established method used to study differences in protein expression. Conventional methods rely on comparing images from at least 2 different gels. Due to the high variation between gels, detection and quantification of protein differences can be problematic. Two-dimensional difference gel electrophoresis (Ettan trade mark DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. In the application of DIGE different samples are labelled with mass and charge matched spectrally resolvable fluorescent dyes and are then separated on the same 2-D gel. Using an Escherichia coli lysate "spiked" with varying amounts of four different known proteins, we have tested a novel experimental design that exploits the sample multiplexing capabilities of DIGE, by including a standard sample in each gel. The standard sample comprises equal amounts of each sample to be compared and was found to improve the accuracy of protein quantification between samples from different gels allowing accurate detection of small differences in protein levels between samples.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2003

Estradiol control of expression and levels of estradiol-binding proteins in the medial preoptic area, medial hypothalamus and pituitary.

The brains of mammals have at least three estradiol-binding proteins: estradiol receptor-alpha (ERalpha), ERbeta, and sex hormone-binding globulin (SHBG). In this study we compare the effects of estradiol treatment on the expression of mRNA for these three estradiol-binding proteins in two reproductively important brain areas, the medial preoptic area-anterior hypothalamus (MPOA-AH) and medial hypothalamus (MH) as well as in the hippocampus in ovariectomized rats, using the reverse transcriptase-polymerase chain reaction (RT-PCR). We also used surface-enhanced laser desorption ionization time of flight (SELDI-TOF) mass spectrometry (MS) to analyze the effects of estradiol in ovariectomized rats on SHBG levels in the MPOA-MH as well as the neurohypophysis. In vivo estradiol treatment in ovariectomized rats eliminated or significantly reduced expression of all three estradiol-binding proteins in both the MPOA-AH and MH. This change in ERalpha, ERbeta, and SHBG expression did not occur in the hippocampus. Both Northern blot and DNA sequence analysis confirmed the results of the RT-PCR for SHBG. SELDI-TOF MS analysis demonstrated that in vivo estradiol treatments resulted in dramatically decreased levels of SHBG in the hypothalamus and that a reduction in SHBG mRNA by estradiol treatment also resulted in a reduction in SHBG protein levels. Estradiol treatment also eliminated detectable SHBG from the neurohypophysis, suggesting that estradiol controls SHBG levels in this release site. That in vivo estradiol treatments had the same inhibitory effects on mRNA levels for SHBG and both ERs suggests similar translational control mechanisms for all three steroid-binding proteins in the brain. That estradiol treatments also reduced pituitary SHBG suggests that such treatment releases SHBG from the neurohypophysis.

Animals↗

PubMed2003

Detection of protease activities with the mass-spectrometry-assisted enzyme-screening (MES) system.

The MALDI-MES provides a rapid, sensitive and reproducible alternative approach to existing analytical techniques for the detection of enzymatic activities that does not require a chromophore or radiolabeling. An improved method is presented, by which enzymes with defined substrate specificities can be detected with a MALDI mass spectrometer in complex protein fractions. In order to demonstrate the utility of the new method, in this study we describe the use of MALDI-MES to detect proteolytic activities in a protein extract from porcine renal tissue, which contained several thousand proteins as visualized by 2D electrophoresis. The analytical procedure is based on covalent immobilization of proteins to beads. By immobilizing proteins, autolytic and proteolytic degradation is prevented and the removal of those molecules from the protein fraction is achieved, which otherwise would interfere with the mass spectrometric detection of the enzymatic reaction products. The enzymatic activity is determined by incubating the immobilized proteins with a reaction-specific probe, followed by the analysis of the reaction mixture with the MALDI-MS after defined incubation times. The presence of the target enzyme is validated by locating a signal, which fits the molecular mass of the expected reaction product in the mass spectrum. To demonstrate how to detect proteolytic activities in this system, the reactions catalyzed by endopeptidase, angiotensin-converting enzyme, kallikrein, renin, and urotensin-converting enzyme were monitored. The experiments showed that the MALDI-MES method is sufficient according the quantification to investigate the effects of inhibitors. This is demonstrated using a specific renin inhibitor to inhibit an angiotensin-I generating enzyme activity in a renal protein extract.

Animals↗

PubMed2003

Proteomic analyses in Waldenstrom's macroglobulinemia and other plasma cell dyscrasias.

The proteomic analysis of tumor cells emerges as a key complement to gene expression profiling, primarily because regulation of protein expression (at the translational and post-translational levels) can buffer the magnitude of changes occurring at the gene transcription level, in order to fine tune cellular functions. Herein we describe the concept of proteomic analysis of the signaling state of tumor cells, as well as its application in the study of signaling pathways in plasma cell dyscrasias, such as Waldenstrom's macroglobulinemia (WM) and multiple myeloma (MM). Comparative studies of WM versus MM cells at baseline and in the setting of drug treatment reveals proteomic profiles of the signaling state with significant overlap (that could reflect a putative B-cell lineage-related proteomic signature), but also distinct differences, possibly associated with differential features in the biologic behavior and drug sensitivity of these diseases. These proteomic studies pave the way for a more comprehensive insight into the molecular basis of WM versus other B-cell malignancies.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2003

Growth factor array fabrication using a color ink jet printer.

We have developed a novel method for growth factor analysis using a commercial color ink jet printer to fabricate substrata patterned with growth factors. We prepared substrata with insulin printed in a simple pattern or containing multiple areas of varying quantities of printed insulin. When we cultured the mouse myoblast cell line, C2C12, on the insulin-patterned substrata, the cells were grown in the same pattern with the insulin-printed pattern. Cell culture with the latter substrata demonstrated that quantity control of insulin deposition by a color ink jet printer is possible. For further applications, we developed substrata with insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF) spotted in 16 different areas in varying combinations and concentrations (growth factor array). With this growth factor array, C2C12 cells were cultured, and the onset of muscle cell differentiation was monitored for the expression of the myogenic regulator myogenin. The ratio of cells expressing myogenin varied with the doses of IGF-I and bFGF in the sections, demonstrating a feasibility of growth factor array fabrication by a color ink jet printer. Since a printer manipulates several colors, this method can be easily applied to multivariate analyses of growth factors and attachment factors affecting cell growth and differentiation. This method may provide a powerful tool for cell biology and tissue engineering, especially for stem cell research in investigating unknown conditions for differentiation.

Animals↗

PubMed2003

A data review and re-assessment of ovarian cancer serum proteomic profiling.

BACKGROUND: The early detection of ovarian cancer has the potential to dramatically reduce mortality. Recently, the use of mass spectrometry to develop profiles of patient serum proteins, combined with advanced data mining algorithms has been reported as a promising method to achieve this goal. In this report, we analyze the Ovarian Dataset 8-7-02 downloaded from the Clinical Proteomics Program Databank website, using nonparametric statistics and stepwise discriminant analysis to develop rules to diagnose patients, as well as to understand general patterns in the data that may guide future research. RESULTS: The mass spectrometry serum profiles derived from cancer and controls exhibited numerous statistical differences. For example, use of the Wilcoxon test in comparing the intensity at each of the 15,154 mass to charge (M/Z) values between the cancer and controls, resulted in the detection of 3,591 M/Z values whose intensities differed by a p-value of 10-6 or less. The region containing the M/Z values of greatest statistical difference between cancer and controls occurred at M/Z values less than 500. For example the M/Z values of 2.7921478 and 245.53704 could be used to significantly separate the cancer from control groups. Three other sets of M/Z values were developed using a training set that could distinguish between cancer and control subjects in a test set with 100% sensitivity and specificity. CONCLUSION: The ability to discriminate between cancer and control subjects based on the M/Z values of 2.7921478 and 245.53704 reveals the existence of a significant non-biologic experimental bias between these two groups. This bias may invalidate attempts to use this dataset to find patterns of reproducible diagnostic value. To minimize false discovery, results using mass spectrometry and data mining algorithms should be carefully reviewed and benchmarked with routine statistical methods.

Artificial Intelligence↗

PubMed2002

Application of tissue microarray technology to the study of non-Hodgkin's and Hodgkin's lymphoma.

The immunohistochemical analysis of lymphoid neoplasms has led to refined classification schemes based on the profile of antigen expression and correlation with morphological, cytogenetic, molecular, and clinical features. Tissue microarrays (TMAs) are a powerful tool to rapidly characterize the phenotypic profile of a large number of samples. We show that this technique can be readily applied to the study of lymphoma by examining the expression profile of a series of 193 B-cell non-Hodgkin's lymphomas (NHLs) and 29 Hodgkin's lymphomas (HLs) using immunohistochemistry and in situ hybridization (ISH). The NHL cases were studied for the expression of commonly used markers-including CD3, CD5, CD10, CD20, CD23, CD30, CD43, Bcl-2, and cyclin D1 by immunohistochemical staining of TMAs-and these results were compared with whole sections (WS) of the same cases. We found a high degree of correlation between the results achieved with TMAs or WS (86% to 100% of cases). P53 and MIB-1 staining were studied, and the results were similar to that reported in the literature. HL cases were stained for CD20, CD30, CD15 (LeuM1), and latent membrane protein 1 expression, and ISH was performed using probes for EBER-1 and-2 transcripts. The results from HL cases on TMA sections matched exactly with those of WS. We correlated cytogenetic results with immunohistochemical stains and morphology in cases of mantle cell lymphoma [t(11;14)(q13;q32)] and follicular lymphoma [t(14;18)(q32;q24)]. This extensive expression profile of B-cell NHLs and HL tissues discloses the ability of TMAs to rapidly screen a large series of cases and represents the first report of method validation for this technique in the study of lymphoma.

Antigens, CD↗

PubMed2003

White matter changes in schizophrenia: evidence for myelin-related dysfunction.

Numerous lines of inquiry implicate connectivity as a central abnormality in schizophrenia. Myelination and factors that affect myelination, such as the function of oligodendroglia, are critical processes that could profoundly affect neuronal connectivity, especially given the diffuse distribution of oligodendrocytes and the widespread distribution of brain regions that have been implicated in schizophrenia. Multiple lines of evidence now converge to implicate oligodendroglia and myelin in schizophrenia. Imaging and neurocytochemical evidence, similarities with demyelinating diseases, age-related changes in white matter, myelin-related gene abnormalities, and morphologic abnormalities in the oligodendroglia demonstrated in schizophrenic brains are all examined in light of the hypothesis that oligodendroglial dysfunction and even death, with subsequent abnormalities in myelin maintenance and repair, contribute to the schizophrenic syndrome.

Age Factors↗

PubMed2002

G-protein-coupled receptor microarrays.

Membrane-bound proteins represent the single most important class of drug targets. Arraying these proteins is difficult because they typically need to be embedded in membranes to maintain their correctly folded conformations. We describe here the fabrication of microarrays consisting of G-protein-coupled receptors (GPCRs)--the single largest family of membrane-bound proteins-by robotic pin-printing on slides, and demonstrate assays for screening of ligands on these arrays.

Binding, Competitive↗

PubMed2002

Application of self-assembly techniques in the design of biocompatible protein microarray surfaces.

This review focuses on the application of novel technologies for generating biocompatible surfaces for high-throughput screening (HTS) of proteins. Various methods of coupling and spotting proteins on self-assembled monolayer (SAM) surfaces will be described along with the protein chip challenges pertaining to spot homogeneity, morphology, biocompatibility and reproducibility.

Biocompatible Materials↗

PubMed2002

Application of surface-enhanced laser desorption/ionization technology to the detection and identification of urinary parvalbumin-alpha: a biomarker of compound-induced skeletal muscle toxicity in the rat.

In toxicity studies, compound-induced changes are typically evaluated using a combination of endpoints and there are often a number of potential markers in biological fluids which can indicate toxic change in tissues and organs. However, some biomarkers are not specific to the organ of injury and therefore there is a continuing search for more sensitive and specific indicators of target organ toxicity. In experiments to assess the potential diagnostic usefulness of surface-enhanced laser desorption/ionization (SELDI) ProteinChip technology, skeletal muscle toxicity was induced in Wistar Han rats by administering 2,3,5,6-tetramethyl-p-phenylenediamine (TMPD). The skeletal muscle toxicity was monitored using established endpoints such as increase in serum aldolase (Aldol), aspartate aminotransferase (AST) and histopathology, and also using SELDI retentate chromatography mass spectrometry of urine samples. Clear differences in urinary protein patterns between control and TMPD-treated animals were observed on the ProteinChip surfaces. Additionally a specific urine marker protein of 11.8 kDa was identified in TMPD-dosed rats, and the detection of the marker was related to the degree of skeletal muscle toxicity assessed by recognized clinical pathology endpoints. The 11.8 kDa protein was identified as parvalbumin-alpha. These experiments demonstrated the potential of urinary parvalbumin-alpha as a specific, noninvasive, and easily detectable biomarker for skeletal muscle toxicity in the rat and the potential of SELDI technology for biomarker detection and identification in toxicology studies.

Animals↗

PubMed2003

Recent developments in protein microarray technology.

The sequencing of the human genome and the advent of DNA chips and sophisticated bioinformatics platforms have enabled molecular biologists to take a more global view of biological systems and to analyze naturally occurring genetic variation. Microarrays of antibodies can measure the concentrations of many proteins quickly and simultaneously. Microarrays of genomically encoded proteins allow scientists to screen entire genomes for proteins that interact with particular factors, catalyze particular reactions, or act as substrates for protein-modifying enzymes or as targets of autoimmune responses. The new protein microarray platforms will prove invaluable to basic biological research, and will dramatically accelerate the pace of discovery of drug targets and diagnostic biomarkers.

Drug Design↗

PubMed2002

Therapeutic cancer targeting peptides.

Antitumor monoclonal antibodies have shown clinical promise as cancer cell surface targeting agents. More tumor targeting antibodies are likely to be approved by the FDA in the next few years. However, there are two major limitations in antibody-targeted therapy: large size and nonspecific uptake of the antibody molecules by the liver and the reticuloendothelial system. These result in poor tumor penetration of antibody pharmaceuticals and dose-limiting toxicity to the liver and bone marrow. Peptides are excellent alternative targeting agents for human cancers, and they may alleviate some of the problems with antibody targeting. In the last decade, several investigators have successfully used combinatorial library methods to discover cell surface binding peptides that may be useful for cancer targeting. The phage-display library technique and the "one-bead one-compound" combinatorial library method are the two approaches that have been used. Cancer cell surface receptors or endothelial cell surface receptors of the neovasculature are the two popular therapeutic targets for cancer. Results from preclinical studies with some peptides are encouraging in their targeting potential.

Amino Acid Sequence↗