Micropatterning of proteins and mammalian cells on biomaterials.
(73/1981)
Controlling the spatial organization of cells is vital in engineering tissues that require precisely defined cellular architectures. For example, functional nerves or blood vessels form only when groups of cells are organized and aligned in very specific geometries. Yet, scaffold designs incorporating spatially defined physical cues such as microscale surface topographies or spatial patterns of extracellular matrix to guide the spatial organization and behavior of cells cultured in vitro remain largely unexplored. Here we demonstrate a new approach for controlling the spatial organization, spreading, and orientation of cells on two micropatterned biomaterials: chitosan and gelatin. Biomaterials with grooves of defined width and depth were fabricated using a two-step soft lithography process. Selective attachment and spreading of cells within the grooves was ensured by covalently modifying the plateau regions with commercially available protein resistant triblock copolymers. Precise spatial control over cell spreading and orientation has been observed when human microvascular endothelial cells are cultured on these patterned biomaterials, suggesting the potential of this technique in creating tissue culture scaffolds with defined chemical and topographical features. (+info)
Saccharomyces cerevisiae glutaredoxin 5-deficient cells subjected to continuous oxidizing conditions are affected in the expression of specific sets of genes.
(74/1981)
The Saccharomyces cerevisiae GRX5 gene codes for a mitochondrial glutaredoxin involved in the synthesis of iron/sulfur clusters. Its absence prevents respiratory growth and causes the accumulation of iron inside cells and constitutive oxidation of proteins. Null Deltagrx5 mutants were used as an example of continuously oxidized cells, as opposed to situations in which oxidative stress is instantaneously caused by addition of external oxidants. Whole transcriptome analysis was carried out in the mutant cells. The set of genes whose expression was affected by the absence of Grx5 does not significantly overlap with the set of genes affected in respiratory petite mutants. Many Aft1-dependent genes involved in iron utilization that are up-regulated in a frataxin mutant were also up-regulated in the absence of Grx5. BIO5 is another Aft1-dependent gene induced both upon iron deprivation and in Deltagrx5 cells; this links iron and biotin metabolism. Other genes are specifically affected under the oxidative conditions generated by the grx5 mutation. One of these is MLP1, which codes for a homologue of the Slt2 kinase. Cells lacking MLP1 and GRX5 are hypersensitive to oxidative stress caused by external agents and exhibit increased protein oxidation in relation to single mutants. This in turn points to a role for Mlp1 in protection against oxidative stress. The genes of the Hap4 regulon, which are involved in respiratory metabolism, are down-regulated in Deltagrx5 cells. This effect is suppressed by HAP4 overexpression. Inhibition of respiratory metabolism during continuous moderately oxidative conditions could be a protective response by the cell. (+info)
Proteomics techniques and their application to hematology.
(75/1981)
The recent sequencing of a number of genomes has raised the level of opportunities for studies on proteins. This area of research has been described with the all-embracing term, proteomics. In proteomics, the use of mass spectrometric techniques enables genomic databases to be used to establish the identity of proteins with relatively little data, compared to the era before genome sequencing. The use of related analytical techniques also offers the opportunity to gain information on regulation, via posttranslational modification, and potential new diagnostic and prognostic indicators. Relative quantification of proteins and peptides in cellular and extracellular material remains a challenge for proteomics and mass spectrometry. This review presents an analysis of the present and future impact of these proteomic technologies with emphasis on relative quantification for hematologic research giving an appraisal of their potential benefits. (+info)
Development of gene ontology tool for biological interpretation of genomic and proteomic data.
(76/1981)
We have designed and developed a Gene Ontology based navigation tool, GoMiner, which organizes lists of interesting genes from a microarray or a protein array experiment for biological interpretation. It provides quantitative and statistical output files and useful visualization (e.g., a tree-like structure) to map the list of genes to its biological functional categories. It also provides links to other resources such as pubmed, locuslink, and biological molecular interaction map and signaling pathway packages. (+info)
Cell protection, resistance and invasiveness of two malignant mesotheliomas as assessed by 10K-microarray.
(77/1981)
Malignant pleural mesothelioma (MPM) is an aggressive serosal tumor, strongly associated with former exposure to asbestos fibers and for which there is currently no effective treatment available. In human, MPM is characterized by a high local invasiveness, poor prognosis and therapeutic outcomes. In order to assess molecular changes that specify this phenotype, we performed a global gene expression profiling of human MPM. Using a 10,000-element microarray, we analyzed mRNA relative gene expression levels by comparing a mesothelioma cell line to either a pleural cell line or tumor specimens. To analyze these gene expression data, we used various bioinformatics softwares. Hierarchical clustering methods were used to group genes and samples with similar expression in an unsupervised mode. Genes of known function were further sorted by enzyme, function and pathway clusters using a supervised software (IncyteGenomics). Taken together, these data defined a molecular fingerprint of human MPM with more than 700 up- or down-regulated genes related to several traits of the malignant phenotype, specially associated with MPM invasiveness, protection and resistance to anticancer defenses. This portrait is meaningful in disease classification and management, and relevant in finding new specific markers of MPM. These molecular markers should improve the accuracy of mesothelioma diagnosis, prognosis and therapy. (+info)
Altered E-cadherin and epidermal growth factor receptor expressions are associated with patient survival in lung cancer: a study utilizing high-density tissue microarray and immunohistochemistry.
(78/1981)
E-cadherin (E-cad) and epidermal growth factor receptor (EGFR) are important cell adhesion and signaling pathway mediators. This study aimed to assess their expression in lung adenocarcinoma (AdC) and squamous cell carcinoma (SCC) and their association with clinicopathologic variables. In all, 130 resectable lung cancers (stages I-IIIA) were studied using a high-density tissue microarray. Two to three cores from each case were arrayed into three blocks using a Beecher system. Immunohistochemistry was performed using an avidin-biotin complex method and monoclonal antibodies against E-cad and EGFR. Unequivocal membrane staining in >10% of tumor cells was considered as a positive expression of E-cad and EGFR. Markers expression and coexpression were analyzed against clinicopathologic variables (age, gender, smoking status, performance status, weight loss, histology, grade, stage, and lymph node involvement) and patient survival. There were 118, 126, and 115 cases that were fully assessable for E-cad, EGFR, and both markers, respectively. For E-cad, 65 cases (55%) were positive (+), 53 (45%) were negative (-); 23 cases of the negative group had only cytoplasmic staining. For EGRF, 43 cases (34%) were (+), and 83 (66%) were (-). There was no significant association between E-cad or EGFR, and any of the clinicopathologic variables except for an association between EGFR(+) and SCC histologic type. Both negative and cytoplasmic staining of E-cad correlated with shorter patient survival with P=0.008 and 0.002, respectively. EGFR expression did not correlate with patient survival; however, patients with E-cad(-)/EGFR(+) phenotype had poorer survival than those with E-cad(+)/EGFR(-) (P=0.026). Our study suggests that lung AdC and SCC may be stratified based on expression of E-cad and EGFR with the E-cad(-)/EGFR(+) expression having a worse disease outcome. Moreover, the cytoplasmic expression of E-cad may represent an altered localization of this protein in association with tumorigenicity. (+info)
How industry is approaching the search for new diagnostic markers and biomarkers.
(79/1981)
In the diagnostic and the pharmaceutical industry there is a constant need for new diagnostic markers and biomarkers with improved sensitivity and specificity. During the last 5 years, only a few novel diagnostic markers have been introduced into the market. Proteomics technologies are now offering unique chances to identify new candidate markers. Before a marker can be introduced into the market, three successive developmental phases have to be completed: the discovery phase, in which a variety of proteomics technologies are applied to identify marker candidates; the prototype developmental phase, in which immunological assays are established and validated in defined sample collectives; and finally the product development phase, with assay formats suitable for automated platforms. The hurdles that a potential candidate marker has to pass in each developmental phase before reaching the market are considerable. The costs are increasing from phase to phase, and in industry a number of questions concerning the medical need and the potential return on investment have to be answered before a proteomics discovery project is started. In this review, we will cover aspects of all three developmental phases including the repertoire of discovery tools for protein separation as well as giving an outline of modern principles of mass spectrometry for the identification of proteins. (+info)
Enhanced protein profiling arrays with ELISA-based amplification for high-throughput molecular changes of tumor patients' plasma.
(80/1981)
PURPOSE: The purpose of this study is to develop a high-throughput approach to detect protein expression from hundreds and thousands of samples and to apply this technology to profile circulating angiogenic factor protein levels in patients with gynecological tumors. EXPERIMENTAL DESIGN: Analytes containing a mixture of protein are immobilized onto antibody-coated surface of support in array format. The presence of protein in analytes is detected with biotin-labeled antibody coupled with an enhanced chemiluminescence or fluorescence detection system. The exact amount of protein can be quantitatively measured. The expression levels of five angiogenic factors (angiogenin, interleukin 8, vascular endothelial growth factor, platelet-derived growth factor, and epidermal growth factor) from 157 samples were quantitatively measured using this novel protein array technology and were statistically analyzed. The expression patterns of angiogenic factors were analyzed using two-way hierarchical cluster analysis approach. RESULTS: A novel protein array technology, which can simultaneously and quantitatively measure few protein levels from hundreds and thousands of samples was developed. Only minute amounts of sample are required for the assay. This approach also features high sensitivity and specificity. Using this novel protein array approach, we analyzed the plasma expression levels of five angiogenic factors in 137 patients diagnosed with a tumor and 20 controls. Statistical analysis reveals different expression levels of angiogenic factors between patients and controls. Cluster analysis suggests a possible classification of normal subjects from patients. CONCLUSIONS: Enhanced protein profiling arrays provide a high-throughput and sensitive system to detect one or few protein from hundreds and thousands of samples. Such an approach should have broad application in biomedical discovery. (+info)