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PubMed1978

Membrane receptors of mouse leukocytes. II. Sequential expression of membrane receptors and phagocytic capacity during leukocyte differentiation.

Analysis of four mature cell markers on mouse bone marrow leukocytes grown in vitro, demonstrated a distinct sequence of marker appearance during the terminal phases of granulocytic cell differentiation. A similar pattern of marker expression was also suggested by analysis of mature neutrophils and macrophages isolated from normal tissues. Among cultured neutrophils, receptors for the Fc portion of IgG (FcR) were first expressed on myelocytes and metamyelocytes, and then subsequently on more mature cells. Morphologically mature colony neutrophils (polymorphs) from agar cultures contained only FcR and complement receptor type two (CR(2)) (C3d receptor), and lacked both complement receptor type one (CR(1)) (C3b receptor) and the capacity to ingest latex, bacteria, or iron particles. Neutrophils from 2 and 3 wk liquid media cultures of marrow cells differed from agar grown neutrophils in that they had phagocytic capacity (particle ingestion) [Pi] in addition to FcR and CR(2). Furthermore, in the 4th and 5th wk of these continuous liquid cultures, CR(1) was also expressed, completing the surface marker profile of normal blood neutrophils. Based on these studies, the following order of appearance of these four markers on cells from the myelocytic series was proposed: FcR {arrow} FcR CR(2) {arrow} FcR CR(2) Pi {arrow} FcR CR(2) Pi CR(1). Differential studies of tissue leukocytes containing these same markers revealed that a heterogeneity existed among morphologically mature neutrophils. Even though 95 percent of blood polymorphs contained all four markers, the same was true of only half of spleen polymorphs and only 20 percent of bone marrow polymorphs. Cells of the monocyte-macrophage series were studies in parallel with neutrophils. Cultured marrow monocytes acquired the four mature cell markers so rapidly that the order of receptor appearance could not be determined. However, it was found that CR2 was lost during the terminal phase of monocyte maturation into activated macrophages.

Animals↗
PubMedDate not supplied

mRNA differential display analysis of nephrotic kidney glomeruli.

BACKGROUND: Differential display RT-PCR (DDRT-PCR) is a new powerful technique for identification and characterization of altered gene expression in eukaryotic cells and tissues. We studied here changes in kidney glomerular gene expression in patients with congenital nephrotic syndrome of the Finnish type (CNF), an inherited kidney disease with heavy proteinuria already in utero. METHODS: Using the DDRT-PCR approach and isolated glomeruli from removed human kidneys, we compared the gene expression patterns of normal human and CNF glomeruli. Differential expression of candidate genes was verified by Northern blotting, and the corresponding PCR fragments were sequenced and compared to known sequences in databanks. RESULTS: We found several genes and sequence tags with altered expression in nephrotic glomeruli including fragments with close homologies to cytochrome c oxidase subunit I, integrin-linked kinase, insulin-like growth factor II receptor and eotaxin, and also clones resembling anchyrin and cadherin-like consensus sequences. CONCLUSION: All the sequences identified are of interest in respect to pathogenesis of proteinuria. Furthermore, this study reveals potentially new members to known gene families with tissue and cell type-specific expression.

Adult↗
PubMed1993

Isozyme specific polymerase chain reaction analysis of differential gene expression: a general method applied to lignin peroxidase genes of Phanerochaete chrysosporium.

Analysis of differential expression of closely related genes is a general problem. The lignin peroxidase genes of Phanerochaete chrysosporium represent a typical case. They are differentially expressed according to the conditions encountered during growth. We show that the expression of two such genes, LIG1 and LIG5, can be differentiated at the mRNA level using a highly specific form of the polymerase chain reaction. This generally applicable method allows rapid and accurate analysis of complex and closely related mRNA populations.

Base Sequence↗
PubMed1998

High throughput analysis of differential gene expression.

Elucidation of the changes in gene expression associated with biological processes is a central problem in biology. Advances in molecular and computational biology have led to the development of powerful, high-throughput methods for the analysis of differential gene expression. These tools have opened up new opportunities in disciplines ranging from cell and developmental biology to drug development and pharmacogenomics. In this review, the attributes of five commonly used differential gene expression methods are discussed: expressed sequence tag (EST) sequencing, cDNA microarray hybridization, subtractive cloning, differential display, and serial analysis of gene expression (SAGE). The application of EST sequencing and microarray hybridization is illustrated by the discovery of novel genes associated with osteoblast differentiation. The application of subtractive cloning is presented as a tool to identify genes regulated in vivo by the transcription factor pax-6. These and other examples illustrate the power of genomics for discovering novel genes that are important in biology and which also represent new targets for drug development. The central theme of the review is that each of the approaches to identifying differentially expressed genes is useful, and that the experimental context and subsequent evaluation of differentially expressed genes are the critical features that determine success.

Animals↗
PubMed1984

A differential thermal analysis (DTA) for the heats of reaction and temperature rises produced during the setting of tooth coloured restorative materials.

Differential thermal analysis (DTA) has been used to obtain the temperature rises produced during the setting of a range of tooth coloured restorative materials, which included silicates, glass ionomers and composites. These have been compared on the basis of the temperature rise per unit volume of material. Calibration of the areas under the reaction peaks (produced during this isothermal operation) to obtain heats of reaction is discussed and a method described. As for the temperature rises, values are given per unit volume of material. The results are compared with those reported in other studies from which DTA is seen to be a technique suited to the determination of these parameters and which might be considered for use a standard method.

Chemical Phenomena↗
PubMed1998

Elevated expression of S100P, CAPL and MAGE 3 in doxorubicin-resistant cell lines: comparison of mRNA differential display reverse transcription-polymerase chain reaction and subtractive suppressive hybridization for the analysis of differential gene expression.

Subtractive suppressive hybridization (SSH) and mRNA differential display reverse transcription-polymerase chain reaction (DDRT-PCR) were compared for their ability to detect the expression of drug-resistance associated genes in a doxorubicin-resistant and -sensitive colon carcinoma cell line (LoVo H67P). The expression pattern of more than 9000 bands obtained by DDRT-PCR were identical in both cell lines by more than 95%. Of the remaining differentially expressed DDRT-PCR products, 21 cDNA fragments were further analyzed after cloning. A total of 210 clones were sequenced resulting in 40 different sequences of which only five were differentially expressed as revealed by Northern blot analysis. SSH, on the other hand, resulted in 30 different sequences of 37 clones analyzed. Thirteen of 30 sequences (43%) could be identified by databank analysis (excluding expressed sequence tags) in contrast to nine of 40 clones (23%) obtained by DDRT-PCR. Of the clones identified by SSH, 60% exhibited a differential expression comparing the doxorubicin-resistant and -sensitive cell line, respectively, as compared to only 13% of the DDRT-PCR derived clones. The application of SSH resulted in the identification of differentially expressed genes in three doxorubin-resistant cell lines (LoVo DxR, ARH D60 and KB-V1) as compared to the sensitive parental cell lines. A significant higher expression of S100P, a protein involved in calcium metabolism, as well as MAGE 3 (melanoma antigen gene) was found in the resistant cell lines using this methodology. The expression of CAPL, a second protein involved in calcium metabolism, was only moderately elevated in the doxorubicin-resistant cells. We found that subtractive suppressive hybridization proved to be a more rapid and reliable method for the detection of differentially expressed mRNAs in our system.

Antigens, Neoplasm↗
PubMed2003

Pain dynamics observed by functional magnetic resonance imaging: differential regression analysis technique.

PURPOSE: To observe the dynamic responses of the cortical areas related to the pain processing by using the differential regression analysis (DRA) technique in functional magnetic resonance imaging (fMRI) and investigation of pain mechanisms. MATERIALS AND METHODS: For pain studies, thermal stimulation was applied by immersing the index finger into a hot bath of water with a temperature of 50-52 degrees C. Motor (finger tapping) and visual (flickering light) stimulation experiments were conducted to elucidate the physiological differences between the simple sensory tasks and pain tasks. To obtain dynamic responses, T values (regression analysis) were sequentially estimated by using a series of shifted differential window functions (narrow width). RESULTS: By using the DRA technique, well-defined prompt responses were observed for both motor and visual stimuli. On the other hand, in the pain experiment, a set of sequentially varying responses was observed for the thalamus (Thal), the dorsal anterior cingulate cortex (dACC), the caudal ACC (cACC), and the rostral ACC (rACC). This time-dependent response suggests the dynamics of pain signal processing in cortical areas. CONCLUSION: The results support the hypothesis that the activated areas are similar to the previously reported pain processing areas; however, new sequential responses were observed, suggesting that the technique may reveal dynamics of pain perception and their pathway, important elements in understanding the mechanism of pain. The DRA technique can provide a new opportunity for many spatiotemporal analyses, for example, the physiologically complex and little-studied physiological phenomena, such as pain dynamics.

Cerebral Cortex↗
PubMed2004

Profiling analysis of differential gene expression between hematogenous and peritoneal metastatic sublines of human pancreatic cancer using a DNA chip.

We established the novel sublines HPC-1H5, HPC-3H4, HPC-4H4, and Panc-1H5, which have a high potential of liver metastasis, and HPC-1P5a, HPC-3P4a, HPC-4P4a, and Panc-1P5a, which have a high potential of peritoneal dissemination, derived from low metastatic HPC-1, HPC-3, HPC-4, and Panc-1cell lines, respectively. To clarify the molecular mechanisms of cancer metastasis and of the different levels of gene expression in a variety of metastatic potentials in pancreatic cancer, we performed a broad analysis of differential gene expression analysis between parental cell lines and metastatic sublines. In comparison with the parental cell lines, 65 and 36 genes were overexpressed and underexpressed in highly liver-metastatic sublines. On the other hand, 43 and 45 genes were overexpressed and underexpressed in highly peritoneal-metastatic sublines. uPAR and Serin protease were overexpressed, and E2A and IGF1R were underexpressed in both metastatic sublines. Hierarchical clustering analysis revealed 22 genes classifying liver, peritoneal metastatic sublines and low-metastatic parental cell lines. These genes might be targeted genes separating those two major metastatic forms after surgery. A greater number of cell line samples and more genes will have to be utilized in future studies in order to understand the involvement of genes in cancer metastasis more thoroughly. However, these results will help to clarify the molecular mechanisms of pancreatic cancer metastasis.

Cell Line, Tumor↗
PubMed2004

Multi-class cancer subtype classification based on gene expression signatures with reliability analysis.

Differential diagnosis among a group of histologically similar cancers poses a challenging problem in clinical medicine. Constructing a classifier based on gene expression signatures comprising multiple discriminatory molecular markers derived from microarray data analysis is an emerging trend for cancer diagnosis. To identify the best genes for classification using a small number of samples relative to the genome size remains the bottleneck of this approach, despite its promise. We have devised a new method of gene selection with reliability analysis, and demonstrated that this method can identify a more compact set of genes than other methods for constructing a classifier with optimum predictive performance for both small round blue cell tumors and leukemia. High consensus between our result and the results produced by methods based on artificial neural networks and statistical techniques confers additional evidence of the validity of our method. This study suggests a way for implementing a reliable molecular cancer classifier based on gene expression signatures.

Artificial Intelligence↗
PubMed2006

Differential proteomic analysis of nuclear extracts from thyroid cell lines.

Nuclear proteins play a major role in controlling cell functions. Differential proteomic analysis of nuclear proteins by combined 2D gel electrophoresis (2D-E) and mass spectrometry procedures can provide useful information to understand the control of cell proliferation and differentiation. To identify proteins involved in dedifferentiation, we used a differential proteomics approach by comparing nuclear extracts from the differentiated rat thyroid cell line FRTL-5 and the derived undifferentiated Ki-mol cell line, obtained by transformation with the Ki-ras oncogene. Thirteen proteins were identified as differently expressed in the nuclear compartment between the two cell lines. RT-PCR analysis performed on seven differently expressed genes showed that only in two cases the difference may be ascribable to a transcriptional mechanism. Since one of the identified proteins, namely apurinic apyrimidinic endonuclease/redox effector factor-1 (APE1/Ref-1), is suspected to play a role in thyroid tumorigenesis, we used a glutathione S-transferase (GST)-pulldown assay coupled to a 2D electrophoretic/matrix assisted laser desorption ionization-time of flight (MALDI-TOF)-mass spectrometry (MS) analysis to detect and identify its interacting partners. We show here that beta-actin directly interacted with APE1/Ref-1, as confirmed by co-immunoprecipitation assays and that this interaction was enhanced by oxidative stress on FRTL-5 cells.

Animals↗
PubMed2006

Comparative analysis of differentiation and behavior of human neural and mesenchymal stem cells in vitro and in vivo.

Comparative analysis of differentiation of human neural and mesenchymal stem cells in tissue culture and after transplantation into the brain was carried out using the same antibody set. Neural stem cells differentiated into all types of neural cells, are retained after transplantation, migrate, and form reciprocal relationships with the recipient brain. Mesenchymal stem cells were incapable of neural development under conditions of common culturing or after transplantation and retained the fibroblast-like status. Recipient filaments grew into mesenchymal stem cell transplants containing no neural cells due to local changes in the extracellular matrix at the site of transplantation.

Animals↗
PubMed2002

Differential display analysis of gene expression in yeast.

RNA differential display (DD) is a powerful and straightforward method that employs random reverse-transcription polymerase chain reaction amplification of mRNA species with electrophoresis for comparative analysis of two or more transcriptomes. The small yeast genome represents a convenient model for studying basic functions of the eukaryotic genome and simultaneously provides valuable information towards further refinement of this technique. Several examples discussed below illustrate how DD coupled with classical yeast genetic approaches may be used for studying transcriptionally regulated genetic systems.

Blotting, Northern↗
PubMed2025

Analysis of differentially expressed genes in schizophrenia based on bioinformatics and corresponding mRNA expression levels.

OBJECTIVE: This study aimed to use bioinformatics analysis to identify differentially expressed genes (DEGs) involved in the pathogenesis of schizophrenia and validate their mRNA expression levels through real-time quantitative PCR (qPCR). MATERIAL/METHODS: Datasets from the publicly available Gene Expression Omnibus (GEO) database were analyzed using R software to identify DEGs. Functional enrichment analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, were conducted. A protein-protein interaction (PPI) network was constructed using Cytoscape software to identify key genes with notable expression changes. The expression levels of these key genes were subsequently validated in schizophrenia patients using qPCR to assess potential susceptibility genes. RESULTS: In total, 813 DEGs were identified, with six key genes highlighted through GO analysis and PPI network screening. Among these, HDAC1, UBA52, and FYN demonstrated statistically significant differences in mRNA expression between schizophrenia patients and healthy controls (P&#xa0;<&#xa0;0.05). CONCLUSIONS: This study identified several DEGs potentially linked to the pathogenesis of schizophrenia, suggesting that HDAC1, UBA52, and FYN could serve as candidate susceptibility genes and diagnostic biomarkers. These findings provide new insights and directions for future schizophrenia research.

Humans↗
PubMed1982

Diatomite as a source of cristobalite for dental investments a differential thermal analysis study.

Cristobalite can be obtained from diatomite by heat-treatment at relatively low temperatures and short times without the use of flux. The differential thermal analysis method used to examine the alpha leads to beta inversion was not as satisfactory as it might have been, and to determine the amount and crystallinity of cristobalite formed any such method should be supported by other techniques, in particular, X-ray diffraction. To determine the suitability or otherwise of cristobalite produced from diatomite for use in dental investments, dialatometric studies are indicated since the property of thermal expansion is of fundamental importance to a dental investment.

Chemical Phenomena↗
PubMed2003

Proteomic analysis of the systemic immune response of Drosophila.

Improvements in two-dimensional gel electrophoresis, mass spectrometry, and bioinformatics provide new tools to characterize proteins involved in a physiological process, such as the immune response of the insect model Drosophila melanogaster. Profiling of the proteins present in the hemolymph (insect blood) of noninfected flies versus flies infected with bacteria or fungi was performed by two-dimensional gel electrophoresis, silver or Coomassie staining, and image analysis. Through this differential analysis, more than 70 out of 160 spots were up- or down-regulated by at least 5-fold after microbial infection. Coomassie staining, in-gel digestion, and database searches yielded the identity of a series of proteins that are directly involved in the Drosophila immune system. This included proteases, protease inhibitors, and recognition molecules such as prophenoloxydase-activating enzymes, serpins, and Gram-negative binding protein-like. Proteins with a potential function in the immune response were also identified, such as an odorant binding protein, peptidylglycine alpha-hydroxylating monooxygenase, and transferrin, affording new candidates for further investigation of innate immune mechanisms. Moreover, several molecules resulting from the cleavage of proteins were detected after the fungal infection. Altogether, this first differential proteomic analysis of the immune response of Drosophila paves the way for the study of proteins affected during innate immunity.

Animals↗
PubMed2005

EDGE: extraction and analysis of differential gene expression.

EDGE (Extraction of Differential Gene Expression) is an open source, point-and-click software program for the significance analysis of DNA microarray experiments. EDGE can perform both standard and time course differential expression analysis. The functions are based on newly developed statistical theory and methods. This document introduces the EDGE software package.

Algorithms↗