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At least 433 records · Page 24Linked to original sources
PubMed2005

Differential expression of immunoglobulin kappa chain constant region in human abdominal aortic aneurysm.

BACKGROUND: A number of the research into the pathogenesis of the abdominal aortic aneurysm (AAA) have focused on the alteration of gene expression. The current technique for elucidating alterations of gene expression has a setback in that many artifact complementary DNA (cDNA) products present abnormal polymerase chain reaction (PCR) amplification. Our study was designed to identify differentially expressed genes in AAA using the annealing control primer (ACP) system, which was recently developed to identify only authentic genes. MATERIALS AND METHODS: The tissues of the human abdominal aorta were obtained from the patients of AAA and aortic occlusive disease (AOD), and normal abdominal aorta (NA) from brain death donors. Total RNAs were isolated from three groups of human abdominal aorta (10 AAA, five NA, three AOD) and then reverse transcribed into complementary DNA (cDNA). The ACP method was done to screen the difference in the expression pattern of the mRNA (mRNA). RESULTS: One differentially expressed cDNA band was detected in AAA but not in NA and AOD. This cDNA was sequenced and computer searching against the GenBank revealed that the cDNA had more than 90% identity with the immunoglobulin kappa chain constant region (Ig kappa-C). DISCUSSION: Our finding suggests that differentially expressed Ig kappa-C gene only in AAA is a candidate gene that may play a pivotal role in the pathogenesis of AAA formation. The correlation of mRNA level and protein level is, however, not clear. Thus, to directly identify the role of Ig light chains in the pathogenic event of AAA, the further study comparing the level and kinds of expressed protein with the corresponding Ig kappa-C gene will be required.

Aortic Aneurysm, Abdominal↗
PubMed2006

Signal regulatory protein molecules are differentially expressed by CD8- dendritic cells.

A normalized subtracted gene expression library was generated from freshly isolated mouse dendritic cells (DC) of all subtypes, then used to construct cDNA microarrays. The gene expression profiles of the three splenic conventional DC (cDC) subsets were compared by microarray hybridization and two genes encoding signal regulatory protein beta (Sirpbeta1 and Sirpbeta4) molecules were identified as differentially expressed in CD8(-) cDC. Genomic sequence analysis revealed a third Sirpbeta member localized in the same gene cluster. These Sirpbeta genes encode cell surface molecules containing extracellular Ig domains and short intracytoplasmic domains that have a charged amino acid in the transmembrane region which can potentially interact with ITAM-bearing molecules to mediate signaling. Indeed, we demonstrated interactions between Sirpbeta1 and beta2 with the ITAM-bearing signaling molecule Dap12. Real-time PCR analysis showed that all three Sirpbeta genes were expressed by CD8(-) cDC, but not by CD8(+) cDC or plasmacytoid pre-DC. The related Sirpalpha gene showed a similar expression profile on cDC subtypes but was also expressed by plasmacytoid pre-DC. The differential expression of Sirpalpha and Sirpbeta1 molecules on DC was confirmed by staining with mAbs, including a new mAb recognizing Sirpbeta1. Cross-linking of Sirpbeta1 on DC resulted in a reduction in phagocytosis of Leishmania major parasites, but did not affect phagocytosis of latex beads, perhaps indicating that the regulation of phagocytosis by Sirpbeta1 is a ligand-dependent interaction. Thus, we postulate that the differential expression of these molecules may confer the ability to regulate the phagocytosis of particular ligands to CD8(-) cDC.

Amino Acid Sequence↗
PubMed1996

Connexins and E-cadherin are differentially expressed during trophoblast invasion and placenta differentiation in the rat.

We have characterized the spatial and temporal expression pattern of six different connexin genes and E-cadherin during trophectoderm development in the rat. During the initial phase of trophoblast invasion at 6 days postcoitum (dpc), the trophoblast expressed E-cadherin but no connexin expression could be observed. With progressing invasion of the polar trophoblast into the maternal decidua, from 7 dpc onwards E-cadherin expression in the ectoplacental cone cells was lost and was now restricted to the extraembryonic ectoderm. In the ectoplacental cone and extraembryonic ectoderm instead connexin31 mRNA and protein could be found. This pattern was maintained up to day 10 postcoitum. The start of labyrinthine trophoblast differentiation from day 11 postcoitum onwards was characterized by persisting expression of E-cadherin in the extraembryonic ectoderm and its derivative, the chorionic plate. In addition to E-cadherin, from 10 dpc onwards, connexin26 started to be expressed in the chorionic plate, and both molecules remained coexpressed in the labyrinthine trophoblast of the mature placenta. During this differentiation process connexin31 remained expressed mainly in the proliferating spongiotrophoblast. From day 14 postcoitum onwards, the expression of connexin31 in the spongiotrophoblastic cells decreased, and in parallel they started to express connexin43. The trophoblastic giant cells, first characterized by connexin31, lost all of the investigated connexins during midgestation on day 12 postcoitum but started to express connexin43 from day 18 postcoitum onwards. Our studies suggest that loss of E-cadherin and induction of connexin31 expression is correlated with the proliferative and invasive stages of the ectoplacental cone, whereas appearance of connexin26, E-cadherin and connexin43 reflects the switch to the differentiated phenotypes of the mature placenta.

Animals↗
PubMed2000

Testing for differentially-expressed genes by maximum-likelihood analysis of microarray data.

Although two-color fluorescent DNA microarrays are now standard equipment in many molecular biology laboratories, methods for identifying differentially expressed genes in microarray data are still evolving. Here, we report a refined test for differentially expressed genes which does not rely on gene expression ratios but directly compares a series of repeated measurements of the two dye intensities for each gene. This test uses a statistical model to describe multiplicative and additive errors influencing an array experiment, where model parameters are estimated from observed intensities for all genes using the method of maximum likelihood. A generalized likelihood ratio test is performed for each gene to determine whether, under the model, these intensities are significantly different. We use this method to identify significant differences in gene expression among yeast cells growing in galactose-stimulating versus non-stimulating conditions and compare our results with current approaches for identifying differentially-expressed genes. The effect of sample size on parameter optimization is also explored, as is the use of the error model to compare the within- and between-slide intensity variation intrinsic to an array experiment.

Galactose↗
PubMed2003

Cloning of differentially expressed genes following lipopolysaccharide stimulation in human umbilical vein endothelial cells.

OBJECTIVE: To clone the differentially expressed genes in human umbilical vein endothelial cells (HUVEC) stimulated by lipopolysaccharide (LPS). METHODS: Two-directional (forward and backward) suppression subtractive hybridization (SSH) was performed on HUVEC cultured in either standard media or treated for 6 hours with LPS (100 ng/ml). To restrict the number of false-positive clones, colony dot hybridization was used to further verify the differentially expressed cDNA clones. Positive clones were sequenced. RESULTS: These analyses have identified both novel and known genes whose expression is influenced by LPS. The known genes include a group related to proinflammatory events, a group related to cellular apoptosis and proliferation, a group related to protein synthesis and cytoskeletal rearrangment, and a group related to energy metabolism and signal transduction. CONCLUSIONS: SSH is a powerful technique of high sensitivity for the detection of differential gene expression in HUVEC stimulated by LPS.

Cells, Cultured↗
PubMed2005

Hotelling's T2 multivariate profiling for detecting differential expression in microarrays.

The most widely used statistical methods for finding differentially expressed genes (DEGs) are essentially univariate. In this study, we present a new T(2) statistic for analyzing microarray data. We implemented our method using a multiple forward search (MFS) algorithm that is designed for selecting a subset of feature vectors in high-dimensional microarray datasets. The proposed T2 statistic is a corollary to that originally developed for multivariate analyses and possesses two prominent statistical properties. First, our method takes into account multidimensional structure of microarray data. The utilization of the information hidden in gene interactions allows for finding genes whose differential expressions are not marginally detectable in univariate testing methods. Second, the statistic has a close relationship to discriminant analyses for classification of gene expression patterns. Our search algorithm sequentially maximizes gene expression difference/distance between two groups of genes. Including such a set of DEGs into initial feature variables may increase the power of classification rules. We validated our method by using a spike-in HGU95 dataset from Affymetrix. The utility of the new method was demonstrated by application to the analyses of gene expression patterns in human liver cancers and breast cancers. Extensive bioinformatics analyses and cross-validation of DEGs identified in the application datasets showed the significant advantages of our new algorithm.

Algorithms↗
PubMed2025

Bayesian identification of differentially expressed isoforms using a novel joint model of RNA-seq data.

We develop a Bayesian approach, BayesIso, to identify differentially expressed isoforms from RNA-seq data. The approach features a novel joint model of the sample variability and the deferential state of isoforms. Specifically, the within-sample variability and the between-sample variability of each isoform are modeled by a Poisson-Lognormal model and a Gamma-Gamma model, respectively. Using a Bayesian framework, the differential state of each isoform and the model parameters are jointly estimated by a Markov Chain Monte Carlo (MCMC) method. Extensive studies using simulation and real data demonstrate that BayesIso can effectively detect isoforms of less differentially expressed and differential transcripts for genes with multiple isoforms. We applied the approach to breast cancer RNA-seq data and uncovered a unique set of isoforms that form key pathways associated with breast cancer recurrence. First, PI3K/AKT/mTOR signaling and PTEN signaling pathways are identified as being involved in breast cancer development. Further integrated with protein-protein interaction data, pathways of Jak-STAT, mTOR, MAPK and Wnt signaling are revealed in association with breast cancer recurrence. Finally, several pathways are activated in the early recurrence of breast cancer. In tumors that occur early, members of pathways of cellular metabolism and cell cycle (such as CD36 and TOP2A) are upregulated, while immune response genes such as NFATC1 are downregulated.

Humans↗
PubMed2006

Intensity-based hierarchical Bayes method improves testing for differentially expressed genes in microarray experiments.

BACKGROUND: The small sample sizes often used for microarray experiments result in poor estimates of variance if each gene is considered independently. Yet accurately estimating variability of gene expression measurements in microarray experiments is essential for correctly identifying differentially expressed genes. Several recently developed methods for testing differential expression of genes utilize hierarchical Bayesian models to "pool" information from multiple genes. We have developed a statistical testing procedure that further improves upon current methods by incorporating the well-documented relationship between the absolute gene expression level and the variance of gene expression measurements into the general empirical Bayes framework. RESULTS: We present a novel Bayesian moderated-T, which we show to perform favorably in simulations, with two real, dual-channel microarray experiments and in two controlled single-channel experiments. In simulations, the new method achieved greater power while correctly estimating the true proportion of false positives, and in the analysis of two publicly-available "spike-in" experiments, the new method performed favorably compared to all tested alternatives. We also applied our method to two experimental datasets and discuss the additional biological insights as revealed by our method in contrast to the others. The R-source code for implementing our algorithm is freely available at http://eh3.uc.edu/ibmt. CONCLUSION: We use a Bayesian hierarchical normal model to define a novel Intensity-Based Moderated T-statistic (IBMT). The method is completely data-dependent using empirical Bayes philosophy to estimate hyperparameters, and thus does not require specification of any free parameters. IBMT has the strength of balancing two important factors in the analysis of microarray data: the degree of independence of variances relative to the degree of identity (i.e. t-tests vs. equal variance assumption), and the relationship between variance and signal intensity. When this variance-intensity relationship is weak or does not exist, IBMT reduces to a previously described moderated t-statistic. Furthermore, our method may be directly applied to any array platform and experimental design. Together, these properties show IBMT to be a valuable option in the analysis of virtually any microarray experiment.

Animals↗
PubMed2006

Bioinformatic screening of human ESTs for differentially expressed genes in normal and tumor tissues.

BACKGROUND: Owing to the explosion of information generated by human genomics, analysis of publicly available databases can help identify potential candidate genes relevant to the cancerous phenotype. The aim of this study was to scan for such genes by whole-genome in silico subtraction using Expressed Sequence Tag (EST) data. METHODS: Genes differentially expressed in normal versus tumor tissues were identified using a computer-based differential display strategy. Bcl-xL, an anti-apoptotic member of the Bcl-2 family, was selected for confirmation by western blot analysis. RESULTS: Our genome-wide expression analysis identified a set of genes whose differential expression may be attributed to the genetic alterations associated with tumor formation and malignant growth. We propose complete lists of genes that may serve as targets for projects seeking novel candidates for cancer diagnosis and therapy. Our validation result showed increased protein levels of Bcl-xL in two different liver cancer specimens compared to normal liver. Notably, our EST-based data mining procedure indicated that most of the changes in gene expression observed in cancer cells corresponded to gene inactivation patterns. Chromosomes and chromosomal regions most frequently associated with aberrant expression changes in cancer libraries were also determined. CONCLUSION: Through the description of several candidates (including genes encoding extracellular matrix and ribosomal components, cytoskeletal proteins, apoptotic regulators, and novel tissue-specific biomarkers), our study illustrates the utility of in silico transcriptomics to identify tumor cell signatures, tumor-related genes and chromosomal regions frequently associated with aberrant expression in cancer.

Algorithms↗
PubMed2006

Annealing control primer system identifies differentially expressed genes in blastocyst-stage porcine parthenotes.

There is very little information available on stage-specific gene expression during early embryo development, particularly in the pig. Here, we accurately identified the genes that are specifically or prominently expressed in parthenogenetic porcine blastocysts as compared with 2-cell stage embryos. We accomplished this by using a PCR technology regulated by annealing control primers (ACPs). By utilizing 120 ACPs, a total of 46 expressed sequence tags (ESTs) of genes that are differentially expressed in blastocysts as compared with 2-cell stage embryos were cloned and sequenced. The cloned genes or ESTs all exhibited significant sequence similarity with known genes or ESTs of other species. Of the known genes, six genes [renin-binding protein (RNBP), BMDP, solute carrier family 25 (SLC25A6), MTHFD1, TRK-fused gene (TFG), spermidine synthase (SRM)] were selected and their stage-specific expression levels in porcine parthenotes were determined by real-time quantitative polymerase chain reaction at the 1-, 2-, 4-cell, morula and blastocyst stages. While RNBP, BMDP, SLC25A6, MTGFD1 and SRM were highly expressed only at the blastocyst stage, TFG was highly expressed at the 1-cell stage, then declined after genomic activation, high levels of expression being again detected at the morula and blastocyst stages. This analysis suggests that the ACP system is an effective tool for use in the identification of stage-specific genes in small numbers of porcine parthenotes. Examination of the genes differentially expressed in the blastocyst, which we have identified here, will provide insight into the molecular basis of preimplantation development.

Animals↗
PubMed2003

Profiling of genes differentially expressed between fetal liver and postnatal liver using high-density oligonucleotide DNA array.

The liver is an essential organ in humans not only for the production and storage of energy but also for detoxification of chemical compounds, but knowledge about changes in the gene expression profile in the human liver during the prenatal and postnatal periods is limited. Profiling of genes differentially expressed between the fetal liver (FL) and the postnatal liver (PNL) is one of the methods to investigate candidates affecting the difference in biological characteristics between FL and PNL. To identify genes differentially expressed between FL and PNL (childhood and adult liver), we analyzed the gene expression profiles across 9 FL and 14 PNL samples using a high-density oligonucleotide DNA array. Using Mann-Whitney U test followed by k-nearest-neighbors (supervised learning method) and hierarchical clustering (unsupervised learning method) algorithms, we found 33 genes clearly discriminating between the FL group and PNL group. The functional classification of the 33 genes identified was related to several kinds of biological pathways, regulating the cell cycle (PCNA, CDC7L1, CCND3, YWHA1, PKMYT1), DNA replication and repair (RFC4, RECQ2, PCNA, NAP1L1), cell growth (IGF2, IGFBP2, PRSS11), hormonal signals (AR, SRD5A1, NR1I3), and cellular metabolism (E2-EPF, WWP1, CYP2C9, CYP2E1, CYP2A6, CYP2A7, CYP2A13, CYP4F2, CYP3A4, DDT). The results presented herein provide evidence of a differential expression profile of genes regulating the cell cycle, DNA replication and repair, cell growth, regulation of hormonal signals, and cellular metabolism, between FL and PNL in humans. The 33 genes identified in this study are suggested to be useful markers clearly discriminating between FL and PNL using the gene expression profile.

Adult↗
PubMed2001

Identification of genes differentially expressed by putrescine in HepG2 hepatoblastoma cells.

In order to identify genes differentially expressed by putrescine, a polyamine, which play important roles in the regulation of cell proliferation and the development of cancer, we performed mRNA differential display analysis using total RNA extracted from HepG2 cells (human hepatoblastoma cell line) treated with a specific inhibitor of polyamine biosynthesis, alpha-difluorometylornithine (DFMO). A total of 25 genes were up-regulated and 32 genes down-regulated by putrescine. Of the genes differentially expressed by putrescine, we chose three that were related to the respiratory chain and oxidative phosphorylation and analyzed them by Northern blot analysis. Cytochrome oxidase subunit 1, low molecular mass ubiquinone-binding protein, and NADH dehydrogenase subunit 2 were found to be down-regulated by putrescine. We examined intracellular ATP level in HepG2 cells, and found that ATP level in DFMO-treated cells was increased by exogenous putrescine.

Journal Article↗
PubMed2004

Identification of genes differentially expressed in T cells following stimulation with the chemokines CXCL12 and CXCL10.

BACKGROUND: Chemokines are involved in many biological activities ranging from leukocyte differentiation to neuronal morphogenesis. Despite numerous reports describing chemokine function, little is known about the molecular changes induced by cytokines. METHODS: We have isolated and identified by differential display analysis 182 differentially expressed cDNAs from CXCR3-transfected Jurkat T cells following treatment with CXCL12 or CXCL10. These chemokine-modulated genes were further verified using quantitative RT-PCR and Western blot analysis. RESULTS: One hundred and forty-six of the cDNAs were successfully cloned, sequenced, and identified by BLAST. Following removal of redundant and non-informative clones, seventeen mRNAs were found to be differentially expressed post treatment with either chemokine ligand with several representing known genes with established functions. Twenty-one genes were upregulated in these transfected Jurkat cells following both CXCL12 and CXCL10, four genes displayed a discordant response and seven genes were downregulated upon treatment with either chemokine. Identified genes include geminin (GEM), thioredoxin (TXN), DEAD/H box polypeptide 1 (DDX1), growth hormone inducible transmembrane protein (GHITM), and transcription elongation regulator 1 (TCERG1). Subsequent analysis of several of these genes using semi-quantitative PCR and western blot analysis confirmed their differential expression post ligand treatment. CONCLUSIONS: Together, these results provide insight into chemokine-induced gene activation and identify potentially novel functions for known genes in chemokine biology.

Blotting, Western↗
PubMed1996

Combining the technique of RNA fingerprinting and differential display to obtain differentially expressed mRNA.

We have modified recently developed methods of RNA fingerprinting and differential display based on arbitrarily primed PCR which can be used for detection and cloning of differentially expressed mRNAs. Our protocol requires only a single cDNA synthesis for each different RNA sample, in contrast to the multiple cDNA reactions required for differential display method, followed by selective amplification of cDNA sequence fraction by arbitrary and oligo(dT) primers. We have shown that the longer primers (25-29-mers) allow the use of optimal dNTP concentration and higher stringency PCR. Further improvements include using TaqStart antibody for hot start PCR and thermostable enzyme mixes suitable for long-distance PCR. Long-distance PCR enables the method to display bands of up to 2 kb and should result in a higher fidelity of PCR products to the original RNA template. When these improvements are combined the resulting method is highly reproducible, and more than 85% of the differentially expressed bands can be confirmed by Northern blot analysis.

Adult↗
PubMed2002

Differentially expressed gene profiles between multidrug resistant gastric adenocarcinoma cells and their parental cells.

To better understand the molecular mechanisms of multidrug resistance (MDR) of human cancers, we isolated differentially expressed genes from drug-resistant human gastric adenocarcinoma cell lines using a polymerase chain reaction-based subtractive hybridization technique. Sixty three genes were identified to be differentially expressed in the drug-resistant human gastric adenocarcinoma cell lines. Among the 63 up-regulated genes, 27 were known genes, of which two have been reported to be associated with MDR. The remaining ones were undefined genes or expressed sequenced tags. The results suggest that this strategy is efficient for large-scale cloning of differentially expressed genes in drug-resistant cells. Further characterization of these genes will shed more light on the understanding of molecular mechanisms of MDR of human cancer cells.

Adenocarcinoma↗
PubMed2003

Differential expression of the tetracycline-controlled transactivator-driven human CYP1B1 gene in double-transgenic mice is due to androgens: application for detecting androgens and antiandrogens.

Differential expression of the tetracycline-controlled transactivator (tTA)-driven human cytochrome p450 (CYP) 1B1 gene was found in the livers of male mice, at high levels in neonates, but at low levels in adults. The goals of this study were to determine whether the differential expression of the tTA-driven human CYP1B1 (hCYP1B1) gene in neonates and adults was testosterone dependent and whether flutamide, a representative potent antiandrogen, led to the induction of hCYP1B1. This was tested by treating castrated transgenic mice with testosterone propionate and musk extracts. It was concluded that: (i). the levels of expression of both tTA and hCYP1B1 gradually declined, with clear changes being apparent between 2 and 4 weeks of age, (ii). castration of adult males resulted in the increased expressions of both tTA and hCYP1B1 to levels similar to those found in adult females, (iii). treatment of castrated male and adult female mice with testosterone propionate and musk extracts led to the restoration of the levels of expression of hCYP1B1 in the adult males, and (iv). treatment of adult males with flutamide caused an increase in the levels of expression of hCYP1B1 in the adult females, as indicated by the antiandrogenic activity. Thus, the differential expression of the tTA-driven hCYP1B1 gene in the transgenic mice was caused by androgen, and it is possible that castrated male and adult female mice expressing the tTA-controlled hCYP1B1 could be used as the basis for a strategy for the detection of androgens and antiandrogens.

Aging↗
PubMed2005

Differential expression of the peripheral benzodiazepine receptor and gremlin during adipogenesis.

This study used the mRNA differential display technique to identify differentially expressed genes during the process of adipogenesis in the preadipocyte cell line, 3T3-L1. 3T3-L1 cells were treated with dexamethasone, isobutyl-1-methylxanthine, and insulin to induce differentiation into mature adipocytes. Cells were collected at three time-points during differentiation: Day 0 (d0), or nondifferentiated; Day 3 (d3), during differentiation; and Day 10 (d10), >90% of the cells had differentiated into mature adipocytes. Initial studies yielded 18 potentially differentially regulated cDNA candidates (8 down-regulated and 10 up-regulated). Reverse Northern and Northern blots confirmed differential expression of six of the candidates. Four of the candidates up-regulated on d3 and d10 were identified by sequence analysis to be lipoprotein lipase, a well-known marker of adipocyte differentiation. A fifth candidate that was expressed in d0, but not d3 or d10, was identified as DRM/gremlin, a bone morphogenetic protein antagonist. Finally, a sixth candidate that was increased at d3 and d10 was identified as the peripheral benzodiazepine receptor, which has been implicated in proliferation, differentiation, and cholesterol transport in cells. This study is the first to show that peripheral benzodiazepine receptor and DRM/gremlin are expressed in preadipocyte cell lines and that they are differentially regulated during adipogenesis.

3T3-L1 Cells↗
PubMed2000

Differential expressions of BMP family genes during chondrogenic differentiation of mouse ATDC5 cells.

Clonal cell line ATDC5 enables the monitoring of the early- and late-phase chondrogenic differentiation in a single culture. Undifferentiated ATDC5 cells differentiate into type II collagen expressing chondrocytes through a cellular condensation stage (early-phase differentiation) and then to type X collagen-expressing hypertrophic chondrocytes (late-phase differentiation). Progression of cellular differentiation was accelerated by the activation of bone morphogenetic protein (BMP) signaling. ATDC5 cells expressed transcripts for at least four members of the BMP family. The BMP-4 transcripts were expressed in all stages of differentiation, as were transcripts for BMP type IA receptor (ALK-3) and BMP type II receptor. In contrast, transcripts for Growth/ Differentiation factor-S (GDF-5) were induced during a cellular condensation, and those for BMP-6 were induced during the formation of cartilage nodules, and declined as the differentiated ATDC5 cells became hypertrophic, and BMP-7 transcripts were only detected after cells became calcified. Exogenously added BMP-4 indeed promoted the early-phase differentiation. Late-phase differentiation of cells was also stimulated by BMP-4 and BMP-6. Thus, the cumulative increase in BMP signaling promoted the sequential transitions of differentiation steps of cells. These results indicate that the coordinated expressions of endogenous BMPs are involved in the progression of chondrogenic differentiation in ATDC5 cells.

Animals↗