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Evaluation of fibronectin gene expression by in situ hybridization. Differential expression of the fibronectin gene among populations of human alveolar macrophages.

Populations of alveolar macrophages recovered from the lower respiratory tract secrete fibronectin, a multi-functional glycoprotein capable of influencing cell migration, attachment, differentiation, and proliferation. Using in situ hybridization of 35S-labeled antisense and sense RNA fibronectin probes, the present study demonstrates that most, but not all, normal alveolar macrophages contain fibronectin mRNA transcripts, and that among those macrophages expressing this gene, the relative amount of fibronectin mRNA transcripts varies from cell to cell. Interestingly, while 66 +/- 3% of normal alveolar macrophages contain fibronectin mRNA transcripts, this is increased to 82 +/- 2% (P less than 0.01) of alveolar macrophages recovered from the lungs of individuals with idiopathic pulmonary fibrosis (IPF), a chronic inflammatory disorder associated with exaggerated amounts of fibronectin in the lower respiratory tract. Furthermore, of the macrophages expressing the fibronectin gene, those from IPF patients contain more fibronectin mRNA transcripts than those from normals. Consistent with this observation, evaluation of tissue samples from IPF patients demonstrated that of all cells present, alveolar macrophages showed the greatest numbers of fibronectin mRNA transcripts per cell. These observations demonstrate that there can be marked cell-to-cell variation in the expression of the gene for a macrophage product such as fibronectin, suggesting that there are processes that modulate similar cells in the same anatomic compartment to vary their expression of the same gene.

Adult↗

Identification of differentially expressed genes in multiple microarray experiments using discrete fourier transform.

Research in the post-genome sequence era has been shifting towards a functional understanding of the roles and relationships between different genes in different conditions. While the advances in genetic expression profiling techniques including microarrays enable detailed and genome-scale measurements, the extraction of meaningful information from large datasets remains a challenging task. Here, we propose a novel method of generating gene differential expression profiles such that gene expression values from one dataset can be directly compared with those of another dataset. A simplified Discrete Fourier Transform is applied to interposed gene expression values, thereby generating the 'spectra' for a pair of conditions. Using this technique, differentially expressed genes produce higher amplitudes at the Nyquist Frequency. By measuring the phase of the 'spectra' generated, the over- and under-expressed nature of the genes can be identified. This method was validated using two sets of GeneChip array data, one from prostate cancer related dataset and the other from macular degeneration related dataset. The genes identified as differentially expressed by our method were found to be similar to those published using their preferred methods. Based on our findings, the proposed DFT method could be used efficiently in identifying differentially expressed genes from multiple-array experiments from two different conditions.

Fourier Analysis↗

Identifying differentially expressed genes in meta-analysis via Bayesian model-based clustering.

A Bayesian model-based clustering approach is proposed for identifying differentially expressed genes in meta-analysis. A Bayesian hierarchical model is used as a scientific tool for combining information from different studies, and a mixture prior is used to separate differentially expressed genes from non-differentially expressed genes. Posterior estimation of the parameters and missing observations are done by using a simple Markov chain Monte Carlo method. From the estimated mixture model, useful measure of significance of a test such as the Bayesian false discovery rate (FDR), the local FDR (Efron et al., 2001), and the integration-driven discovery rate (IDR; Choi et al., 2003) can be easily computed. The model-based approach is also compared with commonly used permutation methods, and it is shown that the model-based approach is superior to the permutation methods when there are excessive under-expressed genes compared to over-expressed genes or vice versa. The proposed method is applied to four publicly available prostate cancer gene expression data sets and simulated data sets.

Algorithms↗

[Analysis of differential expression genes related to different metastasis potential of adenoid cystic carcinoma using restriction fragments differential display PCR].

OBJECTIVE: To construct differential expression profiles of adenoid cystic carcinoma cell lines for screening candidate genes related to metastasis and to verify some candidate genes in adenoid cystic carcinoma. METHODS: Restriction fragments differential display PCR (RFDD-PCR) was used to set up gene expression profiles of adenoid cystic carcinoma cell lines-ACC-M and ACC-2, with high and low metastasis potential respectively. Candidate genes were screened through bioinformatics analysis. Then, a gene family of these candidate genes was checked using semi-quantitative reverse transcription-PCR(RT-PCR). RESULTS: Two gene expression profiles including 5420 gene fragments were constructed, 12 genes of a family called matrix metalloproteinase genes (MMPs) were observed obvious differentially expressed between two cell lines. Results of semi-quantitative RT-PCR also identified this different expression of MMP2,MMP7,MMP9,MMP14,MMP15 and MMP24. CONCLUSION: The construction of gene expression profiles of ACC-M and ACC-2 cell lines makes the foundation for seeking the target genes of adenoid cystic carcinoma. MMP2,MMP7,MMP9 and MMP15 may be relevant with carcinogenesis, development and metastasis of adenoid cystic carcinoma, and different metastasis potential may result from different subtype of MMPs gene family.

Carcinoma, Adenoid Cystic↗

Analysis of differentially expressed genes in human hepatocellular carcinoma using suppression subtractive hybridization.

The genetic basis of hepatocellular carcinoma (HCC) has not yet been fully understood. Although various methods have been developed to detect differentially expressed genes in malignant diseases, efficient analysis from clinical specimens is generally difficult to perform due to the requirement of a large amount of samples. In the present study, we analysed differentially expressed genes with a small amount of human HCC samples using suppression subtractive hybridization (SSH). Total RNA were obtained from the hepatitis C virus-associated HCC and adjacent non-HCC liver tissues. cDNA was synthesized using modified RT-PCR, and then tester cDNA was ligated with 2 different kinds of adaptors and hybridized with an excess amount of driver cDNA. Tester specific cDNA was obtained by suppression PCR and the final PCR product was subcloned and sequenced. We identified 7 known genes (focal adhesion kinase, deleted in colon cancer, guanine binding inhibitory protein alpha, glutamine synthetase, ornithine aminotransferase, M130, and pepsinogen C) and 2 previously unknown genes as being overexpressed in HCC, and 1 gene (decorin) as suppressed in HCC. Quantitative analysis of gene expression using quantitative RT-PCR demonstrated the differential expression of these genes in the original and other HCC samples. These findings demonstrated that it is possible to identify the previously unknown, differential gene expression from a small amount of clinical samples. Information about such alterations in gene expression could be useful for elucidating the genetic events in HCC pathogenesis, developing the new diagnostic markers, or determining novel therapeutic targets.

Amino Acid Sequence↗

[Differential expression of cardiac immediate early and late response gene in exercise-induced and hypertensive cardiac hypertrophic remodeling].

Our previous studies have shown that the myocardial phenotypes between exercise-induced and hypertensive cardiac hypertrophy are different. However, alteration of cardiac gene expression in the two kinds of hypertrophy is not clear. We therefore studied the expression of immediate early and late responsive gene in Wistar rats subject to a 12-weeks swimming program and spontaneously hypertensive rats (SHR) by Northern blot. The autoradiographs were analyzed by scanning densitometry. When compared with matched sedentary control, swimming rats and SHR increased their heart/body weight by 29% and 61% respectively (P < 0.01). After a series of swimming program, levels of c-fos and atrial natriuretic factor (ANF) mRNA were instantly increased 203% and 214% respectively in the swimming rats. However in 24 h after swimming, ANF mRNA was decreased to the level of control but c-fos mRNA was still higher than than in control. The levels of c-fos and ANF mRNA were increased by 80% and 298% in SHR respectively. The expression of beta-myosin heavy chain (MHC) mRNA was increased by 117% in SHR but decreased by 12% in swimming rats. The expression of alpha-MHC mRNA was decreased by 42% in SHR but unchanged in swimming rats. The abundance alpha/beta-MHC mRNA in swimming rats is 336% higher than in SHR. These results showed differential expression of the immediate early and late responsive gene in exercise-induced and SHR hypertrophied heart remodeling. It is suggested that (1) the differential expression of c-fos gene may differently regulate remodeling of these two cardiac hypertrophies; (2) the differential expression of ANF gene may be a molecular marker for distinguishing the two kinds of cardiac hypertrophy; and (3) the differential expression of alpha/beta-MHC gene ratio may regulate exercise-induced and hypertensive hypertrophied myocardial cell changing to contractile phenotype and synthetic phenotype respectively, at the gene transcription level.

Animals↗

A spline function approach for detecting differentially expressed genes in microarray data analysis.

MOTIVATION: A primary objective of microarray studies is to determine genes which are differentially expressed under various conditions. Parametric tests, such as two-sample t-tests, may be used to identify differentially expressed genes, but they require some assumptions that are not realistic for many practical problems. Non-parametric tests, such as empirical Bayes methods and mixture normal approaches, have been proposed, but the inferences are complicated and the tests may not have as much power as parametric models. RESULTS: We propose a weakly parametric method to model the distributions of summary statistics that are used to detect differentially expressed genes. Standard maximum likelihood methods can be employed to make inferences. For illustration purposes the proposed method is applied to the leukemia data (training part) discussed elsewhere. A simulation study is conducted to evaluate the performance of the proposed method.

Algorithms↗

Differential expression of G protein alpha and beta subunit genes during development of Phytophthora infestans.

A G protein alpha subunit gene (pigpa1) and a G protein beta subunit gene (pigpb1) were isolated from the oomycete Phytophthora infestans, the causal agent of potato late blight. Heterotrimeric G proteins are evolutionary conserved GTP-binding proteins that are composed of alpha,beta, and gamma subunits and participate in diverse signal transduction pathways. The deduced amino acid sequence of both pigpa1 and pigpb1, showed the typical conserved motifs present in Galpha or Gbeta proteins from other eukaryotes. Southern blot analysis revealed no additional copies of Galpha or Gbeta subunit genes in P. infestans, suggesting that pigpa1 and pigpb1 are single copy genes. By cross-hybridization homologues of gpa1 and gpb1 were detected in other Phythophthora species. Expression analyses revealed that both genes are differentially expressed during asexual development, with the highest mRNA levels in sporangia. In mycelium, no pigpa1 mRNA was detected. Western blot analysis using a polyclonal GPA1 antibody confirmed the differential expression of pigpa1. These expression patterns suggest a role for G-protein-mediated signaling during formation and germination of asexual spores of P. infestans, developmental stages representing the initial steps of the infection process.

Amino Acid Sequence↗

[Preliminary study on differential expressed genes in T lymphocyte of chronic obstructive pulmonary disease patients with Fei-qi deficiency syndrome].

OBJECTIVE: To study T lymphocyte related genes with differential expression in patients with chronic obstructive pulmonary disease (COPD) of Fei-qi deficiency (FQD) syndrome type by gene chips. METHODS: Lymphocytes in peripheral blood were isolated by Ficoll technique from blood samples collected from COPD patients of FQD syndrome type, Fei-yin deficiency (FYD) syndrome type, and also from healthy subjects for control. They were sorted and purified by flow cytometry, and the different expressed genes were screened from them by gene chip technique. RESULTS: There were 15 genes with high differential expression between patients of FQD type and those of FYD syndrome type, and between patients of FQD type and healthy subjects. CONCLUSION: Gene chip technique could be used for studying the gene expression profiles of TCM syndrome, and the T-lymphocyte related genes with differential expression in COPD patients with FQD were screened preliminarily.

Adult↗

Differentially expressed genes in adult familial myelodysplastic syndromes.

The precise genetic events leading to myelodysplastic syndromes (MDSs) and leukemic transformation remain poorly defined. Even less is known about adult familial MDS. We report an adult MDS family in whom enriched tissue-specific transcripts were derived by subtractive hybridization of cDNA from the mononuclear and CD34+ cells of affected and unaffected family members. These expression libraries were then hybridized to Genome Discovery arrays containing 18 404 genes and expressed sequence tags, and several clusters of differentially expressed genes were identified. A group of 21 genes was underexpressed (>5-fold) in affected vs unaffected family members, and among these were transcription factors and genes involved in myeloid differentiation, such as ZNF140 and myeloid nuclear differentiation antigen (MNDA). Another group of 36 genes was overexpressed (>5-fold), and these encoded proteins belonging to signaling pathways, such as Ras- and Fos-related genes. The top two genes downregulated in this MDS family, ZNF140 and MNDA, were similarly altered in another MDS family, and in some cases of sporadic MDS. Our data suggest that we have identified genes differentially expressed in adult familial MDS, and that alteration of some of these genes may also be important for the evolution of different stages or severity of sporadic MDS.

Adolescent↗

Identification of differentially expressed genes in chemically induced skin tumors.

Previous studies have demonstrated a role for the fos gene in promoting malignant conversion of mouse skin tumors. In the study reported here, differential display was performed to identify fos- and jun-regulated genes that are differentially expressed during premalignant progression. Total RNA isolated from variants of the papilloma cell line SP-1 transduced with retroviral vectors expressing v-jun and v-fos alone or in tandem was analyzed for the presence of differentially expressed transcripts by using 35 different primer combinations. Differentially expressed clones were rescreened by dot-blot analysis by using cDNA from chemically induced tumors with a high or low risk of malignant conversion. Three differentially displayed fragments were isolated in this analysis. Homology searches indicated that these fragments shared significant homology with the apoptosis inhibitor bcl-2, human alternative splicing factor/splicing factor 2 (ASF/SF2), and a novel gene not present in the GenBank or EMBL databases. In situ hybridization indicated that the expression levels of the bcl-2 homolog increased with malignant potential in chemically derived mouse skin tumors. A similar analysis indicated that expression of the ASF/SF2 homolog was greater in papillomas than in normal skin or in squamous cell carcinomas. Transcripts for this gene were most abundant in the granular layer. The expression pattern of the third differential display fragment was consistent with that of a tumor suppressor gene. This gene was expressed at very high levels in normal skin and benign papillomas but was essentially undetectable in squamous cell carcinomas. Through this approach, we identified known and novel genes that may contribute to malignant progression in epidermal tumors.

Animals↗

[IFN alpha- and IFN gamma-induced differentially expressed genes].

OBJECTIVES: It was previously found that IFN alpha could reduce, while IFN gamma could enhance metastatic potential of a mouse mammary adenocarcinoma cell line MA891. The aim of this study was to identify the sequence of cDNA fragments that were differentially expressed by the two types of IFN, respectively. METHODS: Total RNA was extracted from MA891 cells treated with IFN alpha or IFN gamma. Northern blotting was used to confirm the differentially expressed gene fragments. DNA sequencing was then carried out. RESULTS: The Northern blots indicated that among the 10 cDNA fragments obtained by mRNA differential display technique, 4(G15 gamma, C4 alpha, T2 gamma, and T11 gamma) were shown to be unequivocally differentially expressed. Among them, the nucleotide sequence of G15 gamma did not show homology with any of the existing genes, while that of C4 alpha, T2 gamma and T11 gamma was highly homolgous to mouse minor histocompatility complex gene, mouse beta-actin gene and hamster high blood pressure gene, respectively. CONCLUSIONS: Four differentially expressed cDNA fragments by IFN alpha- and IFN gamma-treated MA891 cells have been ascertained. Regardless of sequence homology, none of them is reportedly related to tumor cell metastasis. The results herein reported provide basis for the elucidation of new metastasis-associated gene(s).

Adenocarcinoma↗

Screening differentially expressed genes in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis.

AIM: To screen genes differentially expressed in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis. METHODS: A subtracted cDNA library of mouse hepatocarcinoma cell line with high potential of lymphatic metastatic Hca-F and its synogenetic cell line Hca-P with a low metastatic potential was constructed by suppression subtracted hybridization(SSH) method. The screened clones of the subtracted library were sequenced and GeneBank homology search was performed. RESULTS: Fourteen differentially expressed cDNA fragments of Hca-F were obtained with two novel genes. CONCLUSION: SSH is a useful technique to detect differentially expressioned genes and an effective method to clone novel genes.

Animals↗

[Differentially expressed genes in vascular endothelial cell line ECV 304 induced by high density lipoprotein].

OBJECTIVE: To isolate and clone the differentially expressed genes in vascular endothelial cell induced by high density lipoprotein (HDL) so as to study the anti- atherogenesis molecular mechanism of HDL. METHODS: Differential display reverse transcription PCR method was used to analyze the differentially expressed cDNA in vascular endothelial cell line ECV 304 induced by HDL. After sequencing and homology research, several differentially expressed cDNA fragments were confirmed by Northern blot analysis. RESULTS: Up-regulated and down-regulated cDNA fragments in ECV 304 induced by HDL were isolated. Nine cDNA fragments were highly homologous to the known human genes and four were fragments of novel genes. The known up-regulated genes included genes of human STE20-like kinase, PBK1 protein, transglutaminase, myosin alkali light chain, apobec-1 binding protein 1, and death- associated protein (DAP). The known down-regulated genes included genes of ribosomal protein L7a (RPL7A), voltage-dependent anion channel isoform 2, and glycinamide ribonucleotide transformylase. Northern blot analysis revealed that the expression levels of transglutaminase and apobec-1 binding protein 1 were upregulated by 50% and 70% respectively. CONCLUSION: HDL upregulates the expression of apobec-1 binding protein 1 and transglutaminase in endothelial cells. The high level expression of transglutaminase and apobec-1 binding protein 1 in endothelial cells induced by HDL may be related to anti-atherogenesis function of HDL.

Blotting, Northern↗

[Differentially express genes in human embryonic lung fibroblasts with damage tolerance induced by low-dose hydroquinone].

OBJECTIVE: To observe the differentially expressed genes in human embryonic lung fibroblasts (HELF) induced by small-dose hydroquinone (HQ) using fluorescence differential display-PCR (DD-PCR). METHODS: According to the dose-effect relation of HQ toxicity we established previously, HQ dose that did not induce observed cell damage or proliferation arrest was defined as low dose (100 pmol/L), and that causing obvious cell damage as the high dose (100 micrommol/L). The cells were then treated with low or high dose of HQ, or exposed to high-dose HQ following pretreatment with low-dose HQ for some time, respectively. Fluorescence DD-PCR was performed and 33 differentially expressed genes were identified in the cells with different treatments, and 8 of the identified genes were amplified, cloned, sequenced and blasted. RESULTS: Seven of the 8 amplified genes were unknown genes, and the left one was identified as a known gene highly homologous to that encoding Homo sapiens Rap1 interacting factor 1 (RIF1). CONCLUSION: Low-dose HQ can induce damage tolerance in HELF, and identification of the differentially expressed genes may provide valuable sight into the mechanism of HQ-induced damage tolerance.

Adaptation, Physiological↗

Separation and identification of differentially expressed nuclear matrix proteins between human esophageal immortalized and carcinomatous cell lines.

AIM: To separate and identify differentially expressed nuclear matrix proteins (NMPs) between the immortalized human esophageal epithelial cell line (SHEE) and the malignantly transformed esophageal carcinoma cell line (SHEEC), and to provide new ways for finding specific markers and the pathogenesis of esophageal carcinoma. METHODS: SHEE and SHEEC cell lines were used to extract NMPs. The quality of NMPs was monitored by Western blot analysis including DNA topoisomerase IIalpha, proliferation cell nuclear antigen (PCNA) and histone. NMPs of SHEE and SHEEC were analyzed by two-dimensional electrophoresis (2-DE), silver staining and PDQuest6.2 image analysis software. Three spots in which the differentially expressed NMPs were more obvious, were selected and analyzed with matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI- TOF-MS) and database search. RESULTS: Western blot analysis revealed that DNA topoisomerase IIalpha and PCNA were detected, and the majority of histones were deleted in NMPs of SHEE and SHEEC. After 2-DE image analysis by PDQuest6.2 software, the 2-DE maps were detected with an average of 106+/-7.1 spots in SHEE and 132+/-5.0 spots in SHEEC. Most of them were matched one another (r=0.72), only 16 protein spots were found differing in intensity. Three NMPs including cytoskeletal tropomyosin, FK506-binding protein 6, similar to retinoblastoma binding protein 8 were preliminarily identified by MALDI- TOF-MS. CONCLUSION: These differentially expressed NMPs may play an important role during malignant transformation from SHEE to SHEEC. Their separation and identification will contribute to searching for specific markers and probing into the pathogenesis of esophageal carcinoma.

Blotting, Western↗

Identification of differentially expressed genes in mouse hepatocarcinoma ascites cell line with low potential of lymphogenous metastasis.

AIM: To identify genes differentially expressed in mouse hepatocarcinoma ascites cell line with low potential of lymphogenous metastasis. METHODS: A subtracted cDNA library of mouse hepatocarcinoma cell line with low potential of lympho-genous metastasis Hca-P and its synogenetic cell line Hca-F with high metastatic potential was constructed by suppression subtracted hybridization (SSH) method. The screened clones of the subtracted library were sequenced and GenBank homology search was performed. RESULTS: Fifteen differentially expressed cDNA fragments of Hca-P were obtained which revealed 8 known genes, 4 expressed sequence tags (ESTs) and 3 cDNAs showed no homology. CONCLUSION: Tumor metastasis is an incident involving multiple genes. SSH is a useful technique to detect differentially expressed genes and an effective method to clone novel genes.

Animals↗

Expression analysis of subtractively enriched libraries (EASEL): a widely applicable approach to the identification of differentially expressed genes.

A variety of methods for high throughput analysis of differential gene expression has been developed over the past years. We have implemented the EASEL technique that adds flexibility, efficiency and wide-applicability to these methods. The EASEL procedure is unique as it integrates several well established techniques and thereby offers a combination of subtractive hybridization of 3' cDNA ends with macroarrays analysis and Serial Analysis of Gene Expression (SAGE). In addition, once a set of interesting, differentially expressed genes is identified, the material required for follow up studies to test the hypothesis that the gene is truly involved in the process of interest is readily available. In this report, we first present a step-by-step validation of the procedure, since several of the incorporated steps had to be tailored to meet specific requirements and implied drastic modifications of the original methods. Secondly, we applied EASEL to the identification of up-regulated gene products in the outflow tract region of the embryonic rat heart. Here we provide evidence that at least two among the differentially expressed genes detected, follistatin-like protein gene and membrane type 1-metallo proteinase gene, are selectively up-regulated in the outflow tract, suggesting their involvement in the development of this region during embryogenesis.

Animals↗