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At least 469 records · Page 26Linked to original sources
PubMed2004

Two-step differential expression analysis reveals a new set of genes involved in thyroid oncocytic tumors.

Thyroid oncocytic adenomas are a class of tumors characterized by the presence of abundant mitochondria. We performed a differential display RT-PCR analysis on two oncocytic adenomas and their paired controls. We then carried out a microarray analysis using the 460 selected, differentially expressed clones on four other oncocytomas and their paired controls. Thirty genes, 12 encoded by mitochondrial DNA and 18 nuclear-encoded, were overexpressed by a factor of at least 2 in the tumors compared with the controls. Seven of the 18 nuclear-encoded genes are involved in protein metabolism: DKFZP434I116, B3GTL, SNX19, RP42, SENP1, UBE2D3, and the CTSB gene, which is known to be particularly deregulated in most thyroid tumors. Other genes are implicated in signal transduction (ITGAV) or tumorigenesis (AF1q). Immunohistochemistry allowed us to confirm overexpression of the ITGAV and CTSB genes at the protein level and showed a marked relocation of the CTSB protein. We confirmed the overexpression of the AF1q oncogene in 56% of 18 oncocytic tumors by quantitative RT-PCR analysis, which attested to the heterogeneity of these tumors. Our results show an increased expression of genes involved in protein metabolism in oncocytoma, the significance of which requires investigation.

Adenine Nucleotide Translocator 2↗
PubMed2002

Gene expression analysis with universal n-mer arrays.

Gene expression profiling is one of the many applications that have benefited from the massively parallel nucleic acid detection capability of DNA microarrays. Current expression arrays, however, are expensive and inflexible. They are custom-designed for each organism and they do not offer the possibility of incorporating updated genomic information without production of a new chip. One possible solution is the development of a universal chip, consisting of all 4n possible DNA sequences of length n. Studying different organisms or new genes would simply require modifications to the hybridization pattern analysis software. The key problem is to find a value of n that is large enough to afford sufficient specificity, yet is small enough for practical fabrication and readout. We developed an analytical model, supported by computer-assisted calculation with yeast and mouse transcript data, to argue that it is both practical and useful to fabricate n-mer arrays with 10 < or = n < or = 16.

Alternative Splicing↗
PubMed2004

Molecular cloning and expression analysis of three genes encoding starch synthase II in rice.

Three starch synthase (SS) genes, OsSSII-1, OsSSII-2 and OsSSII-3, were identified in rice (Oryza sativa L.) and localized to chromosomes 10, 2 and 6, respectively. The three OsSSII full-length cDNAs were cloned, and the predicted amino acid sequences were found to share 52-73% similarity with other members of the plant SSII family. The SS activity of each OsSSII was confirmed by expression and enzyme activity assay in Escherichia coli. Expression profile analysis revealed that OsSSII-1 was expressed in endosperms, leaves and roots; OsSSII-2 was mainly expressed in leaves, while OsSSII-3 was mainly expressed in endosperms. Similar to the OsSSI proteins, the OsSSII-2 and OsSSII-3 proteins were found in the soluble as well as the starch-granule-bound fractions in rice. The roles of the OsSSII proteins in starch biosynthesis in rice and the evolutionary relationships of the genes encoding monocotyledonous and dicotyledonous class-II SS enzymes are discussed.

Amino Acid Sequence↗
PubMed2006

Use of routinely collected amniotic fluid for whole-genome expression analysis of polygenic disorders.

BACKGROUND: Neural tube defects related to polygenic disorders are the second most common birth defects in the world, but no molecular biologic tests are available to analyze the genes involved in the pathomechanism of these disorders. We explored the use of routinely collected amniotic fluid to characterize the differential gene expression profiles of polygenic disorders. METHODS: We used oligonucleotide microarrays to analyze amniotic fluid samples obtained from pregnant women carrying fetuses with neural tube defects diagnosed during ultrasound examination. The control samples were obtained from pregnant women who underwent routine genetic amniocentesis because of advanced maternal age (>35 years). We also investigated specific folate-related genes because maternal periconceptional folic acid supplementation has been found to have a protective effect with respect to neural tube defects. RESULTS: Fetal mRNA from amniocytes was successfully isolated, amplified, labeled, and hybridized to whole-genome transcript arrays. We detected differential gene expression profiles between cases and controls. Highlighted genes such as SLA, LST1, and BENE might be important in the development of neural tube defects. None of the specific folate-related genes were in the top 100 associated transcripts. CONCLUSIONS: This pilot study demonstrated that a routinely collected amount of amniotic fluid (as small as 6 mL) can provide sufficient RNA to successfully hybridize to expression arrays. Analysis of the differences in fetal gene expressions might help us decipher the complex genetic background of polygenic disorders.

Amniotic Fluid↗
PubMed2005

Effects of RNA degradation on gene expression analysis of human postmortem tissues.

The Affymetrix GeneChip platform was used to build a gene expression database of the normal human body. Postmortem human tissues represent a valuable source of biological materials for this type of study, but their use entails some delays before harvesting such tissues. We first evaluated the RNA quality obtained from tissues obtained 3-5 h postmortem and found variations that were both tissue and donor-dependent. RNAs extracted from brain regions were of higher quality than those obtained from the gut, while the cause of death was a significant factor in donor-dependent differences. To avoid these variables, we used rat duodenum to determine the effects of RNA degradation on the analysis of gene expression. Surprisingly, even samples exhibiting significant RNA degradation yielded robust gene expression results, comparable to those obtained using intact samples at a certain signal intensity cutoff. We extended these findings to our human expression database and obtained similar results, indicating that the Affymetrix platform, which is biased to the 3' end of transcripts for detection, can tolerate significant RNA degradation, while still yielding high quality expression data. Our resulting body index expression database is a valuable research tool. As examples of potential uses, we report novel expression sites for four potential therapeutic targets--CCL27, GPR22, GPR113 and GPR128--and as well as a set of thymus-specific genes, including three not previously associated with the thymus.

Animals↗
PubMed2005

Optimization of the PAXgene blood RNA extraction system for gene expression analysis of clinical samples.

One major problem associated with collecting whole blood from patients for use as a source of RNA in gene expression studies is that the RNA degrades during collection and storage. Preservation of RNA quality is vital in such studies because the stability of the RNA ultimately affects analysis of gene expression. In this study the PAXgene blood collection system was compared with a standard erythrocyte lysis method for isolating RNA from blood samples. The methods were compared in terms of RNA yield, RNA stabilization, and DNA contamination. The study also included the downstream application to RT-PCR analysis for relative mRNA expression levels of the ribonucleotide reductase subunits R1 and R2. The results show that blood collection in conventional collection tubes, and leukocyte isolation by erythrocyte lysis lead to significant degradation of RNA. Our findings confirm the ability of PAXgene to stabilize RNA in whole blood; however, RNA extracted by the PAXgene method contained significant DNA contamination. Given the low basal expression of the target genes analyzed in this study, contaminating DNA could potentially affect accurate interpretation of RT-PCR data. As a result, the PAXgene protocol was optimized to include off-column DNase treatments, which yielded high-quality RNA suitable for gene expression studies. Furthermore, the results suggest that RNA isolation with PAXgene is advantageous compared to traditional extraction methods for RT-PCR analysis of large or different-sized amplicons.

Actins↗
PubMed2002

Multiplex gene expression analysis for high-throughput drug discovery: screening and analysis of compounds affecting genes overexpressed in cancer cells.

Drug discovery strategies are needed that can rapidly exploit multiple therapeutic targets associated with the complex gene expression changes that characterize a polygenic disease such as cancer. We report a new cell-based high-throughput technology for screening chemical libraries against several potential cancer target genes in parallel. Multiplex gene expression (MGE) analysis provides direct and quantitative measurement of multiple endogenous mRNAs using a multiplexed detection system coupled to reverse transcription-PCR. A multiplex assay for six genes overexpressed in cancer cells was used to screen 9000 chemicals and known drugs in the human prostate cancer cell line PC-3. Active compounds that modulated gene expression levels were identified, and IC50 values were determined for compounds that bind DNA, cell surface receptors, and components of intracellular signaling pathways. A class of steroids related to the cardiac glycosides was identified that potently inhibited the plasma membrane Na(+)K(+)-ATPase resulting in the inhibition of four of the prostate target genes including transcription factors Hoxb-13, hPSE/PDEF, hepatocyte nuclear factor-3alpha, and the inhibitor of apoptosis, survivin. Representative compounds selectively induced apoptosis in PC-3 cells compared with the nonmetastatic cell line BPH-1. The multiplex assay distinguished potencies among structural variants, enabling structure-activity analysis suitable for chemical optimization studies. A second multiplex assay for five toxicological markers, Hsp70, Gadd153, Gadd45, O6-methylguanine-DNA methyltransferase, and cyclophilin, detected compounds that caused DNA damage and cellular stress and was a more sensitive and specific indicator of potential toxicity than measurement of cell viability. MGE analysis facilitates rapid drug screening and compound optimization, the simultaneous measurement of toxicological end points, and gene function analysis.

Actins↗
PubMed2005

Characterization and expression analysis of bactericidal permeability-increasing protein (BPI) antimicrobial peptide gene from channel catfish Ictalurus punctatus.

Antimicrobial peptides are important components of host defenses against microbial invasions. Bactericidal permeability-increasing protein (BPI) is an antimicrobial peptide belonging to the lipid transfer/LPS-binding protein family. It serves important roles in defending against Gram-negative bacteria in the innate immune system. Here we report cloning of complete BPI cDNA from channel catfish (Ictalurus punctatus) by 5' RACE after obtaining the partial BPI cDNA sequence from EST analysis. The channel catfish BPI cDNA is 1640 bp in length with a 1428-bp open reading frame that encodes a protein of 475 amino acids. Catfish BPI gene shows high similarity with the BPI/LBP gene isolated from other teleost fish. As part of ongoing efforts in comparative genome analysis, we have assigned the catfish BPI gene to bacterial artificial chromosome (BAC) clones. Southern blot analysis on multiple BPI BAC clones indicated the presence of a single copy of the BPI gene in the catfish genome. Reverse transcription-polymerase chain reaction (RT-PCR) analysis on healthy tissues showed that BPI was expressed in a wide range of tissues including head kidney, gill, skin, trunk kidney, brain, intestine, liver, muscle, ovary, spleen and stomach. The BPI gene was not developmentally expressed until 48 h after fertilization. Quantitative real time PCR (QRT-PCR) analysis indicated that the BPI gene expression was induced after challenge with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC). BPI upregulation peaked 3 days after challenge, mirroring the expression pattern of inflammatory chemokines in catfish, suggesting that it plays a role in the innate defense response.

Amino Acid Sequence↗
PubMed2002

Gene expression analysis reveals chemical-specific profiles.

The application of gene expression profiling technology to examine multiple genes and signaling pathways simultaneously promises a significant advance in understanding toxic mechanisms to ultimately aid in protection of public health. Public and private efforts in the new field of toxicogenomics are focused on populating databases with gene expression profiles of compounds where toxicological and pathological endpoints are well characterized. The validity and utility of a toxicogenomics is dependent on whether gene expression profiles that correspond to different chemicals can be distinguished. The principal hypothesis underlying a toxicogenomic or pharmacogenomic strategy is that chemical-specific patterns of altered gene expression will be revealed using high-density microarray analysis of tissues from exposed organisms. Analyses of these patterns should allow classification of toxicants and provide important mechanistic insights. This report provides a verification of this hypothesis. Patterns of gene expression corresponding to liver tissue derived from chemically exposed rats revealed similarity in gene expression profiles between animals treated with different agents from a common class of compounds, peroxisome proliferators [clofibrate (ethyl-p-chlorophenoxyisobutyrate), Wyeth 14,643 ([4-chloro-6(2,3-xylidino)-2-pyrimidinylthio]acetic acid), and gemfibrozil (5-2[2,5-dimethylphenoxy]2-2-dimethylpentanoic acid)], but a very distinct gene expression profile was produced using a compound from another class, enzyme inducers (phenobarbital).

Animals↗
PubMed2006

Expression analysis of PCNA gene in chronic myelogenous leukemia--combined application of siRNA silencing and expression arrays.

Imatinib metylase is the first choice treatment for BCR/ABL positive chronic myelogenous leukemia (CML). However, as some CML patients develop resistance to imatinib therapy, there is a significant interest in development of alternative treatment strategies, such as identifying targets other than BCR/ABL that may participate in CML. Previously, we demonstrated strong PCNA up-regulation in CML patients. To further study its role in CML pathogenesis, we performed silencing of PCNA expression followed by array experiments. PCNA inhibition led to down-regulation of CDK1, CDK4, PLK1, ERK3, JNK1, STAT5, and several inhibitors of apoptosis (DAXX, Mdm2, survivin). The following genes were up-regulated: CDK inhibitors p21 and p19-INK4D, pro-apoptotic FAST kinase, fibronectin, etc. However, as PCNA affects cell growth in naturally proliferating cells as well as in cancerous cells, it seems to act a secondary role relating to proliferation activity of leukemic cells.

Apoptosis↗
PubMed2005

A tiling microarray expression analysis of rice chromosome 4 suggests a chromosome-level regulation of transcription.

The complete genome sequence of cultivated rice (Oryza sativa) provides an unprecedented opportunity to understand the biology of this model cereal. An essential and necessary step in this effort is the determination of the coding information and expression patterns of each sequenced chromosome. Here, we report an analysis of the transcriptional activity of rice chromosome 4 using a tiling path microarray based on PCR-generated genomic DNA fragments. Six representative rice organ types were examined using this microarray to catalog the transcribed regions of rice chromosome 4 and to reveal organ- and developmental stage-specific transcription patterns. This analysis provided expression support for 82% of the gene models in the chromosome. Transcriptional activities in 1643 nonannotated regions were also detected. Comparison with cytologically defined chromatin features indicated that in juvenile-stage rice the euchromatic region is more actively transcribed than is the transposon-rich heterochromatic portion of the chromosome. Interestingly, increased transcription of transposon-related gene models in certain heterochromatic regions was observed in mature-stage rice organs and in suspension-cultured cells. These results suggest a close correlation between transcriptional activity and chromosome organization and the developmental regulation of transcription activity at the chromosome level.

Chromosome Mapping↗
PubMed2002

Expression analysis of neuroglobin mRNA in rodent tissues.

Neuroglobin is a respiratory protein which was reported to be preferentially expressed in the vertebrate brain. Here we present the first detailed analysis of the expression of neuroglobin in mouse and rat tissues. Neuroglobin mRNA was detected in all brain areas studied. Most, but not all, nerve cells were labeled, suggesting differential expression of Ngb. Neuroglobin mRNA was detected in the peripheral nervous system, explaining previous northern hybridization signals in organs other than the brain. Substantial neuroglobin expression was also found in metabolically active endocrine tissues such as the adrenal and pituitary glands. The granule localization of neuroglobin transcripts in various neuronal extensions let us speculate that peripheral translation of neuroglobin protein occurs. This could have important functional consequences for synaptic plasticity, an active metabolic process that needs large amounts of oxygen. The hybridization signals suggest that the local concentration of neuroglobin is sufficient for its putative primary function as an oxygen-supplying protein.

Adrenal Glands↗
PubMed2002

Profiling novel sulfonamide antitumor agents with cell-based phenotypic screens and array-based gene expression analysis.

A series of small molecules from sulfonamide-focused libraries have been evaluated in these laboratories to discover novel antitumor agents. Cell-based screens using flow cytometric analysis revealed the presence of two distinct classes of cell cycle inhibitors in this series; one (including E7010 and ER-67865) arrested mitosis by preventing tubulin polymerization; and the other (including E7070 and ER-68487) caused a decrease in the S-phase fraction along with cell cycle perturbation in G1 and/or G2 via an unknown mechanism(s). To further characterize both classes of antitumor sulfonamides with respect to their effects on gene expression, we used oligonucleotide microarray analysis for representative compounds. Consistent with the phenotypic observations, essentially the same transcription profiles were found between E7010 and ER-67865 and also between E7070 and ER-68487. However, there was very little overlap between genes affected by E7010 and E7070. As a characteristic expression change for microtubule-depolymerizing agents, the down-regulation of alpha-tubulin transcripts was evident in both E7010- and ER-67865-treated cells. On the other hand, E7070 and ER-68487 repressed significantly the expression of a variety of genes involved in metabolic processes, cell cycle progression, immune response, and signal transduction. Of the compounds examined, E7010 and E7070 have progressed to clinical trials, demonstrating some objective responses in the Phase I setting. Described herein is profiling of novel anticancer drug candidates from the sulfonamide class based on phenotypic screens and gene expression analysis. This includes a translational research that may suggest potentially useful markers for pharmacodynamic drug assessment in clinic.

Antineoplastic Agents↗
PubMed2005

Isolation and expression analysis of genes encoding DNA methyltransferase in wheat (Triticum aestivum L.).

DNA methylation of cytosine residues, catalyzed by DNA methyltransferases, is suggested to play important roles in regulating gene expression and plant development. In this study, we isolated four wheat cDNA fragments and one cDNA with open reading frame encoding putative DNA methyltransferase and designated TaMET1, TaMET2a, TaMET2b, TaCMT, TaMET3, respectively. BLASTX searches and phylogenetic analysis suggested that five cDNAs belonged to four classes (Dnmt1, Dnmt2, CMT and Dnmt3) of DNA methyltransferase genes. TaMET2a encoded a protein of 376 aa and contained eight of ten conserved motifs characteristic of DNA methyltransferase. Genomic sequence of TaMET2a was obtained and found to contain ten introns and eleven exons. The expression analysis of the five genes revealed that they were expressed in developing seed, during germination and various vegetative tissues, but in quite different abundance. It was interesting to note that TaMET1 and TaMET3 mRNAs were clearly detected in dry seeds. Moreover, the differential expression patterns of five genes were observed between wheat hybrid and its parents in leaf, stem and root of jointing stage, some were up-regulated while some others were down-regulated in the hybrid. We concluded that multiple wheat DNA methyltransferase genes were present and might play important roles in wheat growth and development.

Amino Acid Sequence↗
PubMed2004

Multi-tissue gene-expression analysis in a mouse model of thyroid hormone resistance.

BACKGROUND: Resistance to thyroid hormone (RTH) is caused by mutations of the thyroid hormone receptor beta (TRbeta) gene. To understand the transcriptional program underlying TRbeta mutant-induced phenotypic expression of RTH, cDNA microarrays were used to profile the expression of 11,500 genes in a mouse model of human RTH. RESULTS: We analyzed transcript levels in cerebellum, heart and white adipose tissue from a knock-in mouse (TRbetaPV/PV mouse) that harbors a human mutation (referred to as PV) and faithfully reproduces human RTH. Because TRbetaPV/PV mice have elevated thyroid hormone (T3), to define T3-responsive genes in the context of normal TRbeta, we also analyzed T3 effects in hyperthyroid wild-type gender-matched littermates. Microarray analysis revealed 163 genes responsive to T3 treatment and 187 genes differentially expressed between TRbetaPV/PV mice and wild-type littermates. Both the magnitude and gene make-up of the transcriptional response varied widely across tissues and conditions. We identified genes modulated in T3-dependent PV-independent, T3- and PV-dependent, and T3-independent PV-dependent pathways that illuminated the biological consequences of PV action in vivo. Most T3-responsive genes that were dysregulated in the heart and white adipose tissue of TRbetaPV/PV mice were repressed in T3-treated wild-type mice and upregulated in TRbetaPV/PV mice, suggesting the inappropriate activation of T3-suppressed genes in RTH. CONCLUSIONS: Comprehensive multi-tissue gene-expression analysis uncovered complex multiple signaling pathways that mediate the molecular actions of TRbeta mutants in vivo. In particular, the T3-independent mutant-dependent genomic response unveiled the contribution of a novel 'change-of-function' of TRbeta mutants to the pathogenesis of RTH. Thus, the molecular actions of TRbeta mutants are more complex than previously envisioned.

Adipose Tissue↗
PubMed1998

Identifying markers for pancreatic cancer by gene expression analysis.

To begin to identify new tumor markers, we recently performed a systematic study of gene expression in cancers of the colon and pancreas. Of the 45,000 genes identified, 183 were found to be expressed at significantly elevated levels in pancreatic cancer. One of the genes was tissue inhibitor of metalloproteinase type I (TIMP-1), which encodes a secreted protein. Analysis of TIMP-1 serum levels revealed significant increases in pancreatic cancer patients, but TIMP-1 by itself was inadequate as a serum marker for cancer. However, a combination of individually suboptimal markers (TIMP-1, CA19-9, and carcinoembryonic antigen) detected 60% of 85 patients with pancreatic cancers in a highly specific manner. These results suggest that a systematic analysis of gene expression can reveal novel serum markers and that individually suboptimal markers can be combined to yield higher sensitivity and specificity.

Biomarkers, Tumor↗
PubMed2005

Molecular characterization and expression analysis of vitellogenin in the marine crab Portunus trituberculatus.

As a precursor of the major yolk protein vitellin (Vn), vitellogenin (Vg) has been studied in crustacean for decades, mainly in shrimp. However, little is known about the Vg molecule in crabs. In the present study, we report a cDNA encoding Vg in the marine crab Portunus trituberculatus, an important commercial species in aquaculture. The cDNA sequence is 7,846 bp in length, containing a 7,680 bp open reading frame, that encodes 2,560 amino acid residues. The deduced amino acid sequence showed 32-75% identity with other known crustacean Vgs. Seven potential cleavage sites (RXXR) were identified in the deduced Vg precursor. SDS-PAGE and Western blotting analysis demonstrated that Vns in the ovary of the mature crab consisted of three major polypeptides (102, 100 and 85 kDa). Northern blotting analysis revealed that Vg was mainly expressed in hepatopancreas in vitellogensis females. A Vg transcript was also identified in the ovary of the crab by reverse transcription-polymerase chain reaction analysis.

Amino Acid Sequence↗