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At least 721 records · Page 40Linked to original sources
PubMed2006

Influence of surgical manipulation on prostate gene expression: implications for molecular correlates of treatment effects and disease prognosis.

PURPOSE: Measurements of tissue gene expression are increasingly used for disease stratification, clinical trial eligibility, and assessment of neoadjuvant therapy response. However, the method of tissue acquisition alone could significantly influence the expression of specific transcripts or proteins. This study examines whether there are transcript alterations associated with surgical resection of the prostate gland by radical retropubic prostatectomy. MATERIALS AND METHODS: Twelve patients with clinically localized prostate cancer underwent immediate in situ prostate biopsy after induction of anesthesia for radical prostatectomy. Ex vivo prostate biopsies were performed immediately after surgical removal. Prostate epithelium was acquired by laser-capture microdissection, and transcript abundance levels were quantitated by cDNA microarray hybridization and confirmed by quantitative polymerase chain reaction. Data were analyzed by paired, two-sample t test using Statistical Analysis of Microarray algorithms, and linear models were fit as a function of clinical characteristics. RESULTS: Of 5,753 cDNAs with measurable expression in prostate epithelium, 88 (1.5%) were altered as a result of surgery (false-discovery rate < or = 10%), representing 62 unique genes. These included transcripts encoding acute phase response proteins, IER2 and JUNB, and regulators of cell proliferation, p21Cip1 and KLF6. Of the clinical characteristics examined, including patient age, prostate volume, serum prostate-specific antigen, blood loss, and operative time, only gland volume was significantly and negatively associated with the magnitude of gene expression difference between pre- and postsurgical specimens. CONCLUSION: Surgical manipulation results in significant gene expression changes. Molecular analyses of surgical samples should recognize that transcript alterations occur rapidly, and these results are important when designing and analyzing molecular correlates of clinical studies.

Aged↗
PubMed1994

An alternatively spliced form of HLA-G mRNA in human trophoblasts and evidence for the presence of HLA-G transcript in adult lymphocytes.

The HLA-G monomorphic, nonclassical class I gene encodes the major histocompatibility complex (MHC) molecule, which is the only MHC antigen expressed on cytotrophoblast cells of placenta. In this work, we have investigated expression of the HLA-G gene in fetal tissues and adult peripheral blood cells by using a sensitive hot-start reverse transcriptase PCR technique. PCR amplification with HLA-G primers specific for exon 3 has enabled us to demonstrate an alternatively spliced form of HLA-G mRNA present in fetal first trimester trophoblasts and lacking exon 4 (HLA-G.3-5). This low abundance transcript (approximately 1:200) in comparison to full-length mRNA may encode the protein that excludes the alpha 3 domain and by conformational changes may present a different ability to bind to peptides. Moreover, expression of the HLA-G transcript was found in adult peripheral lymphocytes and equally in B- and T-cell populations. These results are discussed in the context of the fetal-maternal relationship presented by HLA-G gene products.

Adult↗
PubMed2005

Gene expression profile studies of human keratoconus cornea for NEIBank: a novel cornea-expressed gene and the absence of transcripts for aquaporin 5.

PURPOSE: To increase the database of genes expressed in human cornea and to gain insights into the molecular basis of keratoconus (KC). METHODS: A cDNA library was constructed from KC corneas harvested at keratoplasty and used for expressed sequence tag (EST) analysis. Data were analyzed using grouping and identification of sequence tags (GRIST). Expression of selected clones was examined by RT-PCR. RESULTS: A total of 7680 clones was sequenced from the 5' end. After bioinformatics analysis, 4090 clusters of clones, each potentially representing individual genes, were identified. Of these, 887 genes were represented by more than one clone. The five most abundant transcripts, represented by >60 clones each, were for keratin-12, TGFBI (BIGH3), decorin, ALDH3, and enolase 1, all known markers for cornea. Many other markers for epithelial, stromal, and endothelial genes were also present. One cluster of six clones came from an apparently novel gene (designated KC6) located on chromosome 18 at p12.3. RT-PCR of RNA from several human tissues detected KC6 transcripts only in cornea. In addition, no clones were observed for the usually prominent corneal epithelial cell marker aquaporin 5 (AQP5), a water channel protein. Semiquantitative RT-PCR confirmed that expression of AQP5 is much lower in KC cornea than in non-KC cornea. CONCLUSIONS: This analysis increases the database of genes expressed in the human cornea and provides insights into KC. KC6 is a novel gene of unknown function that shows cornea-preferred expression, whereas the suppression of transcripts for AQP5 provides the first clear evidence of a molecular defect identified in KC.

Amino Acid Sequence↗
PubMed1992

Bronze-2 Gene Expression and Intron Splicing Patterns in Cells and Tissues of Zea mays L.

A large fraction of the transcripts of the Bronze-2 (Bz2) gene of maize (Zea mays L.) are unspliced in purple husk tissues. The accumulation of unspliced messages could have destructive potential if the intron-bearing mRNAs are translated into aberrant proteins. Our initial studies suggested that both genetic and physiological factors may influence the degree of splicing failure. Nuclear background rather than cis-sequence effects is shown to contribute to the genetic component. The accumulation of unspliced message does not appear to be directly influenced by diurnal effects on transcript abundance, by the expression level of the Bz2 gene, or by thermal stress. We also show that maize cell cultures (Black Mexican Sweet, BMS) can be used to examine the molecular details involved in splicing failure. Much like whole maize plants, the BMS cells excise the Bz2 intron with varying degrees of efficiency. In contrast with heterologous constructs containing plant introns, splicing of the native Bz2 intron can appproach 100% in BMS cells. Splicing of transcripts from a marked, introduced gene can be compared to the endogeneous Bz2 gene facilitating analysis of the impact of sequence changes.

Journal Article↗
PubMed2004

Auxin activates KAT1 and KAT2, two K+-channel genes expressed in seedlings of Arabidopsis thaliana.

The transcript abundance of the K+-channel gene ZMK1 (Zea mays K+ channel 1) in maize coleoptiles is controlled by the phytohormone auxin. Thus, ZMK1 is thought to function in auxin-regulated coleoptile elongation, as well as during gravitropism and phototropism. To investigate related growth phenomena in the dicotyledonous plant Arabidopsis thaliana, we screened etiolated seedlings for auxin-induced K+-channel genes. Among the members of the Shaker-like K+ channels, we thereby identified transcripts of the inward rectifiers, KAT1 (K+ transporter of Arabidopsis thaliana) and KAT2, to be upregulated by auxin. The phloem-associated KAT2 was localised in cotyledons and the apical part of etiolated seedlings. In contrast, the K+-channel gene KAT1 was expressed in the cortex and epidermis of etiolated hypocotyls, as well as in flower stalks. Furthermore, KAT1 was induced by active auxins in auxin-sensitive tissues characterised by rapid cell elongation. Applying the patch-clamp technique to protoplasts of etiolated hypocotyls, we correlated the electrical properties of K+ currents with the expression profile of K+-channel genes. In KAT1-knockout mutants, K+ currents after auxin stimulation were characterised by reduced amplitudes. Thus, this change in the electrical properties of the K+-uptake channel in hypocotyl protoplasts resulted from an auxin-induced increase of active KAT1 proteins. The loss of KAT1-channel subunits, however, did not affect the auxin-induced growth rate of hypocotyls, pointing to compensation by residual, constitutive K+ transporters. From gene expression and electrophysiological data, we suggest that auxin regulation of KAT1 is involved in elongation growth of Arabidopsis. Furthermore, a role for KAT2 in the auxin-controlled vascular patterning of leaves is discussed.

Arabidopsis↗
PubMed1996

Differential regulation of the growth hormone receptor gene: effects of dexamethasone and estradiol.

GH receptor (GHR) expression differs during development between central and peripheral tissues. Peripheral GHR expression is known to be sensitive to gonadal and adrenal steroids, but little is known about their effects on GHR in the central nervous system. We have now studied the effects of estradiol (E2) or dexamethasone on GHR expression in rat arcuate nucleus (ARC) and hippocampus, using quantitative in situ hybridization. Dexamethasone, which strongly down-regulates hepatic GHR expression, had no effect on central GHR transcript abundance, whereas E2 treatment, which stimulates hepatic GHR expression, significantly reduced ARC GHR messenger RNA (mRNA) levels. E2 also increased somatostatin (SS) expression significantly in both ARC and periventricular nuclei but did not reduce ARC GH-releasing hormone (GHRH) mRNA levels. Ovariectomy stimulated GHR and GHRH mRNA levels in the ARC, whereas it lowered ARC SS expression. E2 replacement in ovariectomized animals restored GHRH and SS mRNA levels to control values. Hippocampal GHR mRNA transcripts showed the same response to these endocrine manipulations as seen in the ARC. The induction of hepatic GHR expression by E2 is known to involve the transcription of an alternate 5' untranslated first exon, GHR1. This was readily detectable in the liver using a specific GHR1 probe but could not be detected in any CNS area. Our results show that GHR expression in the CNS is sensitive to regulation by peripheral steroids but that CNS and hepatic expression of GHR is differentially regulated by the same treatments.

Animals↗
PubMed2004

cDNA cloning of a rat orthologue of SH2D2A encoding T-cell-specific adaptor protein (TSAd): expression in T and NK cells.

The T-cell-specific adapter protein (TSAd), encoded by the SH2D2A gene, has been implicated in modulation of proximal signaling events as well as in transcriptional regulation in human T cells. We have isolated its rat homologue ( rSH2D2A) from an NK cell cDNA library and mapped the corresponding gene to chromosome 2 with a hamster-rat radiation hybrid cell panel. rSH2D2A encodes a 376 amino acid protein (rTSAd) which shows greater homology to mouse than human TSAd. In rats, rTSAd was specifically expressed by NK cells and T cells but not by other leukocytes tested. Similarly, in humans we observed abundant transcripts for TSAd in NK cells and T cells. The data suggest that TSAd may have a regulatory role in cellular activation of T and NK cells.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Activation of gibberellin biosynthesis and response pathways by low temperature during imbibition of Arabidopsis thaliana seeds.

Exposure of imbibed seeds to low temperature (typically 4 degrees C) is widely used to break seed dormancy and to improve the frequency of germination. However, the mechanism by which temperature accelerates germination is largely unknown. Using DNA microarray and gas chromatography-mass spectrometry analyses, we found that a subset of gibberellin (GA) biosynthesis genes were upregulated in response to low temperature, resulting in an increase in the level of bioactive GAs and transcript abundance of GA-inducible genes in imbibed Arabidopsis thaliana seeds. Using a loss-of-function mutant, the cold-inducible GA biosynthesis gene, AtGA3ox1, was shown to play an essential role in mediating the effect of low temperature. Besides temperature, AtGA3ox1 also is positively regulated by active phytochrome and negatively regulated by GA activity. We show that both red light and GA deficiency act in addition to low temperature to elevate the level of AtGA3ox1 transcript, indicating that multiple signals are integrated by the AtGA3ox1 gene to control seed germination. When induced by low temperature, AtGA3ox1 mRNA was detectable by in situ RNA hybridization in an additional set of cell types relative to that in red light-induced seeds. Our results illustrate that the GA biosynthesis and response pathways are activated during seed imbibition at low temperature and suggest that the cellular distribution of bioactive GAs may be altered under different light and temperature conditions.

Arabidopsis↗
PubMed2004

Investigation of different transcript quantitation tools for high-throughput mapping of brain gene expression using voxelation.

Voxelation is a new approach for genome scale acquisition of brain gene expression patterns. The method employs high-throughput analysis of spatially registered voxels (cubes) to create multiple volumetric images of brain gene expression, similar to those obtained from biomedical imaging systems. The spatial resolution of voxelation depends on voxel size, with smaller voxels giving higher resolution. An important question is the applicability of different transcript profiling tools for the various levels of resolution that can be employed. Here, we describe the use of three methods to analyze voxel transcript abundance: real-time PCR, microarray analysis and linear amplification coupled with microarrays. We show statistically significant concordance between real-time PCR and microarray analysis for the myelin basic protein gene in human brain specimens at differing levels of spatial resolution. In addition, we also demonstrate the feasibility of using linear amplification coupled with microarray analysis to create voxelation maps from the mouse brain at high resolution, 1 microl. These data indicate the suitability of a number of transcript profiling tools for various levels of spatial resolution in voxelation.

Brain↗
PubMed2003

Amplification of representative cDNA pools from single human oocytes and pronucleate embryos.

In the human embryo, gene expression studies have been hindered by the scarcity of material and the fact that in vitro fertilisation (IVF) embryos available for research are usually of poor quality and are, therefore, not representative of normal development. This has led most authors to study individual human embryos, using conventional RT-PCR strategies, which permit analysis of only a few genes. Variability in the expression of genes between individual embryos is characteristic of these studies. In this study, a global RT-PCR strategy has been used, allowing the analysis of an almost infinite number of genes from a single embryo. We have used oocytes, which failed to fertilise and representative pronucleate embryos donated from cycles in which the patient conceived, to investigate possible variability in transcript abundance between individual embryos. We have screened oocytes and embryos for a panel of genes including beta-actin (expressed in 24/28 oocytes, 6/6 pronuclear embryos), the integrins beta1 (17/28 oocytes, 6/6 pronuclear embryos) and beta5 (8/28 oocytes, 5/6 pronuclear embryos), and the apoptotic regulators BCL-2 (20/28 oocytes, 2/6 pronuclear embryos) and BAX (21/28 oocytes, 5/6 pronuclear embryos). The expression of the pro-apoptotic regulator BAX increased in human oocytes following prolonged periods of culture. Overall, patterns of gene transcript presence showed variation between embryos and this was independent of either zona removal or lysis conditions. Pronucleate embryos showed less variation, however, even sibling embryos from the patient did not express an identical subset of genes.

DNA, Complementary↗
PubMed2000

Carbohydrate regulation of leaf development: Prolongation of leaf senescence in Rubisco antisense mutants of tobacco.

Previous investigations have shown that increased source strength as a result of elevated CO(2) can alter the timing of the phases of change that occur in photosynthetic rates during dicot leaf ontogeny [Miller et al. (1997) Plant Physiol 115: 1195-1200]. To evaluate the converse situation of decreased source strength, we examined leaf development in rbcS antisense mutants of tobacco. These mutants have depressed Rubisco levels and decreased rates of carbohydrate production. We found that antisense leaves are longer-lived than wild type leaves and that this appeared to be due to a prolongation of the senescence phase of development, as monitored by photosynthetic rates, chlorophyll content, and the abundance and activity of Rubisco. Declines in these parameters during leaf ontogeny in both the wild type and mutant plants were generally accompanied by coordinate reductions in the levels of rbcS mRNA and rbcL mRNA, as well as by reductions in chloroplast rRNA, chloroplast DNA and total protein. We suggest that the prolongation of senescence in the antisense leaves is due to an impact of source strength on leaf developmental programming that occurs, at least in part, at the level of transcript abundance of nuclear and chloroplast genes for chloroplast rRNAs and proteins. We hypothesize that plants are capable of sensing a range of source strength conditions to initiate and modulate leaf developmental programming.

Journal Article↗
PubMed2005

Transcript copy number estimation using a mouse whole-genome oligonucleotide microarray.

The ability to quantitatively measure the expression of all genes in a given tissue or cell with a single assay is an exciting promise of gene-expression profiling technology. An in situ-synthesized 60-mer oligonucleotide microarray designed to detect transcripts from all mouse genes was validated, as well as a set of exogenous RNA controls derived from the yeast genome (made freely available without restriction), which allow quantitative estimation of absolute endogenous transcript abundance.

Animals↗
PubMed2005

Discovery of immune-related genes expressed in hemocytes of the tarantula spider Acanthoscurria gomesiana.

The present study reports the identification of immune related transcripts from hemocytes of the spider Acanthoscurria gomesiana by high throughput sequencing of expressed sequence tags (ESTs). To generate ESTs from hemocytes, two cDNA libraries were prepared: one by directional cloning (primary) and the other by the normalization of the first (normalized). A total of 7584 clones were sequenced and the identical ESTs were clustered, resulting in 3723 assembled sequences (AS). At least 20% of these sequences are putative novel genes. The automatic functional annotation of AS based on Gene Ontology revealed several abundant transcripts related to the following functional classes: hemocyanin, lectin, and structural constituents of ribosome and cytoskeleton. From this annotation, 73 transcripts possibly involved in immune response were also identified, suggesting the existence of several molecular processes not previously described for spiders, such as: pathogen recognition, coagulation, complement activation, cell adhesion and intracellular signaling pathway for the activation of cellular defenses.

Amino Acid Sequence↗
PubMed1994

Reduced hepatic growth hormone (GH) receptor gene expression and increased plasma GH binding protein in experimental uremia.

In uremia, reduced longitudinal growth and decreased hepatic insulin-like growth factor-I (IGF-I) secretion despite elevated GH serum levels point to an insensitivity to the action of GH. The molecular basis that accounts for this insensitivity could comprise decreased GH receptor expression in the target organs for GH or binding of GH in the circulation to substances that compete with the receptor. To address this hypothesis, the abundance of hepatic GH receptor mRNA was measured by solution hybridization RNase protection assay in uremic female Sprague-Dawley rats, following two-stage 5/6 nephrectomy, and in pair-fed and in ad libitum-fed sham-operated controls; rat GH binding protein (GHBP) plasma concentration was measured by a sensitive direct RIA. Uremia was associated with a 50% decrease of hepatic GH receptor expression compared to pair-fed controls, which themselves showed a 25% reduction of hepatic GH receptor mRNA abundance when compared to ad libitum-fed controls. Plasma GHBP levels in uremia were markedly higher than in both control groups. Treatment with recombinant human GH (rhGH) (10 IU/kg body wt per day s.c. for 10 days) led to a comparable induction of IGF-I plasma levels and weight gain in uremia and pair-fed controls, indicating that the insensitivity to GH in uremia can be overcome by large rhGH doses. Subcutaneous rhGH injections did not significantly alter the hepatic GH receptor transcript abundance or plasma GHBP levels in any of the groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Spatial and temporal pattern of cellular myc oncogene expression in developing human placenta: implications for embryonic cell proliferation.

We have analyzed staged human placentas by Northern, dot blot, and in situ hybridization to human c-myc probes. Placental RNA exhibits a stage-specific appearance of a 2.4 kb transcript of the c-myc gene. The frequency of this transcript varies 20 to 30 fold over the course of placental development, showing a peak at 4-5 weeks after conception, where the myc transcripts comprise about 0.05% by weight of the total placental mRNA. A clear decline in placental c-myc transcription is seen before the end of the first trimester of pregnancy. In situ hybridization to 125I-labeled myc probes demonstrates an unequal spatial distribution of myc transcripts in placental with particularly high expression in the cytotrophoblastic shell of early placenta. Labeling of placental explants with 3H-thymidine, the localization of myc transcripts to cytotrophoblasts, and the temporal pattern of myc expression all support a strong correlation between myc transcript abundance and cytotrophoblast proliferation. We argue for a role for the c-myc gene in the proliferation of normal cells in this tissue.

Cell Division↗
PubMed2006

A molecular correlate to the Gleason grading system for prostate adenocarcinoma.

Adenocarcinomas of the prostate can be categorized into tumor grades based on the extent to which the cancers histologically resemble normal prostate glands. Because grades are surrogates of intrinsic tumor behavior, characterizing the molecular phenotype of grade is of potential clinical importance. To identify molecular alterations underlying prostate cancer grades, we used microdissection to obtain specific cohorts of cancer cells corresponding to the most common Gleason patterns (patterns 3, 4, and 5) from 29 radical prostatectomy samples. We paired each cancer sample with matched benign lumenal prostate epithelial cells and profiled transcript abundance levels by microarray analysis. We identified an 86-gene model capable of distinguishing low-grade (pattern 3) from high-grade (patterns 4 and 5) cancers. This model performed with 76% accuracy when applied to an independent set of 30 primary prostate carcinomas. Using tissue microarrays comprising >800 prostate samples, we confirmed a significant association between high levels of monoamine oxidase A expression and poorly differentiated cancers by immunohistochemistry. We also confirmed grade-associated levels of defender against death (DAD1) protein and HSD17 beta4 transcripts by immunohistochemistry and quantitative RT-PCR, respectively. The altered expression of these genes provides functional insights into grade-associated features of therapy resistance and tissue invasion. Furthermore, in identifying a profile of 86 genes that distinguish high- from low-grade carcinomas, we have generated a set of potential targets for modulating the development and progression of the lethal prostate cancer phenotype.

Adenocarcinoma↗
PubMed2006

Molecular characterization of a conserved archaeal copper resistance (cop) gene cluster and its copper-responsive regulator in Sulfolobus solfataricus P2.

Using a comparative genomics approach, a copper resistance gene cluster has been identified in multiple archaeal genomes. The cop cluster is predicted to encode a metallochaperone (CopM), a P-type copper-exporting ATPase (CopA) and a novel, archaea-specific transcriptional regulator (CopT) which might control the expression of the cop genes. Sequence analysis revealed that CopT has an N-terminal DNA-binding helix-turn-helix domain and a C-terminal TRASH domain; TRASH is a novel domain which has recently been proposed to be uniquely involved in metal-binding in sensors, transporters and trafficking proteins in prokaryotes. The present study describes the molecular characterization of the cop gene cluster in the thermoacidophilic crenarchaeon Sulfolobus solfataricus. The polycistronic copMA transcript was found to accumulate in response to growth-inhibiting copper concentrations, whereas copT transcript abundance appeared to be constitutive. DNA-binding assays revealed that CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the copMA gene cluster.

Amino Acid Sequence↗
PubMed2006

Within-tree transcriptome profiling in wood-forming tissues of a fast-growing Eucalyptus tree.

Despite the availability of high-throughput transcript profiling technology, little is known about tissue-specific gene expression patterns in the wood-forming tissues of Eucalyptus plantation tree species. We used cDNA-amplified fragment length polymorphism (AFLP) analysis in combination with infrared fragment detection and semi-automated band quantification to profile gene expression in a 6-year-old, fast- growing Eucalyptus tree. The expression profiles of 6385 transcript-derived fragments (TDFs) were analyzed across four major woody tissues (mature xylem, immature xylem, phloem and cork) collected from two stem positions, to provide a global view of transcript abundance and variability in the Eucalyptus stem. About 21% of the TDFs were differentially expressed and could be grouped into clusters representing co- expressed genes. A total of 71 TDFs representing different gene clusters were isolated and characterized. These included genes implicated in cell fate, signal transduction and cell wall biosynthesis, processes closely associated with xylogenesis. Analysis of the expression levels of selected TDFs by quantitative RT-PCR corroborated the TDF quantification and confirmed that cDNA-AFLP analysis is a highly efficient and accurate tool for transcript profiling and gene discovery in wood-forming tissues of tree species.

Cluster Analysis↗