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At least 325 records · Page 18Linked to original sources
PubMed2005

Human-antigen R (HuR) expression in hypertension: downregulation of the mRNA stabilizing protein HuR in genetic hypertension.

In aged spontaneously hypertensive rats (SHR), vasorelaxant responses to NO are attenuated compared with normotensive control rats (Wistar-Kyoto [WKY]) because of a decreased expression of the important NO receptor soluble guanylyl cyclase (sGC). Because the expression of sGC subunits alpha1 and beta1 is controlled at the post-transcriptional level by the mRNA-binding protein human-antigen R (HuR), we now assessed whether or not altered expression of HuR could account for downregulation of sGCalpha1 and sGCbeta1 in genetic hypertension. The expression of HuR (and sGCalpha1 and sGCbeta1) in aortas from aged SHR was significantly decreased at the mRNA and protein level compared with age-matched WKY rats, whereas expression of HuR was not different in prehypertensive young (2 months of age) SHR and age-matched WKY rats. The mRNA-binding activity of HuR in native aortic protein extracts from aged SHR was markedly reduced compared with normotensive WKY rats, as detected by RNA electrophoretic mobility shift analysis, using biotin-labeled adenine and uracil (AU)-rich element (ARE)-containing RNA probes from the 3'-untranslated region of sGCalpha1 and sGCbeta1. In contrast, ARE-binding activity was not different between prehypertensive young SHR and young WKY rats. In vitro RNA degradation assays using the same AU-rich sGC mRNA probes revealed an accelerated sGCalpha1 and sGCbeta1 mRNA decay in the presence of native protein extract from hypertensive SHR, which was less rapid with aortic protein from normotensive WKY rats. These findings suggest that in this animal model of genetic hypertension, the reduced expression of sGC subunits is mediated by downregulation of the sGC mRNA-stabilizing protein HuR.

3' Untranslated Regions↗
PubMed1999

Salpalpha and Salpbeta, growth-arresting homologs of Sam68.

Sam68, a nuclear RNA-binding protein, is a major substrate of the Src tyrosine kinase in mitotic cells. In addition to a tyrosine-rich C-terminal region, Sam68 also has six poly-proline (SH3-binding) sites, many of which are located in an amino-terminal region. Sam68 appears to act as an adaptor protein, associating with many SH2- and SH3-containing signal-transducing proteins (Richard et al., Mol. Cell. Biol. 15:186-197, 1995). Here we describe a novel 55kDa protein, Salpalpha, which has sequence similarity to Sam68 throughout its length. Salpalpha lacks the amino-terminal region found in Sam68, and has only a single poly-proline site, which binds the SH3 domain of the p85 subunit of PI 3-kinase. Salpalpha is tyrosine-phosphorylated when expressed in Rous sarcoma virus-infected chicken embryo fibroblasts (RSV-CEF); unlike Sam68, however, Salpalpha does not co-precipitate with v-Src. Salpbeta, an alternatively spliced isoform lacking the C-terminal tyrosine-rich region, is also tyrosine-phosphorylated in RSV-CEF, and also binds the SH3 domain of p85. We further show that expression of either Salpalpha or Salpbeta down-regulates the expression of Sam68 in CEF, and arrests the growth of these cells. Our results suggest that Salp may function as a negative regulator of cell growth.

3T3 Cells↗
PubMed2005

RNA interference targeted to multiple P2X receptor subtypes attenuates zinc-induced calcium entry.

A postulated therapeutic avenue in cystic fibrosis (CF) is activation of Ca(2+)-dependent Cl(-) channels via stimulation of Ca(2+) entry from extracellular solutions independent of CFTR functional status. We have shown that extracellular zinc and ATP induce a sustained increase in cytosolic Ca(2+) in human airway epithelial cells that translates into stimulation of sustained secretory Cl(-) transport in non-CF and CF human and mouse airway epithelial cells, cell monolayers, and nasal mucosa. On the basis of these studies, the Ca(2+) entry channels most likely involved were P2X purinergic receptor channels. In the present study, molecular and biochemical data show coexpression of P2X(4), P2X(5), and P2X(6) subtypes in non-CF (16HBE14o(-)) and CF (IB3-1) human bronchial epithelial cells. Other P2X receptor Ca(2+) entry channel subtypes are expressed rarely or not at all in airway epithelia, epithelial cell models from other CF-relevant tissues, or vascular endothelia. Novel transient lipid transfection-mediated delivery of small interference RNA fragments specific to P2X(4) and P2X(6) (but not P2X(5)) into IB3-1 CF human airway epithelial cells inhibited extracellular zinc- and ATP-induced Ca(2+) entry markedly in fura-2 Ca(2+) measurements and "knocked down" protein by >65%. These data suggest that multiple P2X receptor Ca(2+) entry channel subtypes are expressed in airway epithelia. P2X(4) and P2X(6) may coassemble on the airway surface as targets for possible therapeutics for CF independent of CFTR genotype.

Animals↗
PubMed2004

Identification and partial characterization of a variant of human CXCR3 generated by posttranscriptional exon skipping.

Chemokines are recognized as functionally important in many pathological disorders, which has led to increased interest in mechanisms related to the regulation of chemokine receptor (CKR) expression. Known mechanisms for regulating CKR activity are changes in gene expression or posttranslational modifications. However, little is known about CKR with respect to a third regulatory mechanism, which is observed among other seven-transmembrane receptor subfamilies, the concept of differential splicing or processing of heteronuclear RNA. We now report on the discovery of a variant human CKR, CXCR3, resulting from alternative splicing via exon skipping. The observed RNA processing entails a drastically altered C-terminal protein sequence with a predicted four- or five-transmembrane domain structure, differing from all known functional CKR. However, our data indicate that that this splice variant, which we termed CXCR3-alt, despite its severe structural changes still localizes to the cell surface and mediates functional activity of CXCL11.

Alternative Splicing↗
PubMed2004

From silencing to gene expression: real-time analysis in single cells.

We have developed an inducible system to visualize gene expression at the levels of DNA, RNA and protein in living cells. The system is composed of a 200 copy transgene array integrated into a euchromatic region of chromosome 1 in human U2OS cells. The condensed array is heterochromatic as it is associated with HP1, histone H3 methylated at lysine 9, and several histone methyltransferases. Upon transcriptional induction, HP1alpha is depleted from the locus and the histone variant H3.3 is deposited suggesting that histone exchange is a mechanism through which heterochromatin is transformed into a transcriptionally active state. RNA levels at the transcription site increase immediately after the induction of transcription and the rate of synthesis slows over time. Using this system, we are able to correlate changes in chromatin structure with the progression of transcriptional activation allowing us to obtain a real-time integrative view of gene expression.

Animals↗
PubMed2003

Single molecule profiling of alternative pre-mRNA splicing.

Alternative pre-messenger RNA splicing is an important mechanism for generating protein diversity and may explain in part how mammalian complexity arises from a surprisingly small complement of genes. Here, we describe "digital polony exon profiling,"a single molecule-based technology for studying complex alternative pre-messenger RNA splicing. This technology allows researchers to monitor the combinatorial diversity of exon inclusion in individual transcripts. A minisequencing strategy provides single nucleotide resolution, and the digital nature of the technology allows quantitation of individual splicing variants. Digital polony exon profiling can be used to investigate the physiological and pathological roles of alternately spliced messenger RNAs, as well as the mechanisms by which these messenger RNAs are produced.

Acrylamide↗
PubMed2002

Experience-dependent coincident expression of the effector immediate-early genes arc and Homer 1a in hippocampal and neocortical neuronal networks.

The transcription of the immediate-early genes Arc and Homer 1a (H1a) is dynamically regulated in response to synaptic activity; their protein products function at the postsynaptic sites of excitatory synapses. Previous studies demonstrate a role for Arc in the maintenance of long-term potentiation and in memory consolidation processes and indicate a role for H1a in modifying glutamatergic signaling pathways. Using double-label fluorescence in situ hybridization, we demonstrate that Arc and H1a RNA expression is induced strongly in the same neurons of rat hippocampus and neocortex after exploration of a novel environment. These findings support the view that novel experience activates a cell-specific genomic program and that Arc and H1a may function in concert in the structural and functional modifications of dendrites that lead to long-term changes in synaptic efficacy.

3' Untranslated Regions↗
PubMed1999

Two homologous cytochrome b5s are expressed in both neurons and glial cells of the rat brain.

To avoid the possibility of blood contamination and of gene rearrangement during library construction we isolated total RNA directly from cultured rat embryonic neuronal cells and glioma C6 cells to be used as template for RT-PCR. By using specific primers for both membrane-bound b5 and soluble b5, DNA bands of appropriate size were clearly amplified indicating that both neurons and glial cells expressed b5s, although soluble b5 seemed to be less expressed in these cells. Nucleotide sequence of the internal exon for soluble b5 was reinvestigated and confirmed to be 58 bp containing genetic codons for His-Ser-Ala-Leu and stop.

Alternative Splicing↗
PubMed2001

Expression of the Pib rice-blast-resistance gene family is up-regulated by environmental conditions favouring infection and by chemical signals that trigger secondary plant defences.

The rice blast resistance gene Pib is a member of the nucleotide binding site (NBS) and leucine-rich repeat (LRR) class of plant disease resistance (R) genes and belongs to a small gene family. We describe here the isolation and characterization of a Pib homologue (PibH8), and extensive investigation of the expression of the Pib gene family (Pib, PibH8, HPibH8-1, HPibH8-2) under various environmental and chemical treatments. PibH8 shows 42% identity and 60% similarity to Pib and, like Pib, has a duplication of the kinase 1a, 2, and 3a motifs of the NBS region in the N-terminal half of the protein. Interestingly, genes of the Pib family exhibit a diurnal rhythm of expression. RNA gel blot analysis revealed that their expression was regulated dramatically by environmental signals. such as temperature, light and water availability. Their expression was also induced by chemical treatments, such as jasmonic acid, salicylic acid, ethylene and probenazole. Our findings suggest that expression of the Pib gene family is up-regulated by environmental conditions that would favour pathogen infection. This may reflect the evolution of anticipatory control of R gene expression.

Abscisic Acid↗
PubMed2001

Differential expression of cdc25 cell-cycle-activating phosphatases in human colorectal carcinoma.

cdc25 is a family of cell-cycle phosphatases that activate the cyclin-dependent kinases. cdc25A and B, but not C, have oncogenic potential in vitro. In this study, we analyzed the possible implication of cdc25 genes in the progression of colorectal tumors. RNA and DNA were extracted from 34 paired tumor and normal colorectal tissues and examined by Northern blot, RT-PCR, and Southern blot, respectively. Protein expression was analyzed by Western blot in a subset of normal and tumor samples. The expression levels were correlated with the clinicopathologic characteristics and survival of the patients. cdc25B mRNA was overexpressed in 19 carcinomas (56%). A significant correlation was observed between high cdc25B mRNA levels and the relapse-free, overall, and cancer-related survival of the patients. The cdc25B2 splicing variant was detected in 27 carcinomas (79%) but only in 9 normal samples (26%) and was associated with the grade of the differentiation of the tumors. cdc25A mRNA was overexpressed in four tumors (12%) and cdc25C1 mRNA was overexpressed in nine tumors (26%). A new cdc25C2 splicing variant lacking exon 4 and 5 was identified in all of the tumors and in 56% of the normal samples. No amplifications or gene rearrangements of these genes were detected. In conclusion, these findings indicate that cdc25 isoforms and splicing variants are differentially regulated in colorectal carcinomas and may participate in the development of these tumors. Additionally, the correlation between cdc25B mRNA levels and the survival of the patients also suggest that the cdc25B isoform may be involved in the progression of the disease.

Adult↗
PubMed2002

Genomic structure and embryonic expression of estrogen receptor beta a (ERbetaa) in zebrafish (Danio rerio).

Estrogenic steroid hormones mediate complex actions important in both embryonic and adult life. The hormones signal through ligand-inducible transcription factors known as estrogen receptors (ERs). In this study, we have isolated a zebrafish estrogen receptor with homology to human estrogen receptor beta (ERbeta). This zebrafish ERbeta (ERbetaa) has a conserved genomic structure of eight coding exons with boundaries similar to those of human ERbeta. The coding exon structures of two other zebrafish estrogen receptors (ERalpha and ERbetab) are presented as well. We also analyzed 3.3 kb of the promoter region and identified numerous putative transcription factor binding sites, including SP1 and ER half sites. Zebrafish ERbetaa message RNA is maternally loaded, but quickly degraded after fertilization, as detected by reverse transcriptase polymerase chain reaction. ERbetaa transcripts are detected again between 24 and 48 h post fertilization. These results indicate that ERbeta has been highly conserved during evolution and is likely used during later embryogenesis in zebrafish. Future identification of the expression levels and patterns of this and other estrogen receptors in zebrafish will allow a better understanding of estrogen signaling during embryogenesis.

Amino Acid Sequence↗
PubMed2000

Differential expression of p73 splice variants and protein in benign and malignant ovarian tumours.

The p73 gene encodes a protein with substantial structural and functional similarities to the tumour-suppressor p53. Alternative splicing of p73 mRNA leads to expression of 6 known RNA species and proteins (alpha, beta, gamma, delta, epsilon, zeta). We analysed the expression of these splice variants in ovarian adenocarcinoma by RT-PCR followed by detection of amplicons with the Southern technique and by immunoblot in 32 malignant and benign epithelial ovarian tumour specimens and 3 ovarian adenocarcinoma cell lines (A2780, 2008, OVCAR-3). p73alpha mRNA was expressed in all 17 ovarian cancer specimens, and 14 of 17 expressed at least 3 splice variants. In contrast, a different expression pattern was present in the ovarian adenomas: p73alpha was detected in 6 of 12 benign tumours, and only 1 adenoma expressed 3 splice variants. p73 protein was expressed in 9 of 16 ovarian cancer specimens, in all cell lines and in 1 of 3 borderline tumours. In contrast, none of 9 ovarian adenomas expressed detectable amounts of p73 protein. Expression of p73 mRNA and protein was not correlated with FIGO stage and histological grade, but we observed a significant correlation with over-expression of p53 protein. In summary, epithelial ovarian cancers express a more complex p73 isoform pattern and higher levels of p73 mRNA and protein than ovarian adenomas.

Adenocarcinoma↗
PubMed1996

Alternative RNA splicing-generated cardiac troponin T isoform switching: a non-heart-restricted genetic programming synchronized in developing cardiac and skeletal muscles.

An alternative RNA splicing-generated cardiac troponin T (cTnT) isoform switching has been found during both avian and mammalian heart development. The present study demonstrates that the mouse cTnT isoform switching is approximately 10 days earlier than that in rat during perinatal heart development. The mouse cTnT isoform switching is also approximately 10 days earlier than the transcriptionally regulated troponin I (TnI) isoform switching, a transition that showed similar timing during mouse and rat heart development. In addition, synchronized cTnT and TnI switches are observed during both rat and chicken heart development. More interestingly, the expression of cTnT in both mouse and rat neonatal skeletal muscles showed an isoform transition pattern synchronized with the cTnT isoform switching in the neonatal heart of the corresponding species. The results suggest that the alternative RNA splicing-generated cTnT isoform switching during development is (a) not a direct response to the functional changes in perinatal myocardium and (b) not a restricted regulation in the heart, but is controlled by a species-specific programming that is synchronized in developing cardiac and skeletal muscles.

Alternative Splicing↗
PubMed2002

Function of the transcriptional regulating protein of 132 kDa (TReP-132) on human P450scc gene expression.

Cytochrome P450scc catalyzes the important first step in the steroid synthesis pathway; however, it is clear that additional factors regulating the temporal and spacial specific expression of the CYP11A1 gene remain to be identified. To isolate novel transcription factors that regulate this gene, a cis-acting element of the 5'-flanking region from nucleotides -155 to -131 (-155/-131) was used to screen a human placental lambda gt11 cDNA expression library, and an interacting clone was isolated. The open reading frame of the cDNA encodes several domains that are characteristic of transcription factors including an acidic region, a region rich in prolines and three zinc-finger motifs. Expression of the cDNA by in vitro transcription/translation and by transient transfection in HeLa cells yielded a protein of 132 kDa, which concurs with the predicted size. Transfection of the cDNA in placental JEG-3 and adrenal NCI-H295 cells, stimulate expression of a reporter construct controlled by the P450scc gene 5'-flanking region from nucleotides -1676 to +49. This transcriptional regulating protein of 132kDa (TReP-132) when expressed in HeLa cells was demonstrated to interact with the -155/-131 region in bandshift analysis, and tandem copies of this region was shown to confer activation of the heterologous HSV thymidine kinase minimal promoter. Coexpression of CBP/p300 with TReP-132 further increased promoter activity, and the proteins were demonstrated to interact physically. RNA analysis demonstrated the highest levels of expression in the adrenal cortex and testis; and transcript expression is also found in the steroidogenic JEG-3, NCI-H295, and MCF-7 cell lines, but not in non-steroidogenic HepG2 and HK293 cells. Subsequently it has been shown that TReP-132 interacts with steroidogenic factor-1 (SF-1) through specific domains; and along with the interaction with CBP/p300 these factors are postulated to form a complex to regulate expression of the P450scc gene.

Cholesterol Side-Chain Cleavage Enzyme↗
PubMed2005

Nuclear receptor expression in fetal and pediatric liver: correlation with CYP3A expression.

The mechanisms underlying interindividual variation and developmental changes in cytochrome P450 3A (CYP3A) expression and activity are not fully understood. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) methods were used to detect, during human fetal and pediatric development, mRNA expression of nuclear receptors involved in the regulation of CYP3A genes. Quantitative RT-PCR was conducted on RNA extracted from prenatal (n = 60, 76 days to 32 weeks estimated gestational age) and pediatric (n = 20, 4 days to 18 years of age) liver tissue with primers for nuclear receptors implicated in regulating CYP3A gene expression. Pregnane X receptor (PXR) and constitutive androstane receptor (CAR) were expressed at low (and highly variable) levels in pre- and neonatal liver relative to liver tissue derived from older children. CAR was expressed at higher levels relative to PXR in prenatal liver (757 +/- 480 molecules CAR/ng of RNA versus 271 +/- 190 molecules PXR/ng of RNA after correction for 18S rRNA). In contrast, mRNA expression of the heterodimer partner RXRalpha was less variable (33-fold) and did not differ appreciably between pre- and postnatal liver samples (219 +/- 101 molecules/ng of RNA prenatal versus 253 +/- 232 molecules/ng of RNA postnatal). Expression of HNF4alpha1 mRNA was similar to that of RXRalpha. Log CYP3A7 mRNA expression was significantly correlated with PXR (r2 = 0.372) and CAR (r2 = 0.380) mRNA in fetal liver, but associations were weaker than those observed with CYP3A4 mRNA in postnatal liver (r2 = 0.610 and 0.723 for PXR and CAR, respectively). In conclusion, nuclear receptor mRNA expression demonstrates considerable interindividual variability in human fetal and pediatric liver and is significantly correlated with CYP3A expression.

Age Factors↗
PubMed2004

Assembly of splicing complexes on exon 11 of the human insulin receptor gene does not correlate with splicing efficiency in-vitro.

BACKGROUND: Incorporation of exon 11 of the insulin receptor gene is both developmentally and hormonally-regulated. Previously, we have shown the presence of enhancer and silencer elements that modulate the incorporation of the small 36-nucleotide exon. In this study, we investigated the role of inherent splice site strength in the alternative splicing decision and whether recognition of the splice sites is the major determinant of exon incorporation. RESULTS: We found that mutation of the flanking sub-optimal splice sites to consensus sequences caused the exon to be constitutively spliced in-vivo. These findings are consistent with the exon-definition model for splicing. In-vitro splicing of RNA templates containing exon 11 and portions of the upstream intron recapitulated the regulation seen in-vivo. Unexpectedly, we found that the splice sites are occupied and spliceosomal complex A was assembled on all templates in-vitro irrespective of splicing efficiency. CONCLUSION: These findings demonstrate that the exon-definition model explains alternative splicing of exon 11 in the IR gene in-vivo but not in-vitro. The in-vitro results suggest that the regulation occurs at a later step in spliceosome assembly on this exon.

Alternative Splicing↗
PubMed2004

Novel small GTPase subfamily capable of associating with tubulin is required for chromosome segregation.

The small GTPase superfamily, which includes the Ras, Rho/Rac, Rab, Arf and Ran subfamilies, serves as a signal transducer to regulate cell proliferation and differentiation, actin cytoskeleton, membrane trafficking, and nuclear transport. Here, we identify novel GTPases (human Gie1 and Gie2) that form a distinct subfamily of the small GTPases in terms of their sequences and intracellular function. Gie stands for 'novel GTPase indispensable for equal segregation of chromosomes', and this subfamily is conserved in multicellular organisms. Expression of dominant-negative Gie mutants in mammalian cells or knockdown of Gie transcripts using RNA interference in Drosophila S2 cells induced abnormal morphology in the chromosome segregation. Gie protein has ability to bind to tubulin and localizes with microtubules on the spindle mid-zone in late mitosis. Furthermore, overexpression of Gie mutants that lack putative effector domains but have tubulin-binding ability induced micronucleus formation. Thus, this is the first report showing that a small GTPase subfamily capable of associating with microtubules might be involved in chromosome segregation.

ADP-Ribosylation Factors↗
PubMed2003

Modulation of allergic response in nasal mucosa by antisense oligodeoxynucleotides for IL-4.

BACKGROUND: The cytokine IL-4 is highly involved in T(H)2 inflammation, such as that seen in allergic rhinitis. IL-4 can induce IgE synthesis and eotaxin. Recent studies have shown that stimulation of allergic nasal tissue with ragweed allergen can induce local IL-4 mRNA production. OBJECTIVE: We set out to determine whether IL-4 antisense phosphorothioate-modified oligodeoxynucleotides (PS-ODNs) could inhibit IL-4 production and downstream events of IL-4. METHODS: Nasal mucosa biopsy specimens were obtained from patients with seasonal ragweed allergic rhinitis out of season, incubated ex vivo with or without PS-ODNs, and challenged with ragweed. FITC-labeled oligonucleotides were used to determine tissue uptake. By using immunocytochemistry, IL-4-, IL-13-, eotaxin 1-, and IFN-gamma-producing cells were enumerated, and by using in situ hybridization, epsilon germline transcript RNA- and IL-4 mRNA-positive cells were examined. RESULTS: The antisense PS-ODN was taken up by the tissue, and preincubation of the tissue with the IL-4 antisense PS-ODN caused a decrease in allergen-induced IL-4 mRNA and a decrease in the amount of IL-4 immunoreactivity (n = 7, P <.001). PS-ODNs had inhibitory effects on allergen-induced epsilon germline transcript RNA expression (n = 7, P <.001) and mucosa eotaxin 1 immunoreactivity (n = 7, P <.05). In contrast, the PS-ODNs increased the amount of IFN-gamma immunoreactivity (n = 7, P <.05), suggesting a nonspecific mechanism for reduced synthesis of IgE and eotaxin. CONCLUSIONS: Our results show that the IL-4 antisense PS-ODN effectively inhibits IL-4, IgE synthesis, and eotaxin, principal mediators of allergic inflammation, suggesting that PS-ODNs might offer a possible topical treatment for allergic rhinitis.

Allergens↗