Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Differentiation choices in the haemopoietic and nervous systems are controlled in part by instructive factors. The cholinergic differentiation factor (CDF, also known as leukaemia inhibitory factor, LIF) affects the development of cultured cells from both systems. To understand the role of CDF/LIF during normal development in vivo, we have begun to localize its mRNA in the late fetal and postnatal rat. Application of reverse transcriptase-polymerase chain reaction and RNase protection methods reveals that CDF/LIF mRNA levels are developmentally modulated in both haemopoietic and neural tissues. A target tissue of cholinergic sympathetic neurons, the footpads that contain the sweat glands, express high levels of this mRNA (relative to mRNA for actin and beta 2-microglobulin). Levels in targets of noradrenergic neurons are lower, but do undergo significant changes during development. Signals are also detected in selective regions of the adult brain, and in embryonic skeletal muscle. This finding in muscle may be significant for motor neurons, because CDF/LIF is a trophic factor for these neurons in culture. Embryonic liver, neonatal thymus and postnatal spleen express CDF/LIF mRNA, and expression in gut is the highest of all tissues examined. The selective tissue distribution and developmental modulation of CDF/LIF mRNA expression support a role for this factor in the normal development of several organ systems.
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PMID:Further studies of the distribution of CDF/LIF mRNA. 142 9

We have examined the expression of mRNAs encoding five major neurotransmitter-synthesizing enzymes in MAH cells, a clonal cell line derived by retroviral immortalization of a rat embryonic sympathoadrenal progenitor cell. These mRNAs include tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), tryptophan hydroxylase (TpH), and glutamic acid decarboxylases (GADs) 1 and 2. We find that MAH cells express high levels of TH mRNA and low levels of ChAT and TpH mRNAs. Neither GAD1 nor GAD2 mRNAs are detectable using an RNase protection assay with a detection limit of less than one transcript per cell. A similar pattern of mRNA expression is observed in postnatal superior cervical ganglia, adrenal medulla, and in PC12 cells. Transmitter synthesis and accumulation assays indicate that MAH cells can synthesize both catecholamines and acetylcholine. Thus the TH and ChAT mRNAs detected in these cells are likely to be translated into active enzyme. To corroborate these data obtained using MAH cells, we performed similar transmitter synthesis and accumulation assays on sympathoadrenal progenitors directly isolated from E14.5 fetal adrenal glands by fluorescence-activated cell sorting. These progenitor cells also synthesize and accumulate both catecholamines and acetylcholine, albeit to different extents than MAH cells. Both MAH cells and their nonimmortal counterparts are able to increase slightly their cholinergic function upon short-term exposure to CDF/LIF, a factor known to induce acetylcholine synthesis in postmitotic sympathetic neurons. Taken together, these data suggest that progenitor cells in the sympathoadrenal lineage acquire the ability to simultaneously transcribe several different neurotransmitter enzyme genes early in development, prior to their choice of final cell fate. At the same time, the progenitors possess receptors which regulate expression of these genes in response to environmental factors. This ability may permit the cells to choose from several different transmitter phenotypes in response to different environments, as they migrate through the embryo. The persistent transcription of these genes in adult cells, moreover, may in part account for the phenotypic plasticity of cells in this lineage.
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PMID:Co-expression of multiple neurotransmitter enzyme genes in normal and immortalized sympathoadrenal progenitor cells. 168 90

Differentiation inhibiting activity/leukemia inhibitory factor (DIA/LIF) is a glycoprotein that controls differentiation of pluripotential stem cells. Alternative transcription generates both diffusible and matrix-associated forms of DIA/LIF. Transcriptional analysis using a sensitive ribonuclease protection assay revealed that the two messages are expressed independently, consistent with the proposition that the two forms of DIA/LIF have distinct biological roles. DIA/LIF expression was found to be activated early during differentiation of embryonic stem (ES) cells, providing a mechanism for feedback regulation of stem cell renewal. Expression of DIA/LIF by mesenchymal cells was shown to be controlled in a paracrine manner by polypeptide regulatory factors. Specific expression of the two forms of DIA/LIF was also demonstrated in the egg cylinder-stage mouse embryo. The combination of cell type-specific and signal-specific regulation enables very precise control over DIA/LIF expression and may represent an important component of the regulatory networks that govern stem cell proliferation and differentiation during mammalian development.
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PMID:Developmentally programmed induction of differentiation inhibiting activity and the control of stem cell populations. 170 81

Differentiation inhibiting activity/leukaemia inhibitory factor (DIA/LIF) is a pleiotropic cytokine which has been implicated in a variety of developmental and physiological processes in mammals due to its broad range of biological activities in vitro. A role in very early development is suggested by the requirement for DIA/LIF to support the self-renewal of cultured embryonic stem (ES) cells. Other data point to potential roles in the establishment and maintenance of primordial germ cells, in osteogenesis and in haematopoiesis, and possibly in neuronal specification. DIA/LIF may also act as a mediator of the hepatic acute phase response. In the present study the expression of DIA/LIF transcripts during murine development and in adult mice has been determined using a highly sensitive ribonuclease protection analysis. In contrast to previous reports, it is apparent that DIA/LIF transcripts are present at low levels in many adult mouse tissues. Higher levels of expression are observed in skin, lung, intestine, and uterus. Elevated amounts of mRNA are also found in certain foetal tissue during late gestation and neonatally. In earlier embryogenesis, however, DIA/LIF mRNA is produced primarily in extraembryonic tissues. The alternative transcripts which produce either soluble or matrix-associated DIA/LIF exhibit overlapping but non-identical patterns of expression, consistent with the proposition that the two isoforms may have distinct biological functions. These findings are suggestive of widespread roles for DIA/LIF in vivo and are discussed in the light of available data on the phenotype of homozygous DIA/LIF-deficient mice.
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PMID:Expression of alternative forms of differentiation inhibiting activity (DIA/LIF) during murine embryogenesis and in neonatal and adult tissues. 768 30

The neuropoietic cytokine cholinergic differentiation factor/leukemia inhibitory factor (CDF/LIF) acts as a trophic factor, enhancing neuronal survival, and as a differentiation factor, altering neuronal gene expression. There is also evidence that its plays a role in the response of adult neural tissue to injury. We have examined this possibility further in rats by analyzing changes in the levels of mRNAs for CDF/LIF and its two receptor subunits in response to peripheral nerve damage in culture and in vivo. Using a quantitative RNase protection assay, we find that CDF/LIF mRNA increases dramatically (176-fold) in adult, but not neonatal, sympathetic ganglia and in adult dorsal root ganglia and sciatic nerve after organ culture for 24 hr. This mRNA is clearly detectable by in situ hybridization only in the nonneuronal cells of these structures. When the sciatic nerve is transected in vivo, CDF/LIF mRNA increases significantly in the regions immediately proximal and distal to the lesion site. The mRNA for the ligand binding subunit of the CDF/LIF receptor complex decreases somewhat upon culture and nerve section. The dramatic rise in CDF/LIF mRNA after nerve injury is further evidence that this cytokine is involved in the response to damage, a function that overlaps with its postulated role in wounding or infection in several nonneural tissues.
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PMID:Major changes in the expression of the mRNAs for cholinergic differentiation factor/leukemia inhibitory factor and its receptor after injury to adult peripheral nerves and ganglia. 804 54

There has been a dramatic expansion of DNA sequence information compiled over the past several years for a variety of eukaryotic and prokaryotic genomes. Accompanying this increase in knowledge of genomic structure and organization has been a growing interest in studying the function of individual genes including regulation of their expression. A number of methods such as Northern blotting, ribonuclease protection assay, and hybridization arrays have been developed to analyze gene expression at the transcriptional (mRNA) level. Although quantitative estimates of mRNA transcripts can be obtained from each of these methods, oftentimes they lack sufficient sensitivity or the methodology is too costly or too labor-intensive to be applied to the analysis of a large number of samples. The most sensitive method for analyzing gene expression at the mRNA level involves the combination of reverse transcription and polymerase chain reaction (RT-PCR). However, in order to provide accurate quantitative estimates of gene expression, a rapid and efficient method is required for separation and detection of the double-stranded DNA (dsDNA) products of RT-PCR. Recent advances in capillary electrophoresis with laser-induced fluorescence detection (CE/LIF) have made this method suitable for the automated analysis of large numbers of RT-PCR samples. An overview of the application of CE/LIF to quantitative analysis of gene expression by RT-PCR is presented along with selected protocols and examples. Both relative-quantitative (RQ) and quantitative-competitive (QC) approaches to RT-PCR are discussed in conjunction with the use of CE/LIF for rapid and accurate quantitative analysis of PCR products.
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PMID:Quantitative analysis of gene expression by reverse transcription polymerase chain reaction and capillary electrophoresis with laser-induced fluorescence detection. 1198 56

The influence of seminal plasma on the mRNA expression of cytokines in human endometrial epithelial and stromal cells and the cytokine production of spermatozoa were investigated in vitro. Seminal plasma and spermatozoa were collected from healthy volunteers and were screened by enzyme-linked immunosorbent assay for cytokines. Epithelial and stromal cells from fertile women were cultured on matrigel or polystyrol and incubated with pooled seminal plasma or with transforming growth factor beta1 (TGF-beta1), interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF), which were found to be significantly concentrated in seminal plasma. Endometrial cytokine expression was analysed by RNase protection assay and supported by RT-PCR. Supernatants of highly purified spermatozoa did not contain detectable levels of IL-1beta, IL-6 and VEGF. Screening of seminal plasma revealed concentrations >10-fold above the serum level for TGF-beta1, IL-8 and VEGF. Incubation of epithelial cells with 0.1, 1 and 10% seminal plasma resulted in concentration-dependant stimulation of IL-1beta, IL-6 and LIF mRNA expression. Maximum stimulation was found in epithelial cells from tissue samples taken in the mid secretory phase. Epithelial mRNA expression of IL-1beta, IL-6 and LIF increased by stimulation with TGF-beta1 and IL-8, but not with VEGF. In conclusion, seminal plasma stimulates expression of pro-inflammatory cytokines in endometrial epithelial cells in vitro. This effect might at least in part be exerted by TGF-beta1 and IL-8, abundantly present in seminal plasma. The in-vivo physiological relevance of these in-vitro studies remains to be determined.
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PMID:Seminal plasma induces mRNA expression of IL-1beta, IL-6 and LIF in endometrial epithelial cells in vitro. 1461 40

Quantification of gene expression provides valuable information regarding the response of cells or tissue to stimuli and often is accomplished by monitoring the level of messenger RNA (mRNA) being transcribed for a particular protein. Although numerous methods are commonly used to monitor gene expression, including Northern blotting, real-time polymerase chain reaction, and RNase protection assay, each method has its own drawbacks and limitations. Capillary electrophoresis with laser-induced fluorescence (CE-LIF) can reduce protocol time, eliminate the need for radioactivity, and provide superior sensitivity and dynamic range for quantification of RNA. In addition, CE-LIF can be used to directly determine the amount of an RNA species present, something that is difficult and not normally accomplished using current methods. Gene expression is detected using a fluorescently labeled riboprobe specific for a given RNA species. This direct approach was validated by analyzing levels of 28S RNA and also used to determine the amount of discoidin domain receptor 2 mRNA in cardiac tissue.
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PMID:Direct quantification of gene expression using capillary electrophoresis with laser-induced fluorescence. 1711 23

A simple, sensitive, and rapid quantitative LC-MS/MS assay was designed for the simultaneous quantification of free and glycoprotein bound monosaccharides using a multiple reaction monitoring (MRM) approach. This study represents the first example of using LC-MS/MS methods to simultaneously quantify all common glycoprotein monosaccharides, including neutral and acidic monosaccharides. Sialic acids and reduced forms of neutral monosaccharides are efficiently separated using a porous graphitized carbon column. Neutral monosaccharide molecules are detected as their alditol acetate anion adducts [M + CH(3)CO(2)](-) using electrospray ionization in negative ion MRM mode, while sialic acids are detected as deprotonated ions [M - H](-). The new method exhibits very high sensitivity to carbohydrates with limits of detection as low as 1 pg for glucose, galactose, and mannose, and below 10 pg for other monosaccharides. The linearity of the described approach spans over three orders of magnitudes (pg to ng). The method effectively quantified monosaccharides originating from as little as 1 microg of fetuin, ribonuclease B, peroxidase, and alpha(1)-acid glycoprotein human (AGP) with results consistent with literature values and with independent CE-LIF measurements. The method is robust, rapid, and highly sensitive. It does not require derivatization or postcolumn addition of reagents.
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PMID:Multiple-reaction monitoring liquid chromatography mass spectrometry for monosaccharide compositional analysis of glycoproteins. 1931 80

Monolithic immobilized pH gradient (M-IPG) materials were prepared in microchannles by photoinitiated polymerization of acrylamide, glycidylmethacrylate and Bis, followed by the attachment of focused Ampholine onto the surface of porous monoliths via epoxide groups. With M-IPG materials as matrix, FITC-labeled ribonuclease B, myoglobin and alpha-casein were well separated by microchip isoelectric focusing (muCIEF) without carrier amphocytes (CAs) added in the buffer. Both chemical and pressure mobilization were applied to drive focused zones for LIF detection. Our experimental results showed that pressure mobilization was preferable with neglectable band broadening, and good peak shape and high detection sensitivity were obtained. All these results demonstrate that muCIEF with M-IPG materials is not only an efficient mode for protein enrichment and separation but also attractive to couple with other CE modes to achieve multi-dimensional separation or MS for further identification, without the interference of mobile CAs.
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PMID:Microchip isoelectric focusing with monolithic immobilized pH gradient materials for proteins separation. 1996 Apr 63


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