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At least 667 records · Page 37Linked to original sources
PubMed1987

Atrial natriuretic polypeptide (ANP) in human ventricle. Increased gene expression of ANP in dilated cardiomyopathy.

Tissue levels of atrial natriuretic polypeptide (ANP) messenger RNA (ANPmRNA) and ANP in the human atrium and ventricle were measured simultaneously by the blot hybridization technique and the specific radioimmunoassay for ANP. Hearts were obtained from two patients without cardiac complications and from a patient with dilated cardiomyopathy (DCM) at autopsy. Total RNA extracted from ventricles contained a hybridizing RNA band of the same size as atrial ANPmRNA in both control and DCM hearts. The ANPmRNA level in the control ventricle was 0.2% of that in the atrium. The ANPmRNA level in the DCM ventricle increased to about 7% of that in the corresponding atrium and was approximately 40 times higher than that in the control ventricle, although the ANPmRNA level in the DCM atrium was comparable to that in the control atrium. The total content of ANPmRNA in the DCM ventricle reached about 30% of that in the corresponding atrium and was much the same as that in the control atrium. The ANP level in the DCM ventricle was approximately 1.0 microgram/g and much higher than that in the control ventricle (0.02 microgram/g).

Atrial Natriuretic Factor↗
PubMed1993

Human IM-9 lymphoblasts as a model of the growth hormone-insulin-like growth factor axis: gene expression, and interactions of ligands with receptors and binding proteins.

Human IM-9 lymphoblasts bind growth hormone (hGH) and insulin-like growth factors (IGFs). We have systematically examined the IM-9 cells as a valuable model of the interaction of hGH and the IGFs at the cellular level. Cells were cultured in medium with 10% serum and for a subset of experiments cultured in serum-free medium. Binding of [125I]hGH and [125I]IGF-I and -II to intact IM-9 cells was measured: unlabeled hGH inhibited binding of [125I]hGH (half max. 20 ng/ml). Binding of [125I]IGF-I was inhibited by IGF-I (half max. 7.5 ng/ml), IGF-II (half max. 60 ng/ml), and insulin and anti IGF-I receptor antibody (alpha IR3). [125I]IGF-II was inhibited by IGF-II (half max. 15 ng/ml), IGF-I (half max. 500 ng/ml), insulin (half max. 250 ng/ml) but not by alpha IR3. Crosslinking experiments with [125I]IGF-II and DSS as the crosslinking agent and analysis of radioligand-receptor complexes by SDS-PAGE under reducing conditions revealed that [125I]IGF-II bound to a 250 kDa and a 135 kDa receptor species. The latter possibly represents an insulin-type receptor whereas the 250 kDa species had the characteristics of the IGF-II/M6P receptor. When IM-9 cell conditioned medium was analyzed in ligand blotting experiments with either [125I]IGF-I or -II a 30 kDa IGFBP species was detected on the autoradiographs. Also, IGF-II immunoreactivity (approx. 1 ng/ml medium) was measured in the cell conditioned medium using an IGF-BP blocked RIA employing [125I]IGF-II. In a subset of experiments IM-9 cells were homogenized in 4 M guanidinium-thiocyanate and RNA extracted in 5.7 M CsCl. Denatured RNA was electrophoresed on 0.8% agarose gels and transferred to a nylon membrane, fixed and the blots hybridized with cDNA probes. Probes were labeled with [32P]dCTP using a random prime labeling procedure: a Pst I 700 bp fragment of the human IGF-I cDNA, a 554 bp Pst I-Sal I fragment of the IGF-II cDNA, a 614 bp Pst I fragment of the IGF-I receptor cDNA and a 663 bp Pst I fragment of the IGF-II/M6P receptor. Autoradiographs of Northern blots showed specific hybridization with the IGF-I probe at 3.7 kb and with the IGF-II probe at 5.3 kb. No signal was detected with the IGF-I receptor cDNA probe. Hybridization with the IGF-II/M6P receptor probe yielded a 9 kb RNA species.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding, Competitive↗
PubMed1988

Atrial natriuretic peptide gene expression and its secretion by pneumocytes derived from neonatal rat lungs.

Using a primary culture of pneumocytes derived from neonatal rat lungs, we investigated the synthesis and secretion at transcriptional and peptide levels of pulmonary rat(r) atrial natriuretic peptide (ANP). Total RNA extracted from pneumocytes contained a hybridizing RNA band of the same size as atrial rANP mRNA. Immunoreactive (IR)-rANP content in pneumocytes was 0.5% of that in atrial myocytes, and 8.6% of that in ventricular myocytes, while the secretory rate from pneumocytes was about 7% of atrial and ventricular myocytes. Triiodothyronine (T3, 5 x 10(-10) to 5 x 10(-8) M), dexamethasone and testosterone (5 x 10(-9) to 5 x 10(-8) M) significantly stimulated the synthesis of IR-rANP by pneumocytes in a dose-dependent manner. However, the stimulatory effect exerted by T3 on rANP synthesis, unlike in the case of cardiocytes, was much more potent than that of dexamethasone, as evidenced by the significant difference in potency at both transcriptional and peptide levels. The present study suggests that ANP secreted from lungs may at least in part contribute to circulating ANP pool, and that the tissue-dependent difference of sensitivity to thyroid hormone may play an important role in the regulation of developmental ANP gene expression in mammalian lungs.

Animals↗
PubMedDate not supplied

Human placental norepinephrine transporter mRNA: expression and correlation with fetal condition at birth.

The purpose of this study was to determine the primary form of human placental norepinephrine transporter (hNET) mRNA expressed in the human placenta and to compare the level of expression in normal pregnancies and in pregnancies complicated by drug exposure or other forms of physiological derangement. We used the hNET cDNA to measure RNA extracted from placenta and examined placental RNA following complicated and uncomplicated pregnancies. To compare transporter expression and its relation to fetal condition at birth, umbilical arterial plasma catecholamine levels, umbilical arterial blood gases and placental transporter mRNA level were compared by linear regression analysis. Uncomplicated pregnancies had a higher level of placental norepinephrine transporter mRNA than complicated pregnancies. An inverse relationship between umbilical cord norepinephrine level and transporter expression was demonstrated. We conclude that placental transporter expression represents an important and newly described metabolic function of the placenta. Placental catecholamine clearance mediated via the placental NET may be important in the pathophysiology of disorders associated with placental dysfunction, impaired placental blood flow or intrauterine growth retardation. This may also explain the adverse effects of drugs, such as cocaine, which block catecholamine transport.

Apgar Score↗
PubMed1989

Tobacco mosaic virus RNA enters chloroplasts in vivo.

Several lines of evidence are presented to allow us to conclude that tobacco mosaic virus (TMV) RNA enters the chloroplast in vivo. Chloroplasts were prepared from either directly inoculated or systemically infected leaves of tobacco plants inoculated with one of several strains of the virus and from uninfected control plants. Intact chloroplasts were isolated on Percoll gradients and treated with pancreatic RNase and thermolysin to destroy potential TMV virions and RNA on the outside or bound to their surfaces. Northern blot analysis of RNA extracted from these chloroplasts demonstrated that full-length TMV RNA was present within the chloroplasts prepared from both directly inoculated and systemically invaded leaves. Only genomic length, but not subgenomic length, RNA was found in the chloroplast extracts, indicating a selectivity of the transport of the viral RNA into the chloroplast. A temperature-sensitive TMV mutant (Ts 38), in which no virions are formed at 35 degrees C, was used to demonstrate that at that restrictive temperature viral RNA is detected in the chloroplast, indicating that free viral RNA can enter the chloroplast rather than intact virions. To our knowledge, the transport of a foreign RNA species into chloroplasts has not been reported previously.

Journal Article↗
PubMed1978

The relationship between the activities of different pools of RNA polymerases I and II during PHA-stimulation of human lymphocytes.

Following PHA-stimulation of lymphocytes in culture, it is known that nuclear RNA synthesis and the amount of extractable RNA polymerase activity rise in these cells. The relationship between these two phenomena has been examined. Using an in vitro assay system which discriminates between polymerase activity which is "engaged" in nuclear RNA synthesis and a pool of "free" enzyme, the data suggest that the factors regulating the interaction between these two pools of enzyme activity are different for forms I and II RNA polymerases.

Adult↗
PubMed1978

Characterization of an RNA-dependent RNA polymerase activity associated with measles virus.

An RNA-dependent RNA polymerase activity has been found copurifying with measles virus infectivity and complement-fixing antigen in three Vero cell-grown variants of measles virus: the attenuated Edmonston B strain, the natural non-attenuated Edmonston strain, and a subacute sclerosing panencephalitis isolate, IP-3. Incubation of purified measles virions with immunoglobulin G derived from sera of monkeys hyperimmunized against measles specifically removes activity sedimenting in the density region of measles virions. The requirements of the reaction, which is RNase sensitive, are similar to those reported for other paramyxovirus-associated activities, including detergent, divalent cation, ribonucleoside triphosphates, and a reducing agent. The size classes of RNA synthesized correspond to those found in measles-infected cells, including 50, 35, and 16 to 20S. The product RNA of the Edmonston B virus-stimulated reaction was rendered RNase resistant by annealing with RNA extracted from purified Edmonston B virions. RNA from uninfected Vero cells was ineffective in the annealing reaction.

Antigen-Antibody Reactions↗
PubMed2001

Stimulation of intracellular free calcium in GH3 cells by gamma3-melanocyte-stimulating hormone. Involvement of a novel melanocortin receptor?

The melanocortin (MC) gamma3MSH is a peptide that can be generated from the N-terminal domain of POMC and is believed to signal through the MC3 receptor. We recently showed that it induces a sustained rise in intracellular free calcium levels ([Ca(2+)](i)) in a subpopulation of pituitary cells, particularly in the lactosomatotroph lineage. In the present study we report that gamma3MSH and some analogs increase [Ca(2+)](i) in the GH- and PRL-secreting GH3 cell line and evaluate on the basis of pharmacological experiments and gene expression studies which MC receptor may be involved. A dose as low as 1 pM gamma3MSH induced an oscillating [Ca(2+)](i) increase in a significant percentage of GH3 cells. Increasing the dose recruited an increasing number of responding cells; a maximum was reached at 0.1 nM. gamma2MSH, alphaMSH, and NDP-alphaMSH displayed a similar effect. SHU9119, an MC3 and MC4 receptor antagonist, and an MC5 receptor agonist, did not affect the number of cells showing a [Ca(2+)](i) rise in response to gamma3MSH. SHU9119 had also no effect when added alone. MTII, a potent synthetic agonist of the MC3, MC4, and MC5 receptor as well as an N-terminally extended recombinant analog of gamma3MSH showed low potency in increasing [Ca(2+)](i) in GH3 cells, but high potency in stimulating cAMP accumulation in HEK 293 cells stably transfected with the MC3 receptor. In contrast, a peptide corresponding to the gamma2MSH sequence of POMC-A of Acipenser transmontanus increased [Ca(2+)](i) in GH3 cells, but was about 50 times less potent than gamma2- or gamma3MSH in stimulating cAMP accumulation in the MC3 receptor expressing HEK 293 cells. By means of RT-PCR performed on a RNA extract from GH3 cells, the messenger RNA of the MC2, MC3, and MC4 receptor was undetectable, but messenger RNA of the MC5 receptor was clearly present. These data suggest that the GH3 cell line does not mediate the effect of gamma3MSH through the MC3 receptor. The involvement of the MC5 receptor is unlikely, but cannot definitely be excluded. The findings animate the hypothesis that there exists a second, hitherto unidentified, MC receptor that displays high affinity for gamma3MSH.

Adrenal Glands↗
PubMed1988

Novel electrophoretic approach to study of poly(A)+RNA pool heterogeneity in different areas of human postmortem brain.

A simple method for fast fractionation of human postmortem brain total poly(A)+RNA has been developed. Guanidine thiocyanate extractable RNA was directly applied on the membranes of cellulose nitrate with immobilized oligo(dT) chains. After the electrophoresis in 35 mM tris-acetete buffer (pH 7.90) containing 10 mM EDTA and 35% formamide, the membranes were washed with the same buffer for removal of nonbinding poly(A)-lacking RNAs. Then, poly(A)+RNA fractions were selectively stained by 3,5-dimethylphenol in the presence of FeCl3 at pH 2.35. Finally, the resulting electrophorograms were scanned at 500 nm for quantitative evaluation of the data.

Brain↗
PubMed2003

[Aspiration biopsy RNA diagnosis].

Recent advances in molecular-based techniques, especially RNA analyses such as differential display, serial analysis of gene expression(SAGE) and DNA arrays, have enabled us to detect the specific changes in mRNA in cancer tissues rapidly and easily. We have established a new method of preoperative molecular-based diagnosis of thyroid carcinomas, one of the most common cancers in females. This technique, Aspiration Biopsy RNA Diagnosis, ABRD, allows us to perform preoperative RNA analysis of the tumors by extracting RNA from tumor cells obtained by fine needle aspiration biopsies(FNABs). For example, by ABRD detection of oncofetal fibronectin or calcitonin mRNA in FNABs, three types of thyroid carcinomas, papillary, anaplastic and medullary carcinomas, were accurately diagnosed preoperatively without the help of cytological or pathological examinations by a skillful pathologist. Further, by real-time monitoring RT-PCR measuring the expression levels of these cancer-specific mRNAs, a fully automated system was established. Because ABRD can be performed without any severe invasion to the patients, in the near future, when more reliable systems of quantitative RNA analysis are developed, ABRD will probably become one of the standard tests for preoperative diagnosis of cancer.

Biopsy, Needle↗
PubMed2002

[Introduction of a simple and effective method for plant total RNA isolation].

Wheat leaf and seeds at different development stages had been squashed in liquid nitrogen, then lysed by urea buffer which contains 0.1% SDS and 0.1% LDS, denatured protein had been removed by NaAc and chloroform precipitation, total RNA was further purified by LiCl. The RNA we obtained had sharp bands of 28S and 18S after agarose gel electrophoresis, 23S and 16S RNA bands can also be seen clearly in leaf RNA extract, the value of OD260/OD280 of RNA was 2.05-2.105 mg RNA can been isolated from 10 g leaf of wheat. This method can also been used in high molecular weight DNA isolation but the concentration of SDS and LDS must be increased to 1%.

English Abstract↗
PubMed1990

Detection of Philadelphia chromosome-positive cells from glass slide smears using the polymerase chain reaction.

Southern and Northern blot hybridization studies and the polymerase chain reaction (PCR) have been used to analyze the bcr-abl gene complex in chronic myelogenous leukemia (CML). Because fresh or cryopreserved cells may not always be available for molecular analyses, we investigated the possibility of using routinely prepared glass slide smears of blood or bone marrow as our source of cellular material. Cellular RNA was prepared directly from the blood or bone marrow smears using a modified RNA extraction procedure. cDNA was synthesized from RNA and amplified with PCR using bcr and abl-specific primers. Using this procedure, the bcr-abl fusion gene was detected by PCR in 21 of 21 patients with CML. Three patients who had undergone allogenic bone marrow transplantation (BMT) for CML were also studied by PCR. bcr-abl was identified transiently in one patient, persisted in one patient after BMT for 2 years until relapse occurred, and was absent in one patient to 18 months after BMT. We have shown that PCR can detect the bcr-abl gene of CML using material from glass-slide smears. This technique may be useful as a general approach in evaluating archival hematologic specimens for the expression of critical gene products.

Bone Marrow Transplantation↗
PubMed1993

An improved strategy and a useful housekeeping gene for RNA analysis from formalin-fixed, paraffin-embedded tissues by PCR.

Specific amplification of nucleic acid sequences by PCR has been extensively used for the detection of gene rearrangements and gene expression. Although successful amplification of DNA sequences has been carried out with DNA prepared from formalin-fixed, paraffin-embedded (FFPE) tissues, there are only a few reports regarding RNA analysis in this kind of material. We describe a procedure for RNA extraction from different types of FFPE tissues, involving digestion with proteinase K followed by guanidinium-thiocyanate acid phenol extraction and DNase I digestion. These RNA preparations are suitable for PCR analysis of mRNA and even of intronless genes. Furthermore, the universally expressed porphobilinogen deaminase mRNA proved to be useful as a positive control because of the lack of pseudogenes.

Base Sequence↗
PubMed1995

Noninvasive detection of putative biomarkers for colon cancer using fecal messenger RNA.

Deaths from colon cancer number over 60,000 each year in the United States. Because early detection results in a high cure rate, development of noninvasive techniques for detection of colon cancer has received much interest. The ability to detect early changes in colonocyte genes and gene expression would provide valuable information. We have shown previously that alterations in protein kinase C (PKC) isoform expression are associated with changes in colonic cell proliferation, a key intermediate marker for the prediction of tumorigenesis. Here, we describe a method for the quantitative detection of mRNAs for select PKC isoforms isolated from rat feces containing exfoliated colonocytes. After total RNA extraction from fresh fecal material, polyadenylated RNA was selectively purified and quantitated with slot blotting and hybridization to oligodeoxythymidylic acid. Fecal polyadenylated RNA was used for semiquantitative (mimic) RT-PCR to quantitate PKC isoform mRNA expression. We detected mRNA for PKC-alpha, PKC-delta, PKC-epsilon, and PKC-sigma, but not for PKC-beta or PKC-gamma, which is consistent with the profile of isoforms detected previously in scraped colonic mucosa using immunoblot analysis. This noninvasive method, utilizing feces containing exfoliated colonocytes, is a sensitive noninvasive technique for quantitating luminal mRNAs. This provides a means to monitor gene expression of colonic epithelial cells, which may have predictive value in monitoring the neoplastic process.

Animals↗
PubMedDate not supplied

Quantitative analysis of rat thyroglobulin messenger RNA in FRTL-5 cells by competitive polymerase chain reaction with human thyroglobulin messenger RNA.

To measure relative expression level of mRNA in a small number of cultured rat thyroid cells (FRTL-5), we developed a system of a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. Human thyroglobulin mRNA in total RNA extracted from a human thyroid tissue was used as an internal control. FRTL-5 cells in a 24 well dish were lysed with denaturing solution containing human RNA. Total RNA was extracted followed by reverse transcription and polymerase chain reaction. After digestion with a restriction enzyme, PCR products were separated by electrophoresis and stained with Sybr Green I, then their fluorescence was measured with fluorescent image analyser. Increase of thyroglobulin mRNA in FRTL-5 cells stimulated by thyroid stimulating hormone (TSH) was observed by this technique. Because this method does not require a large number of cells or radioactive isotopes, it is as useful for the analysis of the relative expression level of mRNAs in the cells as the conservative methods such as Northern Blot.

Animals↗
PubMed2005

Comparison of 16S rRNA and 16S rDNA T-RFLP approaches to study bacterial communities in soil microcosms treated with chromate as perturbing agent.

Transcripts of ribosomal RNA have been used for assessing the structure and dynamics of active bacterial populations; however, it remains unclear whether the information provided by community profiling derived from RNA is different from that derived from DNA, particularly when a selective pressure is applied on the bacterial community. In the present work, terminal-restriction fragment length polymorphism (T-RFLP) community profiles based on DNA and RNA extracted from soil microcosms treated with a toxic concentration of chromate were compared. Microcosms of a nonpolluted agricultural soil and of a heavy-metal-rich soil (serpentine) were treated with chromate and DNA and RNA were extracted. T-RFLP analysis was performed on amplified and retro-amplified 16SrRNA gene sequences, and band profiles obtained from samples of DNA and of RNA were compared. Some of the T-RFLP bands, identified as peculiar peaks in the profiles, were cloned and sequenced for taxonomic interpretation. Results indicated that: (1) community profiles derived from RNA and DNA were partly overlapping; (2) there was a strong correlation between the dynamics shown by RNA- and DNA-based T-RFLP profiles; (3) chromate addition exerted a clear effect on both agricultural and serpentine soil bacterial communities, either at the DNA and at the RNA level; however, the profiles derived from RNA showed sharper differences between treated and control samples than that of DNA-based profiles.

Agriculture↗
PubMed2000

Evaluation of RNA isolation methods and reference genes for RT-PCR analyses of rare target RNA.

Reverse transcription polymerase chain reaction (RT-PCR) analysis is increasingly becoming part of the diagnostic and prognostic evaluation for hematologic and oncologic disorders. Currently, different RNA isolation methods are used in the diagnostic laboratories. No data are available on their suitability for sensitive detection of breakpoint cluster region-abelson (BCR-ABL) gene transcripts. We have extracted RNA from mononuclear cell (MNC) fractions and from lysed blood samples of 4 patients (1 with leukocytosis, 1 with chronic myelogeneous leukemia (CML) under interferon treatment, and 2 CML patients after bone marrow transplantation) with 3 RNA isolation reagents (TRIzol, RNAzol, FastTube reagent). RNA yield was slightly higher with RNAzol than with TRIzol as indicated by agarose gel electrophoresis and spectrophotometric measurement at 260 nm. The FastTube reagent was unsuitable for RNA isolation from MNC, and was not evaluated for lysed blood. Quantitative competitive RT-PCR amplification of the ABL gene showed comparable results for RNA isolated with RNAzol and TRIzol. In RNA samples extracted from lysed whole blood, the presence of amplifiable RNA/cDNA was confirmed by amplification of 4 selected reference genes (porphobilinogen deaminase (PBGD), ABL, the gene spanning the BCR on chromosome 22 and retinoic acid receptor alpha (RARA)) in a multiplex PCR. High quality, DNA-free RNA was obtained with RNAzol, and 1 BCR-ABL-positive (specific for translocation t [9; 221) cell among 2x10(4) normal cells was successfully detectable by single step RT-PCR. In RNA isolated with TRIzol, major contaminations with genomic DNA were observed which significantly impaired the interpretation of the results of RT-PCR analysis.

Base Sequence↗
PubMed2001

Preparations of a RNA-lipid complex film and its physical properties.

We extracted RNA (aver. 70-80 bases) from yeasts and prepared a RNA-lipid complex by replacing sodium counter cations with cationic amphiphiles (2C12N+2C1). The RNA-lipid complex was soluble only in organic solvents and formed a self-standing film by casting from organic solutions. The RNA film was physically strong, although its molecular weight is small and the RNA molecules are oriented in one direction by stretching the film, in which RNAs form intermolecular hydrogen bonds and show the physical strength of the film.

DNA↗