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At least 793 records · Page 44Linked to original sources
PubMed2004

Profiling and functional annotation of mRNA gene expression in pediatric rhabdomyosarcoma and Ewing's sarcoma.

Using Affymetrix oligonucleotide microarrays, we analyzed mRNA gene expression patterns of 12 primary pediatric rhabdomyosarcomas (RMS) and 11 Ewing's sarcomas (EWS), which belong to the small round blue cell tumors (SRBCTs). Diagnostic classification of these cancers is frequently complicated by the highly similar appearance in routine histology, and additional molecular markers could significantly improve tumor classification. A combination of three independent statistical approaches (t-test, SAM, k-nearest neighborhood analysis) resulted in 101 highly significant probe sets that clearly discriminate between EWS and RMS. We identified novel marker transcripts that have not been previously associated with either RMS or EWS yet, including CITED2, glypican 3 (GPC3), and cyclin D1 (CCND1). Expression levels for selected candidate genes were validated by quantitative real-time reverse-transcription PCR. Furthermore, to identify biologically meaningful trends, functional annotations were assigned to 946 genes differentially expressed between EWS and RMS (t-test). Genes involved in protein biosynthesis (n = 28) and complex assembly (n = 9), lipid metabolism (n = 23), energy generation (n = 22), and mRNA processing (n = 11) were expressed significantly higher in EWS. Thus, functional annotation of tumor-specific genes reveals detailed insights into tumor biology and differentiation-specific expression patterns and gives important clues related to the possible cellular origin of these pediatric tumors. Supplementary material for this article is available at the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.

Biomarkers, Tumor↗
PubMed1975

Pilot experiments with EB virus in owl monkeys (Aotus trivirgatus). III. Serological and biochemical findings in an animal with reticuloproliferative disease.

Nucleic acid hybridization with EB virus complementary RNA has demonstrated unequivocally that EB virus DNA is present in the cells of a continuous lymphoblastoid line established in culture from a pathological lymph node of an owl monkey with reticuloproliferative disease after inoculation with EB virus. There were 48 to 49 EB virus genome equivalents per cell. In addition, serological studies showed that the diseased owl monkey developed specific antibodies to EB virus capsid antigens. The antibodies were first detected by indirect immunofluorescence in serum sampled 13 weeks after inoculation, and were not found in sera from other animals without the disease. None of the monkeys developed heterophile antibodies. The significance of the biochemical and serological findings is discussed in relation to the nature of the reticuloproliferative disease and the possibility of tumour-induction by EB virus.

Animals↗
PubMed1977

Transformation of lymphocytes by Herpesvirus papio.

Cotton-topped (CT) or white-lipped (WL) marmoset lymphocytes were transformed in vitro with herpesvirus papio (HVP) into permanently growing lymphoblastoid cell lines (LCL). Five of 9 HVP-transformed CT cell lines contained cells with antigens reacting with antibodies to Epstein-Barr virus (EBV) capsid antigen (VCA) and/or to EBV-induced early antigens (EA). None of 12 WL LCL revealed such antigen-producing cells. Cells from both groups of cultures failed to react with antibodies to the EBV-specified nuclear antigen (EBNA). Exposure of baboon circulating lymphocytes to X-irradiated HVP or EBV-carring cells, or to suspensions of EBV resulted in establishment of LCL which all contained VCA and/or EA-positive, but no EBNA-positive cells. Nuclear antigens were undetectable also with anti-VCA-positive sera from baboons, chimpanzees, or other non-human primates. DNA-complementary RNA (cRNA) filter hybridization with EBV cRNA showed that with one exception transformed CT or WL marmoset cells contained at least 1-2 virus genome equivalents per cell, while at least 12-25 virus genome equivalents per cell were detected in transformed baboon cells. These data need confirmation by DNA-DNA reassociation kinetics.

Animals↗
PubMed1978

Somatic cell hybrids between human lymphoma cell lines. V. IdUrd inducibility and P3HR-1 superinfectability of Daudi/HeLa (DAD) and Daudi/P3HR-1 (DIP-1) cell lines.

We have studied two types of somatic cell hybrid with regard to expression of the Epstein-Barr virus (EBV) cycle and its regulation. The first, DIP-1, a hybrid formed between two human lymphoma EBV producers (Daudi and P3HR-1), contained EBV DNA, expressed the virus-determined nuclear antigen (EBNA), andwas a producer of the EBV-associated antigens EA (early antigen) and VCA (viral capsid antigen). The second, DAD, a hybrid series of clones formed between Daudi and a HeLa cell derivative (D98), differed with regard to the expression of EBNA, EA, VCA and the content of EBV DNA. EA was regularly induced in the EBV DNA-containing hybrids following treatment with iododeoxyuridine (IdUrd). This induction was greater in lines spontaneously expressing EA. In two hybrids, DIP-1 and DAD10, VCA and virus DNA synthesis were also induced in the presence of IdUrd, the latter being detected by in situ hybridization with P3HR-1 EBV complementary RNA. Finally, while DIP-1 was superinfectable by the P3HR-1 EBV strain, the DAD series of hybrids were refractory to P3HR-1 superinfection and lacked EBV receptors.

Antigens, Viral↗
PubMed1979

Herpesvirus papio: state and properties of intracellular viral DNA in baboon lymphoblastoid cell lines.

Herpesvirus papio (HVP) is an indigenous B-lymphotropic virus of baboons (Papio sp.) present in latent form in baboon lymphoblastoid cell lines. It shares cross-reacting viral capsid and early antigens with the Epstein-Barr virus (EBV), and HVP DNA and EBV DNA show partial sequence homology. EBV-specific complementary RNA was employed here as a probe to investigate the physical state of the HVP DNA component in baboon lymphoblastoid cells after fractionation of cellular DNA by density gradient centrifugation. Five virus-producing cultures contained both free and integrated HVP DNA sequences while one non-producing cell line had two or three viral genome equivalents per cell in an apparently integrated form. Further analysis of one virus-producing line showed that the free HVP DNA fraction was composed of both linear and circular viral DNA. Contour length measurements of HVP circular DNA molecules by electron microscopy revealed that they were similar in length to the EBV circular DNA present in human lymphoblastoid cells.

Animals↗
PubMed1982

Simian virus 40 cRNA is processed into functional mRNA in microinjected monkey cells.

Monkey cells, microinjected with simian virus 40 (SV40) in vitro synthesized cRNA produce full-size tumor (T)-antigen. This was verified by analyzing immunoprecipitates of microinjected cells by polyacrylamide gel electrophoresis. Early SV40 DNA contains an intron within the large T-antigen coding sequences. Therefore, cRNA copied in vitro from the early DNA strand requires removal of the intron in order to become a functional mRNA. Polyadenylation of the cRNA in vitro by Escherichia coli poly(A)-polymerase increased the biological activity of the RNA. Detection of T-antigen by gel electrophoresis required as little as 50 poly(A)-cRNA injected cells. Splicing of the microinjected cRNA appears to be a nuclear process. Cells enucleated by cytochalasin B prior to injection do not synthesize large T-antigen. However, small t-antigen, a protein with a continuous sequence, is synthesized in these cells. Finally, it is shown that the process of splicing is not required for the transport of mRNA from the nucleus into the cytoplasm. Authentic T-antigen mRNA, isolated from virus infected cells, induced T-antigen synthesis with similar efficiency after either nuclear or cytoplasmic injection.

Animals↗
PubMed1983

An improved in situ hybridization method for the detection of cellular RNAs in Drosophila tissue sections and its application for localizing transcripts of the homeotic Antennapedia gene complex.

An improved method for the detection of cellular RNAs in tissue sections has been developed. It involves in situ hybridization of tritium-labeled cloned DNA probes to tissue sections and autoradiography. The method was calibrated by using a cloned DNA probe complementary to transcripts abundant in the midgut cells of Drosophila larvae. The improved method also permitted the detection of these transcripts in sectioned embryos where they are much less abundant. The sensitivity of the method can be approximated by quantifying the signal intensities over the hybridizing embryonic midgut cells relative to the larval midgut cells for which the number of transcripts has been estimated. Based on these calculations we estimate that the method is sensitive enough to detect 100 complementary RNA molecules per cell after 3 days of autoradiographic exposure with a signal-to-noise ratio of 10. The method has been successfully applied to detect transcripts of the homeotic gene Antennapedia. Serial sections allow us to study the spatial pattern of gene expression in the course of development.

Journal Article↗
PubMed1989

Anion transport in oocytes of Xenopus laevis induced by expression of mouse erythroid band 3 protein--encoding cRNA and of a cRNA derivative obtained by site-directed mutagenesis at the stilbene disulfonate binding site.

A vector was constructed containing a cDNA for mouse band 3 obtained from Demuth et al. (1986, EMBO J., 5, 1205-1214), a synthetic linker (containing 5'-non-translated region, start codon and a coding region for the first 12 N-terminal amino acids), and RNA polymerase promoters suitable for in vitro transcription of cRNA. After injection of the cRNA into the cytoplasm of Xenopus oocytes and incubation for 16 h, expression of mouse band 3 was demonstrated by immunoprecipitation, immunohistochemical methods and influx or efflux measurements with 36Cl-. Antisense cRNA inhibits the expression. Lysines 558 and 561 were replaced by asparagines using oligonucleotide-directed mutagenesis. Like the original band 3, the mutant shows stilbene disulfonate-inhibitable anion exchange. However, in contrast to the original band 3, inhibition by 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonate (H2DIDS) is no longer irreversible. This indicates that thiourea bond formation between H2DIDS and band 3 involves one of the two modified lysine residues. It also shows that the two lysine residues are not essential for the execution of the anion transport function of band 3. The results described suggest that the cDNA clone of Demuth et al. (1986) encodes a protein with properties that are representative for the properties of the bulk of the band 3 protein in the plasma membrane of the red cell of the mouse.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Cloning, expression, pharmacology and regulation of a delayed rectifier K+ channel in mouse heart.

Neonatal mouse cardiac poly(A)+ mRNA microinjection into Xenopus oocytes directed the expression of a delayed rectifier K+ current. A cDNA encoding this channel, called mIsK, was cloned from a neonatal mouse heart cDNA library whose properties were studied after expression of the complementary RNA in Xenopus oocytes. Among the different known K+ channel blockers, only the class III antiarrhythmic clofilium inhibited mIsK in the 10-100 microM range. The channel was completely insensitive to other antiarrhythmics such as quinine, quinidine, sotalol or amiodarone. mIsK was enhanced by increasing intracellular Ca2+ and by microinjected Ca(2+)-calmodulin dependent protein kinase II. These stimulations were reversed by the calmodulin antagonist W7. Conversely, the phorbol ester PMA, the diacylglycerol analog OAG and microinjected purified protein kinase C inhibited mIsK. This inhibitory effect could be prevented by the protein kinase C inhibitor staurosporine. These results were consistent with the presence of consensus sequences for kinase II and kinase C in the mIsK structure. Cultured newborn mouse ventricular cardiac cells exhibited a delayed rectifier K+ current which had biophysical properties similar to those of cloned mIsK and which was inhibited by clofilium and protein kinase C activators. In situ hybridization experiments revealed that mIsK mRNA was homogeneously distributed in the cardiac tissue. Neonatal mouse heart expressed the most mIsK mRNA compared with various other rat and mouse tissues. Since this K+ channel generates a current which appears to be involved in the control of both the action potential duration and the beating rate, these results suggest an important role for the mIsK channel in cardiac cell physiology and cardiac pathology.

Amino Acid Sequence↗
PubMed2003

Human cardiomyocytes express high level of Na+/glucose cotransporter 1 (SGLT1).

We have quantitatively measured gene expression for the sodium-dependent glucose cotransporters 1 and 2 (SGLT1 and SGLT2) in 23 human tissues using the method of real time PCR. As predicted, our results revealed that the expression of SGLT1 was very high in the small intestine (1.2E + 6 molecules/microg total RNA) relative to that in the kidney (3E + 4 molecules/microg total RNA). Surprisingly, we observed that the expression of SGLT1 in human heart was unexpectedly high (3.4E + 5 molecules/microg total RNA), approximately 10-fold higher than that observed in kidney tissue. DNA sequencing confirmed that the PCR amplified fragment was indeed the human SGLT1 gene. Moreover, in situ hybridization studies using a digoxigenin (DIG)-labeled antisense cRNA probe corresponding to human SGLT1 cDNA confirm that human cardiomyocytes express SGLT1 mRNA. In contrast, the expression of SGLT2 in human tissues appears to be ubiquitous, with levels ranging from 6.7E + 4 molecules/microg total RNA (in skeletal muscle) to 3.2E + 6 molecules/microg total RNA (in kidney), levels 10-100-fold higher than the expression of SGLT1 in the same tissues. Our finding that human cardiomyocytes express high levels of SGLT1 RNA suggests that SGLT1 may have a functional role in cardiac glucose transport. Since several SGLT inhibitors are currently in development as potential anti-diabetic agents, it may be important to assess the functional consequences of inhibition of SGLT1 in the heart.

Humans↗
PubMed2005

Hantaviruses in Central South America: phylogenetic analysis of the S segment from HPS cases in Paraná, Brazil.

We sequenced the complete S segments of hantaviruses detected from 12 HPS patients living in southern of Brazil. Samples were obtained from patients diagnosed in different years, in distinct areas, and with a broad spectrum of clinical signs. Despite these differences, all the S proteins of hantavirus from Paraná were identical, except for one amino acid substitution. Phylogenetic analyses of the complete S segment nucleotide and amino acid sequences indicated that hantaviruses from Paraná form a distinct clade from those circulating in South and North America. Other hantaviruses from Brazil were not placed in the same clade. The Oligoryzomys nigripes-associated strains ITA37 and ITA38 from Paraguay were found to belong to the same clade as the hantaviruses from Paraná. Paraguay and Paraná state are located at the same latitude and some ecosystems are similar in both places. The geographic position and common rodent hosts could explain this phylogenetic relationship.

Brazil↗
PubMed1994

Gonadal steroid control of preprocholecystokinin mRNA expression in the limbic-hypothalamic circuit: comparison of adult with neonatal steroid treatments.

The neuropeptide cholecystokinin (CCK) is involved in the regulation of female, but not male, reproductive behavior. In both sexes, estrogen regulates the expression of CCK in adulthood within the bed nucleus of the stria terminalis and medial amygdaloid nucleus. These areas are parts of an interconnected limbic system-hypothalamic circuit, the development of which is influenced by estrogen during the early postnatal period. This is the same period during which central nervous system (CNS) expression of CCK is dramatically increased, suggesting that the male and female patterns of CCK expression may be the result of early postnatal exposure to estrogen. In the present experiment, the expression of preprocholecystokinin (pCCK) mRNA was determined by in situ hybridization with an isotopically labeled pCCK complementary RNA and emulsion autoradiography in animals whose neonatal and adult gonadal steroid levels had been manipulated. The number of pCCK-expressing cells in animals that were gonadectomized as adults was determined by neonatal estrogen, but stimulation with steroids in adulthood induced a similar number of pCCK-expressing cells in both sexes in the medial amygdala and bed nucleus of the stria terminalis. Neonatal treatment of females with estrogen or testosterone, followed by ovariectomy in adulthood, eliminated the sex difference in pCCK mRNA expression. Males treated neonatally with the aromatase inhibitor androstenedione (to block metabolism of testosterone to estrogen) and orchidectomized in adulthood had a level of pCCK mRNA expression that was similar to that of ovariectomized females. These data suggest that, during neonatal development, estrogen determines the constitutive expression of pCCK mRNA in the medial amygdala and bed nucleus of the stria terminalis, resulting in higher levels of pCCK mRNA expression in males than in females. However, exogenous gonadal steroids induce the same levels of pCCK mRNA expression in adult females, indicating that the levels of gonadal steroids and the patterns of their secretion are the predominant influences on the sexually dimorphic adult levels of pCCK mRNA expression.

Aging↗
PubMed1990

Expression of a sperm protein gene during spermatogenesis in mammalian testis: an in situ hybridization study.

In previous work a specific membrane protein with an estimated Mr of 20.1 kDa was purified from rabbit sperm tails and designated as rSMP-B protein. Antibodies were raised against rSMP-B protein and used to isolate and identify the cDNA coding the rSMP-B protein from a rat testis lambda gt11 expression library. The nucleotide sequence of the cDNA was determined in a previous study. Single-stranded 35S-labeled RNA probes were prepared. With the techniques of in situ hybridization, rSMP-B mRNA was detected in spermatids of rat and rabbit testis. The present results support our previous observation that immunization of male rabbits with the rSMP-B protein results in the arrest of spermatogenesis at the spermatid stage. Overall, rSMP-B protein appears to be involved in spermiogenesis, and the synthesis of the mRNA encoding the protein occurs in germ cells during the postmeiotic haploid phase of spermatogenesis.

Animals↗
PubMed1990

Detection of endothelin immunoreactivity and mRNA in pulmonary tumours.

Paraffin sections of 66 surgically resected lung tumours were immunostained with antisera to human endothelin-1 and to the C-terminal peptide of big endothelin. With both antisera, strong immunoreactivity was demonstrated in 11 of 15 squamous cell carcinomas and 11 of 16 adenocarcinomas. Focal immunoreactivity was seen in small cell carcinoma (2/12), large cell carcinoma (2/5), and carcinoid tumours (2/11). Four lymphomas and three sarcomas did not show endothelin immunoreactivity. Cryostat sections of 22 of the 66 tumours were hybridized with radiolabelled complementary RNA probes prepared from the 3' non-coding region of endothelin-1 cDNA, and the chromosomal genes encoding endothelin-2 and -3. In situ hybridization demonstrated the presence of endothelin mRNAs in 4 of 7 squamous cell carcinomas and in 5 of 8 adenocarcinomas, in a pattern similar to that shown by immunocytochemistry. No hybridization signals were obtained from the other types of tumours. In lung tissue adjacent to the tumours, endothelin-like immunoreactivity and mRNA were detected in pulmonary endocrine cells and, in some cases, other epithelial cells, and in alveolar capillary endothelial cells. This study demonstrates the expression of endothelin in a number of pulmonary tumours and suggests a possible role for this peptide in the growth and/or differentiation of these tumours.

Adenocarcinoma↗
PubMed1993

Molar quantification of specific messenger ribonucleic acid expression in northern hybridization using cRNA standards.

Procedures were developed for estimation of specific mRNA expression as measured by Northern hybridization in terms of moles of message present in tissue samples. These procedures use an externally added cRNA pseudomessage standard with cRNA concentration curves for both the standard and the message of experimental interest in order to convert hybridization signals into moles of message per milligram wet weight of tissue. We present a test of these procedures using hybridization to the message for the glucocorticoid-inducible enzyme tyrosine aminotransferase in rat liver. These procedures accommodate for variations in both RNA yield and hybridization signal intensities, and are applicable to the measurement of changes in message expression of twofold or more using triplicate determinations of a single RNA preparation. This approach allows for the integration of data from many individual hybridization experiments, and may be useful in experimental situations that involve population studies and/or long-term comparisons of treatments.

Animals↗
PubMed1993

A quantitative polymerase chain reaction assay for interleukin 5 messenger RNA.

The quantitation of low abundance messenger (m)RNA species such as that for interleukin 5 (IL-5) cannot be achieved by Northern blotting. Polymerase chain reaction (PCR) technology can be used to overcome this paucity of target molecules, but in its standard format it is semiquantitative. We describe the synthesis, purification, and use of a synthetic IL-5 mRNA (cRNA) molecule which, when included in the PCR reaction, enabled the amplification of IL-5 mRNA molecules from clinical samples to be made quantitative. A stringent protocol involving analysis of the purified transcript by PCR (to ensure its freedom from template molecules) is required. Preliminary experiments indicate that IL-5 mRNA levels are elevated in asthmatics.

Base Sequence↗