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At least 1,333 records · Page 74Linked to original sources
PubMed1981

Expression of simian virus 40-rat preproinsulin recombinants in monkey kidney cells: use of preproinsulin RNA processing signals.

The complete rat preproinsulin gene I was cloned into a simian virus 30 (SV 40) vector. Most of the late region of the viral vector, including the SV40 intervening sequences (introns) and all of the major splice junctions, was deleted and replaced by the entire rat insulin gene. The recombinant molecules and a temperature-sensitive helper virus (tsA28) were inoculated into monkey kidney cultures. The formation of stable transcripts of the insulin insert was as efficient as the production of late SV40 mRNA. Analysis of these transcripts indicated that the rat preproinsulin gene nucleotide signals involved in RNA splicing and poly(A) addition were used. Examination of the 5' ends of the mRNAs showed several classes, one of which was the same size as the authentic rat insulinoma mRNA. This suggests that a portion of the transcripts may be initiated or processed faithfully, or both, at their 5' ends within rat insulin sequences. Significant quantities of a protein identified as rat proinsulin were synthesized. Detection of most of the proinsulin in the tissue culture medium suggests that this protein was secreted.

Animals↗
PubMed1989

Northern analyses using single-stranded probes do not support a role for GATA/GACA repeats in sex determination in mice and men.

The possible role of GATA/GACA repeated sequences in mammalian sex determination was investigated using Northern analyses of mouse and human RNA. Brain, liver, and gonadal RNA from three developmental stages of mice of both sexes and also human fetal RNA from various tissues were hybridized to both sense and antisense Bkm riboprobes as well as to the synthetic oligonucleotide (GATA)5. At low levels of stringency, putative transcripts of various sizes were observed in all tissue samples with all probes. At high stringency, only a putative transcript of approximately 12 kb was observed, but this was later shown to consist of contaminating DNA. No sex-specific differences were observed in any tissue or developmental stage. Thus, we find no evidence that the GATA/GACA repeated sequences are specifically expressed in quantities detectable by Northern analyses in a manner important to mammalian sex determination.

Animals↗
PubMed1991

Demonstration of osteonectin mRNA in megakaryocytes: the use of the polymerase chain reaction.

Platelets have been shown to release osteonectin on thrombin stimulation. The origin of platelet osteonectin was unclear as it may have been synthesized by megakaryocytes or it may have been endocytosed from plasma as other platelet alpha-granule constituents are. Platelet osteonectin has a larger apparent molecular size than the bone species, although the molecular basis for this difference has not been elucidated. These two issues have been addressed here by (1) examining the potential for osteonectin biosynthesis in human megakaryocytes by demonstrating the presence of osteonectin mRNA in purified megakaryocytes, and (2) comparing the coding portion of osteonectin transcript in megakaryocytes to the size of its bone counterpart. Because of the limitations of cell population purity and in obtaining sufficient numbers of megakaryocyte cells for Northern analysis, we have used the polymerase chain reaction (PCR) to detect the presence of human osteonectin mRNA in megakaryocyte and megakaryocyte-depleted bone marrow cells. Isolation of RNA, cDNA synthesis, and PCR were performed on human osteosarcoma SaOS-2 cells, enriched megakaryocytes, and megakaryocyte-depleted cells. Restriction enzyme analysis of PCR DNA products confirmed the identity of the products as those encoding osteonectin for all three cell populations studied. In addition, the sizes of DNA indicate that osteonectin genomic DNA, nuclear RNA, or altered transcript were not amplified, and that the transcript from megakaryocytes is the same size as that from bone cells. These data suggest that the difference in protein size between platelet and bone osteonectin is due to posttranslational modification. To overcome the possibility that megakaryocyte signal originated from contaminating cells (less than 5% by cell count), all three cell populations were diluted to less than one cell per tube and PCR amplification was performed. Limiting dilution analyses demonstrated the presence of osteonectin mRNA in single megakaryocytes as well as in single cells from the cell population depleted of megakaryocytes, suggesting the capacity for osteonectin biosynthesis in all cells studied. The procedure we describe in this report can be used to examine specific characteristics of mRNA molecules in heterogeneous cell populations and in situations where only small quantities of cells can be obtained.

Blotting, Southern↗
PubMed1980

Malignancies of metastatic murine lymphosarcoma cell lines and clones correlate with decreased cell surface display of RNA tumor virus envelope glycoprotein gp70.

Variant sublines of the murine lymphosarcoma RAW117 have been derived by sequential cycles of intravenous inoculation of cells and harvesting of solid liver tumors in syngeneic BALB/c mice [Brunson K. W. & Nicolson, G. L. (1978) J. Natl. Cancer Inst. 61, 1499-1503] and also by sequential removal of lectin-reactive cells via repeated adsorption on immobilized-lectins [Reading, C. L. Belloni, P. N. & Nicolson, G. L. (1980) J. Natl. Cancer Inst. 64, 1241-1249]. These cell sublines and their clones were analyzed for abilities to form gross liver tumor metastases after injection intravenously or subcutaneously into syngeneic mice, and this response was related to certain cell surface properties including quantities of viral antigens and lectin-binding sites, exposure of specific cell surface proteins, and quantities of cell surface glycoproteins visualized in gels with 125I-labeled lectins or antibodies. Consistent differences were obtained between RAW117 sublines of low and high malignancy with respect to the amounts or exposures of cell surface glycoprotein components of Mr approximately 70,000 or 69,000 and 71,000, depending on the gel system. Competition radioimmunoassays for RNA tumor virus antigens in the RAW117 lines and clones indicated the presence of Moloney murine leukemic virus antigens gp70, p30, and p12. Enhanced malignancy and metastasis to liver was accompanied by decreases in the cellular contents of viral antigens and loss of gp70 cell surface exposure. Analysis of several clones obtained from sublines selected in vivo and in vitro for high or low malignancy confirmed the inverse relationship between metastasis and expression of gp70 in this system.

Animals↗
PubMed1981

Excessive reversible phenobarbital induced nuclear DNA-polyploidization in the growing mouse liver.

Phenobarbital was injected intraperitoneally into male white NMRI mice aged 0.5, 1. 1.5, 3, 6 and 12 months at a dose of 120 mg/kg body weight for 10 consecutive days. The 0.5 month-old mice did not tolerate the phenobarbital dose and died. The experimental animals and one of the controls were sacrificed 1, 3, 5, 10, 15 and 20 days after phenobarbital administration was started. Liver weights were recorded and liver cells were isolated. The number of nuclei per cell was determined and the DNA-content of each single nucleus was measured by Feulgen fluorescence cytophotometry. Liver weights showed an increase of 25--30% during phenobarbital treatment and returned slowly to lower values after cessation of drug application. The hepatic enlargement was accompanied by an excessive and likewise reversible nuclear and whole cell DNA-polyploidization, i.e. polyploidization beyond the physiological age-dependent ploidy level; for example, mean values of 7.7 c per nucleus (versus 4.2 c in the controls) and 14.3 c for whole liver cells (versus 7.5 c in the controls) were found in 3 months-old animals after 5 days of treatment. As with the induction of microsomal enzymes, the augmentation of smooth endoplasmic reticulum, and the increase of RNA- and protein-synthesis, excessive DNA-polyploidization of liver cell nuclei appears to be an expression of hepatocellular hypertrophy due to the functional or metabolic stress imposed upon the liver by large quantities of phenobarbital. After cessation of drug administration the abnormally high ploidy cells are eliminated - probably by necrobiosis - and the liver cells return to their normal age-dependent DNA-ploidy level. The liver cells of the one-month-old animals revealed the physiological polyploidization to be slightly inhibited. This is probably due to some toxic effect of phenobarbital. Phenobarbital did not alter the number of nuclei per liver cell.

Aging↗
PubMed1979

Identification and characterization of a 19-S complex containing a 27 000-Mr protein in Artemia salina.

The cytoplasm of the cryptobiotic Artemia salina gastrula contains a large quantity of a unique 19-S complex. This particle is a specific aggregated form of a 27 000-Mr protein, having a molecular weight of 10(6) and an apparent buoyant density of 1.25 -- 1.26 g/cm3 in sucrose and 1.31 g/cm3 in CsCl. The relative quantity of this 19-S complex decreases significantly with respect to 80-S monoribosomes during the postgastrula development. Biochemical and immunological studies indicate that the 27 000-Mr protein is one of the RNA-binding proteins [Ovchinnikov et al., FEBS Lett. 88, 21 -- 26 (1978)] but is absent in the protein components associated with poly(A)-containing ribonucleoprotein complexes. The possibility is also suggested that the 27 000-Mr protein and Artemia elongation factor eEF-Ts might be related to each other on the basis of amino acid composition and immunological cross-reactivity.

Alkylation↗
PubMed1988

Regulation of food intake and hepatic protein synthesis by recombinant-derived cytokines.

During inflammation, activated monocytes and lymphocytes synthesize and release many soluble protein mediators, such as interleukin (IL) 1, tumor necrosis factor-alpha, and IL-2. It is presently unclear which cytokines, if any, contribute to the anorexia and hepatic protein changes frequently seen during inflammation. To evaluate their potential role, food intake and liver and plasma protein synthesis were determined in both endotoxin-sensitive C57Bl/6j mice and endotoxin-resistant C3H/HeJ mice given either crude secretory products of Staphylococcus albus-stimulated human blood monocytes or murine recombinant IL-1-alpha, human recombinant IL-1-alpha or -beta, human recombinant tumor necrosis factor-alpha, or human IL-2. When given intraperitoneally to healthy animals, 2,000 lymphocyte-activating factor U/day of secretory products of activated human blood monocytes or recombinant murine IL-1-alpha depressed spontaneous food intake by 42 and 53%, respectively. Human IL-1-alpha and -beta and human tumor necrosis factor-alpha produced smaller reductions in food intake. In contrast, human IL-2, when given in equimolar quantities, had no appreciable effect on food intake or body weight. Administration of crude secretory products of activated blood monocytes, recombinant IL-1, or tumor necrosis factor-alpha increased liver weight, protein, and RNA content. In addition, plasma protein synthesis was significantly increased, as were serum amyloid P concentrations. Administration of recombinant tumor necrosis factor-alpha resulted in IL-1 production by peritoneal adherent cells. However, IL-2 had no effect on any hepatic parameter.

Animals↗
PubMed2003

Chronic active Epstein-Barr virus infection associated with mutations in perforin that impair its maturation.

Chronic active Epstein-Barr virus infection (CAEBV) is a rare disease in which previously healthy persons develop severe, life-threatening illness. Mutations in the perforin gene have been found in familial hemophagocytic lymphohistiocytosis, which shares some features with CAEBV. We studied a patient who died at age 18, 10 years after the onset of CAEBV. The patient had high titers of antibodies to EBV, EBV RNA in lymph nodes, T-cell lymphoproliferative disease, and hemophagocytic lymphohistiocytosis. DNA sequencing showed novel mutations in both alleles of the perforin gene that resulted in amino acid changes in the protein. The quantity of the native form of perforin from the patient's stimulated peripheral blood mononuclear cells (PBMCs) was extremely low and immunoblotting showed accumulation of an uncleaved precursor form of perforin. Stimulated PBMCs from the patient were defective for Fas-independent cytotoxicity. These data imply that mutations in this patient resulted in reduced perforin-mediated cytotoxicity by his lymphocytes. This is the first case in which perforin mutations have been shown to result in accumulation of the uncleaved, immature form of perforin. Mutations in the perforin gene are associated with some cases of CAEBV with hemophagocytic lymphohistiocytosis.

Adolescent↗
PubMed1986

Strand displacement applied to assays with nucleic acid probes.

This novel method for the detection of specific nucleic acid sequences has potential applications to clinical diagnosis. During hybridization, a signal-bearing nucleic acid strand is displaced by the target nucleic acid from a partially single-stranded complementary probe strand of nucleic acid. The probe:signal strand complex is prepared by hybridizing single-stranded probe that is entirely complementary to the target nucleic acid with a shorter signal sequence that is complementary to a portion of the probe strand. The sample nucleic acid is added to this hybrid complex under hybridization conditions. The target sequence, if present in the sample, will hybridize first to the unoccupied probe sequences, and then will displace the labeled strand by branch migration. By this "strand displacement" the signal strands are freed in solution, where they may be separated from those still hybridized; the quantity of label measured is directly proportional to the amount of analyte sequences in the sample. This method, demonstrated here for model and synthetic DNAs, can easily be adapted for the detection of any RNA or DNA sequence and obviates the need for immobilization of sample. A wide variety of labeling techniques can be used, and the displacement can be performed in solution or with the hybrid complex attached to a solid support. This assay circumvents nonspecific binding of label to the filter matrix and the laborious washing steps inherent in other assays involving nucleic acid probes.

Animals↗
PubMed1995

[Transition from the latent to the active enzymatic form as a model of regulation of extracellular matrix degradation in the chorioamnion during human labor].

Matrix metallo proteinases (MMP) are the physiological mediators of collagen degradation and its participation in physiopathogenesis of premature rupture of membranes has been suggested by our group. With the idea of defining if some MMP become active active in a coordinated way with labor in fetal membranes, we analyzed enzymatic activity and immunoreactive protein present in extracts of amnion and chorion. It was possible to identify the presence of MMP-9 in extracts of membranes obtained during cesarean sections, without labor, although its activity/quantity was faintly detectable. Instead, extracts of fetal membranes obtained during active labor showed large activity/quantity of this MMP. With a monoclonal antibody, it was possible to show that the active form of MMP-9 could only be found in samples with labor. MMP-9 and its messenger RNA, were localized by immunohistochemistry and in situ hybridization in amniotic epithelium, in some fibroblasts of the compact layer and in trophoblast-like cells in chorion. It is concluded that: 1. Activity and quantity of MMP-9 increase selectively associated to labor; and 2. That this enzyme is expressed by different cellular populations of fetal membranes.

Amnion↗
PubMed1994

Changes in the messenger ribonucleic acid for insulin-like growth factor-I and -II in the porcine testis during and between two waves of testicular development.

The boar testis was used as a model for examining the possible role of production of the insulin-like growth factors (IGF) in steroidogenesis and/or testicular growth as testicular development occurs in waves. Blood and testes were sampled from boars at different ages (100-102 days of gestation; 7, 19, and 30 days; and 10 and 25 wk), selected to occur during and between the last two waves of testicular development. Serum was analyzed for testosterone and RNA was extracted from the testes for Northern and dot-blot analysis of IGF mRNA. Testosterone concentrations declined (p = 0.01) from 7 days to 10 wk of age and rebounded at 25 wk, indicating completion of the second and third waves of testicular development. The quantity of testicular mRNA for IGF-I increased gradually with age, and that for IGF-II decreased. We therefore conclude that regulation of the expression of the mRNAs for IGF-I and -II in the pig testis is not a function of either the waves of testicular development or the level of steroidogenesis.

Actins↗
PubMedDate not supplied

Characterization of S-[2-(N1-adenyl)ethyl]glutathione as an adduct formed in RNA and DNA from 1,2-dibromoethane.

The major DNA adduct derived from 1,2-dibromoethane is known to be S-[2-(N7-guanyl)-ethyl]glutathione; minor nucleic acid DNA adducts were characterized in view of the possibility that some might be unusually persistent or biologically active. RNA was modified in vitro by treatment with 1,2-dibromoethane and glutathione in the presence of rat liver cytosol, and bases were released by mild acid hydrolysis, which liberated greater than 99% of the bound radioactivity. One of the minor adducts was identified as S-[2-(N1-adenyl)ethyl]glutathione on the basis of its UV, mass, and NMR spectra. This adduct could be synthesized by reaction of S-(2-chloroethyl)-glutathione with adenosine. The material was desulfurized by treatment with Raney Ni to give N1-ethyladenine in low yield. The Raney Ni reaction was accompanied by considerable formation of the corresponding N6-adenine derivative via Dimroth rearrangement. Another adduct was identified as S-[2-(N7-guanyl)ethyl]cysteinylglycine by its UV, mass, and NMR spectra, but the material was demonstrated to be formed from the major DNA adduct, S-[2-(N7-guanyl)-ethyl]glutathione under conditions of mild acid hydrolysis. The imidazole ring opened derivative of S-[2-(N7-guanyl)ethyl]glutathione was synthesized and found not to be formed in DNA in vitro or in vivo. The two remaining minor adducts account for 1-2% of the total binding, but insufficient quantities were recovered to allow for structure determination; however, neither of these (uncharacterized) minor products are seen after the reaction of S-(2-chloroethyl)glutathione with guanosine or adenosine. S-[2-(N1-Adenyl)ethyl]glutathione was formed in rat liver RNA and DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

A universal reference sample derived from clone vector for improved detection of differential gene expression.

BACKGROUND: Using microarrays by co-hybridizing two samples labeled with different dyes enables differential gene expression measurements and comparisons across slides while controlling for within-slide variability. Typically one dye produces weaker signal intensities than the other often causing signals to be undetectable. In addition, undetectable spots represent a large problem for two-color microarray designs and most arrays contain at least 40% undetectable spots even when labeled with reference samples such as Stratagene's Universal Reference RNAs. RESULTS: We introduce a novel universal reference sample that produces strong signal for all spots on the array, increasing the average fraction of detectable spots to 97%. Maximizing detectable spots on the reference image channel also decreases the variability of microarray data allowing for reliable detection of smaller differential gene expression changes. The reference sample is derived from sequence contained in the parental EST clone vector pT7T3D-Pac and is called vector RNA (vRNA). We show that vRNA can also be used for quality control of microarray printing and PCR product quality, detection of hybridization anomalies, and simplification of spot finding and segmentation tasks. This reference sample can be made inexpensively in large quantities as a renewable resource that is consistent across experiments. CONCLUSION: Results of this study show that vRNA provides a useful universal reference that yields high signal for almost all spots on a microarray, reduces variation and allows for comparisons between experiments and laboratories. Further, it can be used for quality control of microarray printing and PCR product quality, detection of hybridization anomalies, and simplification of spot finding and segmentation tasks. This type of reference allows for detection of small changes in differential expression while reference designs in general allow for large-scale multivariate experimental designs. vRNA in combination with reference designs enable systems biology microarray experiments of small physiologically relevant changes.

Calibration↗
PubMed1988

Endothelial cell hyperplasia in human glioblastoma: coexpression of mRNA for platelet-derived growth factor (PDGF) B chain and PDGF receptor suggests autocrine growth stimulation.

The genes for platelet-derived growth factor (PDGF) A chain, B chain/c-sis, and the PDGF receptor are expressed in human malignant glioma cell lines. In the present investigation we have studied the expression of these genes in biopsy specimens from human glioblastomas. Hyperplasia of the vascular endothelium is a prominent characteristic of human glioblastoma multiforme and simian sarcoma virus-induced gliomas in primates. RNA transfer blot analysis of biopsies from glioblastoma multiforme showed transcripts for PDGF A and B chains and the PDGF receptor. Tissue sections from this tumor examined by in situ hybridization techniques revealed that the proliferating vascular endothelial cells contained large quantities of mRNA for PDGF B chain/c-sis and its receptor and, to a lesser extent, for PDGF A chain. In contrast, the tumor cells expressed more mRNA for PDGF A chain than for PDGF B chain and PDGF receptor. The latter two were also expressed at higher levels in glioma cells than in glial cells of nontumorous human brain tissue. Thus, an autocrine stimulation by the PDGF B chain/c-sis product via its receptor, evoked by interaction with surrounding glioma cells, could be the mechanism behind the pathological proliferation of endothelial cells characteristically found in this type of malignancy.

Animals↗
PubMed1997

Detection of plasmids using DNA and RNA probes and the light-addressable potentiometric sensor.

Intact plasmids, plasmid fragments, and cDNA were detected using two DNA or RNA probes of varying lengths, each containing only biotin or fluorescein molecules. The probes were hybridized with the target plasmid/cDNA, bound with streptavidin, captured on nitrocellulose membranes, and detected using the urease-conjugate of an anti-fluorescein antibody via the light-addressable potentiometric sensor. The output of the silicon-chip sensor is in the form of rate, microV/s, and is directly proportional to the quantity of hybridized DNA captured on the membrane. Use of the larger probes (for beta-actin cDNA target) results in detection of intact plasmid at the level of approximately 106 target molecules; complementary DNA could be detected at similar levels. When the smaller probes are utilized (pGEM or pBSActB targets), plasmid fragmented targets could be detected only at levels 200 times greater than those observed when using the larger RNA probes.

Biotin↗
PubMed1995

Quantitation of APC mRNA in human tissues.

The aim of this study was to develop a method for quantitation of APC mRNA and to measure APC mRNA levels in human tissues. This was done using a competitive reverse transcription PCR with a synthetic RNA control. APC mRNA (pg/microgram total RNA) was quantitated in the following tissues: normal colon 133.6 +/- 69.0; normal duodenum 16.6 +/- 15.7; normal thyroid 30.4 +/- 20.3; normal esophagus 65.2 +/- 58.8; colonic carcinoma 66.5 +/- 30.3; esophageal carcinoma 54.7 +/- 26.5; papillary thyroid carcinoma 55.8 +/- 32.9 and colonic adenomas from patients with familial adenomatous polyposis 46 +/- 15. APC mRNA levels were significantly lower in the duodenum and thyroid than in the colon and esophagus. The quantity of APC mRNA was lower in colonic adenomas from FAP patients than in normal colonic mucosa (p < 0.05).

Base Sequence↗
PubMed2002

Rapid microorganism identification with on-slide proteolytic digestion followed by matrix-assisted laser desorption/ionization tandem mass spectrometry and database searching.

A simple method for microorganism identification is proposed in this paper. In this method, no isolation or fractionation of microorganisms and no special search algorithm or database are needed. Experimentally, a sample of the unfractionated intact microorganism is subjected to brief on-slide proteolytic digestion and the digestion product ions detected in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) are selected for tandem mass spectrometry (MS/MS). Database searching using the MS/MS spectrum identifies the microorganism protein, and thereby, according to the source of the protein, the microorganism. The method was demonstrated to be feasible with enterobacteriophage MS2 as the model microorganism. The novel method is simple, rapid, and requires a small quantity of sample.

Amino Acid Sequence↗
PubMed1989

Homologous interference of lymphocytic choriomeningitis virus involves a ribavirin-susceptible block in virus replication.

Depending on the multiplicity of infection (MOI), infection of L929 cells results in either productive lymphocytic choriomeningitis virus replication or homologous interference M. Bruns, A. Gessner, H. Lother, and F. Lehmann-Grube, Virology 166:133-139, 1988). As shown in this communication, productive lymphocytic choriomeningitis virus replication as observed at a low MOI was effectively inhibited by ribavirin. In contrast, virus yields increased if cells were infected with a high MOI and in the presence of 5 microM of the antiviral compound. This drug-dependent release of infectious virus was preceded by enhanced nucleoprotein (NP) synthesis, a change in intracellular NP distribution, and by an onset of glycoprotein synthesis. It is therefore proposed that this block in viral replication is brought about by a posttranslational effect on a viral gene product, probably the NP, present in reasonably large quantities both during homologous interference as well as persistent infection.

Animals↗