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PubMed1985

Activation of type I collagen genes in cultured scleroderma fibroblasts.

Fibroblasts cultured from affected skin areas of five patients with cutaneous scleroderma were found to produce increased amounts of collagen when compared with nonaffected control cells. Total RNA was isolated from the cultures and analyzed for its level of pro alpha 1 (I)collagen mRNA by hybridization of RNA blots with a cloned cDNA probe. The levels of pro alpha 1 (I)collagen mRNAs relative to total RNA were two- to sixfold higher in the samples from affected cells, accounting for the increased synthesis of type I collagen. Cytoplasmic dot hybridizations were performed to measure the cellular content of pro alpha 1 (I)collagen mRNA: up to ninefold increases in the level of this mRNA per cell were found. Upon subculturing, scleroderma fibroblasts were found to reduce gradually the increased synthesis of collagen to the level of nonaffected controls by the tenth passage. The levels of type I collagen mRNAs were also reduced, but more slowly. The results suggest that in scleroderma fibroblasts the genes for type I collagen are activated at procollagen mRNA level or that they are more stable and that the activating factors are lost during prolonged cell culture because cells from affected areas lose their activated state.

Adolescent↗
PubMed2006

Localization and diurnal variations of carbonic anhydrase mRNA expression in the inner ear of the rainbow trout Oncorhynchus mykiss.

Physiological studies have suggested that carbonic anhydrase (CA) plays a central role in otolith biomineralization via ion transport. However, the presence and exact function of CA in the inner ear have not been determined. In the present study, to investigate the localization of CA and its involvement in otolith calcification, we cloned two cDNAs encoding CAs from the rainbow trout sacculus. These two cDNAs, designated rainbow trout CAa (rtCAa) and rtCAb, both had an open reading frame encoding 260 amino acids with a sequence identity of 78%. Remarkably, rtCAb has a high degree of homology (82%) with "high activity CA" in the zebrafish, and its mRNA expression showed variation in the range 1.9-11.4 x 10(4) copies/ng total RNA in the sacculus. In contrast, rtCAa mRNA was constantly expressed at approximately 3 x 10(4) copies/ng total RNA. In situ hybridization revealed that rtCAb mRNA was strongly expressed in the distal squamous epithelial cells and transitional epithelial cells, except the mitochondria-rich cells, whereas, rtCAa was localized in extrasaccular tissue. These results suggest that the rtCAb isozyme is involved in the daily increment formation and calcification of otoliths via phase and spatial differences of the bicarbonate supply to the endolymph.

Amino Acid Sequence↗
PubMed1991

The expression of glycogen phosphorylase in normal and dystrophic muscle.

Specific cofactor labelling was employed to determine the degradation rate of glycogen phosphorylase in normal adult C57BL/6J mice and their dystrophic counterparts (C57BL/6Jdy/dy). The rate constant for the decay of phosphorylase-bound label was 0.125 day-1 in normal muscle and 0.49 day-1 in dystrophic muscle, i.e. a lower rate of catabolism of phosphorylase in dystrophic muscle. Quantitative Northern-blot analyses of total RNA isolated from normal and dystrophic muscle indicated that the abundance of phosphorylase mRNA as a percentage of total RNA was approx. 40% lower in dystrophic muscle. The specific activity of phosphorylase in dystrophic muscle is approx. 60% lower than in normal muscle, and is elicited by a lower rate of turnover of the enzyme, i.e. both synthesis and degradation are decreased.

Animals↗
PubMed2003

Peroxisome proliferator-activated receptor-gamma gene expression in orbital adipose/connective tissues is increased during the active stage of Graves' ophthalmopathy.

The mechanisms involved in the increase of orbital retro-ocular adipose tissue that occurs in Graves' ophthalmopathy (GO) are still unclear. In this condition, the orbital tissue shows glycosaminoglycans deposition produced by activated fibroblasts capable of undergoing adipocytic differentiation. Many genes are involved in adipogenic mechanisms including the transcription factor peroxisome proliferator-activated receptor-gamma (PPAR-gamma). We evaluated the level of expression of the PPAR-gamma gene in normal and GO orbital adipose/connective tissue specimens using a quantitative and sensitive reverse transcription (RT) competitive polymerase chain reaction (PCR) assay. Our results show that the expression of PPAR-gamma was significantly greater in adipose/connective tissue from patients in the active stage of GO than in controls (150.8 +/- 103.9 and 24.0 +/- 4.9 amol/micro g of total RNA respectively, p < 0.05), while there was no significant difference between patients with inactive GO (58.8 +/- 40.6 aM/microg total RNA) and controls. These results suggest that increased PPAR-gamma gene expression in the active stage of GO may be dependent on the inflammatory process in this disease. We speculate that the increased orbital fat tissue observed in GO may be a consequence of the anti-inflammatory PPAR-gamma action.

Adipose Tissue↗
PubMed1992

Molecular characterization of the soybean alcohol dehydrogenase gene family amplified in vitro by the polymerase chain reaction.

Soybean (Glycine max) alcohol dehydrogenase (ADH) cDNAs were amplified in vitro from total RNA by the polymerase chain reaction (PCR). The amplification strategy involved first strand cDNA synthesis from anaerobic cotyledon total RNA using an 18-thymidine primer. The second strand cDNA primer was a conserved sequence near the 5' end of known plant ADH transcripts. The PCR products were ligated into a plasmid vector and unique clones were isolated on the basis of size and restriction pattern. Sequence analysis revealed three distinct classes of soybean ADH cDNAs, all of which showed high homology to Adh genes from maize and peas. RNA blot hybridization analyses showed differential expression patterns for these genes. One gene, expressed constitutively in all seedling organs, was inducible by anaerobiosis, one gene was expressed only in anaerobic organs, and the third gene was expressed predominantly in anaerobic roots.

Journal Article↗
PubMed2006

Suspension array analysis of 16S rRNA from Fe- and SO(4)2- reducing bacteria in uranium-contaminated sediments undergoing bioremediation.

A 16S rRNA-targeted tunable bead array was developed and used in a retrospective analysis of metal- and sulfate-reducing bacteria in contaminated subsurface sediments undergoing in situ U(VI) bioremediation. Total RNA was extracted from subsurface sediments and interrogated directly, without a PCR step. Bead array validation studies with total RNA derived from 24 isolates indicated that the behavior and response of the 16S rRNA-targeted oligonucleotide probes could not be predicted based on the primary nucleic acid sequence. Likewise, signal intensity (absolute or normalized) could not be used to assess the abundance of one organism (or rRNA) relative to the abundance of another organism (or rRNA). Nevertheless, the microbial community structure and dynamics through time and space and as measured by the rRNA-targeted bead array were consistent with previous data acquired at the site, where indigenous sulfate- and iron-reducing bacteria and near neighbors of Desulfotomaculum were the organisms that were most responsive to a change in injected acetate concentrations. Bead array data were best interpreted by analyzing the relative changes in the probe responses for spatially and temporally related samples and by considering only the response of one probe to itself in relation to a background (reference) environmental sample. By limiting the interpretation of the data in this manner and placing it in the context of supporting geochemical and microbiological analyses, we concluded that ecologically relevant and meaningful information can be derived from direct microarray analysis of rRNA in uncharacterized environmental samples, even with the current analytical uncertainty surrounding the behavior of individual probes on tunable bead arrays.

Bacteria↗
PubMed2002

Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines.

BACKGROUND: We describe an alternative method to determine mRNA half-life (t1/2) based on the Real-Time RT-PCR procedure. This approach was evaluated by using the beta-actin gene as a reference molecule for measuring of mRNA stability. RESULTS: Human leukemia Nalm-6 and CCRF-CEM cells were treated with various concentrations of Actinomycin D to block transcription and aliquots were removed periodically. Total RNA was isolated and quantified using the RiboGree&ncircledR; fluorescent dye with the VersaFluor Fluorometer System. One &mgr;g of total RNA was reverse transcribed and used as template for the amplification of a region of the beta-actin gene (231 bp). To generate the standard curve, serial ten-fold dilutions of the pBactin-231 vector containing the cDNA amplified fragment were employed, beta-actin mRNAs were quantified by Real-Time RT-PCR using the SYB&RcircledR; Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the beta-actin mRNA exhibited a half-life of 6.6 h and 13.5 h in Nalm-6 and CCRF-CEM cells, respectively. The t1/2 value obtained for Nalm-6 is comparable to those estimated from Northern blot studies, using normal human leukocytes (5.5 h). CONCLUSIONS: We have developed a rapid, sensitive, and reliable method based on Real-Time RT-PCR for measuring mRNA half-life. Our results confirm that beta-actin mRNA half-life can be affected by the cellular growth rate.

Journal Article↗
PubMed2003

Activation of the MAP kinase pathway induces chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) expression in human breast cancer cell lines.

Growth factors are essential for cellular growth and differentiation in both normal and malignant human breast epithelial cells. In the present study we investigated the effect of epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha) and phorbol myristate acetate (PMA) on chicken ovalbumin upstream promoter-transcription factor (COUP-TF) expression in human breast cancer cells. The orphan receptors COUP-TFI and COUP-TFII are members of the nuclear receptor superfamily. The high degree of evolutionary conservation of these proteins strongly argues for an important biological function. COUP-TF expression was highest in SK-BR3 cells (approximately 130 amol/ micro g total RNA), while the lowest COUP-TF expression was observed in MCF-7 cells (3.5 amol/ micro g total RNA). While treatment of EGF, TGFalpha and PMA induced expression of COUP-TFII, COUP-TFI did not respond to these agents. Oncostatin M (OSM) is known to exert an antiproliferative effect in breast cancer cells. Treatment of MCF-7 cells with OSM resulted in an approximately 90% reduction of COUP-TFII mRNA expression. In SK-BR3 cells, treatment with the MEK inhibitor UO126 resulted in a profound suppression of endogenous COUP-TFII expression. Furthermore, cotreatment with UO126 prevented induction of COUP-TFII expression by EGF in MCF-7 cells. In conclusion, our data provide evidence, for the first time, that mitogenic substances which activate the MAP kinase pathway, can induce COUP-TFII expression. Our results strongly suggest that an active MAP kinase pathway is essential for COUP-TFII expression in human breast cancer cells.

Blotting, Western↗
PubMed2005

[Study of genes that differentially expressed in eosinophils of patients with asthma by suppression subtractive hybridization technology].

OBJECTIVE: To study genes that differentially expressed in peripheral blood eosinophils of patient with asthmatic. METHODS: Total RNA extracted from eosinophils of patient at the time of exacerbation was taken as the tester and the total RNA from eosinophils of the same patient after improvement as driver. The ds cDNA was synthesized by SMART PCR cDNA Synthesis technology. The differentially expressed genes were obtained by suppression subtractive hybridization (SSH) technology and were further identified by Southern blot. RESULTS: Twelve differentially expressed genes including transformation growth factor beta activated kinase like (TAK1L) protein, cGMP gated channel protein were obtained. CONCLUSION: Increased expression of these genes were involved in the regulation of proinflammatory response, signal transduction, energy metabolism and cell apoptosis, which may help to clarify the molecular mechanism of eosinophils in asthma and may provide the theoretical base for finding out the new medicines towards eosinophils.

Asthma↗
PubMed2004

[Construction of cDNA library from human colorectal cancer cell HRT-18].

OBJECTIVE: To construct a cDNA library from human colorectal cancer cell HRT-18. METHODS: The total RNA was extracted from human CRC (colorectal cancer) cell HRT-18 and the mRNA was isolated from the total RNA by MagneSphere technique, and then the first-strand cDNA was synthesized with oligo (dT) primer containing sfi I site while the double-strand cDNA was amplified through LD-PCR (long-distance PCR) by SMART technique. The double strand cDNA was digested by sfi I ( I A & I B) restriction enzyme before cDNA size fractionation, and the double-strand cDNA fractionated was ligated into the lambdaTripIEX2 vector and then was packaged into lambda phage in vitro. RESULTS: The unamplified human CRC cell HRT-18 cDNA library consisted of 6. 5 x 10(6) independent clones, recombinants clones was more than 96%. The titer of the amplified cDNA library was 1.1 x 10(9) pfu/ml and the average insert of the recombinants was 1.1 kb. CONCLUSION: The quality of the constructed cDNA library from human CRC cell HRT-18 is excellent and it is helpful to screen CRC specific antigen.

Colorectal Neoplasms↗
PubMedDate not supplied

[Reactions of rat fetal hepatocytes to hormones in primary cultures grown in selective media with an addition of glucocorticoid and barbiturate].

After growing of fetal rat hepatocytes in the medium containing cortisol and sodium barbital for 4-8 days and subsequent cultivation in the medium without glucocorticoid and barbiturate for 1-4 days insulin retained its ability to stimulate total RNA and protein synthesis, while human growth hormone kept its enhancing action on RNA biosynthesis. However, cortisol did not change protein synthesis and inhibited paradoxically incorporation of 3H-uridine into total RNA, after preincubation of cells in the selective medium. This suggests that prolonged exposure of cultured fetal rat liver cells to cortisol and sodium barbital may cause phenomena similar to those of hormonal and/or enzymatic imprinting.

Animals↗
PubMed1976

[Composition of nucleic acids and concentration of methylated bases in the DNA of Bifidobacterium bifidum var. Tissier].

Nucleotide composition of the sum total DNA of B. bifidum, biotype III, was determined by paper chromatography in combination with ultraviolet spectrophotometry. DNA of B. bifidum was referred to the GC-type (GC -- 62.6 mol%). Two additional nitrogen bases were present in the DNA composition; 5-methylcytosine and 6-methylaminopurine -- 0.45 mol% and 0.20 mol%, respectively. Nucleotide composition of the sum total RNA was studied with the aid of high-voltage electrophoresis in combination with ultraviolet spectrophotometry. The sum total RNA was referred to the high GC-type (GC -- 64.9 mol%). These data permit to consider it reasonable to refer bifidobacteria to the Bifidobacterium genus.

2-Aminopurine↗
PubMed2004

Abundance of mRNA encoding for components of the somatotropic axis and insulin receptor in different layers of the jejunum and ileum of neonatal calves.

Insulin-like growth factors-1 and -2, IGFBP-2 and -3, and receptors for IGF type-1 and type-2 (IGF-1R, IGF-2R), growth hormone (GHR), and insulin (InsR) in neonatal calves are variably expressed among gastrointestinal sites and thought to exert site-specific physiological functions. We studied by real-time reverse-transcription PCR, whether there are differences in the abundance of mRNA coding for IGF-I, IGF-2, IGFBP-2, IGFBP-3, IGF-1R, IGF-2R, GHR, and InsR in compartmentalized layers (fractions) of jejunum and ileum of 5-d-old calves fed colostrum. Samples of jejunum consisted primarily of villi and crypts; samples from ileum consisted mainly of villus tips, crypts, and lamina propria (LP; containing mainly Peyer's patches). After slaughter, segments of middle areas of jejunum and ileum were flushed with 154 mM NaCl. Pieces (5 mm x 5 mm) of jejunal (n = 9) and ileal walls (n = 5) were placed on glass slides and snap-frozen in liquid N before being cut horizontally into 10-mum-deep slices using a cryotome at -20 degrees C. Fifteen consecutive and morphologically similar slices were collected as fractions of villus, crypt, and LP layers, respectively. Fractions were characterized by use of 5'-bromo-2-deoxyuridine (BrdU) that labeled proliferating cells, and by expression of lactase mRNA. The BrdU-labeled cells were present in crypts and LP, but not in tips of villi. Lactase mRNA levels were greater in villus than crypt fractions, but lactase mRNA was absent in LP. In jejunum, mRNA levels, relative to levels of housekeeping genes (sum of levels of mRNA coding for ubiquitin, glyceraldehyde phosphate dehydrogenase, beta-actin, and ribosomal RNA), differed (P < 0.05) between fractions for InsR (crypts > villi), IGFBP-2 (crypts > villi), and IGFBP-3 (crypts > villi), and total RNA levels were greater (P < 0.05) in crypt than villus fractions. In ileum, mRNA levels, expressed relative to housekeeping genes, differed (P < 0.05) between fractions for IGF-I (LP > villi, crypts), IGF-2, and IGFBP-3 (villi > crypts, LP), GHR and InsR (crypts > LP), IGFBP-2 (crypts > villi, LP), and total RNA levels were greater (P < 0.05) in LP and crypt than in villus fractions. In conclusion, the tested fractionation technique is quite applicable for gene expression studies in the intestine of calves. Members of the somatotropic axis and of the insulin receptor are not equally expressed in different jejunal and ileal layers of neonatal calves.

Animals↗
PubMed1982

Expression and methylation of the mouse alpha-fetoprotein gene in embryonic, adult, and neoplastic tissues.

Expression of the mouse alpha-fetoprotein gene in embryonic, adult, and neoplastic tissues was assessed by RNA dot hybridization using 32P-labeled alpha-fetoprotein cDNA as probe, alpha-fetoprotein mRNA was present in high levels in total RNA from yolk sac endoderm, fetal liver, and an alpha-fetoprotein-producing hepatoma. In contrast, this mRNA was greatly depleted in total RNA from yolk sac mesoderm and essentially absent in brain, adult liver, and a non-alpha-fetoprotein-producing hepatoma. These results indicated that alpha-fetoprotein gene expression was controlled primarily at the transcriptional level. The presence of the modified base, 5-methylcytosine, in the alpha-fetoprotein gene was studied by comparing hybridization patterns obtained by Southern blot analysis of DNA cleaved with the restriction endonuclease isoschizomers Msp I and Hpa II. The gross sequence organization and reiteration frequency of the alpha-fetoprotein gene were invariant among the DNA samples, whereas, in each case, there was a positive correlation between hypomethylation of six CCGG (Hpa II) sites in the alpha-fetoprotein gene and expression of this gene. These Hpa II sites were distributed throughout a large portion of the alp]a-fetoprotein gene. Patterns of cytosine methylation in this gene were established before day 15 of gestation in yolk sac endoderm and mesoderm.

Animals↗
PubMed1998

Paracetamol increases sensitivity to ultraviolet (UV) irradiation, delays repair of the UNG-gene and recovery of RNA synthesis in HaCaT cells.

We have studied the effect of low levels of paracetamol (0.3 and 1.0 mM) on gene-specific DNA repair, recovery of total RNA synthesis and cytotoxicity after exposure of human keratinocyte cells (HaCaT) to ultraviolet (UV) irradiation. Repair of cyclobutane pyrimidine dimers (CPDs) was measured in the transcriptionally active uracil-DNA glycosylase (UNG) and c-MYC loci. Repair of both strands in the UNG gene was consistently lower in the presence of paracetamol, but this reduction reached significance only at 8 h after irradiation and no dose-response was observed. For the c-MYC gene, we found no significant effect of paracetamol on the repair of CPDs, possibly because UV-irradiation is known to induce transcription of the c-MYC gene and enhanced transcription coupled repair might counteract a negative effect of paracetamol on global genome repair. A dose-dependent delay in the recovery of total RNA synthesis after UV exposure was observed in the presence of paracetamol, which also caused a 20% increase in UV-induced cytotoxicity after 24 h. Paracetamol had no significant effect on either RNA synthesis or cell survival in the absence of UV after 24 h, but reduced cell survival by approximately 10% (at 0.3 mM) and 50%, (at 1.0 mM) after 96 h exposure. Our results demonstrate that paracetamol may inhibit gene-specific repair of CPDs by affecting global genome repair and that different genes may be differentially affected.

Acetaminophen↗
PubMed1995

An improved method for isolating RNA from coccidian oocysts.

A method, based on the disruption of eimerian oocysts in a French pressure cell in the presence of guanidine isothiocyanate, has been developed to isolate large quantities of high quality total RNA efficiently. This procedure results in a 12.5-fold greater number of oocysts broken, and a 22-fold greater yield of total RNA than from disruption by conventional grinding in liquid N2. In addition, the RNA isolated by the French pressure cell method was of equal or superior quality when compared to RNA isolated by grinding. This procedure provides a significant improvement in RNA extraction from eimerian oocysts and will greatly facilitate the study of gene expression in this important group of parasites.

Animals↗
PubMed2000

Quantitative RT-PCR measurement of human cytochrome P-450s: application to drug induction studies.

A quantitative RT-PCR assay has been developed that is able to measure the mRNA content of the major human CYPs (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5). The technique is highly specific, reproducible, rapid, and sensitive enough to quantitate low and high abundant mRNAs. The PCR primers were selected to specifically match each CYP mRNA, to have a very close annealing temperature, and to render PCR products of similar sizes. The PCR conditions were designed to allow the simultaneous measurement of the various human liver CYPs in a single run. To achieve precise and reproducible quantitation of each cytochrome mRNA, a external standard (luciferase mRNA) is added to the probes to monitor the efficiency of the RT step. The degree of amplification is estimated using appropriate cDNA standards and quantitation of the amplified products by fluorescent measurement. This assay can be used to quantify the most relevant CYPs in human liver and cultured human hepatocytes. CYPs 3A4 and 2E1 were the most abundant mRNAs in human liver (2.5 and 1.7 x 10(8) molecules/microgram of total RNA respectively), whereas 1A1 and 2D6 were the least abundant isoforms (1.2 and 2.1 x 10(6) molecules/microgram of total RNA). A similar pattern was also found in short-term cultured human hepatocytes. This technique is also suitable for assessing CYP mRNA induction by xenobiotics. Cells exposed to 3-methylcholanthrene showed a characteristic increased expression of CYP1A2 and 1A1 mRNAs. Upon incubation with phenobarbital and rifampin (rifampicin), human hepatocytes increased CYP 2B6, 3A4, and 3A5 among others.

Actins↗
PubMed2000

The hypoplastic heart in congenital diaphragmatic hernia: reduced expression of basic fibroblast growth factor and platelet-derived growth factor.

Newborn infants with congenital diaphragmatic hernia (CDH) still have high mortality. Recently, the possible role of a cardiac maldevelopment in the high mortality has been suggested. Human and animal studies have demonstrated that heart weight is significantly reduced in the presence of CDH. Basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) are pleiotropic regulatory peptides that are expressed in myocardium in precise developmental and spatial programs. PDGF and bFGF both stimulate cardiac growth by inducing cell proliferation and stimulating the synthesis of extracellular matrix. The aim of this study was to investigate the presence of heart hypoplasia in nitrofen-induced CDH in rats and the role of specific tissue growth factors (bFGF and PDGF) in its genesis. CDH was induced in pregnant rats following administration of 100 mg nitrofen on day 9.5 of gestation (term 22 days). In control animals the same dose of olive oil was given without nitrofen. Cesarean section was performed on day 21 of gestation. The fetuses were divided in two groups: normal controls (n = 8) and nitrofen-induced CDH (n = 8). Total RNA, DNA, and soluble proteins were extracted from the heart in each group and measured. mRNA was extracted from total RNA and a reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate mRNA expression of bFGF and PDGF. The heart/body weight ratio (HBWR) and DNA content were significantly decreased (P < 0.01) in CDH animals compared to controls. RNA and protein content were also reduced in CDH. The expression of bFGF and PDGF mRNA was significantly reduced in the CDH group compared to controls (P < 0.01). The decreased HBWR, DNA, RNA, and protein content in the CDH heart indicates that the heart is hypoplastic in nitrofen-induced left CDH. The downregulation of bFGF and PDGF gene expression in the CDH heart suggests that these regulating peptides may play an important role in the genesis of cardiac hypoplasia in CDH.

Actins↗