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PubMed1982

[Effect of malotilate (diisopropyl 1,3-dithiol-2-ylidenemalonate) on the synthesis and movement of RNA in rat liver (author's transl)].

The effects of malotilate (diisoporpyl 1,3-dithiol-2-ylidenemalonate) on the synthesis and movement of RNA in rat liver were studied in order to elucidate the mechanism of protein synthesis induced by this drug. Incorporation of 3H-cytidine into various RNAs was followed to determine the rates of their synthesis. Oral administration of malotilate to rats enhanced the total RNA synthesis, which reached to maximum level 3 hr after administration. At that time, the increases in the incorporation of radioactivities into the total RNA and the nucleotide pool were closely related to the dosages. Malotilate significantly increased the polyribosomal and t-RNA synthesis as well as nuclear RNA, but not the movement of RNA from the nuclei to the cytosol. It is not likely that malotilate activates specific genes in chromatin because only about 0.1 nmole of malotilate and its metabolites per mg DNA was found in chromatin. From these results, a possible mechanism of protein synthesis induced by malotilate can be proposed as follows: malotilate primarily affects the post-transcription process, promoting the RNA synthesis in the nuclear fraction without any changes of DNA synthesis, and these consequent increases of the polyribosomal RNA and t-RNA synthesis result in the enhancement of protein synthesis.

Animals↗

PubMed2003

Low-level arsenite induced gene expression in HEK293 cells.

Chronic, low-level exposure to arsenic frequently results in skin, lung, bladder, and kidney cancer. Since arsenic is primarily excreted via the kidney, this study focused on this target tissue. Gene array was used as a sensitive low-level monitor of the impact of arsenic on this target tissue. Arsenite [As(III)] was chosen as the chemical species of arsenic since As(III) species are touted as the cellular toxic form of arsenic. Human embryonic kidney cell line HEK293 cells were incubated with 1, 10, and 25 microM arsenite [As(III)] for 6 or 24 h. Total RNA from treated and control cells was isolated, reverse transcribed, and labeled with Cy3 or Cy5, and hybridized to a human cDNA microarray. Hybridizations were performed four times using independent total RNA preparations to ensure reproducibility. Raw data from 10 and 25 microM treated cells exposed for 6 h was normalized within, and between, hybridizations followed by identification of genes affected by arsenite exposure based on practical significance (2-fold change up or down) and reproducibility (affected in four of six measurements). In these studies, 20 genes (HMOX1, MT1E, or FOSL1, etc.) were up-regulated, and 19 genes (MYC, JAK1, or CENPE, etc.) were down-regulated. Genes identified at 10 and 25 microM arsenic exposure were then examined after 1 microM treatment for 6 or 24 h. Expression of affected genes showed a dose-dependent (1-25 microM) trend that was apparently not time-dependent (6 vs. 24 h). The affected genes indicate that even this realistic, low-level arsenite exposure was recognized by the HEK293 cells (e.g. metallothionein genes) and produced an oxidative stress (e.g. heme oxygenase gene). These affected genes were characterized as stress response genes, proto-oncogene, signaling molecules, transcription factors, chemokine receptors, proteolytic enzymes, ESTs, and unknown genes. These findings imply that arsenite induces complex cellular injury and the cellular adaptation to As(III) is associated with alterations in the expression of many genes.

Arsenites↗

PubMed2002

Tyrosine hydroxylase gene expression in ventrolateral medulla oblongata of WKY and SHR: a quantitative real-time polymerase chain reaction study.

The aims of this study were, first, to determine quantitatively the levels of tyrosine hydroxylase (TH) gene expression in both peripheral and central sites related to blood pressure regulation, and to compare the level of expression in Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Second, to see if any relationship exists between TH gene expression and systolic arterial blood pressure. Total RNA was isolated from adrenal glands and from tissue punches taken from the C1 and A1 cell groups in the rostral and caudal ventrolateral medulla oblongata of the brainstem, respectively. Total RNA was reverse-transcribed into cDNA followed by quantitative fluorescence detection polymerase chain reaction for TH cDNA. The levels of TH gene expression measured as a percentage of the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in SHR, were significantly higher ( approximately 2.5-fold) compared to WKY in all sites examined (P<0.01). There was a positive and significant relationship between systolic blood pressure and TH gene expression in the C1 area of the brainstem in both WKY (n = 5, P<0.05) and SHR (n=6, P<0.05). Taken together, these results suggest that elevated gene expression of the TH gene is associated with the phenotypic characteristic of SHR.

Animals↗

PubMed1999

Rat muscle fructose-1,6-bisphosphatase: cloning of the cDNA, expression of the recombinant enzyme, and expression analysis in different tissues.

The 1282 bp cDNA of an isoenzyme of fructose-1,6-bisphosphatase was cloned from rat muscle. It shows 70% positional identity to the cDNA of rat liver fructose-1,6-bisphosphatase and is clearly the product of a gene different from that coding for the liver enzyme. After cloning of the coding region of the rat muscle fructose-1,6-bisphosphatase cDNA in an expression vector, the recombinant enzyme could be detected in E. coli cell-free extracts by activity determination and Western blotting. Overexpressed fructose-1,6-bisphosphatase was found to be allosterically inhibited by AMP comparably to the enzyme isolated from rat muscle. Analysis of steady-state mRNA levels of various rat tissues with reverse-transcriptase polymerase chain reaction (RT-PCR) and Northern blotting revealed one or the two fructose-1,6-bisphosphatase isoenzyme mRNAs in most tissues tested with significant quantitative differences. Quantitative PCR using a homologous competitor showed that 1 microg of total RNA of rat muscle contains 1.7 x 10(6) molecules of rat muscle fructose-1,6-bisphosphatase mRNA. 3 x 10(4) copies of this message were found per microg total RNA of heart and kidney, respectively.

Amino Acid Sequence↗

PubMed2003

Gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells.

OBJECTIVE: To study the gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells. METHODS: Total RNA extracted from human bone marrow derived mesenchymal stem cells and tendon cells underwent reverse transcription, and the products were labeled with alpha-(32)P dCTP. The cDNA probes of total RNA were hybridized to cDNA microarray with 1176 genes, and then the signals were analyzed by Atlas Image analysis software Version 1.01a. RESULTS: Fifteen genes associated with cell proliferation and signal transduction were up-regulated, and one gene that takes part in cell-to-cell adhesion was down-regulated in tendon cells. CONCLUSION: The 15 up-regulated and one down-regulated genes may be beneficial to the orientational differentiation of mesenchymal stem cells into tendon cells.

Bone Marrow Cells↗

PubMed1991

[Quantification of mRNA coding for enterocyte fatty acid binding proteins (FABP) in rats: effect of high lipid diet and starvation].

We have tested in adult rat the specificity and the sensitivity of two cDNA probes derived from recombinant plasmids pJG19 and pJG418. The Northern blot analysis of total RNA extracted from small bowel reveals 0.9 and 0.7 kb mRNA corresponding respectively to the I and L-FABPc mRNA. A linear relation occurs between the amounts of input total RNA and the quantities of specific mRNA found by hybridization with cDNA probes (r = 0.991 and r = 0.998 for I and L-FABPc respectively). We have also evaluated the effect of drastic nutritional status: a high fat diet containing 45% of lipids and a starvation of three days. A two fold increase in I and L-FABPc mRNA occurs in starved rats. By contrast, only the I-FABPc mRNA are slightly enhanced after the hyperlipidic diet (50%). These preliminary results, which must be confirmed by further experiments, suggest a differential nutritional regulation of I and L-FABPc gene expression in rat intestine.

Animals↗

PubMed1988

DNA, RNA and total protein content of leg and breast muscles of White Rock chickens selected for 56-day body weight.

Pectoralis and gastrocnemius muscles from White Rock chickens divergently selected for 29 generations for high or low 56-day body weight were analyzed for DNA, RNA and protein (mg/g) content at 12 ages between hatch and 273 days of age. Dimorphism between lines was maximum at day 18 for both muscle types and then declined with age. High-line chickens generally deposited relatively more muscle tissue than those from the low line. Although nonadditive genetic variation was evident for absolute muscle weights, it was more frequent for muscle weight relative to body weight. For both muscle types, DNA unit number, as measured by DNA content (mg/g), was larger for the high line than the low immediately after hatch and smaller in the high than in the low line from day 10 to 56 after which lines were similar. RNA and protein/g muscle were similar for both lines at most ages. Between days 4 and 56, a period of rapid muscle growth, DNA unit size (protein/DNA) of both muscle types was larger in the high than in the low line. Heterosis was positive for protein, DNA unit number and size, and negative for RNA content. While weight of pectoralis muscle was similar to that of one gastrocnemius muscle on day 1, by day 273 its weight was over 3-fold greater. DNA unit number was higher in pectoralis than gastrocnemius muscle from hatch to day 4, similar on days 7 and 10 and lower for pectoralis muscle beyond day 10. RNA content was similar at all ages except 4, 7 and 10 days. DNA unit size followed the same pattern as DNA unit number; however, greater nuclei number at hatch for the high line corresponded with low DNA unit size. This pattern suggests a higher rate of cellular filling for pectoralis than gastrocnemius muscle.

Aging↗

PubMed2006

Comparative analysis of a 3' end tag PCR and a linear RNA amplification approach for microarray analysis.

BACKGROUND: Various types of amplification techniques have been developed in order to enable microarray gene expression analysis when the amount of starting material is limited. The two main strategies are linear amplification, using in vitro transcription, and exponential amplification, based on PCR. We have evaluated the performance of a linear and an in-house developed exponential amplification protocol that relies on 3' end tag sequences. We used 100 ng total RNA as starting material for amplification and compared the results with data from hybridizations with unamplified mRNA and total RNA. RESULTS: Preservation of expression ratios after amplification was examined comparing log(2) ratios obtained with amplification protocols to those obtained with standard labelling of mRNA. The Pearson correlations were 0.61 and 0.84, respectively, for the two linear amplification replicates and 0.76 and 0.80 for the two exponential amplification replicates. The correlations between repeated amplifications was 0.82 with the exponential method and 0.63 with the linear, indicating a better reproducibility with the PCR-based approach. CONCLUSION: Both amplification methods generated results in agreement with unamplified material. In this study, the PCR-based method was more reproducible than in vitro transcription amplification. Advantages with the in-house developed method are the lower cost since it is non-commercial and that the PCR generated product offers compatibility with both sense and antisense arrays.

3' Untranslated Regions↗

PubMed1996

Quantitative multistandard RT-PCR assay using interspecies polymorphism.

The use of RT-PCR to quantify mRNA is often compromised by the variability of reverse-transcription and amplification reactions as well as by the difficulty of assessing the amount and/or the integrity of input RNA. Use of a competitor RNA or the coamplification of an endogenous standard are widespread methods of monitoring these steps. Taking advantage of both sequence conservation between homologous genes in related animal species and interspecific polymorphism, a protocol that may be regarded as a compromise between these two methods is described here. Total RNA samples, extracted from even minute amounts of tissue belonging to a first animal species, were supplemented with a constant amount of total RNA prepared from a second animal species, which thus acts as a multistandard source. The mixture was reverse-transcribed using hexa-random primers. Separate PCRs were then undertaken so that, for each mRNA of interest, products from both origins could be distinguished. Since the ratio between amplified mRNAs is constant in the standard preparation, an accurate normalization in the assay samples of most variations inherent to PCR is obtained. This protocol allows quantification of several mRNAs species, whose amounts may be very different, in a single cDNA preparation.

Actins↗

PubMed1969

Characterization of ribosomes from the myxomycete Physarum rigidum grown in pure culture.

The plasmodial phase of the myxomycete Physarum rigidum, analyzed during the period of rapid growth, attained a ribonucleic acid (RNA) and protein content of 9.8 and 60.0%, respectively, on a dry weight basis. It possessed ribosomes of the 80S class which, especially in the absence of magnesium ions, partially dissociated to 60S and 40S subunit classes. Electron micrographs of ribosomes treated with uranyl acetate-lead citrate revealed a number of surface features. Nucleotide analyses of both ribosomal and total RNA disclosed that they were composed of 51.0 and 52.5% guanylic and cytidylic acids, respectively. Consistent with most reports on other organisms, guanylic acid was the most abundant nucleotide found in the various types of RNA and cytidylic acid was the least abundant. The S(0) (20,w) values of the total RNA classes, in 0.01 sodium acetate (pH 4.6) containing 0.10 m NaCl, were 5.2, 18.1, and 27.3 in S units. Changing the ionic environment of the RNA (0.017 molal potassium phosphate, pH 7.0, containing 0.01 m disodium ethylenediaminetetraacetate) resulted in a reduction of the S(0) (20,w) values to 4.2, 16.6, and 22.6 in S units, which is indicative of molecular conformational transitions. In general, the amino acid composition of the ribosomal proteins was similar to the data available on ribosomal proteins from other biological sources.

Amino Acids↗

PubMed2005

The quantification of cytochrome P-450 (CYP 3A4) mRNA in the blood of patients with viral liver diseases.

OBJECTIVE: We quantified cytochrome P-450 (CYP) 3A4 mRNA in the blood and liver of patients with viral liver diseases to determine whether CYP 3A4 expression is related to disease progression. DESIGN AND METHODS: Total RNA was extracted from 10 mL of blood from 12 normal volunteers, from 6 patients with acute hepatitis, 17 with chronic hepatitis, 12 with liver cirrhosis, and 16 with hepatocellular carcinoma. Total RNA from 1 mg of liver tissue was extracted simultaneously in 10 patients. CYP 3A4 mRNA was quantified by competitive reverse-transcription polymerase chain reaction and expressed as log copies/microliter. RESULTS: The CYP 3A4 mRNA titer in blood correlated with that of the liver (r = 0.65, P < 0.05). The CYP 3A4 mRNA titer was 1.6 +/- 0.4 in normal controls, 1.0 +/- 0.5 in acute hepatitis, 0.7 +/- 0.2 in chronic hepatitis, 0.5 +/- 0.2 in liver cirrhosis, 0.5 +/- 0.2 in hepatocellular carcinoma, and decreased with progression of liver disease (P < 0.05). CONCLUSION: These data suggest that the CYP 3A4 mRNA level in blood relates to progression of liver disease.

Adult↗

PubMed2005

Rare cancer cell analyzer for whole blood applications: automated nucleic acid purification in a microfluidic disposable card.

One current challenge facing point-of-care cancer detection is that existing methods make it difficult, time consuming and too costly to (1) collect relevant cell types directly from a patient sample, such as blood and (2) rapidly assay those cell types to determine the presence or absence of a particular type of cancer. We present a proof of principle method for an integrated, sample-to-result, point-of-care detection device that employs microfluidics technology, accepted assays, and a silica membrane for total RNA purification on a disposable, credit card sized laboratory-on-card ('lab card") device in which results are obtained in minutes. Both yield and quality of on-card purified total RNA, as determined by both LightCycler and standard reverse transcriptase amplification of G6PDH and BCR-ABL transcripts, were found to be better than or equal to accepted standard purification methods.

Fusion Proteins, bcr-abl↗

PubMed1977

Effect of thiocyanate on mammary gland growth in rats.

In growing rats from 3 weeks to 3 months old, 0.1% potassium thiocyanate (KSCN) decreased the plasma protein-bound iodine level by 47% and the total DNA in the mammary gland by 11%, but had no effect on the total RNA. Administration of thiocyanate in the form of 0.1, 0.3 and 0.5% KSCN during pregnancy and 14 d of lactation lowered the protein-bound iodine level by 48-66%, the total DNA by 11-47% and the total RNA by 14-63%, depending on the KSCN dosage. The 14-d-old litters of rats receiving thiocyanate weighed 4-41% less than those in the control group.

Animals↗

PubMed2002

Amine-modified random primers to label probes for DNA microarrays.

DNA microarrays have been used to study the expression of thousands of genes at the same time in a variety of cells and tissues. The methods most commonly used to label probes for microarray studies require a minimum of 20 microg of total RNA or 2 microg of poly(A) RNA. This has made it difficult to study small and rare tissue samples. RNA amplification techniques and improved labeling methods have recently been described. These new procedures and reagents allow the use of less input RNA, but they are relatively time-consuming and expensive. Here we introduce a technique for preparing fluorescent probes that can be used to label as little as 1 microg of total RNA. The method is based on priming cDNA synthesis with random hexamer oligonucleotides, on the 5' ends of which are bases with free amino groups. These amine-modified primers are incorporated into the cDNA along with aminoallyl nucleotides, and fluorescent dyes are then chemically added to the free amines. The method is simple to execute, and amine-reactive dyes are considerably less expensive than dye-labeled bases or dendrimers.

3T3 Cells↗

PubMed1991

A novel transcriptional response by the cat gene during slow growth of Escherichia coli.

A novel response to growth rate was found with expression of the chloramphenicol acetyltransferase (cat) gene in Escherichia coli. The amount of cat mRNA relative to total RNA increased about 11-fold as growth rates decreased 5- to 6-fold, without an increase in translation. The accumulation of cat mRNA was in contrast to decreased cellular concentrations of total RNA, trxA, ompA, or 23S rRNA as the growth rate decreased and was not due to changes in gene dosage or mRNA stability. Stability of the cat mRNA does not appear to be regulated by growth rate. No significant change in either chemical or functional stability was observed within a five- to sixfold range of growth rates when chemostat-grown cells were used. However, cat mRNA stability was affected by growth medium composition. The half-life of cat mRNA decreased about threefold, with an approximate fourfold increase in generation time due to changes in growth medium. Transcriptional studies have indicated that accumulation of cat mRNA at slow growth rates is the result of a specific transcriptional response to changes in cellular generation times. We propose that increases in the cellular concentration of a specific message at slow growth rates may reflect an additional type of survival response in E. coli.

Cell Division↗

PubMed1990

Regulation of 5'-aminolevulinate synthase activity in overloaded skeletal muscle.

The regulation of the mitochondrial enzyme 5'-aminolevulinate synthase (ALV synthase) activity during chronic weight-bearing activity (overload) in chicken skeletal muscle was investigated. Maximal enzyme activity was increased 2.5- and 4.0-fold after 3 and 7 days of overload. The content of ALV synthase mRNA (ng/mg total RNA) was not changed after 3 days but increased (20%; P less than 0.05) after 7 days of overload. Normalizing the content of ALV synthase mRNA relative to the increase in total RNA indicated that ALV synthase mRNA increased by 1.6- and 2.0-fold at 3 and 7 days, respectively. On this basis, the increase in enzyme activity per gram protein exceeded the increase in mRNA content per gram protein by 60-70%. During overload, the activity of cytochrome oxidase was unchanged after 3 days but increased by 1.5-fold (P less than 0.05) after 7 days of overload. The data indicate that 1) the initial rise in ALV synthase mRNA and activity due to overload occurs in the absence of a prior change in the level of cytochrome oxidase, an enzyme that requires heme for its assembly, and 2) induction of ALV synthase activity is regulated largely by processes at the translational or posttranslational steps.

5-Aminolevulinate Synthetase↗

PubMed2002

Ghrelin expression in islet cell tumors: augmented expression of ghrelin in a case of glucagonoma with multiple endocrine neoplasm type I.

Ghrelin is a 28-amino acid peptide that regulates GH release together with GHRH and somatostatin. The expression of ghrelin has been detected in the stomach, small intestine, hypothalamus, pituitary gland, kidney, placenta, and testis. Recently it was reported that ghrelin is present in pancreatic alpha-cells and that it stimulates insulin secretion. In this study, we examined the ghrelin expression in two cases of glucagonoma and two cases of insulinoma by Northern blot analysis and immunohistochemistry. Ghrelin expression was identified in a case of glucagonoma associated with multiple endocrine neoplasm type I both by Northern blot analysis using total RNA and by immunohistochemistry, although the plasma ghrelin level was not elevated. This is the first case of tumor in which ghrelin gene expression was detected by Northern blot analysis using total RNA.

Adenoma, Islet Cell↗

PubMed2003

Evaluation of RNA messengers involved in lipid trafficking of human intestinal cells by reverse-transcription polymerase chain reaction with competimer technology and microchip electrophoresis.

We developed a semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) procedure based on the combination of competimer technology with microchip electrophoresis. The approach was applied to total RNA extracts from the human colon carcinoma cell line CaCo-2 cultured for 22 days onto tissue plate inserts that allow the polarized cell growth. At time of experiment these cells were incubated for 2 h with lipid micelles containing either cholesterol or a mixture cholesterol/beta-sitosterol or serum-free Dulbecco's modified Eagle medium (DMEM) alone. Total RNA was extracted from cell pellets by silica membrane spin columns and reverse-transcribed by a genetically engineered M-MuLV enzyme and an oligo (dT)(15) primer. The cDNA underwent PCR amplification for the following genes: apolipoprotein A1 (ApoAI), apolipoprotein E (ApoE), ATP-binding cassette A1 (ABCA1), liver X receptor alpha (LXRalpha) and farnesoid X receptor (FXR). The beta-actin gene, used as an endogenous reference, was coamplified with mixtures of primer and competimer at a ratio leading to a reference band of intensity comparable to that of the gene under analysis. Amplification products were separated and quantitated by microchip electrophoresis in order to determine, for each gene, the optimum cycle numbers and primer to competimer ratios for the beta-actin therefore used for evaluating the relative variations of gene expressions in the different experiments. The incubation with cholesterol micelles stimulated both LXR and FXR expression that was accompanied by an increased expression of ABCA1 and ApoAI genes (1.4- and 1.5-fold, respectively) and halved the APOE expression. The effect on ABCA1 and ApoAI expression was even stronger (5.7- and 2.6-fold, respectively) with beta-sitosterol-containing micelles. Similar changes are in line with previous findings and suggest that the short incubation with beta-sitosterol-enriched micelles stimulates a specific mechanism that, via ABCA1 activation, can increase the cholesterol and phytosterol basolateral efflux.

Base Sequence↗