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PubMed2003

Methods to optimize the generation of cDNA from postmortem human brain tissue.

The analysis of gene transcript levels in postmortem human brain is a valuable tool for the study of neurological and psychiatric diseases. Optimization of the methods of RNA extraction and cDNA generation is particularly important in this application because postmortem human brain tissue is in limited supply and generally yields less RNA than many other human tissues. We compared column extraction and solvent extraction for total RNA, reverse transcription (RT) with random hexamers versus oligo-dT priming, and incubation of the RNA with or without DNase for effect on the cDNA product derived from the same homogenized pool of postmortem human frontal cortex. The total RNA obtained from the solvent method was found to be less stable at room temperature and to contain a higher proportion of non-messenger RNA than that obtained from the column method. Evaluating the RT-PCR results per wet weight of tissue extracted, we found that the signal strength was increased >20-fold by a protocol of Qiagen RNeasy column extraction, random hexamer RT priming and omitting DNase treatment of the RNA.

Brain Chemistry↗
PubMed1995

Analysis of viral RNA persistence in seawater by reverse transcriptase-PCR.

It is important to determine the stability of naked viral RNA in seawater, since false-positive results can occur when reverse transcriptase-PCR (RT-PCR) is used to detect viruses if the RT-PCR amplifies free RNA instead of RNA from intact viruses. An acid guanidinium thiocyanate-phenol-chloroform method was used to extract total RNA from a filtered poliovirus cell culture suspension. The sensitivity of detection in this viral RNA study was 600 fg when RT-PCR was used. The extracted total RNA was seeded into filtered and unfiltered seawater, and the resulting preparations were incubated at 4 degrees C and at room temperature (23 +/- 1 degrees C). Our results showed that the seeded RNA was more stable in filtered seawater than in unfiltered seawater at both temperatures. The viral RNA could not be detected by the RT-PCR after 2 days of incubation in unfiltered seawater and after 28 days of incubation in filter-sterilized seawater. Therefore, because of the relatively short life of viral RNA in natural water, the detection of virus in environmental samples by the RT-PCR was mainly due to the presence of well-protected viral particles and not due to the presence of naked viral RNA.

Base Sequence↗
PubMed2001

[Reverse transcriptase PCR (RT-PCR) and quantitative-competitive PCR (QC-PCR)].

Due to its extraordinarily high sensitivity, polymerase chain reaction (PCR) is widely used for amplifying cDNA copies of low abundance mRNA. However, quantitation is unreliable because the amount of PCR product increases exponentially with each cycle of amplification; therefore, minute differences in any of the variables that affect the efficiency of amplification can dramatically alter product yield. Coamplification with a different reporter gene product is a semiquantitative mRNA quantitation method. Thus, we constructed an internal standard with a defined deletion fragment from the target cDNA, and used the same primers to coamplify the unknown and the competitor, allowing us to quantify the amount of specific target cDNA available. In addition, because the efficiency of amplification of the internal control molecules is identical to that of the target template, quantitative PCR can avoid the discrepancies associated with tube-to-tube or sample-to-sample variations in the kinetics of the RT reaction. For RNA quantitation, Northern blots are widely used. However, the Northern blot technique requires at least 10mg of total RNA for semiquantitation while QC-PCR requires only 1mg of total RNA and is useful when only small amounts of tissue are available. QC-PCR is a simple, and inexpensive method in which competitive PCR is used for highly accurate quantitation of mRNA and DNA from a small number of cells.

Blotting, Northern↗
PubMed2002

Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA.

Analysis of transcript representation on gene microarrays requires microgram amounts of total RNA or DNA. Without amplification, such amounts are obtainable only from millions of cells. However, it may be desirable to determine transcript representation in few or even single cells in aspiration biopsies, rare population subsets isolated by cell sorting or laser capture, or micromanipulated single cells. Nucleic-acid amplification methods could be used in these cases, but it is difficult to amplify different transcripts in a sample without distorting quantitative relationships between them. Linear isothermal RNA amplification has been used to amplify as little as 10 ng of total cellular RNA, corresponding to the amount obtainable from thousands of cells, while still preserving the original abundance relationships. However, the available procedures require multiple steps, are labor intensive and time consuming, and have not been shown to preserve abundance information from smaller starting amounts. Exponential amplification, on the other hand, is a relatively simple technology, but is generally considered to bias abundance relationships unacceptably. These constraints have placed beyond current reach the secure and routine application of microarray analysis to single or small numbers of cells. Here we describe results obtained with a rapid and highly optimized global reverse transcription#150;PCR (RT-PCR) procedure. Contrary to prevalent expectations, the exponential approach preserves abundance relationships through amplification as high as 3 x 10(11)-fold. Further, it reduces by a million-fold the input amount of RNA needed for microarray analysis, and yields reproducible results from the picogram range of total RNA obtainable from single cells.

Cluster Analysis↗
PubMed2003

[Expression and purification of the N-domain of human canstatin and its bioactivity].

Total RNA was extracted from placenta umbilical tissue. The canstatin cDNA was amplified from total RNA by net-RT-PCR technique and cloned into pSP72, and the resulted plasmid pSP72C was used as template to amplify its N-domain. The amplified 1-89 aa N-domain was then cloned into pET-3c. The resulted plasmid pET-CN was transformed into E. coli BL21(DE3). The N-domain was efficiently expressed after IPTG induction as a 10 kD band on SDS-PAGE. The expressed product accounted for approximately 35.3% of the total bacterial proteins, as estimated by densitometry and existed mainly as inclusion body. The inclusion bodies were washed, lysed and the reactivated proteins were purified by the Sephadex G-100 gel filtration to a purity of 92.6%. CAM assay showed that N-domain effectively inhibited the angiogenesis of chicken embryo microcapillary vessel.

Angiogenesis Inhibitors↗
PubMed1992

Transcriptional regulation of rat microsomal epoxide hydrolase gene by imidazole antimycotic agents.

The effects of imidazole antifungal agents, including ketoconazole, clotrimazole, miconazole, and econazole, on the expression and regulation of microsomal epoxide hydrolase (mEH) were examined in rat hepatic tissue (doses of agents, 150 mg/kg of body weight/day, orally). Immunoblot analyses revealed that administration of either ketoconazole or clotrimazole caused a approximately 4-5-fold increase in mEH levels, whereas either miconazole or econazole resulted in a approximately 7-fold increase in mEH at day 3 after treatment. RNA hybridization analyses, probed with a 1.3-kilobase mEH cDNA, revealed that administration of these imidazole antifungal agents caused substantial elevation of hepatic mEH mRNA in total RNA. Hepatic mEH mRNA levels in total RNA were elevated approximately 11-, 15-, and 18-fold at 12, 24, and 72 hr, respectively, after ketoconazole treatment, whereas mEH mRNA levels were increased approximately 14-, 19-, and 22-fold, respectively, relative to control, at the same time points after clotrimazole treatment. The rate of increase of mEH mRNA caused by miconazole was more rapid than the rates observed for the other agents examined, with a maximal increase in mRNA being noted at 12 hr after treatment. The degree of mEH mRNA increase after 3 consecutive days of miconazole treatment was appreciably less than that observed at 12 hr after a single treatment. Econazole caused a maximal increase at 24 hr and subsequent decline in mEH mRNA levels after 3 consecutive days of treatment. Elevation of mEH mRNA levels by these antimycotic agents was confirmed in poly(A)+ RNA, as assessed by both Northern and slot blot hybridization analyses. Nuclear run-on analyses revealed that administration of ketoconazole, clotrimazole, or miconazole stimulated the rate of mEH gene transcription at 12 hr after treatment by 11-, 8.5-, and 9-fold, respectively, compared with control, whereas econazole resulted in a 4-fold increase in the rate of mEH gene transcription at the same time point. The transcription rates of mEH mRNA at 24 hr were significantly less than those observed at 12 hr after a single treatment with either ketoconazole, miconazole, or econazole, resulting in 6.5-, 2.5-, and 2-fold increases, respectively, relative to control. Clotrimazole, however, maintained the activated mEH transcription rate at 24 hr after treatment, exhibiting a 11-fold increase, compared with control. These results provide evidence that the imidazole antimycotic agents induce mEH and that the mEH induction involves large increases in mRNA, with transcriptional activation.

Animals↗
PubMed2004

Gene array analysis of macronodular adrenal hyperplasia confirms clinical heterogeneity and identifies several candidate genes as molecular mediators.

Corticotropin (ACTH)-independent macronodular adrenal hyperplasia (AIMAH) is a heterogeneous condition in which cortisol secretion may be mediated by gastrointestinal peptide (GIP), vasopressin, catecholamines and other hormones. We studied the expression profile of AIMAH by genomic cDNA microarray analysis. Total RNA was extracted from eight tissues (three GIP-dependent) and compared to total RNA obtained from adrenal glands from 62 normal subjects. Genes had to be altered in 75% of the patients, and be up- or downregulated at a cutoff ratio of at least 2.0; 82 and 31 genes were found to be consistently up- and downregulated, respectively. Among the former were regulators of transcription, chromatin remodeling, and cell cycle and adhesion. Downregulated sequences included genes involved in immune responses and insulin signaling. Hierarchical clustering correlated with the two main AIMAH diagnostic groups: GIP-dependent and non-GIP-dependent. The genes encoding the 7B2 protein (SGNE1) and WNT1-inducible signaling pathway protein 2 (WISP2) were specifically overexpressed in the GIP-dependent AIMAH. For these, and six more genes, the data were validated by semiquantitative amplification in samples from a total of 32 patients (the original eight, six more cases of AIMAH, and 18 other adrenocortical hyperplasias and tumors) and the H295R adrenocortical cancer cell line. In conclusion, our data confirmed AIMAH's clinical heterogeneity by identifying molecularly distinct diagnostic subgroups. Several candidate genes that may be responsible for AIMAH formation and/or progression were also identified, suggesting pathways that affect the cell cycle, adhesion and transcription as possible mediators of adrenocortical hyperplasia.

Adrenal Cortex Neoplasms↗
PubMed1999

[Relationship between methylation status of gamma-glutamyl transpeptidase (GGT) genes and abnormal expression of its enzyme proteins in tissues of human hepatomas].

OBJECTIVE: To explore the mechanism of expression and alteration of gamma-glutamyl transpeptidase (GGT) genes during the development of human hepatomas. METHODS: The total GGT protein and total RNA were purified in human hepatomas, adjacent paracancerous and distal cancerous tissues. The specific activities of total GGT, membrane-combine GGT and soluble GGT were investigated, the GGT gene of 5'-NC region was amplified by using a nest RT-PCR assay, and methylation status of GGT gene M3 site were analyzed in the present study. RESULTS: An increasing tendency (P < 0.05) of total RNA concentrations was found from cancer in distal cancerous tissues; the specific activities (U/g) of total GGT, membrane-combine GGT and soluble GGT were significantly higher (P < 0.05) in hepatomas than those in adjacent paracancerous or and that distal cancerous tissues; and the frequencies of amplified fragment and hypomethylated M3 site of GGT 5'-NC region genes were 100% and 75% in hepatomas, 85% and 55% in adjacent cancerous, and 75% and 50% in distal cancerous tissues, respectively. CONCLUSIONS: The present data suggest that the abnormal expression of GGT proteins in hepatomas was related to hypomethylation status of GGT genes, and that the fragment analysis of the GGT genes might be a sensitive assay to monitor the hepatic cell canceration.

Carcinoma, Hepatocellular↗
PubMed1980

[RNA-metabolism in aging (author's transl)].

After intraperitoneal application of 14C-orotic acid the tracer distribution and the labelling of total RNA in different organs of two groups of Wistar rats (5 and 22,5 months old) were represented. After 60 min 2/3 of tracer were found in liver and kidney. The uptake by liver of old animals is decreased significantly. The interactions between the tracer distribution in the organism, the time related uptake by the organs, the organ function (influenced by methimazol) and the specific activity of RNA were represented. From the results the conclusion is drawn that the comparison of the specific activities of total RNA alone--especially in comparison of young with old animals--is not sufficient for the examination of RNA synthesis.

Aging↗
PubMed1978

Induction of endogenous guinea pig retrovirus by 5-bromodeoxyuridine: amplification of virus-specific RNA.

Treatment of normal guinea pig embryo cells with 5-bromodeoxyuridine (BUdR) activates endogenous guinea pig retrovirus. In this report the effect of BUdR treatment upon the level of endogenous retroviral RNA in normal guinea pig embryo cells was determined by using hybridization of viral complementary DNA (cDNA) to cellular RNA. We found that 0.0075% (120 copies per cell) of total RNA of untreated cells was virus-specific, whereas 0.32% (5,120 copies per cell) of total cellular RNA obtained from cells 48 h after BUdR treatment was virus-specific. Thus, BUdR causes an approximately 40-fold amplification of virus-specific RNA after 48 h of treatment. Several lines of evidence favor the hypothesis that the amplification of virus-specific RNA observed after BUdR treatment involves enhancement of transcription rather than an alteration of post-transcriptional processing. At different times after BUdR treatment, similar increases in virus-specific RNA concentration occur in both nucleus and cytoplasm. After 48 h of BUdR treatment, nuclear virus-specific RNA increased 99-fold, from 29 copies per cell to 2,880 copies per cell, whereas cytoplasmic virus-specific RNA increased 47-fold from 85 copies per cell to 4,000 copies per cell. Decay rates of virus-specific RNA in the presence of actinomycin D were similar in the presence or absence of BUdR, indicating that BUdR does not stabilize virus-specific RNA. In BUdR-treated cells the t1/2 of virus-specific RNA was 170 min either in the continued presence of BUdR or after the removal of BUdR, and 150 min in untreated cells. The size distribution of nuclear virus-specific RNA sequences, after denaturation with dimethyl sulfoxide, was similar in untreated and BUdR-treated cells, suggesting similar nuclear processing of viral RNA in both untreated and BUdR-treated cells. The accumulation of nuclear precursors to 38S virus-specific RNA was not observed at steady-state levels in untreated or BUdR-treated cells. Similar species of virus-specific RNA (14S 24S, 38S, and 70S) were present in the total cellular RNA of untreated and BUdR-treated cells. Additionally, virus-specific RNA was present in purified polyribosomes of untreated cells. Finally, direct analysis of the amount of radiolabeled virus-specific RNA in nuclear RNA pulse-labeled for 30 min with [3H]uridine was performed by the method of Coffin et al. (J. Mol. Biol. 86:373-396, 1977) for quantitative determination of pulse-labeled virus-specific RNA. It was found that labeled virus-specific RNA comprised 0.0035 to 0.004% of the total pulse-labeled nuclear RNA of cells treated for 48 h with BUdR. This 50-fold increase in radiolabeled virus-specific RNA may full- account for the 40-fold increase in steady-state levels of virus-specific RNA observed after 48 h of BUdR treatment.

Animals↗
PubMed2004

[Effects of 17 beta-estradiol on the gene expression of shear stressed endothelial cells].

OBJECTIVE: To determine the protective effect of 17 beta-estradiol on endothelial cells under shear stress. METHODS: cDNA microarray technology was adopted in the analysis of gene expression of cultured human umbilical vein endothelial cells (HUVECs) that had been incubated by 17 beta-estradiol for 48 h and then exposed to low fluid shear stress (4.20 dyne/cm2) for 2 h, Normal static cultured HUVECs were selected as control. Total RNA from normal static cultured HUVECs was labeled by Cy3-dCTP, and the total RNA of HUVECs incubated by 17 beta-estradiol (10(-7) mol/L) and then exposed to low shear stress was labeled by Cy5-dCTP. The expression ratios reported are the average from the two separate experiments. RESULTS: A total of 384 genes (approximately 0.094%) exhibited differential expression. 226 genes were up-regulated and 158 genes were down-regulated. The transcription protein of up-regulated genes related to synthesis of PGI2, macrophage phagocytosis, and so on. The transcription protein of down-regulated genes related to degradation of fibrinogen, and to production of protooncogene. CONCLUSION: Estrin at the physiological concentration could protect impaired endothelial cells. Its protective effect may be related with some genes.

Cells, Cultured↗
PubMed2002

Upregulation of vasopressin V2 and aquaporin 2 in the inner medullary collecting duct of cardiomyopathic hamsters is attenuated by enalapril treatment.

Previous studies showed that aquaporin 2 (AQP2) is elevated in the kidney of the heart failure rat suggesting that an increased amount of AQP2 contributes to water retention in heart failure. We performed the present study to determine whether angiotensin II play a role in causing an increase in the expression of arginine vasopressin (AVP) V2 and AQP2 mRNA in the kidney of the cardiomyopathic hamster. The expression of AVP V2 and AQP2 mRNA in the inner medullary collecting duct (IMCD) was measured by competitive reverse transcriptase-polymerase chain reaction (RT-PCR) before and after treatment with an angiotensin-converting enzyme inhibitor, enalapril. Our results showed that the expression of AVP V2 (0.53 +/- 0.05 v 1.03 +/- 0.15 amol/microg of total RNA, P <.01) and AQP2 mRNA (0.027 +/- 0.002 v 0.036 +/- 0.002 amol/microg of total RNA, P <.05) in the IMCD of the cardiomyopathic hamster is upregulated. Treating the cardiomyopathic hamster with enalapril for 7 days negated the changes. In situ hybridization experiments confirmed the intensity of the signals for both AVP V2 and AQP2 mRNA was more intense in the IMCD of the cardiomyopathic hamster. Enalapril treatment reduced the signal intensity to a level comparable to the normal hamster. These results suggested that the increases in the expression of AVP V2 and AQP2 mRNA are mediated by angiotensin II.

Angiotensin II↗
PubMed2004

Quantitative evaluation of catecholamine enzymes gene expression in adrenal medulla and sympathetic Ganglia of stressed rats.

Stress-induced changes in mRNA levels of tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) have been expressed as relative arbitrary units compared with a control group. The aim of this study was to quantify basal and stress-induced levels of TH, DBH, and PNMT mRNAs in rat adrenal medulla (AM) and stellate ganglia (SG) by the RT-competitive PCR method using corresponding competitors of known concentration. In rats stressed by immobilization (IMO) once for 2 h, the concentration of mRNAs was determined in various intervals after the end of stress stimulus. In SG, the basal concentration of TH mRNA was 0.017 amol/ng of total RNA, which is approximately 30 times lower than in the AM (0.460 amol/ng RNA). The basal concentration of DBH mRNA in SG was 2.60 amol/ng of total RNA, which is about 150 times more than TH mRNA in SG but only two times less than DBH mRNA in the AM in which PNMT mRNA is present in the highest concentration. After a single 2-h IMO, the peak elevation of TH and DBH mRNA concentration in SG occurred 24 h after the termination of stress stimulus, when their AM mRNA concentrations were already at control values. Presence of PNMT mRNA levels in the SG, of control and stressed rats has been demonstrated for the first time. Repeated IMO (7 days, 2 h daily) did not produce further increase in the mRNA concentrations compared with the elevated values found in adapted control groups. Levels of TH protein were significantly increased only after repeated IMO in SG and AM. Thus, our data show for the first time the exact concentrations of TH, DBH, and PNMT mRNA in SG and AM of rats under control and stress conditions. The lowest concentration of TH mRNA in the AM and SG supports the hypothesis that tyrosine hydroxylation is the rate-limiting step in catecholamine biosynthesis.

Adrenal Medulla↗
PubMed1990

Alteration in the expression of genes for cholesterol side-chain cleavage enzyme and 21-hydroxylase by hypophysectomy and ACTH administration in the rat adrenal.

The changes in steady-state levels of mRNA for cholesterol side-chain cleavage cytochrome P-450 (P-450scc) and steroid 21-hydroxylase cytochrome P-450 (P-450c21) caused by hypophysectomy and ACTH treatment were determined in rat adrenals. Hypophysectomy caused marked decreases in adrenal weight and total RNA per gland. Administration of ACTH resulted in increases in adrenal weight and total RNA. A significant correlation between the amount of RNA and adrenal weight was observed. Both P-450scc and P-450c21 mRNAs were decreased by hypophysectomy and increased by ACTH treatment. P-450scc mRNA decreased to 20% and P-450c21 mRNA to 76% of control values 1 day after hypophysectomy. ACTH caused a significant increase in P-450scc mRNA after 3 h. However, a significant increase in P-450c21 mRNA was observed 12 h after administration of ACTH. These results are concordant with previous studies in vitro utilizing cultured adrenocortical cells. Moreover, the induction of steady-state levels of P-450scc mRNA was faster than that observed by other investigators in studies in vitro. These results may indicate that integrity of the adrenal gland in vivo is important for the action of ACTH.

Adrenal Glands↗
PubMed2001

Gene profiling of human fetal and adult adrenals.

The mechanisms that lead to the steroidogenic differences in the human fetal adrenal (HFA) and adult adrenal gland are not known. However, gene expression clearly plays a critical role in defining their distinct steroidogenic and structural phenotypes. We used DNA microarrays to compare expression levels of several thousand transcripts between the HFA and adult adrenal gland. Total RNA was isolated from 18 HFA and 12 adult adrenal glands. Samples of total RNA were used to make five pools of poly A+ RNA (mRNA). Gene profiling was done using five independent microarrays that contained between 7075 and 9182 cDNA elements. Sixty-nine transcripts were found to have a greater than 2.5-fold difference in expression between HFA and adult adrenals. The largest differences were observed for transcripts that encode IGF-II (25-fold higher in HFA) and 3beta-hydroxysteroid dehydrogenase (24-fold higher in adult). Among the other genes, transcripts related to sterol biosynthesis or to growth and development were higher in the HFA than adult adrenals. Transcripts concerned with cellular immunity and signal transduction were preferentially expressed in the adult adrenal. The vast majority of the 69 transcripts have not been studied with regard to adrenal function. Thus, these gene profiles provide valuable information that could help define the mechanisms that control adrenal function.

3-Hydroxysteroid Dehydrogenases↗
PubMed2001

Expression of insulin target genes in skeletal muscle and adipose tissue in adult patients with growth hormone deficiency: effect of one year recombinant human growth hormone therapy.

Our aim was to investigate the effects of one year recombinant human growth hormone (rhGH) therapy on the regulation by insulin of gene expression in muscle and adipose tissue in adults with secondary GH deficiency (GHD). Six GHD subjects without upper-body obesity were submitted to a 3-h euglycemic hyperinsulinemic clamp before and after one year of rhGH therapy. Muscle and abdominal subcutaneous adipose tissue biopsies were taken before and at the end of each clamp. The mRNA levels of insulin receptor, p85 alpha-phosphatidylinositol-3 kinase (p85 alpha PI-3K), insulin dependent glucose transporter (Glut4), hexokinase II, glycogen synthase, lipoprotein lipase (LPL) in muscle and in adipose tissue, hormone sensitive lipase and peroxisome proliferator-activated receptor gamma (PPAR gamma) in adipose tissue were quantified by RT-competitive PCR. One year treatment with rhGH (1.25 IU/day) increased plasma IGF-I concentrations (54+/-7 vs 154+/-11 ng/ml, P<0.01) but did not affect insulin-stimulated glucose disposal rate measured during the hyperinsulinemic clamp (74+/-9 vs 85+/-5 micromol/kg free fat mass/min). Insulin significantly increased p85 alpha PI-3K, hexokinase II and Glut4 mRNA levels in muscle both before and after rhGH treatment. One year of GH therapy increased LPL mRNA levels in muscle (38+/-2 vs 70+/-7 amol/microg total RNA, P<0.05) and in adipose tissue (2490+/-260 vs 4860+/-880 amol/microg total RNA, P<0.05), but did not change the expression of the other mRNAs. We conclude from this study that GH therapy did not alter whole body insulin sensitivity and the response of gene expression to insulin in skeletal muscle of adult GHD patients, but it did increase LPL expression in muscle and adipose tissue. This result could be related to the documented beneficial effect of GH therapy on lipid metabolism.

Adenoma↗
PubMed1990

Suppression of host mRNA in human smooth muscle cells by a virion competent factor in herpes simplex virus type 1.

Herpes simplex virus type 1 produces an early decrease in protein synthesis in cultured smooth muscle cells isolated from human umbilical artery. To confirm that suppression of host protein synthesis occurs before virus protein expression, cultures were treated with actinomycin D (5 micrograms/ml) continuously from the beginning of infection. In the presence of actinomycin D, by 3.5 hours postinfection, virus-infected cultures showed much less incorporation of [35S]methionine into sodium dodecyl sulfate-polyacrylamide electrophoresis-separated products than did identically treated mock-infected cultures. In the absence of actinomycin D, there was incorporation of radiolabel into viral proteins as early as 4.5 hours postinfection. Hybridization of slot-blotted total RNA demonstrated that virus-infected cultures treated with and without actinomycin D showed a marked decrease in hybridizable RNA by 2 hours PI when compared with mock-infected controls. This decrease was seen with cDNA probes for the mRNA for secreted extracellular matrix macromolecules (fibronectin, thrombospondin and collagen chains alpha 2(I), alpha 1(III] as well as with cDNA probes for RNA of intracellular macromolecules actin and beta-tubulin. In vitro translation of total RNA isolated from infected human smooth muscle cells at 2 and 5 hours postinfection resulted in far less [35S]methionine incorporation into sodium dodecyl sulfate-polyacrylamide electrophoresis-separated products than would be predicted from the amount of hybridizable RNA available. The results suggest that host message is rapidly rendered nonfunctional as well as degraded by means of a virion-competent mechanism(s).

Dactinomycin↗
PubMed1997

Downregulation of IGF-I mRNA expression during postnatal pancreatic development and overexpression after subtotal pancreatectomy and acute pancreatitis in the rat pancreas.

Insulin-like growth factors (IGFs) are important peptides involved in the regulation of cell growth and differentiation in many tissues. The ontogeny of IGF-I was examined in pancreata from 19-day rat fetuses, newborns and 5-, 11-, 26- and 70-day-old rats. For the regeneration studies two models were used: (i) 90% pancreatectomy was carried out and the rats were killed at 1, 2, 3 and 6 days after resection; (ii) acute pancreatitis was induced with caerulein (12 micrograms/body weight three times a day every 8 h for 2 days) and the rats were killed at 1, 2, 5, 7 and 9 days after the first injection. Total RNA was extracted by the guanidinium isothiocyanate method and Northern blots were performed using total RNA and labeled cRNA probes. Abundance of the different mRNA transcripts was estimated by densitometric scanning and normalized to the abundance of 18 S rRNA for each time point. Northern blot analysis during ontogeny showed four (0.8-1.2, 1.9, 4.7 and 7.5 kb) major transcripts in the rat pancreas and liver. Total IGF-I mRNA was 40-fold higher in the adult liver than in the adult pancreas. Moreover, in the liver, IGF-I mRNA levels were higher in the adult than in the fetus, whereas in the pancreas, the highest levels were observed around birth. During the first 3 days after pancreatectomy, a peak of maximal expression was observed after the second day. Densitometric analysis of each IGF-I mRNA species showed concomitant increases in all transcripts. After 6 days, all transcripts had returned to near-control values. IGF-I mRNA expression 2 days after pancreatectomy was 3.5-fold higher than in the newborn. During the first 2 days of acute pancreatitis induction, overexpression of IGF-I mRNA was observed. However, soon after the second day of caerulein treatment, the 7.5 kb transcripts remained elevated whereas those of the others regressed toward control values. Our results show that IGF-I mRNA is overexpressed in both models of pancreatic regeneration but downregulated in the normal adult pancreas.

Acute Disease↗