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PubMed2000

Reverse transcription - 3' rapid amplification of cDNA ends-nested PCR of ACT1 and SAP2 mRNA as a means of detecting viable Candida albicans in an in vitro cutaneous candidiasis model.

The presence of viable cells of Candida albicans, in broth or in a reconstructed living skin equivalent, was determined by the detection of amplicons of partial mRNA sequences of the genes encoding fungal actin (ACT1) and secreted aspartyl proteinase 2 (SAP2). The mRNA of both genes were amplified by reverse transcription-3' rapid amplification of cDNA ends-nested polymerase chain reaction. Single bands of ACT1 (315 bp) and SAP2 (162 bp) mRNA were amplified from total RNA extracts of C. albicans grown in yeast carbon base-albumin broth or in living skin equivalent tissue; only the former was amplified from Sabouraud broth-grown organisms. Primer pairs targeted for ACT1 and SAP2 were Candida genus-specific and C. albicans-specific, respectively. The sensitivity limits of the assay were 100 fg of total RNA or 10 cells of C. albicans, by ethidium bromide staining. When C. albicans-infected living skin equivalent was exposed to amorolfine, amplicons of ACT1 and SAP2 mRNA were not detected in total RNA extracts. Non-amplification of the mRNA correlated with the absence of C. albicans growth in Sabouraud agar cultures of living skin equivalent samples. Reverse transcription-3' rapid amplification of cDNA ends-nested polymerase chain reaction of the mRNA encoding specific proteins of an organism has potential application in determining the viability of the organism in tissue, thus monitoring the efficacy of an antimicrobial therapy, and in detecting mRNA expressed in very little amounts in tissue.

Actins↗
PubMed1994

Collagen type IV gene expression in human optic nerve heads with primary open angle glaucoma.

Previous studies have demonstrated proliferation of basement membranes in the optic nerve head in primary open angle glaucoma (POAG). We used in situ hybridization (ISH) of a radiolabeled riboprobe specific for human collagen IV, a ubiquitous component of basement membranes, to identify cells actively synthesizing basement membranes in the optic nerve head in POAG. In addition, to detect and further characterize the collagen IV mRNA transcripts, we used reverse transcriptase-polymerase chain reaction (RT-PCR) in total RNA extracted from individual optic nerve heads with POAG and from age-matched normal controls. ISH results demonstrate that, in POAG, numerous astrocytes in the prelaminar region expressed collagen IV mRNA. Lamina cribrosa cells and astrocytes in the compressed lamina cribrosa hybridized the probe. Few astrocytes and lamina cribrosa cells hybridized the probe in the optic nerve head of normal age-matched controls. RT-PCR products for collagen IV and for glyceraldehyde-3-dehydrogenase (G3PDH), a reference gene, were detected by agarose electrophoresis as single bands of the expected sizes and positively identified by Southern hybridization using specific cDNA probes in normal and POAG samples. No additional products (bands) were observed in RT-PCR experiments, indicating that there was no genomic DNA contamination in the total RNA extract. The lack of additional bands suggests that, at least in the ten samples used in this study, there were no alternatively spliced RNA products in any of the amplified sequences. Semi-quantitative analyses using densitometry showed a two-fold increase in collagen type IV PCR present in POAG samples. No differences were detected in levels of G3PDH PCR products between POAG and normal samples. This investigation provides evidence of increased biosynthesis of collagen type IV at the mRNA level in optic nerve heads with POAG. Whether this phenomenon represents a response to elevated intraocular pressure or a reparative mechanism to the loss of axons remains to be determined.

Aged↗
PubMed2005

Ribozyme knockdown of the gamma-subunit of rod cGMP phosphodiesterase alters the ERG and retinal morphology in wild-type mice.

PURPOSE: To generate an animal model of retinal degeneration by using AAV-mediated ribozyme knockdown of the gamma-subunit of the rod cGMP phosphodiesterase (PDEgamma) mRNA in the retina of wild-type mice. METHODS: Two hammerhead ribozymes, HRz35 and HRz42, were designed to target the PDEgamma gene in wild-type C57BL/6 mice. The efficiency and specificity of the ribozyme cleavage was tested in vitro against three different types of target: short synthetic RNA oligomers, longer targets transcribed from clones, and full-length mRNA from total retinal RNA extracts. After in vitro validation, the ribozymes were cloned and packaged in a recombinant adenoassociated virus (rAAV) containing a proximal 472-bp murine rod opsin promoter (MOPS) to drive ribozyme synthesis. Three-week-old wild-type C57BL/6 mice were injected subretinally with the vectors. For treated versus partner control retinas, responses to light were measured by full-field electroretinography (ERG), and retinal tissues were examined by light microscopy. Messenger RNA and protein levels of PDEgamma gene were monitored by reverse transcription-polymerase chain reaction (RT-PCR) and Western immunoblot assay. RESULTS: The ribozymes had comparable in vitro kinetic properties in multiple turnover kinetic analyses. Ribozyme HRz35 exhibited a K(cat) of 0.48 minute(-1) and a K(m) of 980 nM, and HRz42 showed a K(cat) of 0.17 minute(-1) and a K(m) of 971 nM. Both ribozymes cleaved at accessible sites in the RNA, as they digested long transcripts transcribed from clones and full-length mRNA from total retinal RNA extracts in vitro. At increasing intervals after subretinal injection with either AAV ribozyme, a 30% to 90% reduction in a- and b-wave amplitudes was observed compared with those in contralateral control eyes that were not injected. Retinal tissue analysis showed that loss of the photoreceptor cells and PDEgamma mRNA and protein paralleled the ERG results. CONCLUSIONS: Ribozyme-mediated somatic knockdown of wild-type PDEgamma mRNA in vivo can efficiently reduce the target RNA leading to a loss in rod photoreceptors and in rod-mediated ERG amplitudes, thus generating an animal model of retinal degeneration resembling human RP in an essentially normal adult retina. This vector ribozyme technique should be applicable to other genes associated with RP and perhaps also to mRNAs of phototransduction genes not yet associated with RP. Application of this approach may be age and species independent.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

Detection of mouse hepatitis virus by the polymerase chain reaction and its application to the rapid diagnosis of infection.

Eight different strains of mouse hepatitis virus (MHV) were analyzed by the polymerase chain reaction (PCR) to see whether two sets of oligonucleotides, which were synthesized based on the published nucleotide sequence of MHV-JHM mRNAs 6 and 7, could be used as universal primers for amplification. Total RNA extracted from virus-infected cells or virus-infected culture fluids was transcribed into cDNA by using reverse transcriptase and oligo(dT) as primer, then the cDNA transcripts were amplified by PCR. The MHV-specific fragments of 199-bp and 241-bp were obtained from all eight strains irrespective of nucleotide differences in the primer regions. The same fragments were also amplified from RNA derived from the liver and brain of MHV-JHM-infected mice as soon as day 1 after intraperitoneal injection, even from the liver from which the virus was not detected. Results of PCR amplification from the liver RNA extracts became positive when more than 10(-2)PFU of MHV-JHM was contained in the PCR reaction mixture. In contrast, anti-MHV antibody was not detected by enzyme-linked immunosorbent assay until day 6 after inoculation. These results suggest that PCR is a very sensitive method to identify a variety of MHV infections in laboratory animals, especially at the early phase of infection.

Animals↗
PubMed2007

Gene expression profiling of women with varying degrees of cervical intraepithelial neoplasia.

OBJECTIVE: A pilot study to determine genomic microarray differences in patients with normal cervical tissue and cervical intraepithelial neoplasia (CIN) 3. MATERIALS AND METHODS: After institutional review board approval, patients referred to the University of Alabama at Birmingham Colposcopy Clinic for a loop electrosurgical excisional procedure were identified. Tissue biopsies of both normal tissue and CIN 3 from fresh loop electrosurgical excisional procedure specimens were obtained and sent to pathology for histological confirmation. Procurement of these 2 types of tissue from the same patient controls for different types of human papillomavirus infection, smoking and nutritional status, age, immunocompetency, and other microenvironment factors (i.e., the patient serves as their own control). Standard RNA extraction techniques (Qiagen, Inc., Valencia, CA) were used to prepare the tissue specimens for microarray analysis with the Affymetrix GeneChip U133A expression array (Affymetrix, Inc., Santa Clara, CA). Paired samples were eligible for microarray analysis only when both normal tissue and CIN 3 were confirmed by pathology. The data were then subsequently subjected to a log-like transformation and analyzed with a t test. For this study, p<.001 was determined to be statistically significant. RESULTS: Twenty-one pairs of both normal and CIN tissues were obtained and underwent histological evaluation followed by RNA extraction. Within the normal group, 86% were confirmed as true normals and 14% as CIN. Within the CIN group, 28.5% were found to have CIN 2, 62% were found to have CIN 3, and 9.5% had no evidence of CIN. Mean RNA content of normal samples was 2.0 microg, whereas the mean RNA content of CIN samples was significantly higher at 7.4 microg (p=006). Ultimately, 5 pairs of normal and neoplastic tissues were subjected to microarray analysis. Using a cutoff of p<.001, 24 candidate genes were identified from more than 18,000 genes. In the CIN 3 group, 14 genes were overexpressed and 10 genes were underexpressed. Of the 14 overexpressed genes, 9 were noted to have identities listed in the National Center for Biotechnology Information public domain. Five (56%) of these 9 genes were directly related to immunity-related pathways, and 3 (33%) of the 9 genes were found to be involved in cell cycle function/control. One overexpressed gene was identified as p53. CONCLUSIONS: The presence of CIN is marked by increased transcriptional activity, evident by an almost 4-fold increase in mean RNA content obtained from our CIN samples versus normal cervical tissue. Furthermore, a number of statistically significant overexpressed genes of interest related to immune function/response and cell cycle control were identified in our pilot microarray study. This data have the ability to direct future research endeavors in cervical neoplasia. Future endeavors include the use of laser capture microdissection to evaluate genomic changes strictly at the epithelial level and, as such, exclude stromal response contributions.

Adult↗
PubMed1991

Lack of evidence for abnormal autocrine or paracrine mechanisms underlying the uncontrolled proliferation of primitive chronic myeloid leukemia progenitor cells.

Previous studies have revealed a consistent defect in the cycling behavior of primitive neoplastic progenitor cells in patients with Philadelphia chromosome (Ph1)-positive chronic myeloid leukemia (CML). This is manifested both in vivo and in long-term cultures of CML cells as an increased rate of turnover amongst Ph1-positive progenitor cell types whose counterparts in normal individuals are mainly quiescent. To determine whether this deregulated proliferative activity of primitive Ph1-positive cells might be explained by a perturbation in the production of growth factors that regulate the turnover of primitive normal cells, the possibility of either autocrine or paracrine mechanisms of Ph1-positive cell stimulation was investigated. Northern blot analysis of total cellular RNA extracted from various CML blood cell populations showed no evidence of increased expression of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-3, IL-6, or tumor necrosis factor-alpha (TNF-alpha) compared with analogous normal peripheral blood cell populations in which transcripts for most of these growth factors are not detectable. A similar analysis of RNA extracted from the adherent layer of 4-week-old long-term cultures established from CML marrow (in which the Ph1-positive cells typically disappear) or from CML blood seeded onto normal marrow adherent layers (in which Ph1-positive cells typically persist) also revealed no difference in growth factor production compared with analogous cultures established with exclusively normal cells. For some of the growth factors studied, the assessment of bioactivity detectable in the medium confirmed the RNA data. There was also no evidence of a decreased production of putative inhibitors of primitive hematopoietic cells, i.e. transforming growth factor-beta and macrophage inflammatory protein-1 alpha by CML versus normal cells or cultures. These results do not support the existence of BCR-ABL induced autocrine or paracrine mechanisms in CML and suggest that constitutive activation of events normally dependent on growth factor receptor stimulation is more likely to underlie the lack of proliferation control exhibited by primitive Ph1-positive cells.

Bone Marrow↗
PubMed2003

Cloning and expression of a functional estrogen receptor-alpha from African catfish (Clarias gariepinus) pituitary.

An African catfish (Clarias gariepinus) estrogen receptor-alpha (cfERalpha) cDNA fragment was amplified by RT-PCR, in combination with a modified 3'-RACE procedure, on total RNA extracted from pituitary. This cDNA fragment was used to screen an African catfish pituitary cDNA library. A clone was obtained that contained an open-reading frame coding for a 620 amino acid cfERalpha protein with a deduced molecular mass of 68.1 kDa. In addition, a partial African catfish estrogen receptor-beta (cfERbeta) cDNA fragment was amplified by RT-PCR on total RNA extracted from testis. Neighbor-joining analysis was used to infer a phylogenetic classification for cfERalpha and cfERbeta. The tree obtained indicated that there are two major clusters of vertebrate ERs: ERalpha and ERbeta. Within each cluster, teleost and tetrapod ER sister clades could be distinguished. The cfERalpha clustered with other teleost ERalphas, whereas cfERbeta clustered with other teleost ERbetas. The ligand-induced transcriptional activity of cfERalpha was demonstrated in a transient gene expression assay using cells in which an acute estrogenic response was created by co-transfecting cultures with recombinant cfERalpha cDNA expression vector constructs in the presence of an estrogen-dependent reporter plasmid. Real-time, quantitative PCR revealed that cfERalpha transcripts were most abundantly expressed in pituitary, while in all other tIssues tested the relative cfERalpha mRNA levels were less than approximately 5% of the level obtained in pituitary. Moreover, we found that, during pubertal development, the relative cfERalpha mRNA levels gradually increased in African catfish pituitary.

Africa↗
PubMed1995

Increased muscle carnitine palmitoyltransferase II mRNA after increased contractile activity.

The capacity of skeletal muscle to oxidize fatty acids increases with endurance training. The oxidation of long-chain fatty acids occurs in mitochondria and is initiated by a carnitine-dependent transport step in which three enzymes help fatty acyl groups enter the matrix compartment. The purpose of this study was to determine whether pretranslational regulation of one of these three enzymes, carnitine palmitoyltransferase II (CPT II), as estimated from the level of CPT II mRNA, plays a role in the doubling of CPT activity in skeletal muscle of rats subjected to daily 2-h bouts of running on treadmills (P. A. Móle, L. B. Oscai, and J. O. Holloszy. J. Clin. Invest. 50: 2323-2330, 1971). After 100 min/day of running on motor-driven treadmills for 2 wk, CPT II mRNA in the plantaris muscle was unchanged when normalized per unit of extracted RNA but was 50% higher (P < 0.05) over sedentary controls when normalized per unit of muscle wet weight. To test whether additional contractile activity would make CPT II mRNA even higher, continuous indirect electrical stimulation was imposed on the tibialis anterior muscles. After 9 days of chronic stimulation, CPT II mRNA was 63, 221, and 137% greater than control (P < 0.001) when normalized to extracted RNA, muscle wet weight, and whole muscle, respectively, compared with the muscle in the control rats. These data indicate that pretranslational regulation of CPT II occurs in response to increased contractile activity in skeletal muscle.

Animals↗
PubMed1990

Detection of infectious pancreatic necrosis virus (IPNV) RNA by hybridization with an oligonucleotide DNA probe.

Marine farming of Atlantic salmon (Salmo salar) is growing rapidly in Norway, and fish diseases have now become one of the biggest obstacles for this new industry. Infectious pancreatic necrosis virus (IPNV) is commonly found in fish from Norwegian sea farms. Diagnosis of IPNV infection is, at present, mainly based on virus isolation in cell cultures and identification by neutralization tests (NT). A DNA-RNA hybridization assay was developed using a 24 base DNA oligonucleotide probe. This is homologous to a part of the nucleotide sequence of the IPNV genome coding for a protease. RNA extracted from IPNV and harvest from infected cell cultures were fixed to nylon filters and hybridized with the 32P end-labelled probe. The results showed that the probe specifically identified IPNV from these two materials, both for the three different virus strains (Ab, Sp and VR-299) used, and for several different field isolates. It did not hybridize with reoviruses or non-infected cell cultures used as controls. These results indicate that the probe is not serotype specific, and furthermore that RNA extraction is not required before hybridization. This method may be a useful alternative to NT for routine identification of IPNV, particularly when non-radioactive labelling of the probe is introduced.

Animals↗
PubMed1978

Effect of RNase III on the size of bacteriophage T7 lysozyme mRNA.

The size of lysozyme mRNA from T7-infected E. coli RNase III+ and RNase III- strains was analyzed by sucrose gradient sedimentation, dimethylsulfoxide (Me2SO) sucorse gradient sedimentation, and preparative gel electrophoresis. Each technique revealed a similar size distribution of multiple lysozyme mRNA's. Analysis by preparative gel electrophoresis of RNA extracted after infection of Escherichia coli Bst (RNase III+) separated lysozyme mRNA into six peaks of activity ranging in size from 0.2 x 10(6) to 1.9 x 10(6) daltons. Four well-resolved major peaks of activity were detected, having apparent molecular weights of approximately 0.61 x 10(6), 0.76 x 10(6), 0.92 x 10(6), and 1.3 x 10(6). A broad band of activity, with a molecular weight range from 0.2 x 10(6) to 0.37 x 10(6), was also present, and a sixth peak of activity was sometimes observed that migrates with a mobility corresponding to a molecular weight of 1.9 x 10(6). Judging from their molecular weight as estimated by electrophoresis, most, if not all, of the lysozyme mRNA's were polycistronic. The RNA extracted after infection of an RNase III- host contained a more heterogeneous collection of lysozyme mRNA's. In addition to lysozyme mRNA activity on RNAs with molecular weights between 0.2 x 10(6) and 1.9 x 10(6), RNA species with molecular weights estimated at 4 x 10(6) to 5 x 10(6) were also detected. The data indicate that RNase III processes at least some of the primary lysozyme transcripts. The multiple lysozyme mRNA's represent discrete RNA species rather than aggregates because analysis of the size of lysozyme mRNA under completely denaturing conditions, in Me2SO, produced a similar size distribution of lysozyme mRNAs. Also, treatment of RNA with 90% Me2SO, which separates the strands of a completely double-stranded RNA, did not significantly alter the electrophoretic mobility of the lysozyme mRNA.

Coliphages↗
PubMed1998

Dystrophin gene analysis on 130 patients with Duchenne muscular dystrophy with a special reference to muscle mRNA analysis.

On dystrophin gene analysis by multiplex polymerase chain reaction (PCR), 76 of 130 (58.5%) Japanese patients with Duchenne muscular dystrophy had a deletion or duplication in genomic DNA. Of the remaining 54 patients who had no identifiable gene mutations, muscle biopsy tissue was available in 16 for RNA extraction. The full length of the coding regions of dystrophin cDNA was amplified in 10 fragments by reverse transcription nested PCR (RT-PCR). Five of 16 patients (31%) had dystrophin cDNA of abnormal size. One patient had a deletion, and two duplications that were not covered by multiplex PCR, one an exon-skipping of exon 51 caused by a 5' consensus splice site mutation of intron 51, and one 172 bp or 202 bp insertion in the cDNA between exon 25 and 26. Nested RT-PCR from the total RNA extracted from muscle biopsy was useful for screening patients who had no identifiable gene abnormality by multiplex PCR.

Adolescent↗
PubMed1995

Isolation and expression pattern of a cDNA encoding a cathepsin B-like protease from Nicotiana rustica.

Sequence analysis of a 1.33 kb clone from a root cDNA library of Nicotiana rustica revealed an open reading frame encoding a protein of 356 amino acids. The deduced protein has high levels of homology to human cathepsin B protease and a cathepsin B-like cysteine protease from wheat but much lower levels of homology with other plant cysteine proteinases. Southern blotting experiments suggest a limited number of cathepsin B-like genes are present in the genome of N. rustica and also that of N. tabacum. RNA analysis involving a range of tissues, harvested from both Nicotiana species 4-5 h after the beginning of a 16 h photoperiod, revealed the cathepsin B-like gene was being expressed strongly in roots, stem and developing flowers but weakly in mature leaves. Further analysis of RNA extracted from leaf tissue of N. tabacum revealed the gene showed rhythmic expression and also that its expression increased in response to wounding. Analysis of leaf tissues harvested during the latter part of a 16 h photoperiod (11 and 16 h after illumination commenced) showed that transcript levels were two three times higher than in leaf tissue harvested either towards the end of the dark period or 5 h after illumination commenced. When leaf tissue was wounded at 11:00 (5 h after plants were illuminated), and harvested for RNA extraction 6 h later, the level of cathepsin B-like transcript in mesophyll tissue was found to be increased ca. 2-fold relative to the level detected in unwounded controls.

Amino Acid Sequence↗
PubMed2002

Modulation of cyclophilin gene expression by N-4-(hydroxyphenyl)retinamide: association with reactive oxygen species generation and apoptosis.

To explore the mechanisms underlying the pro-apoptotic effects of the synthetic retinoid N-4-(hydroxyphenyl)retinamide (4-HPR) on LNCaP human prostate cancer cells, we used the differential display-polymerase chain reaction (DD-PCR) technique to identify 4-HPR-responsive genes. RNA extracted from LNCaP cells that had been treated for 24 h with 4-HPR at a dose (2.5 microM) optimal for apoptosis induction was used for DD-PCR analysis using random primers. A differentially expressed 115 bp fragment was cloned and sequenced and then identified in GenBank as having a high degree of homology with several members of the cyclophilin gene family. Northern blot analyses using specific probes for cyclophilin A, cyclophilin D, and the cloned 115-bp fragment were performed on RNA extracted from LNCaP cells and MCF-7 human breast cancer cells treated with 4-HPR, N-acetylcysteine (NAC, an anti-oxidant), 4-HPR plus NAC, cyclosporin A, R-1881 (a synthetic androgen), dehydroepiandrosterone, all-trans retinoic acid, or prednisone. 4-HPR downregulated the transcript detected by the 115-bp fragment. Expression patterns detected by the 115-bp fragment and cyclophilin D probes were identical in response to each treatment; none of these treatments affected cyclophilin A expression. Furthermore, expression of mRNA transcripts detected by the 115-bp fragment and cyclophilin D probes correlated with the generation of reactive oxygen species (ROS), as detected by measurement of 2,7-dichlorofluorescein oxidation. Therefore, members of the cyclophilin gene family, such as cyclophilin D (a component of the mitochondrial permeability transition pore previously linked with oxidative stress and apoptosis), may play a role in the ROS-mediated apoptotic effects of 4-HPR.

Antineoplastic Agents↗
PubMedDate not supplied

[Induction of immunologic tolerance to transplantation antigens using conditioning of the lymphocyte genome of the recipient with ribonucleic acid from the donor].

The phenomenon of specific immunosuppression which we call "immunologic tolerance" constitutes the final result common to diverse processes partially known. The study of the phenomena inter or intra cellular involved in the divese mechanisms of production, however constitutes a field which is quite obscure. The explanation of these phenomena could offer us some fundamental knowledge towards the better understanding and subsequent manipulation of the process. This present project of investigation proposes to study the possibility of transferring one of these states of tolerance which is better established as is the spontaneous tolerance to the proper antigens of an animal (donor) to a second animal (receptor) genetic and for that reason, antigenically different, through an informational molecule such as the ribonucleo acid (RNA) extracted from the donor animal. This hypothesis is based theoretically on the following points: 1) The state of "self-immunologic tolerance" is probably an active and dynamic phenomenon, in which one can determine the presence of lymphocytes with receptors for proper antigens, without establishing in normal conditions an efferent phase of the immunologic answer, which determines an effect on this antigen. 2) The effective immunologic reactivity (allergic answer) of a determined animal is transferable to a second non-sensitized animal through RNA from the first animal. In this study we present the results of the conditioning of the allogenic reactivity of one strain of mice, through the exposition of the same to RNA extracted from one congenic-resistent strain with respect to first.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1979

Programmed appearance of translatable flagellar tubulin mRNA during cell differentiation in Naegleria.

The programmed de novo synthesis of flagellar tubulin during the hour-long differentiation of Naegleria gruberi from amoebae to flagellates is our paradigm for the study of gene expression during cell differentiation. This paper reports the efficient translation of flagellar tubulin mRNA in the wheat germ cell-free system directed by total or polyadenylated RNA extracted from differentiating cells. The tubulin in the in vitro product has a subunit molecular weight of 55,000, separates into alpha and beta subunits under suitable conditions of polyacrylamide gel electrophoreis and co-polymerizes with calf brain tubulin. At least half of the tubulin synthesized in vitro is precipitated by antibodies specific to flagellar tubulin, and the immunoprecipitated tubulin subunits yield peptide maps similar to those of outer doublet tublin. Flagellar tubulin is the predominant protein synthesized in the cell-free system, and amounts to about 5% of the polypeptides whose synthesis is directed by total RNA from differentiating cells. In contrast, little or no flagellar tubulin is synthesized when the cell-free system is directed by RNA extracted from amoebae prior to differentiation. Translation assays show that at least 92% of the flagellar tubulin mRNA appears during differentiation. The time course of appearance of this mRNA was measured by quantitative immunoprecipitation of the cell-free products. Under conditions where cells from flagella 60 min after initiation of differentiation, translatable flagellar tubulin mRNA was first detected at 20 min, reached a maximum at about 60 min and then declined. An excellent correlation was observed between the amount of translatable flagellar tubulin mRNA and the previously measured rates of flagellar tubulin synthesis in vivo. These results indicate that synthesis of flagellar tubulin is a direct reflection of the abundance of its mRNA, and provide the molecular techniques for dissection of the factors that regulate the rapid appearance of this structural protein during differentiation.

Animals↗
PubMedDate not supplied

[Emergent viruses: SARS-associate coronavirus and H5N1 influenza virus].

Two viral agents with RNA genome are responsible for emerging illnesses: influenza virus A/H5N1 and Severe Acute Respiratory Syndrome virus (SARS). For the diagnosis of SARS virus infection, an epidemiological investigation is necessary to know whether the patient has been exposed to a risk in a country where the SARS virus is circulating or whether the patient had worked in a laboratory handling SARS virus. The detection of SARS virus is possible in various clinical samples (including urine) by viral culture or RT-PCR. The handling of those samples and RNA extraction must be performed in a BSL3 laboratory. The SARS virus RT-PCR is poorly sensitive, therefore the test should be performed on samples collected consecutively for several days. In front of a suspicion of A/H5N1, similar procedures are recommended. An epidemiologic investigation is necessary to specify whether the patient stayed in a country where A/H5N1 virus was circulating. Clinical samples needed for a specific diagnosis are: nasopharyngeal, throat-swab or fecal samples, cerebrospinal fluid and blood. The presence of A/H5N1 virus is confirmed by viral isolation or RNA detection by RT-PCR. RNA extraction must be performed in a BSL3 laboratory. For diagnosis of A/H5N1 virus infection, RT-PCR test amplifies specifically a fragment of H5 gene (Hemagglutinin). In french laboratories of medical virology, procedures are ready to diagnose the first case of A/H5N1 virus infection and cases of reemerging SARS virus infection.

Communicable Diseases, Emerging↗
PubMed1990

Characterization of the corticosteroid-binding globulin messenger ribonucleic acid response in the pregnant hamster.

In this study we measured corticosteroid-binding globulin (CBG) mRNA levels in liver and various nonhepatic tissues of pregnant and nonpregnant hamsters. The N-terminal amino acid sequence (37 residues) of hamster CBG was determined and compared with published cDNA-deduced sequence information for rat and human CBG. Hamster CBG showed considerable sequence homology with both rat (70%) and human (59%) CBG. Because of the high level of homology, we were able to use a cRNA prepared from a rat CBG cDNA as a probe in Northern blot and solution hybridization analyses. Northern blots of hamster and rat liver RNA extracts revealed that the rat CBG cDNA probe hybridized to RNAs that were the same size in rats and hamsters. Further, the Northern blot showed that pregnant hamster liver contained substantially more CBG mRNA than nonpregnant hamster liver. The relative amounts of CBG mRNA in pregnant and nonpregnant hamster livers were compared using a solution hybridization assay. Slope-ratio analysis of the hybridization data revealed that pregnant hamster liver (day 14) contained 40-fold more CBG mRNA than nonpregnant hamster liver. When other tissues (kidney, spleen, small intestine, and decidual tissue) were assayed for CBG mRNA, a small amount of hybridization was detected by solution hybridization. However, Northern blot analysis of RNA extracts from nonhepatic tissues showed that the hybridizable sequences did not migrate at the same position as mature CBG mRNA. These results indicate that the observed increase in serum CBG during hamster pregnancy is largely attributable to an increase in hepatic CBG mRNA.

Amino Acid Sequence↗
PubMed2004

In vitro evaluation of the effects of candidate immunosuppressive drugs: flow cytometry and quantitative real-time PCR as two independent and correlated read-outs.

BACKGROUND: Immune monitoring may use flow cytometry or molecular biology techniques. Flow cytometry assays cells that are phenotypically characterized, whereas TaqMan RT-PCR starts with RNA extraction from unfractionated heterogeneous cell populations. We therefore wondered how the effects of immunosuppressive drugs on cytokine production in stimulated whole blood, as determined by flow cytometry, would correlate with those obtained with quantitative real-time PCR (TaqMan RT-PCR). METHODS: Blood drawn from naive cynomolgus monkeys was exposed to incremental amounts of cyclosporine (CsA; 300, 600, 900 and 1200 ng/ml) or tacrolimus (TRL; 8, 20, 40 and 80 ng/ml) before lectin stimulation in vitro. Blood was then either stained for CD3, IFN-gamma, IL-2, IL-4, and TNF-alpha and analyzed on a flow cytometer with various gating strategies, or submitted to RNA extraction for analysis of the above mentioned cytokines mRNA transcripts using TaqMan RT-PCR. RESULTS: Both methods revealed a parallel dose-dependent inhibition of cytokine production in stimulated blood. The 50% inhibitory concentrations (IC(50)'s) ranged from 511-771 ng/ml (CsA) and 15-29 ng/ml (TRL) with flow cytometry, and from 275-529 ng/ml (CsA) and 11-48 ng/ml (TRL) with TaqMan RT-PCR for T-helper 1 cytokines. Both assays correlated well with a Pearson product moment correlation of 0.76. Extending gating from a CD3(+) gate to a lymphocyte gate improved correlation (r = 0.85) for all cytokines investigated (except IL-2; unchanged) whereas further extending gating resulted, to the contrary, in lower correlations. Independent of gating strategy a high correlation (r = 0.97) was observed when drug IC(50)'s were considered. CONCLUSIONS: Flow cytometry and TaqMan RT-PCR may be used interchangeably to monitor the effects of candidate immunosuppressive drugs on cytokine mRNA production in lectin-stimulated whole blood.

Animals↗