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PubMed2005

An internal reference technique for accurately quantifying specific mRNAs by real-time PCR with application to the tceA reductive dehalogenase gene.

The accuracy of mRNA quantification by reverse transcription (RT) in conjunction with real-time PCR (qPCR) is limited by mRNA losses during sample preparation (cell lysis, RNA isolation, and DNA removal) and by inefficiencies in reverse transcription. To control for these losses and inefficiencies, a technique was developed that utilizes an exogenous internal reference mRNA (ref mRNA) along with mRNA absolute standard curves. The technique was applied to quantify mRNA of the trichloroethene (TCE) reductive dehalogenase-encoding tceA gene in an anaerobic TCE-to-ethene dechlorinating microbial enrichment. Compared to RT-qPCR protocols that utilize DNA absolute standard curves, application of the new technique increased measured quantities of tceA mRNA by threefold, demonstrating a substantial improvement in quantification. The technique was also effective for quantifying the loss of mRNA during specific steps of the sample processing protocol. Analysis revealed that the efficiency of the RNA isolation (56%) step was significantly less than that of the cell lysis (84%), DNA removal (93%), and RT (88%) steps. The technique was applied to compare the effects of cellular exposure to different chlorinated ethenes on tceA expression. Results show that exposure to TCE or cis-1,2-dichloroethene resulted in 25-fold-higher quantities of tceA mRNA than exposure to vinyl chloride or chlorinated ethene starvation.

Anaerobiosis↗
PubMed1995

Site-selected introduction of modified purine and pyrimidine ribonucleosides into RNA by automated phosphoramidite chemistry.

The study of modified nucleoside contributions to RNA chemistry, structure and function has been thwarted by the lack of a site-selected method of incorporation which is both versatile and adaptable to present synthetic technologies. A reproducible and versatile site-selected incorporation of nine differently modified nucleosides into hepta- and octadecamer RNAs has been achieved with automated phosphoramidite chemistry. The 5'-O-(4,4'-dimethoxytrityl-2'-O-tert-butyldimethylsilyl-ribonucleoside- 3'-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite syntheses of m5C, D, psi, riboT, s2U, mnm5U, m1G and m2A were designed for compatibility with the commercially available major and 2'OH methylated ribonucleoside phosphoramidites. The synthesis of the m5C phosphoramidite was uniquely designed, and the first syntheses and incorporation of the two modified purine ribonucleosides are reported in detail along with that of psi, s2U, and mnm5U. Cleavage of RNA product from the synthesis support column, deprotection of the RNA, its purification by HPLC and nucleoside composition analysis are described. Modified nucleoside-containing tRNA domains were synthesized and purified in mumol quantities required for biophysical, as well as biochemical, studies. The anticodon domain of yeast tRNA(Phe) was synthesized with modified nucleosides introduced at the native positions: Cm32, Gm34, m1G37 (precursor to Y), psi 39 and m5C40. The T loop and stem was synthesized with riboT54 and the D loop and stem with D16 and D17. The E coli tRNA(Glu2) anti-codon codon domain was synthesized with mnm5U at wobble position 34, but an attempt at incorporating s2U at the same position failed. The unprotected thio group was labile to the oxidation step of the cyclical process. Chemically synthesized anticodon and T domains have been used in assays of tRNA structure and function (Guenther et al (1994) Biochimie 76, 1143-1151).

Amides↗
PubMed1998

High expression in bacteria and purification of polymorphic mouse interleukin 2 molecules.

Recently, besides the known mouse interleukin 2 (IL-2) molecule, six other IL-2 alleles have been found in different mouse strains. In order to study their in vivo and in vitro biological activities large quantities are required. We cloned the corresponding IL-2 cDNAs into a pET7-7/BL21(DE3) bacterial system, a T7-RNA-polymerase-dependent expression vector, producing between 30 to 100 mg of IL-2 per litre of culture. The purification step is based on the resolution properties of the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) technique and the capability of the IL-2 to regain its activity after SDS denaturation. These purified IL-2 molecules supported the growth of an IL-2-dependent cell line, CTL-L2, in a similar way to a commercial mouse IL-2 control. However, RF IL-2 allele, which is also expressed in NOD mice had a relatively lower growth activity in the CTL-L2 assay. These IL-2 molecules can be obtained in a purified form and totally recovered their activity after elimination of the SDS and 2-mercaptoethanol used in the extraction procedure.

Alleles↗
PubMed1994

Rapid isolation of nuclear transport-competent Xenopus nucleoplasmin produced in Escherichia coli strain BL21(DE3).

Nucleoplasmin is a thermostable karyophilic protein widely used in nuclear transport studies. An expression vector was constructed that contains a string of 10 histidine residues ligated, in frame, to the amino terminal end of the Xenopus nucleoplasmin gene. The vector was then transformed into Escherichia coli strain BL21(DE3). This strain possesses the gene for T7 RNA polymerase under control of the lacUV5 promoter. The induction of the RNA polymerase and subsequent production of nucleoplasmin occurs after exposure to isopropyl-beta-D-thiogalactopyranoside. The nucleoplasmin, produced in milligram quantities per liter of culture, is then isolated by a rapid purification method that includes metal chelation chromatography to purify the oligohistidine-linked nucleoplasmin. Nuclear transport studies indicate that fluorescently labeled nucleoplasmin is translocated to the nuclear interior of permeabilized V79A03 cells, while nucleoplasmin that lacks a nuclear localization signal (core nucleoplasmin) is not imported. The use of this method to produce nuclear transport-competent nucleoplasmin avoids the lengthy purification procedure used to isolate nucleoplasmin from Xenopus laevis oocytes as well as the cost of purchasing and maintaining a toad colony.

Amino Acid Sequence↗
PubMed1999

Quantitative PCR of mycobacterial and propionibacterial DNA in lymph nodes of Japanese patients with sarcoidosis.

BACKGROUND: The causes of sarcoidosis are not known. The DNA of Mycobacterium tuberculosis has been detected in some sarcoid lesions. In Japan, Propionibacterium acnes has been isolated from such lesions, but whether this indigenous bacterium is related to the disease is unclear. We used PCR to estimate the number of genomes of these bacteria in sarcoid lesions, to identify any link between sarcoidosis and these two bacterial species. METHODS: We examined formalin-fixed and paraffin-embedded sections of biopsy and surgical samples from lymph nodes of 15 patients with sarcoidosis, 15 patients with tuberculosis, and 15 patients with gastric cancer (controls). Quantitative PCR was done to amplify segments of 16 S ribosomal RNA of P. acnes and P. granulosum and of insertion sequence 6110 of M. tuberculosis. PCR products were identified and the quantities of the products were estimated in terms of the fluorescence of oligonucleotide reporter probes. The numbers of bacterial genomes in samples were estimated from standard curves of serially diluted bacterial DNA. FINDINGS: Genomes of M. tuberculosis were found in samples from all 15 patients with tuberculosis, from three patients with sarcoidosis, and in one control sample. Genomes of P. acnes were found in 12 of the 15 patients with sarcoidosis, in two tuberculosis patients, and three controls. The difference in the estimated number of P. acnes genomes between individuals with and without sarcoidosis was similar to that in the number of M. tuberculosis between people with and without tuberculosis. There were 5x10(5) P. acnes genomes in sarcoidosis and 3x10(6) M. tuberculosis genomes in tuberculosis, respectively, on average per microg of total DNA. The three patients with sarcoidosis but without P. acnes all had P. granulosum DNA in their biopsy samples; the number of genomes of the bacterium was 5x10(5). INTERPRETATION: These findings suggest that propionibacteria had resided or proliferated ectopically in the sarcoid lesions, whether there was a connection with the disease or not. Propionibacteria are a more likely cause than mycobacteria of sarcoidosis.

DNA, Bacterial↗
PubMed2004

Levels of expression of endothelial markers specific to tumour-associated endothelial cells and their correlation with prognosis in patients with breast cancer.

Tumour endothelial markers (TEMs) are a newly discovered family of endothelial markers associated with tumour specific angiogenesis. This study sought to examine the levels of expression for TEMs in human breast cancer. Breast cancer tissues (n = 120) together with normal background tissues (n = 33) were obtained after surgery. RNA was extracted from frozen sections for gene amplification. The expression of TEMs was assessed using RT-PCR and the quantity of their transcripts was determined using real-time-quantitative PCR (Q-RT-PCR). TEM-7R (P = 0.05) and TEM-8 (P < 0.01) were significantly raised in breast cancer tissues compared with the levels detected in normal background tissues. After a median follow-up of 72.2 months it was found that patients who had recurrent disease and/or who had died from breast cancer had a significantly (P < 0.05) elevated level of TEM-1 compared to those patients who were disease free. In addition, elevated levels of TEM-4, TEM-5, TEM-6, TEM-7 and TEM-7R were also raised in breast cancer tissues. Patients who had developed nodal involvement exhibited significantly (P < 0.05) high levels of TEM-1 and TEM-7R compared to patients who were node negative. Furthermore, the levels of TEMs did not correlate with tumour or histological grade. We conclude that elevated levels of TEM-1, TEM-7R and TEM-8 (but not TEM-2, 4, 5, 6 and 7) are associated with either nodal involvement, and/or disease progression, and may therefore, have a prognostic value in breast cancer.

Base Sequence↗
PubMed2003

Chronic ethanol exposure increases the binding of HuR to the TNFalpha 3'-untranslated region in macrophages.

Tumor necrosis factor alpha (TNFalpha) expression is a key mediator of ethanol-induced liver disease. Increased lipopolysaccharide (LPS)-stimulated TNFalpha expression in macrophages after chronic ethanol feeding is associated with a stabilization of TNFalpha mRNA (Kishore, R., McMullen, M. R., and Nagy, L. E. (2001) J. Biol. Chem. 276, 41930-41937). Here we show that the 3'-UTR of murine TNFalpha mRNA was sufficient to mediate increased LPS-stimulated expression of a luciferase reporter in RAW 264.7 macrophages after chronic ethanol exposure. Further, we show that HuR, a nuclear/cytoplasmic shuttling protein, which binds to TNFalpha mRNA, is required for increased expression of TNFalpha after chronic ethanol. In Kupffer cells, HuR was primarily localized to the nucleus and then translocated to the cytosol in response to LPS in both pair- and ethanol-fed rats. After chronic ethanol feeding, HuR quantity in the cytosol was greater, both at baseline and in response to LPS, compared with pair-fed controls. Using RNA gel shift assays, we found that LPS treatment increased HuR binding to the 65-nucleotide A + U-rich element of the TNFalpha 3'-UTR by 2-fold over baseline in Kupffer cells from pair-fed rats. After chronic ethanol feeding, HuR binding to the TNFalpha A + U-rich element was increased by more than 5-fold at baseline and in response to LPS, compared with pair-fed controls. Down-regulation of HuR expression by RNA interference prevented the chronic ethanol-induced increase in expression of luciferase reporters containing the TNFalpha 3'-UTR. Taken together, these data demonstrate that increased binding of HuR to the TNFalpha 3'-UTR contributes to increased LPS-stimulated TNFalpha expression in macrophages after chronic ethanol exposure.

3' Untranslated Regions↗
PubMed1968

Mass preparation of nuclei from the larval salivary glands of Drosophila hydei.

A method has been developed for isolating gram quantities of salivary glands from late third instar larvae of Drosophila hydei. The isolated glands have a normal appearance and incorporate RNA and DNA precursors normally. Nuclei can be isolated from these glands in 90% yield with the use of detergents. These nuclei contain morphologically normal giant polytene chromosomes.

Animals↗
PubMed2006

Global gene expression profiling of preimplantation embryos.

Preimplantation development is marked by four major events: the transition of maternal transcripts to zygotic transcripts, compaction, the first lineage differentiation into inner cell mass and trophectoderm, and implantation. The scarcity of the materials of preimplantation embryos, both in size (diameter < 100 microm) and in quantity (only a few to tens of oocytes from each ovulation), has hampered molecular analysis of preimplantation embryos. Recent progress in RNA amplification methods and microarray platforms, including genes unique to preimplantation embryos, allow us to apply global gene expression profiling to the study of preimplantation embryos. Our gene expression profiling during preimplantation development revealed the distinctive patterns of maternal RNA degradation and embryonic gene activation, including two major transient waves of de novo transcription. The first wave corresponds to zygotic genome activation (ZGA). The second wave, mid-preimplantation gene activation (MGA), contributes dramatic morphological changes during late preimplantation development. Further expression profiling of embryos treated with inhibitors of transcription or translation revealed that the translation of maternal RNA is required for the initiation of ZGA, suggesting a cascade of gene activation from maternal RNA/protein sets to ZGA gene sets and thence to MGA gene sets. To date, several reports of microarray experiments using mouse and human preimplantation embryos have been published. The identification of a large number of genes and multiple signaling pathways involved at each developmental stage by such global gene expression profiling accelerates understanding of molecular mechanisms underlining totipotency/pluripotency and programs of early mammalian development.

Animals↗
PubMed2006

Synergistic effect of a novel oxymatrine-baicalin combination against hepatitis B virus replication, alpha smooth muscle actin expression and type I collagen synthesis in vitro.

AIM: To study the effect of oxymatrine-baicalin combination (OB) against HBV replication in 2.2.15 cells and alpha smooth muscle actin (alpha SMA) expression, type I, collagen synthesis in HSC-T6 cells. METHODS: The 2.2.15 cells and HSC-T6 cells were cultured and treated respectively. HBsAg and HBeAg in the culture supernatants were detected by ELISA and HBV DNA levels were determined by fluorescence quantitative PCR. Total RNA was extracted from HSC-T6 cells and reverse transcribed into cDNA. The cDNAs were amplified by PCR and the quantities were expressed in proportion to beta actin. The total cellular proteins extracted from HSC-T6 cells were separated by electrophoresis. Resolved proteins were electrophoretically transferred to nitrocellulose membrane. Protein bands were revealed and the quantities were corrected by beta actin. RESULTS: In the 2.2.15 cell culture system, the inhibitory rate against secretion of HBsAg and HBeAg in the OB group was significantly stronger than that in the oxymatrine group (HBsAg, P = 0.043; HBeAg, P = 0.026; respectively); HBV DNA level in the OB group was significantly lower than that in the oxymatrine group (P = 0.041). In HSC-T6 cells the mRNA and protein expression levels of alpha SMA in the OB group were significantly lower as compared with those in the oxymatrine group (mRNA, P = 0.013; protein, P = 0.042; respectively); The mRNA and protein expression levels of type I collagen in the OB group were significantly lower as compared with those in the oxymatrine group (mRNA, P < 0.01; protein, P < 0.01; respectively). CONCLUSION: OB combination has a better effect against HBV replication in 2.2.15 cells and is more effective against alpha SMA expression and type I collagen synthesis in HSC-T6 cells than oxymatrine in vitro.

Actins↗
PubMed1994

Purple acid phosphatase of the human macrophage and osteoclast. Characterization, molecular properties, and crystallization of the recombinant di-iron-oxo protein secreted by baculovirus-infected insect cells.

The purple phosphatases catalyze hydrolysis of phosphate esters (optimum pH approximately 5) and are resistant to inhibition by dextro-rotatory tartrate; their distinctive color is due to Fe(III)-phenolate charge-transfer transitions at their active site. Expression of human purple phosphatase, designated type 5 acid phosphatase, is restricted to osteoclasts and other activated cells of monohistiocytic lineage, but its biological rôle in relation to bone resorption and phagocytosis is unknown. To characterize this enzyme further, we have engineered the human type 5 acid phosphatase into a baculovirus vector expression system that enabled milligram quantities of purple protein to be purified from medium containing Sf9 host cells. The phosphatase cDNA was transcribed as a single RNA species of 1.5 kilobases as in human tissues. Tartrate-resistant acid phosphatase activity reacting with uteroferrin antisera appeared in the culture medium, from which up to 8 mg/liter was purified by two-step cation-exchange chromatography at pH 8.0. Two isoforms of approximately 36 kDa were identified by SDS-polyacrylamide electrophoresis and were converted to a single species of apparent molecular size 34 kDa upon treatment with N-glycosidase F, indicating secreted glycoforms of a single polypeptide. Mass spectroscopy showed that the mean molecular mass of the active, secreted glycoprotein was 35849 Da. The recombinant enzyme (specific activity, 190 mumol p-nitrophenol/min/mg at 37 degrees C) contained 2 iron atoms/molecule and formed purple, monoclinic crystals. Exposure to the ferric chelator, 1,2-dimethyl-3-hydroxypyrid-4-one, rapidly inactivated the enzyme, which was not inhibited by alpha, alpha'-bipyridyl, a ferrous chelator. That ferric iron is essential for enzymatic catalysis, was further indicated by the synergistic effects of the reductant, dithiothreitol, and bipyridyl on phosphatase activity. The recombinant purple phosphatase catalyzed the peroxidation of 5-aminophthalhydrazide (luminol), as evidenced by the induction of chemiluminescence; this reaction was inhibited by alpha, alpha'-bipyridyl at concentrations that did not inhibit phosphatase activity. The divalent iron moiety of human type 5 phosphatase may therefore participate in the generation of free radical species by fluid-phase reactions involving Fenton chemistry that are dissociated from its phosphatase function.

Acid Phosphatase↗
PubMed1988

[Effect of the heat shock reaction on the phenotypic manifestation of mutations in rifampicin resistance affecting the gene of the RNA polymerase beta-subunit under the control of the lactose promotor].

Plasmids bearing the rifampicin-resistant RNA polymerase beta-subunit gene under control of the lac promoter failed to provide Escherichia coli cells with resistance to rifampicin, despite the accumulation of large quantities of the resistant subunit. The effect proved to be connected with the aggregation of the plasmid-borne subunit. Conditions that induce synthesis of heat-shock proteins make the plasmid-containing cells resistant to rifampicin. This finding suggests that heat-shock proteins prevent the formation of protein aggregates.

DNA-Directed RNA Polymerases↗
PubMed1996

Serum hepatitis C RNA titers after liver transplantation are not correlated with immunosuppression or hepatitis.

The degree to which immunosuppression and/or rejection influences recurrent hepatitis C (HCV) after liver transplantation (LT) for end-stage HCV cirrhosis remains poorly defined. We quantified serum HCV-RNA in 84 serum samples from 28 anti-HCV-positive patients taken 223 days prior to and up to 1719 days after liver transplantation to determine if cumulative immunosuppression, rejection, or histologic recurrence correlated with HCV-RNA levels. Histologic, serum chemistry, cumulative steroid, and OKT3 and alpha-interferon (INF) dose data were collected at the time of HCV-RNA sampling. Eighteen of 24 evaluable patients (75%) had HCV-RNA detected in their sera after transplant. Eight patients had 14 rejection episodes, 9 patients received OKT3, and 5 were given INF for histologically moderate hepatitis. Five patients died - two of recurrent hepatitis C - and no retransplants were performed for recurrent hepatitis. Of the 23 survivors, 7 have histologic hepatitis - 2 with persistent ascites, and 2 with mild fibrosis. We could show no correlation between HCV-RNA levels and any of the variables examined although a trend toward increasing HCV-RNA levels with increasing numbers of rejection episodes was observed. In addition, histologic recurrence occurred more frequently for patients treated with OKT3. We conclude that the quantity of circulating viral genome is not influenced by immunosuppressive load and does not correlate with laboratory or histologic signs of recurrence. The roles that rejection, and possibly OKT3, play in the recurrence of HCV after liver transplant need further study.

Female↗
PubMed1992

Enhanced growth of a human keratinocyte cell line induced by antisense RNA for parathyroid hormone-related peptide.

We have used antisense RNA technology to inhibit endogenous parathyroid hormone-related peptide (PTHRP) production in the established human keratinocyte cell line, HPK1A, in order to assess the role of PTHRP as a potential modulator of cell growth. Rat PTHRP cDNA was cloned into the replication defective retroviral vector pYN in an antisense orientation and a stable cell line (HPK1A-AS) was generated after infection by amphotropic virus and selection by the neomycin derivative, G418. Expression of the transfected antisense sequence was confirmed with an RNA sense probe for PTHRP. The effect of the retrovirally mediated gene transfer on the endogenous PTHRP transcript was examined with an RNA antisense probe which demonstrated an absence of the endogenous transcript in HPK1A-AS cells. A 1.6-kilobase transcript was, however, present in equivalent quantities in both uninfected HPK1A and pYN-infected (HPK1A-pYN) cells. Immunocytochemistry and assessment of PTHRP secretion into the medium using an NH2-terminal radioimmunoassay and a UMR 106 adenylate cyclase bioassay confirmed the absence of PTHRP in HPK1A-AS cells. Examination of the inhibition of PTHRP production on cell growth demonstrated a reduction in doubling time and an increase in [3H]thymidine incorporation. Cell cycle analysis showed an increase in the proportion of the cell population in the S phase (relative to G0/G1) in HPK1A-AS cells compared to HPK1A or HPK1A-pYN cells. These data, therefore, indicate that endogenous PTHRP acts as an effective inhibitor of cell growth in this keratinocyte model and that this action occurs, at least in part, by diminishing entry into the S phase of the cell cycle. Furthermore, the antisense RNA method is a potent one to evaluate the cellular actions of PTHRP.

Amino Acid Sequence↗
PubMed1998

Expression, purification and characterization of the recombinant ribonuclease P protein component from Bacillus subtilis.

Ribonuclease P is a ribonucleoprotein complex that catalyzes the essential 5' maturation of all precursor tRNA molecules. The protein component both alters the conformation of the RNA component and enhances the substrate affinity and specificity. To facilitate biochemical and biophysical studies, the protein component of Bacillus subtilis ribonuclease P (RNase P) was overproduced in Escherichia coli using the native amino acid sequence with the initial 20 codons optimized for expression in E.coli . A simple purification procedure using consecutive cation exchange chromatography steps in the presence and absence of urea was developed to purify large quantities of P protein without contaminating nucleic acids. The identity of the recombinant protein as a cofactor of RNase P was established by its ability to stimulate the activity of the RNA component in low ionic strength buffer in a 1:1 stoichiometry. Circular dichroism studies indicate that P protein is a combination of alpha-helix and beta-sheet secondary structures and is quite stable, with a T m of 67 degrees C. The described methods facilitated the large scale purification of homogeneous, RNA-free P protein required for high resolution crystallographic analyses and may be useful for the preparation of other RNA binding proteins.

Amino Acid Sequence↗
PubMed2001

Performance characteristics of the COBAS AmpliScreen HIV-1 test, version 1.5, an assay designed for screening plasma mini-pools.

BACKGROUND: The COBAS AmpliScreen HIV-1 test, version 1.5 (v1.5) (Roche Molecular Systems), is designed for screening pools composed of samples from 24 individual units of blood or plasma. A specimen-processing procedure (Multiprep) simultaneously concentrates and extracts HIV-1, HCV, and HBV particles from plasma and incorporates an HIV-1 internal control (IC) RNA. Processed samples are amplified by RT-PCR using HIV-1-specific primers and detected by hybridization of the amplified products to HIV-1- and IC-specific oligonucleotide probes. STUDY DESIGN AND METHODS: Plasma samples containing known quantities of HIV-1 were used to evaluate analytical sensitivity and precision and to validate a pool testing algorithm. Analytical specificity was evaluated by adding various viruses and bacteria to HIV-1-negative plasma. Seroconversion panels were tested to estimate the window-period reduction achieved by RNA testing. RESULTS: The analytical sensitivity of the test (concentration that yields > or = 95% positive results in a set of replicate tests) was 25 copies of HIV-1 RNA per mL of pooled plasma. Representative strains from all HIV-1 group M subtypes were reproducibly detected (> 95% positive results) at concentrations of 20 to 200 viral particles per mL. The test did not cross-react with a set of 31 viral and 5 bacterial isolates, and it yielded negative results on a panel of 500 blood samples from HIV-1-seronegative donors. Plasma samples containing abnormally high levels of Hb, albumin, triglycerides, or bilirubin did not interfere with the test. HIV-1 RNA was detected 2 to 14 days before HIV-1 antibody and 0 to 28 days before p24 antigen. The test specifically detected pools containing a single positive unit with 2400 HIV-1 RNA copies per mL and correctly identified the positive unit. CONCLUSION: The COBAS AmpliScreen HIV-1 test, v1.5, has sufficient sensitivity to detect a single infected unit containing 600 copies of HIV-1 per mL in a pool with 23 uninfected units and should reduce the window period between infection and seroconversion by at least 2 to 14 days.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Analysis of rat cytochrome P450 isoenzyme expression using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR).

A method was developed using reverse transcriptase-polymerase chain reaction (RT-PCR) to selectively detect and qualitatively determine the levels of mRNA expression of the major isoenzymes of cytochrome P450 (P450 1A1, 1A2, 2B1/2, 2C11, 2E1, 3A1, 3A2, and 4A1) and fatty acyl-CoA oxidase (FACO) in the rat. Total liver RNA was isolated from male Sprague-Dawley rats treated with various inducers of cytochrome P450 (P450) and analyzed for the presence and relative quantities of each P450 isoenzyme mRNA using this technique. The specificity of the oligonucleotide primers used in the detection of each P450 mRNA was tested and confirmed through the simultaneous analysis of liver microsomal protein preparations for the presence of constitutive or inducible P450 apoprotein and enzyme activities using western immunoblotting and specific enzyme activity measures, respectively. This method of P450 expression analysis is proven to be highly specific and readily applicable for the assessment of P450 enzyme induction and down-regulation in the rat during routine toxicology studies when expression of the gene product is regulated by transcriptional activation and/or mRNA stabilization.

Animals↗
PubMed1991

Detection of toxic viral-associated double-stranded RNA (dsRNA) in influenza-infected lung.

While many of the molecular events in viral replication are well studied, the molecular mechanisms by which viral infections trigger such constitutional symptoms as fever and 'malaise' are unknown. The hypothesis that these viral constitutional symptoms can be triggered by the toxic action of dsRNA associated with viral replication was investigated. Total lung RNA from mice acutely infected with PR8 influenza virus, but not from sham-infected mice, was shown to induce fever and altered sleep (excess slow-wave sleep, enhanced amplitudes of electroencephalographic slow waves, and reduced rapid eye movement sleep) when injected into the rabbit brain. Viral-associated dsRNA was shown to be responsible for the rabbit responses by differential nuclease digestion. Influenza viral dsRNA was directly demonstrated in the active lung RNA preparations by reverse transcriptase-polymerase chain reaction techniques. The time course of the responses paralleled those seen in the same model inoculated with nanogram quantities of the synthetic dsRNA polyriboinosinic-polyribocytidylic acid and suggested that they were mediated by induced cytokines. A model for the role of viral-associated dsRNA in eliciting both local cytotoxicity and viral constitutional symptoms is presented.

Animals↗