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Results for “RNA, Competitive Endogenous”

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PubMed1979

Use of molecular hybridization to detect type D retrovirus markers in rhesus placentas and other tissues.

We have shown previously that approximately 20% of the Mason-Pfizer virus (MPV) genome is present as endogenous provirus in rhesus monkeys. We report here that several full-term rhesus placentas examined contain additional MPV proviral sequences in their DNA. Competitive molecular hybridization experiments demonstrated that some of these placentas also contain RNA complementary to the entire MPV 60 to 70S RNA genome. Examination of internal organs of rhesus monkeys captured in the wild also revealed the presence of additional MPV proviral sequences and expression of MPV RNA in some tissues. These results provide further evidence that MPV is being transmitted via a non-germ line mechanism in the rhesus population and now demonstrate the placenta as a good source for the identification of retrovirus transcriptional products and proviral DNA.

Animals↗

PubMed1998

Determination of transgene copy number and expression level using denaturing gradient gel electrophoresis.

Transgenic mice and cell lines are frequently developed to study human disease. Accurate determination of transgene copy number and levels of mRNA are necessary to understand the phenotypic changes observed in these models. Currently, transgene copy number and expression are estimated by Southern blot analysis of genomic DNA and Northern blot analysis of mRNA. We report a novel PCR-based method for determining transgene copy number and levels of transgene expression using competitive PCR between endogenous genomic genes and mutant transgenes followed by denaturing gradient gel electrophoresis (DGGE). We are able to accurately quantify a range of 1-10 copies of transgene incorporated per diploid genome. After reverse-transcribing RNA to cDNA, we are able to quantify levels of transgene mRNA that correlate with biochemical and histological evidence of transgene activity. In conclusion, resolving PCR and reverse transcription-PCR products by DGGE is a rapid and reproducible method that allows for accurate determination of transgene copy number and expression. This technique provides a more complete understanding of transgene effects.

Animals↗

PubMed2002

Application of quantitative competitive polymerase chain reaction for measurements of mRNA from antioxidative enzymes in the diabetic rat retina and kidney.

This present study applied quantitative competitive polymerase chain reaction (QC-PCR) in the analyses of mRNA expression of the endogenous antioxidative enzymes CuZn superoxide dismutase (SOD), MnSOD, catalase, and glutathione peroxidase in tissue samples from the retina and kidney cortex of diabetic rats. RNA was extracted from snap-frozen retinas and pieces of the kidney cortex of male Wistar rats with streptozotocin (STZ)-induced diabetes and control rats. The mRNA levels were analyzed using QC-PCR. The animals were kept in the laboratory for 1 and 6 months, respectively, and fed a normal or probucol- (1% wt/wt) enriched diet. By using QC-PCR, relative mRNA levels of all antioxidative enzymes could be estimated in the retina and kidney cortex. In the retina, the relative catalase mRNA concentration was about 1/10 that of the other enzymes. After 6 months of diabetes, there was a 100% increase of the catalase (median, 0.012 [range, 0.008 to 0.017] v 0.006 [0.005 to 0.010]; P =.011) and a 50% increase of the glutathione peroxidase mRNA levels (0.88 [0.44 to 1.12] v 0.52 [0.31 to 0.79]; P =.044). In the kidney cortex, the relative glutathione peroxidase mRNA level was 10 to 15 times higher, and catalase mRNA level about half of those of CuZnSOD and MnSOD. After 1 month of diabetes, there was an increase only of the glutathione peroxidase mRNA levels, by 170% (17.59 [6.19 to 29.49] v 6.96 [2.34 to 9.04]; P =.047). We conclude that quantification of mRNA can provide difficulties when the amount of sample RNA is limited and/or the gene expression is low. The present study shows QC-PCR to be useful as a tool for measuring expression of mRNA not only in the kidney cortex but also in small tissue samples like the retina. Our results indicate modestly increased mRNA expression of catalase and glutathione peroxidase in the retina and likewise modestly increased mRNA expression of glutathione peroxidase in the kidney cortex of rats with STZ-induced diabetes. Extended studies, also including enzyme activities, are needed before any effect of hyperglycemia on the overall enzyme activity can be established.

Animals↗

PubMed1996

Platelet-derived growth factor-A mRNA expression in fetal, normal adult, and atherosclerotic human aortas. Analysis by competitive polymerase chain reaction.

BACKGROUND: To understand which growth factors are important for growth of atherosclerotic plaques, it is necessary to know the factor's relative abundance and how its gene is regulated in relation to cell proliferation. We tested whether platelet-derived growth factor-A (PDGF-A) mRNA levels correlated with cell proliferation in developing aorta, normal adult aorta, and atherosclerotic plaques. METHODS AND RESULTS: We developed a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay to measure human PDGF-A mRNA levels in small tissue samples. A mutated PDGF-A synthetic RNA was used as an internal standard to compete with endogenous PDGF-A mRNA for amplification. The assay is highly sensitive and much more precise than routine RT-PCR. Correction for heteroduplex pairing between the endogenous and mutant PCR products correlates precisely with synthetic RNA standards and quantitative Northern blotting. Immunostaining with the proliferation marker (proliferating cell nuclear antigen) showed the following rank order of proliferation: fetal aorta >> atherosclerotic plaque > normal aortic media. PDGF-A mRNA levels, however, did not correlate with proliferation. Normal adult aorta contained the most PDGF-A mRNa (34.0+/-7.6 amol/microgram total RNA). Fetal aortas were intermediate (10.2+/-1.6 amol/microgram total RNA); advanced atherosclerotic plaques contained the least PDGF-A mRNA (0.3+/-0.1 amol/microgram total RNA). PDGF-A protein was readily detectable in normal media by immunostaining. Advanced plaques generally had less cell-associated PDGF-A protein, although A-chain was also detected in plaque matrix. CONCLUSIONS: PDGF-A mRNA and protein do not correlate with proliferation among these three groups. The significance of high levels of PDGF-A mRNA in the "quiescent" aortic media is unknown but it clearly does not promote cell replication.

Adult↗

PubMed1999

A receptor activity modifying protein (RAMP)2-dependent adrenomedullin receptor is a calcitonin gene-related peptide receptor when coexpressed with human RAMP1.

Adrenomedullin (ADM) and alpha- and beta-calcitonin (CT) gene-related peptide (alpha-, betaCGRP) are structurally related vasodilatory peptides with homology to CT and amylin. An originally orphan human CT receptor-like receptor (hCRLR) is a Gs protein-coupled CGRP or ADM receptor when coexpressed with recently identified human single transmembrane domain receptor activity modifying proteins 1 (hRAMP1) or -2 (hRAMP2), respectively. Here, the function of the rat CRLR homologue (rCRLR) has been investigated in rat osteoblast-like UMR-106 cells and in COS-7 cells, in the absence and presence of hRAMP1 and -2 and combinations thereof. Transient expression of rCRLR in UMR-106 cells revealed an ADM receptor, and [125I]rat (r) ADM binding was enhanced with hRAMP2 and inhibited by 50% when hRAMP1 was coexpressed. Detectable [125I]h alphaCGRP binding required the presence of hRAMP1, and the expression of CGRP binding sites was unaffected by coexpressed hRAMP2. Specificity of ADM binding sites in [125I]rADM binding inhibition experiments was reflected by an over 1000-fold higher potency of rADM [half-maximal effective concentration = 0.19 +/- 0.05 nM (mean +/- SEM, n = 4)], compared with r alphaCGRP and r betaGRP, to induce a cAMP-responsive luciferase reporting gene (CRE-luc). In rCRLR and hRAMP1 cotransfected cells, expressing predominantly CGRP binding sites, r betaCGRP, r alphaCGRP, and rADM induced CRE-luc with half-maximal effective concentration of 0.27 +/- 0.17 nM, 0.37 +/- 0.27 nM, and 1.4 +/- 0.9 nM, respectively. In COS-7 cells, the results were comparable, but rCRLR required coexpressed hRAMP2 for ADM receptor function. This is consistent with higher levels of endogenous RAMP2 encoding messenger RNA in UMR-106, compared with COS-7 cells. In conclusion, the recognition of RAMP1 and -2 as mediators of CRLR expression as a CGRP or ADM receptor has been extended, with evidence that endogenous RAMP2 is sufficient to reveal an ADM receptor in UMR-106 cells. Inhibition of RAMP2-evoked ADM receptor expression by RAMP1 and generation of a CGRP receptor is consistent with competitive interactions of the different RAMPs with rCRLR.

Animals↗

PubMed1980

Specific antigenic relationships between the RNA-dependent DNA polymerases of avian reticuloendotheliosis viruses and mammalian type C retroviruses.

Immunoglobulin G directed against the DNA polymerase of Rauscher murine leukemia virus (R-MuLV) could bind to 125I-labeled DNA polymerase of spleen necrosis virus (SNV), a member of the reticuloendotheliosis virus (REV) species. Competition radioimmunoassays showed the specificity of this cross-reaction. The antigenic determinants common to SNV and R-MuLV DNA polymerases were shared completely by the DNA polymerases of Gross MuLV, Moloney MuLV, RD 114 virus, REV-T, and duck infectious anemia virus. Baboon endogenous virus and chicken syncytial virus competed partially for antibodies directed against the common antigenic determinants of SNV and R-MuLV DNA polymerases. DNA polymerases of avian leukosis viruses, pheasant viruses, and mammalian type B and D retroviruses and particles with RNA-dependent DNA polymerase activity from the allantoic fluid of normal chicken eggs and from the medium of a goose cell culture did not compete for the antibodies directed against the common antigenic determinants of SNV and R-MuLV DNA polymerases. We also present data about a factor in normal mammalian immunoglobulin G that specifically inhibits the DNA polymerases of REV and mammalian type C retrovirus DNA polymerases.

AKR murine leukemia virus↗

PubMed1998

Interleukin 1beta-stimulated production of nitric oxide in rat hepatocytes is mediated through endogenous synthesis of interferon gamma.

The multiple interlocking regulatory mechanisms that underlie induction of hepatocyte inducible nitric oxide synthase (iNOS) expression are largely unknown. Although previous work has indicated the requirement for multiple proinflammatory cytokines to induce hepatocyte NO production, investigators have recently shown that interleukin-1beta (IL-1beta) alone can initiate iNOS expression. In contrast, interferon gamma (IFN-gamma) serves as the sole initiating factor in other cell systems. On the basis of the known ability of IL-1beta to induce transcription and translation of the IFN family of genes, we hypothesized that IL-1beta-mediated hepatocyte expression of iNOS is dependent on endogenous IFN-gamma synthesis. In a system of rat hepatocytes in primary culture, IL-1beta induced production of both NO and IFN-gamma. Using in situ hybridization and immunoblot analysis, IFN-gamma messenger RNA (mRNA) and protein were detected in hepatocytes exposed to IL-1beta. Inhibition of NO synthesis using the competitive substrate inhibitor N-monomethyl-L-arginine (100 micromol/L) did not alter the extent of IL-1beta-mediated IFN-gamma synthesis. In contrast, anti-IFN-gamma antibody or inhibition of IFN-gamma mRNA translation by addition of antisense IFN-gamma oligodeoxynucleotide probes resulted in undetectable levels of NO metabolites and iNOS protein. Repletion of IFN-gamma to the system restored NO production to levels noted in the presence of IL-1beta alone. Transient transfection analysis using a rat hepatocyte iNOS promoter-reporter gene plasmid construct showed that IL-1beta-induced promoter activation was abolished in the presence of anti-IFN-gamma or antisense IFN-gamma. Again, addition of IFN-gamma to the system restored activity of the iNOS promoter. Parallel experiments examining IL-1beta-mediated endogenous hepatocyte IL-1beta and TNF-alpha synthesis indicated no role for these cytokines in the induction of iNOS expression by IL-1beta. It is concluded that IL-1beta-mediated hepatocyte synthesis of NO is dependent on the simultaneous endogenous synthesis of IFN-gamma.

Animals↗

PubMed1999

Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP.

Raf-1 phosphorylates and activates MEK-1, a kinase that activates the extracellular signal regulated kinases (ERK). This kinase cascade controls the proliferation and differentiation of different cell types. Here we describe a Raf-1-interacting protein, isolated using a yeast two-hybrid screen. This protein inhibits the phosphorylation and activation of MEK by Raf-1 and is designated RKIP (Raf kinase inhibitor protein). In vitro, RKIP binds to Raf-1, MEK and ERK, but not to Ras. RKIP co-immunoprecipitates with Raf-1 and MEK from cell lysates and colocalizes with Raf-1 when examined by confocal microscopy. RKIP is not a substrate for Raf-1 or MEK, but competitively disrupts the interaction between these kinases. RKIP overexpression interferes with the activation of MEK and ERK, induction of AP-1-dependent reporter genes and transformation elicited by an oncogenically activated Raf-1 kinase. Downregulation of endogenous RKIP by expression of antisense RNA or antibody microinjection induces the activation of MEK-, ERK- and AP-1-dependent transcription. RKIP represents a new class of protein-kinase-inhibitor protein that regulates the activity of the Raf/MEK/ERK module.

3T3 Cells↗

PubMed2005

Different MHC class I heavy chains compete with each other for folding independently of beta 2-microglobulin and peptide.

We reported previously that different MHC class I molecules can compete with each other for cell surface expression in F(1) hybrid and MHC class I transgenic mice. In this study, we show that the competition also occurs in transfected cell lines, and investigate the mechanism. Cell surface expression of an endogenous class I molecule in Chinese hamster ovary (CHO) cells was strongly down-regulated when the mouse K(d) class I H chain was introduced by transfection. The competition occurred only after K(d) protein translation, not at the level of RNA, and localization studies of a CHO class I-GFP fusion showed that the presence of K(d) caused retention of the hamster class I molecule in the endoplasmic reticulum. The competition was not for beta(2)-microglobulin, because a single chain version of K(d) that included mouse beta(2)-microglobulin also had a similar effect. The competition was not for association with TAP and loading with peptide, because a mutant form of the K(d) class I H chain, not able to associate with TAP, caused the same down-regulation of hamster class I expression. Moreover, K(d) expression led to a similar level of competition in TAP2-negative CHO cells. Competition for cell surface expression was also found between different mouse class I H chains in transfected mouse cells, and this competition prevented association of the H chain with beta(2)-microglobulin. These unexpected new findings show that different class I H chains compete with each other at an early stage of the intracellular assembly pathway, independently of beta(2)-microglobulin and peptide.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1985

Functional analysis of reverse transcription by a frameshift pol mutant of murine leukemia virus.

Endogenous reverse transcription by wild-type murine leukemia virus (MuLV) was compared to that catalyzed by clone 23, a pol mutant containing a reverse transcriptase protein which lacks the carboxyl-terminal third of the molecule (J. G. Levin, S. C. Hu, A. Rein, L. I. Messer, and B. I. Gerwin (1984), J. Virol. 51, 470-478). Competition immunoassays revealed that mutant virions contain normal amounts of polymerase protein, indicating that the lack of carboxyl-terminal sequences does not alter normal processing of enzyme precursors. Although the mutant enzyme was previously shown to have the ability to copy and degrade RNA:DNA hybrids, the present study demonstrates that it is defective in functions required to generate full-length copies of viral DNA. Analysis of products of endogenous reverse transcription showed that minus-strand strong-stop DNA is formed and that mutant virions synthesize a series of minus-strand DNA intermediates up to 2.2 kb in length. Comparison of mutant and wild-type MuLV reaction products indicated that the 2.2-kb termination site of the mutant corresponds to a normal pausing region for the wild-type enzyme. Computer analysis of sequences and structure within pausing regions suggested the involvement of C-rich consensus sequences plus multibranch loop structures in the general phenomenon of enzyme-pausing during reverse transcription.

DNA, Viral↗

PubMed1983

Studies on the mechanism of anti-inflammatory activities of papyriogenin a and papyriogenin C.

The mechanism of anti-inflammatory action of papyriogenin A and C, obtained from Tetrapanax papyriferum, were investigated by the following methods; the cotton pellet granuloma test in normal and adrenalectomized rats, the blockade by anti-glucocorticoids of vascular permeability caused by serotonin in mice and the competition on 5 beta-reduction of steroidal compounds. The anti-inflammatory activity was observed in the decreasing order; papyriogenin C greater than papyriogenin A on the carrageenin-induced paw edema in mice, as reported in our previous paper. In a serotonin-induced paw edema, pretreatment with progesterone (50, 100 and 200 mg/kg) blocked completely the anti-inflammatory effects of papyriogenin A or C at 10 and 50 mg/kg. When actinomycin D (1 and 2 mg/kg) or cycloheximide (6 mg/kg) was given twice, simultaneously with papyriogenin A or C, during the latent period for the manifestation of the anti-inflammatory effect, the anti-inflammatory effects of papyriogenin A and C were blocked completely by s.c. administration. The effects of papyriogenin A or C, 30 mg/kg p.o., on the cotton pellet granuloma test in adrenalectomized rats were similar to those of normal rats. On the other hand, the competitive effects of papyriogenin A and C on 5 beta-reduction of testosterone and cortisol were recognized to be significant. These activities of papyriogenin A and C are explained by their steroidal actions in the target cell and their competitive effects in endogenous corticoid metabolism in liver.

Animals↗

PubMed1995

Heparin inhibits the expression of interleukin-11 and granulocyte-macrophage colony-stimulating factor in primate bone marrow stromal fibroblasts through mRNA destabilization.

Interactions between different cytokines, extracellular matrix components, and various cell types inside the bone marrow microenvironment are believed to play important roles in the regulation of hematopoiesis. We observed that both interleukin-1 (IL-1) and 12-O-tetradecanoylphorbol-13-acetate (TPA) can stimulate the expression of IL-11 and granulocyte-macrophage colony-stimulating factor (GM-CSF) genes in a primate bone marrow stromal fibroblast cell line, PU-34. We also found that IL-1 or TPA-stimulated IL-11 and GM-CSF expression in PU-34 cells can be abolished by heparin, a class of molecules related to extracellular matrix components, glycosaminoglycans. Because the growth inhibitory signals provided by extracellular factors were less understood, the mechanisms of heparin inhibition of IL-11 and GM-CSF gene expression were further investigated. Our data demonstrate for the first time that heparin did not alter the transcription of endogenous IL-11 and GM-CSF genes or an exogenous IL-11 promoter construct containing an AP-1 sequence. Instead, heparin facilitated the degradation of the corresponding mRNAs. Through RNA gel shift assays, heparin-mediated mRNA destabilization was tentatively linked to its competition for mRNA binding proteins both in the cell-free system and in intact cells. Collectively, our findings suggest that varying degrees of heparin inhibition may provide a novel mechanism for the regulation of cytokine expression during the growth and differentiation of different lineages of hematopoietic cells.

Animals↗

PubMed2000

Agrin fragments differentially induce ectopic aggregation of acetylcholine receptors in myotomal muscles of Xenopus embryos.

Agrin is an extracellular synaptic protein that organizes the postsynaptic apparatus, including acetylcholine receptors (AChRs), of the neuromuscular junction. The COOH-terminal portion of agrin has full AChR-aggregating activity in culture, and includes three globular domains, G1, G2, and G3. Portions of the agrin protein containing these domains bind to different cell surface proteins of muscle cells, including alpha-dystroglycan (G1-G2) and heparan sulfate proteoglycans (G2), whereas the G3 domain is sufficient to aggregate AChRs. We sought to determine whether the G1 and G2 domains of agrin potentiate agrin activity in vivo, as they do in culture. Fragments from the COOH-terminal of a neuronal agrin isoform (4,8) containing G3, both G2 and G3, or all three G domains were overexpressed in Xenopus embryos during neuromuscular synapse formation in myotomal muscles. RNA encoding these fragments of rat agrin was injected into one-cell embryos. All three fragments increased the ectopic aggregation of AChRs in noninnervated regions near the center of myotomes. Surprisingly, ectopic aggregation was more pronounced after overexpression of the smallest fragment, which lacks the heparin- and alpha-dystroglycan-binding domains. Synaptic AChR aggregation was decreased in embryos overexpressing the fragments, suggesting a competition between endogenous agrin secreted by nerve terminals and exogenous agrin fragments secreted by muscle cells. These results suggest that binding of the larger agrin fragments to alpha-dystroglycan and/or heparan sulfate proteoglycans may sequester the fragments and inhibit their activity in embryonic muscle. These intermolecular interactions may regulate agrin activity and differentiation of the neuromuscular junction in vivo.

Agrin↗

PubMed1995

An endothelin-1 mediated autocrine growth loop involved in human renal tubular regeneration.

Renal tubules have the capacity to regenerate following injury. We have investigated the possibility that tubular-derived endothelins, acting as autocrine growth factors, may be involved in this response in human kidney. ET-1 immunoreactivity was demonstrated by immunohistochemical staining in proximal tubules, distal cortical tubules and medullary collecting ducts of human kidney. In cultured human renal proximal tubular cells, RNAase protection assays demonstrated the expression of ET-1 and ET-2 mRNA's, and radioimmunoassay, following separation of conditioned medium by reverse phase HPLC, showed immunoreactive material which co-eluted with ET-1 and ET-2. Competition binding studies revealed the presence of at least two types of endothelin receptor: one with high and one with low affinity for ET-3 relative to ET-1. Analysis of cellular RNA by RT-PCR demonstrated expression of mRNA's for both ETA and ETB receptor subtypes. Combined blockade of ETA and ETB receptors (by PD-145065) but not that of ETA receptors alone (by BQ-123) blocked the mitogenic effect of exogenous or endogenous ET-1 and also profoundly suppressed endogenous ET-1 synthesis. By contrast, incubation with the ETB receptor agonist, BQ-3020, stimulated endogenous ET-1 synthesis. Exposure of the cells to hypoxia (1% O2 for 16 to 24 hr) resulted in specific up-regulation of ET-1 but not ET-2 gene expression. These findings reveal the existence of a hypoxia-inducible, autocrine growth system in human proximal tubular cells, which is mediated by ET-1 through the ETB receptor, and which could function in vivo as an autoregenerative system for restoring tubular integrity after injury. The widespread distribution of ET-1 peptide in different tubular segment suggests that ET-1 mediated tubular regeneration may also occur in other nephron segments.

Binding, Competitive↗

PubMed1978

Isolation and characterization of an endogenous type C RNA virus of mink (Mv1Lu) cells.

Cells of the established MvlLu mink line spontaneously released a reverse transcriptase-containing virus after long-term passage in tissue culture. By molecular hybridization, DNA of normal mink cells was found to possess extensive nucleotide sequence homology with a reverse-transcription product of the viral genome, demonstrating that the new isolate was an endogenous virus of mink origin. The mink virus shared antigenic determinants with the major structural proteins of known mammalian type C viruses. Double-antibody competition radioimmunoassays were developed by utilizing the purified major structural protein, p30, of the mink endogenous virus. The virus was shown to possess antigenic determinants unique from those of other known mammalian type C viruses. It exhibited a higher degree of immunological cross-reactivity with endogenous rat type C and horizontally transmitted feline leukemia viruses than with other mammalian type C viruses tested. The finding that mink cells can remain nonvirus producing for many cell generations argues that there normally exists some cellular restriction to endogenous virus expression in this species.

Animals↗

PubMed1977

Casein and alpha-lactalbumin messenger RNA in experimental breast cancer.

A transplantable rat mammary carcinoma (R3230AC) was previously shown to contain prolactin receptors. Our objective was to determine whether these receptors were functional by measuring specific markers of prolactin action: casein and alpha-lactalbumin (alphaLA) messenger RNA's (mRNA's). Total RNA exacts were translated in a wheat germ cell-free system. Newly synthesized 3H-casein and 3H-alphaLA were separately precipitated with specific antibodies and identified by their mobilities on sodium dodecyl sulfate-acrylamide gels and by competition with nonradioactive casein and alphaLA. Casein and alphaLA mRNA's were both present in unstimulated tumors grown in virgin rats. Casein mRNA but not alphaLA mRNA was markedly stimulated by injections of exogenous ovine prolactin or perphenazine, an agent that stimulates endogenous prolactin. This response was selective in that total mRNA was not significantly altered by prolactin.

Animals↗

PubMed2004

Persistence of MHC DR nonexpression on swine cells by introduction of a mutated MHC class II transactivator gene: a comparison with the effect induced by antisense RNA.

CIITA (class II transactivator) is a coactivator essential for transcription of MHC class II genes. In this study, a construct with a mutated CIITA gene with N-terminal domains depleted was constructed. This mutated CIITA (mCIITA) was able to repress DR and DQ expression in 45.0-60.0% of the mCIITA-transfected clones of swine endothelial cell line PIEC and in B cell line L23, as well as in human cell lines HeLa and Raji. Similarly, 30.0-46.7% of swine cell clones containing the human CIITA antisense RNA also failed to express DR molecules. However, the persistence of the DR repression on the cell lines is quite different. Transfection with mCIITA was persistent for at least 120 days, while with the CIITA antisense RNA, persistence existed for only 35-45 days. To explore the underlying mechanism, Raji cells were transfected with pUHD10-3-mCIITA, a mCIITA-containing, doxycycline-dependent plasmid. The intensity of DR repression is correlated quite well with the efficiencies of the mCIITA expression within the cells in a doxycycline dose-dependent manner. This implicates a competition between the mCIITA and its endogenous full-length counterpart. In addition, we were able to show that purified human CD4 T cells did not respond to the mCIITA-transfected PIECs in xenogeneic mixed lymphocyte endothelial reaction (MLER). The stimulating indices (SI) were only 1.0-1.5, compared with 15.2-18.2 for those transfected with empty vector or an initiation codon-depleted mCIITA that is dysfunctional for protein translation. The results we obtained, especially those for persistent suppression of class II genes, show promise for the possible development of mCIITA-transgenic swine for organ/tissue xenotransplantation.

Animals↗

PubMed1999

Decreased expression of glutathione S-transferase M1 in HPV16-transfected human cervical keratinocytes in culture.

Glutathione S-transferase (GST) M1 is a member of the GST mu family of cytosolic enzymes that have been hypothesized to catalyze the conjugation of glutathione to a large number of hydrophobic substances, including carcinogens such as polynuclear aromatic hydrocarbons present in tobacco smoke, leading to their excretion. Epidemiologic and experimental evidence suggests that the risk of cervical cancer is related to both human papillomavirus (HPV) infection and cigarette smoking. We compared the enzymatic activities and mRNA levels of GSTs in GSTM1-positive human cervical keratinocytes (HCKs) that had been transfected with HPV16 with those in the parental cells. The GSTM1 activity toward the substrate trans-stilbene oxide was 5- to 7-fold lower than in the parental cells. The relative mRNA level in HCK transfected with HPV16 E6/E7, as quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) with normalization against endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, was 6% that of the parental cells. It was 16 and 82%, respectively, in cells that were transfected with HPV16 E6 alone or HPV16 E7 alone. When quantified by competitive RT-PCR using an exogenous nuclease-resistant synthetic cyclophilin RNA transcript as control, the mRNA level in HCK transfected with HPV16 E6 was approximately 10-fold lower that that in the parental cells. It was approximately 5- to 7-fold lower in the HPV16 E7 or HPV16 E6/E7 cells. Our results suggest that viral infections, through the modulation of cellular xenobiotic-metabolizing enzymes, may play a role in the ability of cells to handle environmental carcinogens.

Carcinogens, Environmental↗