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PubMedDate not supplied

[mRNA sequences complementary to the double-stranded regions of pre-mRNA. Hybridization experiments using RNA mercuriation and chromatography on sulfhydryl-Sepharose].

It was demonstrated that 4--6% of the sequences of mouse cell pure polysomal poly (A)mRNA are able to form stable duplexes during hybridization with the excess of double-stranded structures of nuclear pre-mRNA (double-stranded RNA). Hybridization of mRNA with the excess of mercuriated double-stranded RNA and chromatography of the hybrids on sulfhydril-sepharose was investigated. It was found that only 7--11% of the molecules of polysomal poly(A)+mRNA are able to form thermostable hybrids with sequences of double-stranded RNA. It may be supposed that in the population of mRNA there are relatively few molecules (about 5%) which contain sequences originated from long hairpin structures of pre-mRNA. Moreover, probably, there is a number of mRNA molecules (about 5% of the mRNA population) which are complementary in the entire length to "non-hairpin" double-stranded regions of pre-mRNA.

Base Sequence↗
PubMed1983

Elicitor induction of mRNA activity. Rapid effects of elicitor on phenylalanine ammonia-lyase and chalcone synthase mRNA activities in bean cells.

Changes in the activity levels of mRNAs encoding phenylalanine ammonia-lyase and chalcone synthase, two characteristic enzymes of phenylpropanoid biosynthesis, in elicitor-treated cells of dwarf French bean (Phaseolus vulgaris L.) have been investigated by immunoprecipitation of [35S]methionine-labelled enzyme subunits synthesised in vitro in an mRNA-dependent rabbit reticulocyte lysate translation system. Elicitor heat-released from cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes marked rapid increases in the polysomal activities of the mRNAs encoding the two enzymes concomitant with the phase of rapid increase in enzyme activity at the onset of phaseollin accumulation during the phytoalexin defence response. Increased polysomal mRNA activities encoding the two enzymes can be observed 30 min after elicitor treatment. The patterns of induction of the mRNA activities are broadly similar with respect to time and elicitor concentration although small but distinct differences between the enzymes were observed in the elicitor concentration giving maximum induction. There is a close correlation between the induction of polysomal mRNA activity and the induction of enzyme synthesis in vivo by elicitor treatment with respect to both the kinetics of induction and the dependence on elicitor concentration. The data indicate that elicitor stimulation of phenylalanine ammonia-lyase and chalcone synthase synthesis in vivo is largely a result of increased polysomal activity of the mRNAs encoding these enzymes. Similar patterns of induction of polysomal mRNA activity are observed with elicitor preparations from a variety of sources. The marked increases in polysomal mRNA activities encoding phenylalanine ammonia-lyase and chalcone synthase are increases as a proportion of total cellular mRNA activity, indicating that elicitor does not increase these polysomal mRNA activities by stimulation of selective recruitment from the total pool of cellular mRNA.

Acyltransferases↗
PubMed1994

Agonist-mediated destabilization of m1 muscarinic acetylcholine receptor mRNA. Elements involved in mRNA stability are localized in the 3'-untranslated region.

The effects of chronic agonist exposure on receptor number (down-regulation) have been shown, in part, to be due to effects on mRNA levels. Agonist-mediated effects on muscarinic acetylcholine receptor (mAChR) mRNA were investigated in Chinese hamster ovary (CHO) cells stably transfected with m1 mAChR gene constructs containing the open reading frame and a series of deletions of the flanking 3'-untranslated region (3'-UTR). Carbachol (CBC) down-regulated m1 mAChRs encoded by the construct m1C1, an m1 mAChR transcript containing the entire flanking 3'UTR (nucleotides 1526-2622), in a time-dependent fashion with maximal decreases occurring by 12 h. Steady-state levels of m1C1 mRNA declined in a parallel fashion beginning 6 h after CBC pretreatment. Similar findings were obtained with m1C2, a construct which is missing all but 261 bases of flanking 3'-UTR (nucleotides (nt) 1526-1786). Since the rate of mRNA degradation represents an important potential regulatory mechanism to control the level of gene expression, we investigated the effects of CBC treatment on m1C1 and m1C2 mRNA stability. The half-life of either transcript in untreated cells was approximately 14 h, whereas m1C1 and m1C2 transcript half-lives decreased to approximately 3 h in cells treated with CBC. Agonist-induced destabilization of m1C2 mRNA could be mimicked by phorbol esters in a concentration-dependent manner and blocked by the protein kinase inhibitor, H-7. In contrast, m1 mAChR mRNA constructs missing nt 1526-1786 of the 3'-UTR (m1C3 and m1C4) did not undergo agonist- or phorbol ester-induced destabilization. In the neuroblastoma cell line IMR-32, endogenous m1 mAChR mRNA was down-regulated and destabilized following CBC treatment. These results demonstrate that agonist-induced mRNA destabilization is a potential mechanism for regulating m1 mAChR levels. Furthermore, deletion studies identify a 261 base region of the 3'-UTR having the potential to form stable stem-loop structures which likely harbors element(s) responsible for message destabilization.

Animals↗
PubMed2004

The Glc7p nuclear phosphatase promotes mRNA export by facilitating association of Mex67p with mRNA.

mRNA export is mediated by Mex67p:Mtr2p/NXF1:p15, a conserved heterodimeric export receptor that is thought to bind mRNAs through the RNA binding adaptor protein Yra1p/REF. Recently, mammalian SR (serine/arginine-rich) proteins were shown to act as alternative adaptors for NXF1-dependent mRNA export. Npl3p is an SR-like protein required for mRNA export in S. cerevisiae. Like mammalian SR proteins, Npl3p is serine-phosphorylated by a cytoplasmic kinase. Here we report that this phosphorylation of Npl3p is required for efficient mRNA export. We further show that the mRNA-associated fraction of Npl3p is unphosphorylated, implying a subsequent nuclear dephosphorylation event. We present evidence that the essential, nuclear phosphatase Glc7p promotes dephosphorylation of Npl3p in vivo and that nuclear dephosphorylation of Npl3p is required for mRNA export. Specifically, recruitment of Mex67p to mRNA is Glc7p dependent. We propose a model whereby a cycle of cytoplasmic phosphorylation and nuclear dephosphorylation of shuttling SR adaptor proteins regulates Mex67p:Mtr2p/NXF1:p15-dependent mRNA export.

Biological Transport↗
PubMed1976

mRNA guanylyltransferase and mRNA (guanine-7-)-methyltransferase from vaccinia virions. Donor and acceptor substrate specificites.

Characterization of the donor and acceptor specificities of mRNA guanylyltransferase and mRNA (guanine-7-)-methyltransferase isolated from vaccinia virus cores has enabled us to discriminate between alternative reaction sequences leading to the formation of the 5'-terminal m7G(5')pppN-structure. The mRNA guanylyltransferase catalyzes the transfer of a residue of GMP from GTP to acceptors which possess a 5'-terminal diphosphate. A diphosphate-terminated polyribonucleotide is preferred to a mononucleoside diphosphate as an acceptor suggesting that the guanylyltransferase reaction occurs after initiation of RNA synthesis. Although all of the homopolyribonucleotides tested (pp(A)n, pp(G)n, pp(I)n, pp(U)n, and pp(C)n) are acceptors for the mRNA guanylyltransferase indicating lack of strict sequence specificity, those containing purines are preferred. Only GTP and dGTP are donors in the reaction; 7-methylguanosine (m7G) triphosphate specifically is not a donor indicating that guanylylation must precede guanine-7-methylation. The preferred acceptor of the mRNA (guanine-7-)-methyltransferase is the product of the guanylyltransferase reaction, a polyribonucleotide with the 5'-terminal sequence G(5')pppN-. The enzyme can also catalyze, but less efficiently methylation of the following: dinucleoside triphosphates with the structure G(5')pppN, GTP, dGTP, ITP, GDP, GMP, and guanosine. The enzyme will not catalyze the transfer of methyl groups to ATP, XTP, CTP, UTP, or to guanosine-containing compounds with phosphate groups in either positions 2' or 3' or in 3'-5' phosphodiester linkages. The latter specificity provides an explanation for the absence of internal 7-methylguanosine in mRNA. In the presence of PPi, the mRNA guanylyltransferase catalyzes the pyrophosphorolysis of the dinucleoside triphosphate G(5')pppA, but not of m7G(5')pppA. Since PPi is generated in the process of RNA chain elongation, stabilization of the 5'-terminal sequences of mRNA is afforded by guanine-7-methylation.

Guanosine Triphosphate↗
PubMed1996

Partial reversal of increased preproenkephalin messenger ribonucleic acid (mRNA) and decreased preprotachykinin mRNA by foetal dopamine cells in unilateral 6-hydroxydopamine-lesioned rat striatum parallels functional recovery.

In situ hybridization histochemistry was used to investigate the expression of striatal preproenkephalin and preprotachykinin messenger ribonucleic acid (mRNA) in rats with 6-hydroxydopamine lesions of the nigrostriatal pathway followed 4 weeks later by implantation of foetal dopamine cells into the denervated striatum. Striatal dopamine deafferentation caused an (+)-amphetamine-induced rotational asymmetry, an increase in striatal preproenkephalin mRNA message, and a decrease in striatal preprotachykinin mRNA message relative to control animals. Two months after grafting a foetal ventral mesencephalon suspension, there was reversal of the rotational asymmetry to (+)-amphetamine. At this time the increase in striatal preproenkephalin mRNA was significantly attenuated and the decrease in preprotachykinin mRNA was partially reversed compared to animals with a 6-hydroxydopamine lesion alone. Subregional analysis showed the attenuation of the increase in preproenkephalin mRNA to occur in dorsolateral, dorsomedial and ventromedial, but not ventrolateral, striatal subdivisions. The partial reversal of the decreased preprotachykinin mRNA density after grafting was only statistically significant in the DM and VM subdivisions. These results demonstrate graft-induced partial recovery of striatal function, as judged by preproenkephalin and preprotachykinin mRNA levels, within 2 months of transplantation.

Animals↗
PubMed1995

Postnatal rat lung retinoic acid receptor (RAR) mRNA expression and effects of dexamethasone on RAR beta mRNA.

Retinoids exert multiple effects upon lung differentiation and growth. Although the mechanisms involved are presently poorly understood, increasing evidence points to a central role of nuclear retinoic acid receptors (RAR). The purpose of this study was to determine RAR mRNA expression profile during postnatal alveolarization, compared with the expression in prenatal and adult rat lung, and to describe the effects of dexamethasone (DEX) and oxygen on postnatal lung RAR gene expression. Total RNA was isolated from lungs of Sprague-Dawley rats on prenatal day 19, on postnatal days 1, 3, 7, 10, and 14 of life, and from adults. One subgroup of littermate pups was treated with DEX daily for 3 or 7 days. In a second experiment, rats were exposed to room air or to 95% oxygen for 72 hours, and received either DEX or saline. Northern hybridization showed that the levels of all RAR subtypes in fetal lung were 45% or less of levels at postnatal day 1. The 3.7 kb RAR alpha transcript levels were lower than day 1 on days 10 and 14 (relative to day 1, day 10 = 0.54 +/- 0.05; day 14 = 0.54 +/- 0.08), but there was no change in a 2.7 kb RAR alpha transcript over this time period. By contrast, RAR beta mRNA levels were significantly higher at days 3, 10, and 14 compared with day 1 (day 3 = 1.79 +/- 0.19; day 10 = 1.41 +/- 0.14; day 14 = 1.53 +/- 0.05). Similarly, RAR gamma mRNA expression levels were higher on day 10 (1.45 +/- 0.09), but by day 14 there was no difference from day 1. Adult lung 3.7 kb RAR alpha, 2.7 kb RAR alpha, and RAR gamma were lower than day 1, but RAR beta was significantly greater (3.7 alpha = 0.52 +/- 0.05; 2.7 alpha = 0.49 +/- 0.26; gamma = 0.74 +/- 0.06; beta = 1.63 +/- 0.22). Treatment with DEX prevented the rise in RAR beta mRNA occurring on day 3 and significantly lowered (0.65 +/- 0.06) the amount of RAR beta mRNA in day 7 lung. Exposure of rat pups to oxygen caused an increase in RAR beta mRNA (1.21 +/- 0.03). DEX treatment again decreased RAR beta mRNA in both control (0.55 +/- 0.06) and oxygen-exposed pups (0.67 +/- 0.12). In addition, 2.7 kb RAR alpha mRNA was decreased with the combination of DEX and oxygen exposure (0.63 +/- 0.06). The differential gene expression profiles and the response to DEX and oxygen of the various members of the RAR family suggest that each subtype may have a specific role during the period of alveolarization in rat lung.

Air↗
PubMed1996

Differential regulation of mRNA specific for beta 1- and beta 2-adrenergic receptors in human failing hearts. Evaluation of the absolute cardiac mRNA levels by two independent methods.

In human heart failure beta-adrenergic receptors are downregulated which contributes to the reduced responsiveness to positive inotropic beta-agonists in the diseased heart. The present study addressed the question whether the number of beta-adrenergic receptors in the failing human heart is regulated at the level of the mRNA and whether the absolute steady-state levels of subtype-specific mRNAs mirror the expression of receptor-subtype proteins in human heart. In a collaborative effort, two different and independent methods, performed in two independent laboratories, reverse transcription followed by polymerase chain reaction (RT-PCR) and RNase protection assays, were used to determine the absolute steady-state levels of beta 1- and beta 2-adrenergic receptor mRNAs in control (NF) and in failing human hearts. As determined by quantitative RT-PCR the beta 1-mRNA was significantly reduced from 0.98 +/- 0.12 (n = 10) to 0.49 +/- 0.11 pg/microgram total RNA in dilated cardiomyopathy (dCMP, n = 7) and to 0.40 +/- 0.11 pg/microgram total RNA in ischemic cardiomyopathy (iCMP, n = 8). The steady-state levels of mRNA specific for beta 2-adrenergic receptors also tended to be decreased but without reaching significance (NF: 0.16 +/- 0.05, dCMP: 0.11 +/- 0.03, iCMP: 0.13 +/- 0.04 pg/microgram total RNA). RNase protection assays revealed similar values. beta 1-mRNA was found to be significantly reduced from 1.22 +/- 0.22 in NF (n = 10) to 0.63 +/- 0.14 pg/microgram total RNA in dCMP (n = 5) and to 0.52 +/- 0.1 pg/microgram total RNA in iCMP (n = 8). The beta 2-mRNA also tended to be lower in dCMP and in iCMP as compared to NF but again without reaching significance (NF: 0.14 +/- 0.02, dCMP: 0.099 +/- 0.02, iCMP 0.107 +/- 0.02 pg/microgram total RNA). This is the first study to demonstrate in parallel by two different methods performed independently in two laboratories that the ratio of beta 1- and beta 2-adrenergic receptor densities in the left ventricle of the normal human heart of about 80/20 is closely related to the absolute steady state concentrations of their specific mRNA. In addition, the magnitude of the decrease in mRNA-levels of beta 1- and beta 2-adrenergic receptors in the failing human heart closely correlates with the decrease of the respective receptor proteins. These data suggest that the predominant regulation of beta-adrenergic receptors occurs at the mRNA level.

Adult↗
PubMed1998

Increase in the expression of biglycan mRNA expression Co-localized closely with that of type I collagen mRNA in the infarct zone after experimentally-induced myocardial infarction in rats.

Biglycan, a small dermatan sulphate proteoglycan, has been postulated to interact with other components of the extracellular matrix (ECM), specifically collagens. We hypothesized that biglycan messenger ribonucleic acid (mRNA) is increased in the myocardial infarct zone. Biglycan mRNA expression after acute myocardial infarction (AMI) in rats was determined with the use of Northern blotting and in situ hybridization, and its expression pattern was compared to that of type I collagen mRNA [alpha1(I) collagen]. The left coronary artery was ligated in male Sprague-Dawley rats, and the hearts were excised on days 2 and 7. The Northern blot analysis demonstrated that expression of biglycan mRNA in the infarct on days 2 and 7 were 4.0- and 6.8-fold higher, respectively, compared to the sham-operated hearts. The in situ hybridization revealed intense signals for both biglycan and alpha1(I) collagen mRNA on day 2 in the spindle-shaped mesenchymal cells located between the surviving myocytes in the infarct peripheral zone. On day 7, biglycan mRNA signals were observed in the interior of the infarct around the infarct granulation tissue, a distribution that was essentially the same as that of alpha1(I) collagen. These results demonstrated that the increases in the infarct biglycan mRNA expression produced by mesenchymal cells (presumably myofibroblasts and fibroblasts) was closely co-localized with that of type I collagen mRNA, indicating that biglycan contributes to the infarct healing processes.

Animals↗
PubMed1999

Linking mRNA turnover and translation: assessing the polyribosomal association of mRNA decay factors and degradative intermediates.

mRNA decay is a multistep process, often dependent on the active translation of an mRNA and on components of the translation apparatus. In Saccharomyces cerevisiae, several trans-acting factors required for mRNA decay associate with polyribosomes. We have explored the specificity of the interactions of these factors with polyribosomes, using sucrose gradient sedimentation analysis of the yeast UPF1 protein to test whether such interactions are altered when polyribosomes are disrupted by treatment with EDTA, digestion with micrococcal nuclease, or shifting of cells containing a temperature-sensitive eIF3 mutation to the nonpermissive temperature. These experiments, as well as others assaying the strength of factor association in high-salt sucrose gradients, lead us to conclude that Upf1p is tightly bound to the smallest polyribosomes, but not to the 40S or 60S ribosomal subunits. Similar experimental approaches were used to determine whether mRNA decay initiates prior to mRNA release from polyribosomes. Using sucrose gradient fractionation and Northern blotting, we can detect the polysomal association of a PGK1 mRNA decay intermediate and conclude that mRNA decay commences while an mRNA is still being translated.

Genetic Techniques↗
PubMed1991

Differential mRNA stabilities affect mRNA levels in mutant mouse myeloma cells.

A series of mouse myeloma cell lines producing mutant gamma 2b immunoglobin heavy chains, which resemble heavy chain disease proteins, were analyzed for messenger RNA abundance as a function of mRNA alterations. A mutation effectively deleting the gamma 2b-CH1 domain of the mRNA had little or no effect on Ig heavy chain mRNA abundance on half-life (mutant 10.1). A mutation in the gamma 2b-CH2 and CH3 domain, causing premature termination of translation, had more deleterious effects on Ig heavy chain mRNA abundance and half-life (mutant I17). Substitution of the deleted portions of the gamma 2b mRNA with gamma 2a sequences by subclass switching in the cells (mutants K23 and K25) resulted in increased heavy chain abundance and half-life relative to the parent I17. In contrast, kappa light chain mRNA levels and half-lives remain constant among the mutants. The wild-type and mutant cell lines transcribed the Ig heavy chain gamma 2b locus equally when compared with an internal beta-actin standard by transcription run on studies. Therefore, half-life of the Ig heavy chain mRNA seems to be the principal determinant in cytoplasmic mRNA abundance in this system.

Animals↗
PubMed1992

Correlation between up-regulation of lymphokine mRNA and down-regulation of TcR, CD4, CD8 and lck mRNA as shown by the effect of CsA on activated T lymphocytes.

Full activation of different T cell populations via the TcR/CD3 complex leads to transient expression of lymphokine mRNA and to transient and specific down-modulation of TcR, CD4, CD8 and p56lck mRNA (Multi-Receptor Complex or MRC mRNA). This transient down-modulation is due to both a decrease in transcription and stability of these mRNA and is temporally and quantitatively related to lymphokine mRNA induction. We showed that cyclosporin A (CsA), which blocks lymphokine expression also inhibits MRC mRNA down-modulation at the transcriptional level, and does not affect mRNA stability. The fact that CsA inhibits both lymphokine expression and MRC mRNA down-modulation at transcriptional level supports a model in which similar signals trigger the inverse regulation of these two sets of genes via identical transcriptional factors.

Blotting, Northern↗
PubMed1996

Monitoring the temporal and spatial activation pattern of astrocytes in focal cerebral ischemia using in situ hybridization to GFAP mRNA: comparison with sgp-2 and hsp70 mRNA and the effect of glutamate receptor antagonists.

We investigated the temporo-spatial expression of astrocyte glial fibrillary acidic protein (gfap) and sulfated glycoprotein 2 (sgp-2) mRNAs in comparison to 70-kDa heat shock protein (hsp70) mRNA by in situ hybridisation in rats subjected to permanent occlusion of the middle cerebral artery (MCA). Gfap mRNA started to increase in the cingulate cortex of the lesioned hemisphere 6 h after MCA occlusion and gradually spread over the lateral part of the ipsilateral cortex and the striatum from 12 h to 3 days, peaking at 3 days after MCA occlusion. Gfap mRNA also increased in the contralateral cingulate cortex and corpus callosum at 12 and 24 h. Hsp70 mRNA increased markedly in the ipsilateral cortex adjacent to the ischemic lesion, and slightly within the lesion area from 3 to 24 h and disappeared after 3 days. By 7 days, gfap and sgp-2 mRNAs were increased markedly in the peri-infarct area, and in the ipsilateral thalamus parallel with the delayed neuronal damage, whereas the widespread increase of gfap mRNA in the ipsilateral hemisphere declined. Post-occlusion treatment with the glutamate receptor antagonists MK-801 and NBQX slightly attenuate the induction of gfap but did not qualitatively affect the topical expression pattern. Within the cingulate cortex MK-801 treatment resulted in a significant decrease of the signal intensity at all survival times, reflecting most likely an attenuation of lesion-induced spreading depression like depolarization waves by MK-801. The area of hsp70 expression was reduced by both MK-801 and NBQX, most likely reflecting the decrease of the lesion area by both treatment regimens. Our study thus revealed an early and widespread increase of gfap mRNA in the non-ischemic area including the contralateral hemisphere starting between 3 and 6 h, and a delayed circumscribed expression in the peri-infarct border zone after 1 week. Comparison with the expression of hsp70 mRNA suggests that the absence of an early gfap mRNA induction in the peri-lesion zone reflects an impairment of astrocytic function which may be of importance for infarct growth during the early evolution of the pathological process.

Animals↗
PubMed1988

Decline in c-myc mRNA expression but not the induction of c-fos mRNA expression is associated with differentiation of SH-SY5Y human neuroblastoma cells.

The induction of differentiation in SH-SY5Y human neuroblastoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by a rapid and a transient expression of c-fos mRNA and a down-regulation of c-myc mRNA. The TPA-induced expression of c-fos mRNA was inhibited by H-7, a specific inhibitor of protein kinase C (PK-C). Dioctanoylglycerol (DiC8) failed to induce differentiation of SH-SY5Y cells or to down-regulate c-myc mRNA but it did induce the expression of c-fos mRNA. Treatment of IMR-32 human neuroblastoma cells with TPA did not cause differentiation although c-fos mRNA was induced. Since PK-C in SH-SY5Y cells was activated by both TPA and DiC8 it is suggested that the activation of PK-C alone is not sufficient to induce differentiation in SH-SY5Y cells. The down-regulation of c-myc mRNA rather than the induction of c-fos mRNA seems to be associated with differentiation process in SH-SY5Y cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Low hprt mRNA levels and multiple hprt mRNA species in 6-thioguanine-resistant Chinese hamster cell mutants possessing nonsense mutations.

Previous studies suggested that many Chinese hamster ovary (CHO) cell hprt mutants having point mutations in the protein coding region also have low steady-state hprt mRNA concentrations. In addition, polymerase-chain reaction (PCR) amplification of hprt cDNA synthesized from some of these mutants results in multiple products containing deleted exons indicating that these mutants possess multiple species of hprt mRNA. In this study, we have used northern blot analysis to quantify the concentrations of hprt mRNA in 86 mutants known to possess point mutations leading to either missense or nonsense mutations. 28 of 35 nonsense mutants (80%), but only 7 of 51 missense mutants (14%), had < 50% of the hprt mRNA concentration found in parental CHO cells. Furthermore, all the nonsense mutants with premature termination codons in the internal exons of the gene (i.e., exons 3, 4, 5, 6 and 7) showed a significant reduction (averages < 16% of parental) in the steady-state levels of hprt mRNA, while nonsense mutants with termination codons situated in the extreme 5' and 3' regions of the gene had near parental hprt mRNA levels. In the same collection of mutants, the proportion of mutants producing multiple cDNA PCR products was much greater (18/35) for mutants having nonsense mutations than for mutants with missense mutations (2/51). All nonsense mutants with mutations in exons 3, 4 and 5 produced multiple species, while all those with mutations in exons 7, 8 and 9 produced a single PCR product. These results suggest that sequence changes in mammalian genes that affect protein chain length can also affect mRNA concentration and the splicing of pre-mRNA molecules.

Animals↗
PubMed1996

Caffeine-induced expression of c-fos mRNA and NGFI-A mRNA in caudate putamen and in nucleus accumbens are differentially affected by the N-methyl-D-aspartate receptor antagonist MK-801.

Caffeine (100 mg/kg, i.p.) induces a rapid increase in the expression of mRNA for the immediate early genes (IEGs) c-fos and NGFI-A in rat striatum. We have examined how this response is affected by pretreatment with either the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (1 and 3 mg/kg, i.p.), the competitive NMDA receptor antagonist D-CPP (6 mg/kg, i.p.), or the non-selective excitatory amino acid receptor antagonist kynurenic acid (300 mg/kg, i.p). The two NMDA receptor antagonists significantly reduced the caffeine-induced expression of both c-fos mRNA and NGFI-A mRNA in the medial part of the caudate putamen. The effect was less pronounced in the lateral part of the caudate putamen. MK-801 caused an enhancement of c-fos and NGFI-A mRNA expression in nucleus accumbens. Pretreatment with kynurenic acid caused no marked alterations in the caffeine-induced expression of c-fos mRNA and NGFI-A mRNA in any brain region. These findings suggest that glutamatergic transmission via NMDA receptors contributes to the induction of c-fos mRNA and NGFI-A mRNA by caffeine in striatum. In addition we show that MK-801 can either increase or decrease the caffeine effect on IEGs depending on the region studied.

Animals↗
PubMed1996

Analysis of beta-adrenergic receptor mRNA levels in human ventricular biopsy specimens by quantitative polymerase chain reactions: progressive reduction of beta 1-adrenergic receptor mRNA in heart failure.

OBJECTIVES: This study investigated the relation between the severity of heart failure and the extent of the reduction of beta 1-adrenergic receptor messenger ribonucleic acid (mRNA) levels in biopsy specimens from the ventricular septum obtained during cardiac catheterization of patients with various degrees of heart failure. BACKGROUND: Heart failure is accompanied by desensitization of the beta-adrenergic receptor system, which is in part due to downregulation of beta 1-adrenergic receptors. Downregulation of beta 1-adrenergic receptors has been suggested to be caused by reductions in mRNA levels. METHODS: Because biopsy specimens were small and receptor mRNAs not abundant, mRNA levels were determined by quantitative reverse transcription/polymerase chain reactions. This method was validated by measuring synthetic ribonucleic acid (RNA) standards and samples from explanted hearts by solution hybridization assays. Both methods yielded similar results, but the polymerase chain reaction method was approximately 1,000-fold more sensitive. Sources of variations in the polymerase chain reaction were quantitated and found to be best controlled for by determination of the glyceraldehyde phosphate dehydrogenase mRNA as an endogenous control. RESULTS: Beta 1-adrenergic receptor mRNA levels in the biopsy specimens were decreased by 7% in mild (New York Heart Association functional class II), 26% in moderate (functional class III) and > 50% in severe heart failure (functional class IV). There was a good correlation between hemodynamic indicators of heart failure and beta 1-adrenergic receptor mRNA levels. In contrast, beta 2-adrenergic receptor mRNA levels were apparently unaffected by heart failure. CONCLUSIONS: Reduced beta 1-adrenergic receptor mRNA levels occur early in heart failure and can be detected in septal biopsy specimens during right heart catheterization. The reduction in beta 1-adrenergic receptor expression may contribute to further loss of cardiac function.

Adult↗
PubMedDate not supplied

Delta opioid receptor mRNA distribution in the brain: comparison to delta receptor binding and proenkephalin mRNA.

The recent cloning of the mouse delta opioid receptor (Evans et al., 1992; Kieffer et al., 1992) has demonstrated it to be a member of the seven transmembrane G-protein coupled family of neurotransmitter receptors. The present study describes the cellular localization in the central nervous system (CNS) of an mRNA encoding this receptor and compares it with the distribution of delta receptor binding and proenkephalin mRNA using a combination of in situ hybridization and receptor autoradiographic techniques. Delta receptor mRNA was visualized with a cRNA probe (472-903 bp) corresponding to transmembrane domains III-VI of the receptor, while proenkephalin mRNA was labeled with a cRNA probe to exon 3 (139-832 bp). A high level of correspondence was observed between the distribution of delta receptor mRNA and delta receptor binding as defined by the selective ligand [3H]D-Pen2-Pen5-enkephalin. Delta receptor mRNA and binding were expressed in the neocortex, caudate-putamen, nucleus accumbens, olfactory tubercle, diagonal band of Broca, amygdala and the nucleus of the solitary tract. Discrepancies in the distribution of delta receptor mRNA and binding in the olfactory bulb, hippocampus, globus pallidus and substantia nigra pars reticulata, may in part be due to differential receptor synthesis and transport. These results are discussed in relation to the distribution of proenkephalin mRNA and how this may affect our understanding of opioid circuitry in the CNS.

Animals↗