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PubMed1975

Initiation of protein synthesis. Binding of messenger RNA.

Complexes between 30 S ribosomal subunits and fMet-tRNA are formed during incubation of 30 S subunits with fMet-tRNA and all other components for initiation of protein synthesis, except R17 bacteriophage RNA. That these complexes serve as intermediates in the binding of messenger RNA is demonstrated directly by the finding that upon addition of R17 RNA, fMet-tRNA in preformed fMet-RNA-30 S complexes preferentially enters fMet-tRNA-30 S-R17 RNA complexes. On the other hand, incubation of 30 S ribosomal subunits with R17 RNA and all other components for initiation except fMet-tRNA does not yield 30 S-R17 RNA complexes that can act subsequently as functional intermediates in the binding of fMet-tRNA: formation of fMet-tRNA-30 S-R17 RNA complexes does not occur when fMet-tRNA is added and further binding of R17 RNA to 30 S subunits is prevented by specific inhibitors. These experiments lead to an unambiguous order of events in the sequence of initiation, in which binding of fMet-tRNA to the small ribosomal subunit must occur before messenger RNA can be bound and phased correctly. Complexes between fMet-tRNA and 60 S subunits are in rapid equilibrium with the free components, and have a half-life of less than 2 min at 37 degrees. This explains why such complexes are not detected in sucrose gradients, unless they are first fixed with glutaraldehyde. Attachment of R17 RNA, however, results in formation of an fMet-tRNA-30 S-R17 RNA complex that is stabilized greatly; fMet-tRNA in this complex exchanges only very slowly with free fMet-tRNA. Initiation factor IF-3 has two functions in initiation. The first is to direct the binding of messenger RNA to the 30 S-fMet-tRNA complex. This function is not needed when initiation complex formation occurs on ApUpG triplets, in which case the second function of IF-3 is detected, that of providing free 30 S subunits for initiation. The ability of IF-3 to bind directly to R17 RNA may be related to its requirement in messenger RNA recognition. However, since IF-3 exhibits a greater affinity for the 30 S subunit than for R17 RNA, it appears that the recognition function of IF-3 is expressed while IF-3 is associated with the 30 S subunit.

Bacterial Proteins↗
PubMed2000

Differential regulation of Ca(2+)-calmodulin stimulated and Ca(2+)-insensitive adenylyl cyclase messenger RNA in intact and denervated mouse hippocampus.

The Ca(2+)-calmodulin stimulated AC1 and Ca(2+)-insensitive AC2 are major isoforms of adenylyl cyclase, playing an important role in synaptic plasticity in the mammalian brain. We studied the pattern of expression of AC1 and AC2 genes in the hippocampus of C57BL/6 mice. We found that there were differences in their patterns of distribution in the dentate gyrus. AC1 messenger RNA was detected both in the dentate granule cell bodies and the corresponding molecular field whereas AC2 messenger RNA was preferentially distributed in the dentate granule cell layer, suggesting that AC1 and AC2 messenger RNA are differentially regulated in the dentate gyrus. In order to examine the regulation of AC1 and AC2 expression in response to synaptic deafferentation and reinnervation, the distribution patterns of the two AC messenger RNA in the hippocampal fields and the parietal cortex were analysed 2, 5, 9 and 30 days following an unilateral entorhinal cortex lesion. Interestingly, we found significantly reduced levels of AC1 hybridization signal following the lesion whereas the level of AC2 messenger RNA remained unaffected in all lesioned groups. The changes in AC1 messenger RNA were transient, with a maximal reduction at five days postlesion, and were restricted to the granule cell bodies and stratum moleculare of the deafferented dentate gyrus. No significant change in AC1 messenger RNA levels was detected in other hippocampal fields nor for any other postlesion times studied. These findings suggest that, at least in the dentate gyrus, messenger RNA for AC1 and AC2 might be differentially compartmentalized in cell bodies and dendritic fields. The activity-dependent regulation of AC1 messenger RNA levels by afferent synapses may provide an elegant mechanism for achieving a selective local regulation of AC1 protein, close to its site of action.

Adenylyl Cyclases↗
PubMed1993

Fibroblast growth factor receptor-1, interleukin-1 receptor, and glucocorticoid receptor messenger RNA production in the human lacrimal gland.

PURPOSE: To determine whether messenger RNA coding for fibroblast growth factor receptor-1, interleukin-1 receptor, and glucocorticoid receptor messenger RNA are produced in human lacrimal tissue. METHODS: Total cellular RNA was isolated from three specimens of normal human lacrimal tissue and complementary DNA was synthesized. The polymerase chain reaction and sequence-specific primers were used to amplify the sequences of interest from the complementary DNA. Hot blotting and sequence-specific probes were used to demonstrate that the expected amplification products were specific. RESULTS: Data demonstrated that fibroblast growth factor receptor-1, interleukin-1 receptor, and glucocorticoid receptor messenger RNA are produced in human lacrimal tissue. CONCLUSIONS: These results and the previous identification of basic fibroblast growth factor in the lacrimal gland suggest that basic fibroblast growth factor has autocrine or paracrine functions in lacrimal tissue. More study is needed to determine whether the corresponding proteins are produced and, if so, what functions are regulated by fibroblast growth factor receptor-1, interleukin-1 receptor, and glucocorticoid receptor in the lacrimal gland.

Base Sequence↗
PubMed1993

Post-transcriptional regulation of tissue-specific isoforms. A bovine cytosolic RNA-binding protein, COLBP, associates with messenger RNA encoding the liver-form isopeptides of cytochrome c oxidase.

Regulation of the liver isopeptides of bovine cytochrome c oxidase is reported to be post-transcriptional. Extensive interspecies sequence homologies exist in the 3'-untranslated regions for transcripts encoding these liver isoforms, suggesting that these regions may be involved in mediating regulation of mRNA expression. To explore this possibility, several bovine tissue homogenates were assayed for any trans-acting factors that recognized the transcript encoding the liver isoform of subunit VIII (BCOL8). Such a protein factor (COLBP: cytochrome c oxidase L-form transcript-binding protein) was identified in liver, kidney, and lung tissue and was shown to require free sulfhydryl groups for activity. No binding activity, however, was found in muscle-type homogenates. Furthermore, this binding protein also recognized the subunit VIIa-liver transcript but was unable to associate with the mRNA encoding the heart isoform of subunit VIII. Intriguingly, the tissue-specific distribution of COLBP activity parallels the presence of the liver isopeptides in the mature oxidase complex. It is therefore suggested that COLBP may mediate the tissue-specific regulation of cytochrome c oxidase liver isoform mRNA expression.

Animals↗
PubMed1986

Localization of preangiotensinogen messenger RNA sequences in the rat brain.

Angiotensinogen (renin substrate) and its messenger RNA are known to accumulate in the rat brain. We have cloned rat preangiotensinogen cDNAs and used them as probes to measure the accumulation of preangiotensinogen messenger RNA sequences in eight regions of rat brain, as well as in liver and kidney. The brain regions examined were the cerebral cortex, hippocampus, striatum, cerebellum, diencephalon (including basal forebrain structures), midbrain, brainstem, and pituitary. On a tissue weight basis, the accumulation of preangiotensinogen RNA sequences was greatest in the liver, midbrain, and brainstem. The relative concentrations of messenger RNA were ranked as follows: liver, brainstem, midbrain greater than cerebellum, diencephalon greater than hippocampus greater than cortex, striatum, kidney greater than pituitary. Relative RNA concentrations from liver to kidney varied over a 16-fold range. Liver and brain preangiotensinogen RNA sequences were indistinguishable in size as measured by gel electrophoresis; however, the kidney sequences appeared some 100 nucleotides larger. Our data agree with previous measurements of angiotensinogen in the rat brain as assayed by renin-catalyzed angiotensin I release.

Angiotensinogen↗
PubMed1991

Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes.

The product of the yeast PRP22 gene acts late in the splicing of yeast pre-messenger RNA, mediating the release of the spliced mRNA from the spliceosome. The predicted PRP22 protein sequence shares extensive homology with that of PRP2 and PRP16 proteins, which are also involved in nuclear pre-mRNA splicing. The homologous region contains sequence elements characteristic of several demonstrated or putative ATP-dependent RNA helicases. A putative RNA-binding motif originally identified in bacterial ribosomal protein S1 and Escherichia coli polynucleotide phosphorylase has also been found in PRP22.

Adenosine Triphosphate↗
PubMed1977

The distribution of poly(A)+ and poly(A)- protamine messenger RNA sequences in the developing trout testis.

Protamine messenger RNA was isolated in a very pure form from trout testes and used as a template for the synthesis of labeled complementary DNA (cDNA) of high specifiv activity. The cDNA was found to be a full-length transcript of protamine messenger RNA and was used as a probe for hybridization reactions with RNA preparations isolated from three subcellular compartments of differentiating trout testis cells. The RNA populations from the nuclei, polysomes, and postribosomal supernatant of these cells were fractionated into poly(A)-containing [poly(A)+] and poly(A)-free [poly(A)-] RNA to determine the distribution of these two forms of protamine mRNA in these cell compartments. At the early protamine stage of testis development, polysomal and postribosomal supernatant fractions contain almost equal quantities of poly(A)+ protamine mRNA, but poly(A)- protamine mRNA was found almost entirely in the polysomes.

Animals↗
PubMed1986

Identification of renin and angiotensinogen messenger RNA sequences in mouse and rat brains.

Components of the renin angiotensin system have been demonstrated in mouse and rat brains. However, local synthesis of renin has not been documented. In this study, we employed mouse submandibular gland renin complementary DNA (pDD-1D2) and rat liver angiotensinogen complementary DNA (pRang3) to examine whether renin and angiotensinogen RNA sequences exist in mouse and rat brain. Angiotensinogen messenger RNA sequences were readily demonstrable in whole rat and mouse brain using Northern blot hybridization analysis. Using large quantities (greater than 100 micrograms) of brain total RNA and the sensitive complementary RNA probe, we were able to detect low levels of renin RNA sequences in the brains of both species. The relatively low concentration of brain renin messenger RNA and high concentration of angiotensinogen messenger RNA raises several interesting questions about the distribution of these two proteins and their relative contribution to activity of the brain renin-angiotensin system. In summary, our data demonstrate the expression of both renin and angiotensinogen genes in mouse and rat brains and provide definitive evidence for an independent endogenous brain renin angiotensin system.

Angiotensinogen↗
PubMed1996

Glutamate decarboxylase-67 messenger RNA expression in normal human basal ganglia and in Parkinson's disease.

Expression of glutamate decarboxylase-67 messenger RNA was examined in the basal ganglia of normal controls and of cases of Parkinson's disease using in situ hybridization histochemistry in human post mortem material. In controls glutamate decarboxylase-67 messenger RNA expression was detected in all large neurons in both segments of the globus pallidus and in three neuronal subpopulations in the striatum as well as in substantia nigra reticulata neurons and in a small sub-population of subthalamic neurons. In Parkinson's disease, there was a statistically significant decrease of 50.7% in glutamate decarboxylase-67 messenger RNA expression per neuron in the lateral segment of the globus pallidus (controls: mean 72.8 microns2 +/- S.E.M. 8.7 of silver grain/neuron, n = 12; Parkinson's disease: mean 35.9 microns2 +/- S.E.M. 9.7 of silver grain/neuron, n = 9, P = 0.01, Student's t-test). In the medial segment of the globus pallidus, there was a small, but non-significant decrease of glutamate decarboxylase-67 messenger RNA expression in Parkinson's disease (controls: mean 100.6 microns2 +/- S.E.M. 7.2 of silver grain/neuron, n = 11; Parkinson's disease: mean 84.8 microns2 +/- S.E.M. 13.0 of silver grain/neuron, n = 7, P = 0.1, Student's t-test). No significant differences in glutamate decarboxylase-67 messenger RNA were detected in striatal neuronal sub-populations between Parkinson's disease cases and controls. These results are the first direct evidence in humans that there is increased inhibitory drive to the lateral segment of the globus pallidus in Parkinson's disease, as suggested by data from animal models. We therefore provide theoretical support for current experimental neurosurgical approaches to Parkinson's disease.

Aged↗
PubMed1997

The interleukin-1beta-mediated regulation of proenkephalin and opioid receptor messenger RNA in primary astrocyte-enriched cultures.

Opioids have been found to modulate the function of the immune system by regulating the biochemical and proliferative properties of its cellular components. The interaction of opioid and immune systems, however, is not unidirectional, but rather, bidirectional in nature. In the CNS, one cellular target of immune system activation is the astrocytes, glial cells known to synthesize proenkephalin. We have recently shown that these cells also express the messenger RNA transcripts for the opioid receptors mu, delta and kappa, raising the question of the functional significance of this opioid peptide and the related receptors in the astrocytes. That is, why do astrocytes express proenkephalin and opioid receptors, and are these molecules responsive to a factor to which the astrocytes could be exposed in vivo? Furthermore, do these molecules respond to this factor in a region-specific fashion? In the present study, in order to characterize the astrocytic opioid response to an immune factor, we examined the concomitant regulation of mu, delta, kappa and proenkephalin messenger RNAs by interleukin-1beta (1 ng/ml=60 pM, 24 h) in primary astrocyte-enriched cultures derived from the rat (post-natal day 1-2) cortex, striatum, cerebellum, hippocampus and hypothalamus. Interleukin-1beta treatment was found to increase by 55-75% the level of mu receptor messenger RNA in striatal, cerebellar and hippocampal cultures, but not in cultures derived from the cortex or hypothalamus. However, the cytokine had no effect on the level of delta receptor messenger RNA in any of the five cultures examined. In marked contrast to its stimulatory effects on mu receptor messenger RNA levels and its lack of an effect on 6 receptor messenger RNA expression, interleukin-1beta reduced to 10-30% of control levels the kappa receptor messenger RNA levels in all cultures. Interleukin-1beta had no effect on the level of proenkephalin messenger RNA in cortical, striatal, cerebellar and hypothalamic cultures, but did significantly decrease the expression of proenkephalin messenger RNA in hippocampal cultures to 40% of the control level. Therefore, interleukin-1beta differentially regulated opioid receptor messenger RNA in astrocyte-enriched cultures in a manner dependent upon both the receptor type and the brain region from which the culture was derived. The cytokine also differentially regulated proenkephalin messenger RNA in a region-dependent fashion. These findings suggest a capacity for astrocytes to differentially regulate opioid peptide and receptor messenger RNAs in response to an immune factor, supporting the potential existence of a novel immune-opioid system interaction in the CNS.

Animals↗
PubMed1989

Variability in messenger RNA levels in human umbilical vein endothelial cells of different lineage and time in culture.

Levels of seven messenger RNA species were compared in human umbilical vein endothelial cells of different lineage and time in culture. Specifically, cells obtained from the American Type Culture Collection (ATCC) and subcultured were compared to early passage cells from cultures produced in our laboratory. Messenger RNA for tissue plasminogen activator, plasminogen activator inhibitor 1, urokinase, and thrombomodulin were expressed at higher levels in the ATCC cells. Thrombospondin, von Willebrand's Factor, and protein S messenger RNA were expressed at higher levels in the cells that we isolated. In addition, in the ATCC cells a shift in the proportion of plasminogen activator inhibitor messenger RNA from the 3.4 to the 2.4 kilobase species was found. We conclude that specific messenger RNA levels can vary considerably between cultured human umbilical vein endothelial cells. The large variation in mRNA levels which we describe has important implications for experiments involving gene expression in cultured endothelium.

Cell Division↗
PubMed1975

Nucleotide sequences of the 3'-terminal untranslated region of messenger RNA for human beta globin chain.

In normal messenger RNA for the human beta-globin chain, nucleotide sequences have been identified which can be matched to the amino-acid sequence of the abnormally long segment of the beta-chain of hemoglobin Cranston. The finding of these sequences strengthens the hypothesis that the betaCranston chain arose by a frameshift mutation allowing the "readthrough" of the normal termination codon and translation of usually untranslated portions of the messenger RNA for the beta-globin chain. The oligonucleotides which match the amino-acid sequence of hemoglobin Cranston provide a sequence of 36 nucleotides which follows the normal beta-chain termination codon UAA.

Base Sequence↗
PubMed1995

Acute and chronic amphetamine treatments differently regulate neuropeptide messenger RNA levels and Fos immunoreactivity in rat striatal neurons.

Repeated administration of amphetamine results in the well known phenomenon of reverse tolerance or sensitization. However, little is known about cellular and molecular mechanisms underlying acute versus chronic response to amphetamine. In this paper, we investigated the effects of acute (1.5 or 5 mg/kg) and chronic (5 mg/kg/day for 14 days) amphetamine treatment on locomotor activity, stereotypy, Fos immunoreactivity and messenger RNA levels of molecules implicated in dopamine transmission in the rat striatum and substantia nigra. In agreement with other studies, acute amphetamine induced a dose dependent increase in locomotor activity and stereotypy. Also, a comparison between the behavior observed after the first injection and the last injection of amphetamine in chronically treated rats showed sensitization as demonstrated by a higher rating of stereotypy. We have found that acute and chronic amphetamine treatments differently modulate the activity of several output neurons. A double labeling procedure with Fos immunohistochemistry coupled with in situ hybridization demonstrated that acute amphetamine treatment induces Fos immunoreactivity predominantly in striatal neurons expressing substance P messenger RNA (77.07 +/- 1.42%). Only 32.6 +/- 2.07% of Fos immunoreactive neurons expressed preproenkephalin A messenger RNA. In chronic amphetamine treated rats, 56.21 +/- 1.32% of the Fos immunoreactive neurons expressed substance P messenger RNA while 52.12 +/- 1.84% expressed preproenkephalin A messenger RNA. Statistical analysis revealed that this difference is mainly due to a decrease in the density of substance P immunoreactive neurons in chronically treated rats in comparison to acute. Amphetamine treatments induced Fos immunoreactivity in the substantia nigra in non-dopamine neurons. As measured by quantitative in situ hybridization, acute amphetamine induced an increase in substance P, preproenkephalin A and dynorphin messenger RNA levels (+23 +/- 0.05%, +45 +/- 0.07% and +24 +/- 0.05%, respectively). No difference in these increases was observed in relation with the dose injected (1.5 or 5 mg/kg). Chronic amphetamine treatment enhanced only substance P and dynorphin messenger RNA levels (+23 +/- 0.04% and +42 +/- 0.04%, respectively). Neither acute nor chronic amphetamine treatment had any effects on D1 or D2 dopamine receptor messenger RNA levels. Our main conclusions are: (1) in acutely treated rats Fos is essentially expressed by substance P neurons; (2) in chronically treated rats, Fos immunoreactivity is expressed by the two efferent striatal populations (i.e. preproenkephalin A and substance P neurons) and the number of Fos immunoreactive neurons is reduced as compared with acute; (3) neuropeptide messenger RNA levels, but not dopamine receptor messenger RNAs, are affected in the response to acute or chronic treatment with amphetamine.

Amphetamine↗
PubMed1995

Monocular deprivation decreases the expression of messenger RNA for brain-derived neurotrophic factor in the rat visual cortex.

We found that deprivation of pattern vision in one eye, that leaves luminance detection performance unaffected, is sufficient to reduce brain-derived neurotrophic factor (but not trkB) messenger RNA in the visual cortex of young and adult rats. Monocular deprivation by means of eyelids' suture was performed during or after the critical period and the cortical amount of brain-derived neurotrophic factor messenger RNA was analysed by in situ hybridization and RNAase protection after 15-30 days of deprivation. A reduction of brain-derived neurotrophic factor messenger RNA was observed in the visual cortex contralateral to the deprived eye in rats monocularly deprived during the critical period. The same reduction was also found in rats monocularly deprived after the end of the critical period, when anatomical or physiological signs of monocular deprivation are absent. The pharmacological blockade of retinal activity equally affected the expression of brain-derived neurotrophic factor messenger RNA in young and adults. Quantitative RNAase protection assays revealed that the cortical level of brain-derived neurotrophic factor messenger RNA was reduced to the same extent when intraocular injections of tetrodotoxin were performed within or after the critical period. A developmental study of brain-derived neurotrophic factor messenger RNA expression in rat visual cortex showed a marked increase around the time of natural eye-opening followed by a plateau from postnatal day 20 until adult age. Messenger RNA for the kinasic domain of brain-derived neurotrophic factor receptor (trkB) was found in the dorsal lateral geniculate nucleus and the visual cortex during development and in adults. Our results suggest that the reduction of brain-derived neurotrophic factor messenger RNA induced by monocular deprivation is related to the absence of pattern vision rather than to the competitive interactions that underlie the effects of monocular deprivation during the critical period.

Age Factors↗
PubMed1993

Induction of cytochrome P-4502E1 in the human liver by ethanol is caused by a corresponding increase in encoding messenger RNA.

The propensity of centrilobular liver damage to develop in alcohol abusers after exposure to various hepatotoxins, including ethanol itself, has been linked to the induction by ethanol of P-4502E1, a microsomal P-450 enzyme that bioactivates these agents to reactive metabolites. Whereas long-term ethanol consumption elicits a marked increase in hepatic P-4502E1 content, the molecular mechanism by which ethanol produces this effect is the subject of controversy in animals, and it has not been elucidated in human beings. Possible mechanisms include increased enzyme synthesis stemming from elevated 2E1 messenger RNA levels, enhanced translation of preexisting messenger RNA or stabilization of P-4502E1 protein. To determine which, if any, of these mechanisms underlies P-4502E1 induction in human beings, we examined the effects of ethanol intake on the hepatic intralobular distribution of P-4502E1 messenger RNA and the corresponding protein. Liver sections derived from needle biopsy specimens were obtained from five recently drinking alcoholics (last drink no more than 36 hr before) and eight control subjects (five abstaining alcoholics [last drink 96 hr or more before] and three nondrinkers). In situ hybridization of these liver sections with a human P-4502E1 complementary DNA probe was used to localize P-4502E1 messenger RNA transcripts. Quantitative image analysis of hybridized sections from control subjects revealed that P-4502E1 transcript content in perivenular (zone 3) hepatocytes was significantly higher (p < 0.05) than in midzonal (zone 2) and periportal (zone 1) cells (18.3 +/- 1, 9.5 +/- 2 and 3.1 +/- 2 arbitrary density units, respectively; mean +/- S.E.M.). In recent drinkers, acinar regions containing P-4502E1 transcripts were elevated 2.9-fold compared with those in controls (32.8% +/- 7% vs. 11.2% +/- 2%; p < 0.01), with this messenger RNA increase occurring mainly in perivenular cells (29.6 +/- 3 vs. 18.3 +/- 1 units; p < 0.01). P-4502E1 protein distribution, assessed by the immunohistochemical staining of liver sections with P-4502E1 antibodies, was found to be analogous to that of the messenger RNA in control subjects (the level in perivenular cells was greater than that in midzonal cells, which was greater than that in periportal cells), whereas recent drinkers exhibited marked elevations in enzyme content in both perivenular and midzonal hepatocytes. Moreover, cellular levels of P-4502E1 protein and messenger RNA were significantly correlated (rs = 0.79; p < 0.001) in all patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1992

Regionally specific and rapid increases in brain-derived neurotrophic factor messenger RNA in the adult rat brain following seizures induced by systemic administration of kainic acid.

In situ hybridization techniques were used to analyse the spatiotemporal pattern of brain-derived neurotrophic factor messenger RNA elevation associated with kainic acid-induced seizure activity in the rat. Pronounced increases in hippocampal brain-derived neurotrophic factor messenger RNA levels were observed as early as 30 min following the onset of behavioral seizures. The greatest increase (10-fold) occurred in the dentate granule cell layer, while pyramidal layers CA1, CA3, and CA4 exhibited increases of two- to six-fold. Peak elevation of brain-derived neurotrophic factor messenger RNA in CA1 hippocampal region was evident at 4 h in CA3, and in the dentate granule layer at 30 min postseizure. Elevations persisted in the dentate and hilar regions to four days, while the increases in CA1 and CA3 returned to control levels by 16 h following seizure. Significant increases in brain-derived neurotrophic factor messenger RNA were also observed in the superficial layers of cortex (II and III) and in the piriform cortex which reached peak elevations by 8 h. No detectable changes were observed in the dorsomedial thalamus. Although histologically defined pyramidal and granule cell layers displayed relatively uniform increases in brain-derived neurotrophic factor messenger RNA in response to kainate, a closer examination of the labeling patterns using emulsion autoradiography revealed discrete areas of high grain densities overlapping uniform, moderate hybridization densities in the dentate granule cell layer and CA3, suggesting that the capacity to upregulate brain-derived neurotrophic factor messenger RNA in these regions may differ among individual neurons. In conclusion, our studies revealed that brain-derived neurotrophic factor messenger RNA induction in response to systemic kainate administration differs in hippocampal and cortical areas, in magnitude, time of onset and duration. The observed temperospatial pattern does not correspond in a simple way to increases in metabolic or electrical activity associated with seizures or neuronal vulnerability coincident with the seizures.

Animals↗
PubMed1985

Immunological detection of the messenger RNA cap-binding protein.

The 24-kilodalton messenger RNA cap-binding protein (CBP) was purified from the rabbit reticulocyte postribosomal supernatant fraction using an affinity resin consisting of the p-aminophenyl gamma-ester of m7GTP coupled to Sepharose. The affinity-purified CBP was used to raise a goat antiserum. Anti-CBP antibodies were purified by adsorption to CBP coupled to either Controlled-Pore Glass or diazobenzyloxymethyl paper. The affinity-purified antibodies reacted specifically with only the 24-kilodalton polypeptide in whole reticulocyte lysate and in initiation factors prepared from the same source. During a conventional (nonaffinity) purification of CBP from a high salt extract of the ribosomal pellet, immunological reactivity paralleled the ability to reverse cap analogue inhibition of translation, indicating that the 24-kilodalton polypeptide present in the postribosomal supernatant fraction is immunologically cross-reactive with the CBP purified from ribosomes. Fractionation of whole reticulocyte lysate by sucrose gradient sedimentation followed by immunoblotting revealed that CBP was present in the supernatant fraction and the region of the gradient corresponding to ribosomal subunits but not in mono- or polysomes. The CBP to ribosome ratio was found to be approximately 0.02, assuming that the m7GTP-Sepharose retains all of the protein. This is considerably lower than that of other initiation factors and suggests that CBP may be the limiting polypeptide factor involved in the initiation of protein synthesis. The antibodies also inhibited the translation of a capped messenger RNA (globin). Inhibition of the translation of an uncapped RNA (satellite tobacco necrosis virus) was also observed, but to a lesser degree than with globin mRNA.

Animals↗
PubMed1993

Expression of fibronectin messenger RNA in hypertrophic and normal dermal tissues and in vitro regulation by interferon alfa-2b.

After severe thermal injury, hypertrophic scarring which is associated with accumulation of extracellular matrix proteins including fibronectin, frequently develops. We have recently demonstrated that interferon alfa-2b significantly reduces the level of type 1 procollagen messenger RNA expressed by both hypertrophic and normal dermal fibroblasts. In this report, we provide evidence that this cytokine also significantly decreases the expression of fibronectin messenger RNA in human hypertrophic scar and normal dermal fibroblasts. Four dermal fibroblast cell strains were established in cell culture from four human postburn hypertrophic scar tissues with the use of normal dermal fibroblasts from the same patients as controls. These cells were then treated with 2000 U/ml interferon alfa-2b in culture medium at various times. The results of Northern analysis of interferon-treated dermal fibroblasts indicate that this cytokine reduced the expression of fibronectin messenger RNA as early as 12 hours after treatment and reached its lowest level (24% relative to untreated fibroblasts) after 96 hours. When the expression of fibronectin messenger RNA was quantified by densitometry for each individual paired cell strain, a differential response to interferon treatment was found among cell strains. The level of fibronectin messenger RNA expression decreased from 17.2% to 69% in hypertrophic scar fibroblasts and 47% to 83.7% in normal fibroblasts relative to that of untreated control values. Although this decrease was less pronounced in normal fibroblasts than in hypertrophic scar fibroblasts, this reduction was significant in both interferon alfa-2b treated hypertrophic scar fibroblasts (6.39 +/- 0.71 versus 2.88 +/- 0.9, n = 4, p < 0.05) and normal cells compared with untreated controls (5.47 +/- 0.89 versus 3.64 +/- 0.99, n = 4, p < 0.05) as assessed with Student's paired t test. Rehybridization of the RNA blot prepared from interferon alfa-2b treated and untreated hypertrophic scar fibroblasts with a complementary DNA for the tissue inhibitor of metalloproteinase type 2 gelatinase inhibitor showed no significant changes in abundance of this transcript. This result suggests that this cytokine selectively suppresses the expression of fibronectin messenger RNA and that this reduction is not due to RNA loading. A dot blot analysis of total RNA extracted from these tissues was carried out to compare the expression of fibronectin messenger RNA between human hypertrophic scar tissues and normal dermis obtained from the same patients. The blot was initially hybridized with fibronectin complementary DNA and subsequently with a complementary DNA for the tissue inhibitor of metalloproteinase type 2 to correct for RNA loading. When the ratio of fibronectin to tissue inhibitor or metalloproteinase type 2 messenger RNA expression for each hypertrophic scar tissue was compared with its normal control, this ratio was fourfold higher in human hypertrophic scar tissues relative to normal controls. In contrast, the expression of this message in cultured hypertrophic scar fibroblasts was not significantly different from that in normal fibroblasts. The results of this study suggest that hypertrophic scarring developing after thermal injury is associated with an overexpression of fibronectin messenger RNA, and interferon alfa-2b may be of therapeutic value to down-regulate the expression of this transcript.

Journal Article↗