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Two very different components of messenger RNA in an insect cell line.

The messenger RNA lifetimes have been measured in a cell line derived from an invertebrate source, the mosquito Aedes albopictus. The experiments were made possible by a new technique for obtaining undegraded cytoplasmic RNA from cells with high endogenous nuclease levels. There are two components to the decay kinetics of Aedes mRNA. The major fraction of the steady state message population has a half-life of 20 hr which is, as in mammalian cells, comparable to the cell generation time. The short-lived component turns over very rapidly with a half-life estimated to be about 1.2 hr. The difference in lifetime between the short and long-lived components is about 15 fold in these cells, compared to 3-4 fold in mammalian cells. This may reflect the need for a more responsive mRNA regulating system in poikilothermic organisms. The great disparity between the principle messenger lifetimes permits a more definite assignment of a two component behavior to message decay. The data in the case of mammalian cells could not rule out a family of intermediate lifetimes. The long-lived mRNA has a much smaller average sedimentation value than the short-lived material. The effect is similar to, but much larger than, that seen in mammalian cells. Although the lifetime difference is much greater in the insect cells than in human (HeLa) cells, the fast and slow components comprise about the same proportion of the steady state mRNA population: 30 percent and 70 percent, respectively.

Aedes↗

Distribution of metabotropic glutamate receptor 7 messenger RNA in the developing and adult rat brain.

The large number of metabotropic glutamate receptor subtypes suggests diverse roles in brain function, although specific distribution patterns can give clues to subtype-specific functions [Hayashi Y. et al. (1993) Nature 366, 687-690; Nakajima Y. et al. (1993) J. biol. Chem. 268, 11868-11873; Nomura A. et al. (1994) Cell 77, 361-369; Ohishi H. et al. (1993), 1009-1018]. The metabotropic glutamate receptor mGluR7 is sensitive to the agonist L-2-amino-4-phosphonobutyric acid, a presynaptic inhibitor of neurotransmitter release. We examined the anatomic distribution of mGluR7 messenger RNA expression by in situ hybridization in the developing and adult rat central nervous systems. Our results demonstrate that mGluR7 messenger RNA is among the most widely distributed of metabotropic glutamate receptors in both the developing and adult rat nervous system and that mGluR7 messenger RNA is expressed in most neuronal groups known to respond to L-2-amino-4-phosphonobutyric acid, including mitral cells of the olfactory bulb, granule cells of the dentate gyrus and neurons of the entorhinal cortex and dorsal root ganglion. mGluR7 exhibits preferential expression in sensory afferent pathways and is highly represented in the periventricular zone of the hypothalamus, the latter implying a modulatory role for mGluR7 in neuroendocrine pathways. Most strikingly, the majority of neurons at all levels of olfactory circuitry are among the areas of highest mGluR7 messenger RNA content. The anatomic distribution of mGluyR7 messenger RNA suggests that mGluR7 activation may participate in the processing of hippocampal, sensory and olfactory information.

Aging↗

MESSENGER RNA UTILIZATION DURING DEVELOPMENT OF CHICK EMBRYO LENS.

A marked increase in synthesis or availability of messenger RNA which can accommodate eight to ten ribosomes was observed in the chick embryo lens between 11 and 12 days of development. The increase is seen in a shift from polyribosomes consisting of predominantly two to five single ribosomes to polyribosomes of predominantly eight to ten ribosomes in lens cells. Short-lived (half-life, 3 hours) and long-lived (half-life, 30 hours) messenger RNA's coexist in whole lenses on the 14th day of development.

Animals↗

Localization of cytosolic phospholipase A2 messenger RNA mainly in neurons in the rat brain.

Ca2(+)-sensitive 85,000 mol. wt cytosolic phospholipase A2 plays an essential role in the selective and stimulus-dependent release of arachidonic acid from membrane phospholipids. Cytosolic phospholipase A2-catalysed lipid mediators including arachidonic acid and its metabolites have been suggested to be involved in a variety of neuronal functions in the CNS. Since the cellular localization of cytosolic phospholipase A2 is still controversial and obscure, we tried an improved method of rapid processing of each specimens and succeeded in obtaining intense signals of cytosolic phospholipase A2 messenger RNA in the normal rat brain by northern blot analysis and in situ hybridization. Northern blot analysis showed the abundant distribution of cytosolic phospholipase A2 messenger RNA in most regions of the brain, with intense signals observed in the pineal gland and pons. Macroautoradiographs prepared after in situ hybridization with three different antisense riboprobes gave essentially similar patterns of localization; significant signals were widely detected in the gray matter of various regions, i.e. the olfactory bulb, cerebral cortex, hippocampus, amygdala, several thalamic and hypothalamic nuclei and cerebellum. Microautoradiographs showed that most of the intense signals were predominant in neurons, and that faint signals were from glial cells and other non-neuronal cells in the choroid plexus, inner surface cells of veins and the leptomeninges. In addition, the cycloheximide treatment increased the cytosolic phospholipase A2 messenger RNA level in the same cell populations originally possessing messenger RNA signals. Predominant expression of cytosolic phospholipase A2 messenger RNA in neurons may provide the basis for the contribution of cytosolic phospholipase A2-catalysed lipid mediators to a variety of neurotransmission and synaptic functions in the CNS.

Animals↗

Aldose reductase messenger RNA in the lens epithelium in vivo: effects of diabetes mellitus and galactosaemia.

1. Quantitative in situ hybridization histochemistry was used to examine the regulation of aldose reductase messenger RNA in the rat lens after the induction of diabetes mellitus or after feeding a 50% (w/w) galactose diet. 2. Although increased staining for aldose reductase in the lens epithelium has previously been observed by immunohistochemistry after 3 weeks of diabetes or after 7 days of galactose feeding, we have not been able to detect any increase in the amount of aldose reductase messenger RNA in these cells as compared with controls (113 +/- 7%, 105 +/- 9%, 100 +/- 7%, respectively) at these time points (P greater than 0.05). 3. After 15 days of galactose feeding, however, there was a significant increase of 140% (+/- 12%) in the amount of aldose reductase messenger RNA in the lens epithelial cells as compared with controls (P = less than 0.001). 4. These results demonstrate that increased availability of galactose, a high-affinity substrate for the enzyme, leads to increased aldose reductase messenger RNA, which suggests a role for aldose reductase in sugar metabolism in the lens.

Aldehyde Reductase↗

D2 dopamine receptor messenger RNA is altered to a greater extent by blockade of glutamate receptors than by blockade of dopamine receptors.

To study further the molecular mechanisms by which glutamate and dopamine interact to regulate the functions of the basal ganglia, the effects of persistently inhibiting dopamine receptors and glutamate N-methyl-D-aspartate receptors on the density of D1 and D2 dopamine receptors and on the level of their transcripts were examined in mouse brain. To block dopamine receptors, mice were treated with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline once daily for two and six days, or were treated with fluphenazine-N-mustard once daily for five days. To block N-methyl-D-aspartate receptors, mice were treated with dizocilpine by continuous infusion with osmotic mini-pumps for two and six days. The density of D1 and D2 dopamine receptors was measured by receptor autoradiography, and the level of D1 and D2 dopamine receptor messenger RNA was measured by in situ hybridization histochemistry. The results showed that N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline blocked about 90% of both D1 and D2 dopamine receptors, but had no significant effect on the level of either D1 or D2 dopamine receptor messenger RNA. Fluphenazine-N-mustard, which was as effective as N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline in blocking D2 dopamine receptors but had little effect on D1 dopamine receptors, also had no significant effect on the level of D1 and D2 dopamine receptor messenger RNAs. By contrast, continuously infusing dizocilpine significantly decreased the levels of D2 dopamine receptor messenger RNA in striatum, nucleus accumbens and olfactory tubercle. Dizocilpine also caused small decreases in the density of D2 dopamine receptors, but only in posterior striatum was this decrease statistically significant. Dizocilpine slightly and transiently decreased the levels of D1 dopamine receptor messenger RNA in striatum but had no significant effect on the density of D1 dopamine receptors in any region examined. This study demonstrates that persistent blockade of D1 and D2 dopamine receptors has relatively little effect on the levels of D1 and D2 dopamine receptor messenger RNA, but that blockade of N-methyl-D-aspartate receptors produces a rapid and profound decrease in the levels of D2 dopamine receptor messenger RNA and a smaller decrease in the density of D2 dopamine receptors. These results suggest that N-methyl-D-aspartate receptors play an important role in the expression of D2 dopamine receptors in basal ganglia.

Animals↗

Up-regulation of nitric oxide synthase messenger RNA in an integrated forebrain circuit involved in oxytocin secretion.

The hypothalamo-neurohypophysial system contains high levels of neuronal nitric oxide synthase and this increases further during times of neurohormone demand, such as that following osmotic stimulation. Using double in situ hybridization, we demonstrate here an increase in the expression of nitric oxide synthase messenger RNA by oxytocin neurons, but not vasopressin neurons, of the supraoptic nucleus at the time of lactation, when oxytocin is in demand due to another neuroendocrine stimulus, the milk-ejection reflex. In addition, using immunocytochemical retrograde tracing, we show that neurons of the subfornical organ, median preoptic nucleus and organum vasculosum of the lamina terminalis, which project to the supraoptic nucleus, contain nitric oxide synthase. These three structures of the lamina terminalis, together with the hypothalamo-neurohypophysial system, make up the forebrain osmoresponsive circuit that controls osmotically-stimulated release of oxytocin in the rat. The expression of nitric oxide synthase messenger RNA in the lamina terminalis was also shown to increase during lactation. The increases in nitric oxide synthase messenger RNA were not apparent during pregnancy. These results provide evidence for an integrated nitric oxide synthase-containing neural network involved in the regulation of the hypothalamo-neurohypophysial axis. The expression of nitric oxide synthase messenger RNA increases in this circuit during lactation and correlates with a reduction in the sensitivity of the circuit to osmotic stimuli also present in lactation but not pregnancy. As nitric oxide is believed to attenuate neurohormone release, it seems that the increased nitric oxide synthase messenger RNA expression detected here during lactation at a time of high oxytocin demand may be involved in reducing the sensitivity of the whole forebrain circuit to osmotic stimuli.

Animals↗

Transcription-translation and translation-messenger RNA decay coupling: separate mechanisms for different messengers.

Antibiotics were used to inhibit protein synthesis at specific steps in the biosynthetic pathway. In this way, it was possible to study the coupling of protein synthesis to the accumulation of biologically active mRNA in T4-infected Escherichia coli. Functional mRNA for the phage enzymes deoxynucleotide kinase (EC 2.7.4.4; ATP: nucleoside monophosphate phosphotransferase or nucleosidemonophosphate kinase) and alpha-glucosyltransferase (EC 2.4.1.5; 1, 4-alpha-D-glucan: 1, 6-alpha-D-glucan 6-alpha-glucosyltransferase or dextrin dextranase) accumulated during inhibition of protein synthesis irrespective of the step in the synthesis of protein that was blocked. Under these conditions, however, the rate of mRNA synthesis for both enzymes was significantly inhibited. In contrast, the rate of degradation of these mRNAs was markedly dependent on the step in protein synthesis that was inhibited. That is, the site for mRNase action was different for each message. The most important step in protein synthesis required for the stability of deoxynucleotide kinase mRNA is the initiation step. A single ribosome bound to the 5' end of the deoxynucleotide kinase mRNA can stabilize the molecule. On the other hand, the initiation event does not seem to be important for stabilizing the alpha-glucosyltransferase mRNA. Instead, a high ribosome denisty on the alpha-glucosyltransferase messenger is required to achieve significant stability. Therefore, in studying messenger metabolism, it is important to focus on the functional stability of specific mRNAs instead of on total messenger since each mRNA can be metabolized differently.

Anti-Bacterial Agents↗

Time course and localization patterns of interleukin-1beta messenger RNA expression in brain and pituitary after peripheral administration of lipopolysaccharide.

The inflammatory cytokine interleukin-1 has been implicated as a mediator of many centrally controlled responses, such as fever and increased activity of the hypothalamic-pituitary adrenal axis, after systemic infections. To identify the neuroanatomical loci of brain interleukin-1-producing cells during infection, we investigated interleukin-1beta messenger RNA expression by in situ hybridization histochemistry using a 500 nt ribonucleotide probe applied on brain sections from rats injected intraperitoneally with 2.5 mg/kg bacterial lipopolysaccharide or saline. In control animals, interleukin-1beta messenger RNA was not detectable. In the brains of lipopolysaccharide-injected animals, two temporally and spatially distinct waves of interleukin-1beta messenger RNA induction were observed. First, cell labelling appeared at 0.5 h, peaked at 2 h, and declined at 4-8 h. The labelled cells were concentrated in circumventricular organs--organum vasculosum of the lamina terminalis, subfornical organ, median eminence, and area postrema--and in choroid plexus, meninges, and blood vessels. Second, at 8-12 h, scattered small cells became labelled throughout the entire brain parenchyma; the labelling subsided by 24 h. Labelling was not observed in any neurons. In the pituitary, lipopolysaccharide induced strong interleukin-1beta messenger RNA expression initially in the anterior lobe at 0.5-1 h, and later in the neural lobe at 1-2 h, and subsiding thereafter. The results show that at early time points, peripheral lipopolysaccharide induces interleukin-1beta message production at the blood brain barrier and in circumventricular organs where the blood brain barrier is leaky. After a time delay of 6-10 h, however, interleukin-1beta messenger RNA is primarily expressed by non-neuronal cells of the brain in the brain parenchyma. These results suggest that the source of initial brain IL-1 activity after peripheral lipopolysaccharide injection derives from cells of the blood-brain barrier and the circumventricular organs, and the sustained interleukin-1 activity in the central nervous system thereafter is derived from glia.

Animals↗

Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry.

Human immunodeficiency virus type-1 (HIV-1) DNA and messenger RNA sequences in both cell lines and blood obtained directly from HIV-1-infected patients were amplified by polymerase chain reaction and hybridized to fluorescein-labeled probes in situ, and the individually labeled cells were analyzed by flow cytometry. After flow cytometric analysis, heterogeneous cell populations were reproducibly resolved into HIV-1-positive and -negative distributions. Fluorescence microscopy showed that the cellular morphology was preserved and intracellular localization of amplified product DNA was maintained. Retention of nonspecific probe was not observed. Analysis of proviral DNA and viral messenger RNA in cells in the blood of HIV-1-infected patients showed that the HIV-1 genome persists in a large reservoir of latently infected cells. With the use of this technique it is now possible to detect single-copy DNA or low-abundance messenger RNA rapidly and reproducibly in a minor subpopulation of cells in suspension at single-cell resolution and to sort those cells for further characterization.

Base Sequence↗

Differential regulation of alpha-calcitonin gene-related peptide and preprocholecystokinin messenger RNA expression in alpha-motoneurons: effects of testosterone and inactivity induced factors.

alpha-Calcitonin gene-related peptide expression in alpha-motoneurons is regulated by spinal cord transection, axotomy and testosterone, but to date there are no studies which examine the regulation of cholecystokinin expression in motoneurons. In the present study, we compared the regulation of preprocholecystokinin and alpha-calcitonin gene-related peptide messenger RNA levels in motoneurons of the spinal nucleus of the bulbocavernosus. Previously, we demonstrated that manipulations which decrease activity in target muscles of the spinal nucleus of the bulbocavernosus motoneurons increase alpha-calcitonin gene-related peptide message and peptide levels in spinal nucleus of the bulbocavernosus motoneurons. This muscle-nerve interaction is mediated by a soluble factor which is increased by castration. We now report that decreasing plasma testosterone levels decreased preprocholecystokinin messenger RNA levels. Testosterone replacement at the time of castration restored preprocholecystokinin messenger RNA levels to intact values. Injections of crude extracts prepared from denervated bulbocavernosus/levator ani into the homologous muscles of gonadally intact rats increased the levels of alpha-calcitonin gene-related peptide messenger RNA in spinal nucleus of the bulbocavernosus motoneurons. The levels of preprocholecystokinin messenger RNA did not differ in rats injected with denervated bulbocavernosus/levator ani extract or buffer, both of which were significantly higher than in intact, untreated rats. The results of the present experiments imply that levels of preprocholecystokinin and alpha-calcitonin gene-related peptide messenger ribonucleic acid are differentially regulated in spinal nucleus of the bulbocavernosus motoneurons.

Animals↗

Chronic continuous infusion of (-)nicotine reduces basic fibroblast growth factor messenger RNA levels in the ventral midbrain of the intact but not of the 6-hydroxydopamine-lesioned rat.

A negative correlation has been found between smoking and Parkinson's disease. There is evidence to suggest that this correlation appears to be associated with a neuroprotective role of nicotine for dopamine neurons at least in relation to mechanical injury. However, 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) neurotoxicity to dopamine neurons is enhanced by chronic continuous (-)nicotine. More recently, basic fibroblast growth factor has been found to possess neurotrophic activities for many nerve cells including the dopamine cells in vivo and in vitro. Therefore, it is of interest to explore a possible effect of nicotine on basic fibroblast growth factor expression in the ventral midbrain of intact and 6-hydroxydopamine-lesioned rats and how treatment with nicotine can alter the 6-hydroxydopamine-induced injury of the nigral dopamine nerve cells as evaluated by dopamine biochemistry. In the present paper, an analysis of the effects of chronic continuous infusion of (-)nicotine via minipumps was carried out on basic fibroblast growth factor expression in the vental midbrain of the intact male rat and of the 6-hydroxydopamine lesioned rat. A quantitative messenger RNA protection assay analysis was used as well as an immunocytochemical analysis in the substantia nigra. Our findings give evidence that a two-week continuous infusion with (-)nicotine in the intact rat leads to substantial and dose-related (0.03-0.3 mg/kg per h) reductions of basic fibroblast growth factor messenger RNA levels in the ventral midbrain. These changes are not associated with changes in neuronal and glial basic fibroblast growth factor immunoreactivity in this region with the antibodies used. However, a one-week continuous infusion with (-)nicotine (0.125 mg/kg per h) failed to significantly alter the basic fibroblast growth factor messenger RNA levels in the ventral midbrain of solvent and 6-hydroxydopamine-injected rats and thus also the 6-hydroxydopamine-induced increase of basic fibroblast growth factor messenger RNA levels in the ventral midbrain of the lesioned side observed at this time-interval and known to be of astroglial origin [Chadi G. et al. (1994) Neuroscience 61, 891-910]. In agreement, the 6-hydroxydopamine-induced depletion of dopamine in the neostriatum was unaltered by the nicotine treatment (0.125 mg/kg per h). Thus, chronic continuous (-)nicotine treatment may lead to a reduced basic fibroblast growth factor trophic tone in the ventral midbrain of the intact but not of the 6-hydroxydopamine-lesioned rat neither on the lesioned nor on the unlesioned side of the ventral midbrain. It seems possible that chronic nicotine treatment mainly reduces basic fibroblast growth factor messenger RNA levels of neuronal origin, since the astroglial messenger RNA levels dominate after the 6-hydroxydopamine-induced lesions.

Animals↗

Environmental enrichment alters nerve growth factor-induced gene A and glucocorticoid receptor messenger RNA expression after middle cerebral artery occlusion in rats.

Housing rats in an enriched environment after focal brain ischemia improves functional outcome without changes in infarct volume, suggesting neuroplastic changes outside the lesion. In this study, permanent occlusion of the middle cerebral artery was followed by housing in an enriched or a standard environment. Nerve growth factor-induced gene A and glucocorticoid receptor messenger RNA expression were determined by in situ hybridization two to 30 days after middle cerebral artery occlusion. Stroke induced a decrease in nerve growth factor-induced gene A messenger RNA expression in cortical areas outside the ischemic lesion and in the CA1 subregion of the hippocampus two to three days after ischemia. This decrease was more prolonged with environmental enrichment, lasting until 20 days. However, 30 days after focal cerebral ischemia, environmental enrichment increased nerve growth factor-induced gene A expression compared to standard housing. A reduction of hippocampal glucocorticoid receptor (type II) messenger RNA two to 12 days after stroke in standard housed rats was restored by environmental enrichment. These data suggest that improved functional outcome induced by environmental enrichment after middle cerebral artery occlusion is associated with dynamically altered expression of nerve growth factor-induced gene A messenger RNA in brain regions outside the ischemic lesion, and sustained levels of hippocampal glucocorticoid receptor messenger RNA expression.

Animals↗

Cellular localization of messenger RNA for beta-1 and beta-2 adrenergic receptors in rat brain: an in situ hybridization study.

Selective, 35S-labeled, oligonucleotide probes were designed from sequences of the rat beta-1 and beta-2 adrenoceptor messenger RNAs for use in situ hybridization experiments on sections of unfixed rat brain and spinal cord. After hybridized sections were exposed to film or dipped in autoradiographic emulsion, specific and selective labeling patterns characteristic for each receptor messenger RNA and region of the central nervous system were observed. For example, labeling for beta-1 messenger RNA was found in the anterior olfactory nucleus, cerebral cortex, lateral intermediate septal nucleus, reticular thalamic nucleus, oculomotor complex, vestibular nuclei, deep cerebellar nuclei, trapezoid nucleus, abducens nucleus, ventrolateral pontine and medullary reticular formations, the intermediate gray matter of the spinal cord and in the pineal gland, while beta-2 messenger RNA labeling was strongest in the olfactory bulb, piriform cortex, hippocampal formation, thalamic intralaminar nuclei and cerebellar cortex. In some of these regions the beta-1 labeling seemed mainly confined to the cell nucleus. Whether or not this apparently nuclear labeling is specific, i.e. indicates synthesis of beta-1 receptor, remains to be established. However, all labeling patterns described disappeared when excess unlabeled probes were added to their respective radiolabeled probes or when sense probes were employed. Since the in situ method labels only cell bodies that produce the messenger RNA for these two beta receptor subtypes, a comparison between these maps and those of past autoradiographic studies mapping the location of central beta receptors using drugs as radioligands may produce further insights regarding the pre- and postsynaptic localization of these receptors in the various parts of the central nervous system circuitry.

Adrenergic Fibers↗