THE DEGRADATION OF E. COLI MESSENGER RNA BY POLYNUCLEOTIDE PHOSPHORYLASE.
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Adenine↗
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Localization of the messenger RNA for the c-Jun NH2-terminal kinase kinase in the adult and developing rat brain: an in situ hybridization study.
Stress-activated protein kinase/extracellular signal-regulated protein kinase-1/c-Jun NH2-terminal kinase kinase is a dual-specificity kinase which phosphorylates and activates stress-activated protein kinase/c-Jun NH2-terminal kinase, a recently discovered mitogen-activated protein kinase that is stimulated by stressful stimuli and that regulates cellular transcriptional activity. The distribution of the messenger RNA encoding for stress-activated protein kinase/extracellular signal-regulated protein kinase-1 was evaluated in the adult and developing rat central nervous system. In situ hybridization with a 35S-labelled 45mer oligodeoxynucleotide probe was used to map the distribution of the stress-activated protein kinase/extracellular signal-regulated protein kinase-1 messenger RNA in postnatal day 1, 3, 6, 9, 12, 15, 18, 21 and adult rat brains. Specific labelling was generally associated with neuronal profiles. In the adult central nervous system, high hybridization signals were observed in the hippocampus, the granular layer of the cerebellum, the medial habenula, the anterodorsal thalamic nucleus, the red nucleus, the pontine nuclei, the facial nucleus, the motor and mesencephalic nuclei of the trigeminal nerve, the hypoglossal nucleus, the vestibular nucleus and the nucleus ambiguus. Intermediate levels were present in diencephalic and mesencephalic regions and in the neocortex, while basal ganglia displayed a low hybridization signal. In the developing brain, the heterogeneous distribution of the hybridization signal observed in the adult brain was already present, but in the hippocampus and basal ganglia the stress-activated protein kinase/extracellular signal-regulated protein kinase-1 messenger RNA levels were significantly higher at postnatal day 3 and during the second postnatal week than in the adult. The results show that stress-activated protein kinase/extracellular signal-regulated protein kinase-1 is widely expressed in the rat central nervous system and co-localizes with its substrate stress-activated protein kinase. The observed changes in stress-activated protein kinase/extracellular signal-regulated protein kinase-1 messenger RNA levels during postnatal development suggest a role for this protein in the maturation of brain circuits.
Animals↗
Prolonged hypoxia upregulates vascular endothelial growth factor messenger RNA expression in ovine fetal membranes and placenta.
OBJECTIVE: In ovine fetuses, 4 days of hypoxia resulted in a large increase in urine flow, without the development of polyhydramnios, which suggests that intramembranous absorption of the amniotic fluid was enhanced. Because vascular endothelial growth factor is speculated to be a regulator of intramembranous absorption through increases of membrane vascularity and fluid transport, we hypothesized that hypoxia upregulated vascular endothelial growth factor gene expression in the fetal membranes. STUDY DESIGN: Five near-term ovine fetuses that were subjected to 4 days of hypoxia and 5 age-matched time controls were studied. On day 4, the amnion, chorion, and placenta were collected for cellular localization and quantification of vascular endothelial growth factor messenger RNA and for the determination of vascular endothelial growth factor molecular forms that were expressed. The data were analyzed statistically with the use of t tests and 2-factor analyses of variance. RESULTS: Vascular endothelial growth factor messenger RNA was expressed in the fetal membranes localized to the amniotic epithelium and chorionic cytotrophoblast, and to the villous cytotrophoblast of the placenta. In hypoxic fetuses, vascular endothelial growth factor messenger RNA levels in these cell layers were significantly increased compared with the controls. Five vascular endothelial growth factor molecular forms were identified with vascular endothelial growth factor(164) being the most abundant form expressed. The pattern of expression of the forms was not altered by hypoxia. CONCLUSION: In the near-term ovine fetus, hypoxia induced vascular endothelial growth factor messenger RNA expression in the amnion, chorion, and placenta. This was associated with an increase in intramembranous absorption of amniotic fluid. We speculate that the increased intramembranous absorption was mediated by a vascular endothelial growth factor-induced increase in the transport of amniotic fluid into the fetal membranes.
Animals↗
THE DEGRADATION OF ESCHERICHIA COLI MESSENGER RNA BY POLYNUCLEOTIDE PHOSPHORYLASE.
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Adenosine Triphosphate↗
MESSENGER RNA DECAY AND PROTECTION.
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Anti-Bacterial Agents↗
TURNOVER OF NUCLEIC ACID AND OF PROTEIN IN THE MAXIMUM CONCENTRATION OF E. COLI WITH SPECIAL REFERENCE TO MESSENGER RNA ON RIBOSOMES.
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Adenine↗
Expression of alpha1D adrenergic receptor messenger RNA in oxytocin- and corticotropin-releasing hormone-synthesizing neurons in the rat paraventricular nucleus.
The paraventricular nucleus of the hypothalamus contains a number of intermingled populations of neuroendocrine cell groups involved in the hormonal stress response, including cells synthesizing corticotropin-releasing hormone and oxytocin. Ascending noradrenergic afferents to the paraventricular nucleus, acting through alpha1 adrenergic receptors, are thought to play a role in stress-induced activation of the hypothalamic-pituitary-adrenal axis. We have previously demonstrated that, of the three known alpha1 adrenergic receptor subtypes, messenger RNA for the alpha1D subtype is the most prominently expressed in the paraventricular nucleus. Thus, regulation of the expression of this receptor may be important in modulation of the stress response. It is currently unknown, however, which populations of stress-related neuroendocrine cells in the paraventricular nucleus express alpha1 receptors, or whether the excitatory influence of norepinephrine in stress is exerted directly on neurons expressing oxytocin or corticotropin-releasing hormone. Thus, in the present study, we used dual in situ hybridization, combining a digoxigenin-labeled riboprobe encoding the rat alpha1D adrenergic receptor with radiolabeled riboprobes for oxytocin or corticotropin-releasing hormone, to determine the degree to which these neurons in the paraventricular nucleus express alpha1D adrenergic receptors. In sections through the rostral and mid-level paraventricular nucleus, nearly all (>95%) oxytocin neurons also expressed alpha1D messenger RNA. In contrast, the populations of corticotropin-releasing hormone- and alpha1D-expressing cells overlapped only partially, with most alpha1D expression situated more laterally. A subset (37%) of the neurons expressing corticotropin-releasing hormone also expressed alpha1D messenger RNA, and these were found almost entirely within the region of overlap in the lateral aspect of the medial parvocellular region. These observations support a direct role for alpha1 receptors in regulation of oxytocin secretion. Expression of alpha1D messenger RNA in distinct subsets of cells synthesizing corticotropin-releasing hormone may also help to clarify contradictory and inconsistent observations in the literature regarding the role of norepinephrine in the stress response, and may account for a presumed stressor-specific role for norepinephrine in activation of the hypothalamic-pituitary-adrenal axis.
Animals↗
The translation of globin messenger RNA with use of muscle initiation factors.
The ability of embryonic chicken muscle initiation factors to translate rabbit globin messenger RNA in an efficient, fractionated cell-free system has been examined. Although muscle factors stimulate leucine incorporation to only 15--35% the levels achieved with rabbit reticulocyte initiation factors, they synthesize more than one globin chain per mRNA molecule and both alpha and beta globin are produced. Increasing the ribosome concentration and adding the polyamine spermidine to the system produce stimulatory effects which are quantitatively and qualitatively similar for both factor preparations. The lower efficiency of synthesis of muscle factors relative to reticulocyte factors is also apparent when mRNA from encephalomyocarditis virus or embryonic chicken muscle polysomes are used in the cell-free system. These results do not support a specific restriction in the capacity of muscle factors to translate globin mRNA. Furthermore, the similarity of the effects of presumed non-specific components on the activity of muscle and reticulocyte factors suggests that globin synthesis in the cell-free system may be controlled in a similar fashion for both preparations.
Animals↗
PROTECTION OF BETA-GALACTOSIDASE MESSENGER RNA FROM DECAY DURING ANAEROBIOSIS IN ESCHERICHIA COLI.
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Anaerobiosis↗
Differential regional zif268 messenger RNA expression in an escalating dose/binge model of amphetamine-induced psychosis.
Amphetamine-induced psychosis is most often associated with a high-dose multiple binge pattern of stimulant abuse. To simulate these conditions in rats, we used an escalating dose/binge administration paradigm. Animals were pretreated with escalating doses of amphetamine (1.0-8.0mg/kg) over four days, then exposed to nine daily binges (8.0mg/kg every 2h; four injections/day). Other animals received either multiple injections of saline, saline followed by acute amphetamine (8.0mg/kg) or single daily injections of amphetamine (8.0mg/kg) in parallel with the escalating dose/binge treatment. One hour after the last injection, all animals were decapitated and regional brain activation patterns were assessed using in situ hybridization with antisense probes for zif268. Acute amphetamine resulted in a significant elevation of zif268 messenger RNA in both the nucleus accumbens and dorsal striatum. However, whereas after single daily amphetamine treatment this index was no longer elevated above control levels in the dorsal striatum, multiple binge exposures were required for the nucleus accumbens to return to baseline. Agranular insular cortex and medial olfactory tubercle zif268 messenger RNA expression was also markedly increased after acute amphetamine treatment but, unlike the nucleus accumbens and dorsal striatum, this increase was not significantly attenuated by either single daily injection or multiple binge treatment. Zif268 messenger RNA expression in the lateral nucleus of the amygdala also remained elevated above baseline after binge treatment. The possible relationships of these changes in zif268 messenger RNA regional expression patterns to the development of psychosis in high-dose stimulant abusers are discussed.
Amphetamines↗
Characterization of the messenger RNA population coding for components of viperid snake venom.
Total polyadenylated messenger RNA was prepared from the milked venom glands of the South African puff adder (Bitis arietans) and translated in an in vitro translation system. The products of cell free synthesis were immunoprecipitable with puff adder venom antiserum. Treatment of these cell free products with a dog pancreas microsomal membrane preparation demonstrated the presence of signal peptides. Northern blot hybridization of total puff adder venom gland mRNA to its complementary single stranded copy DNA revealed two discrete mRNA populations coding for the major components of puff adder venom. The relative amounts of these polyadenylated mRNA sequences changed during the onset of venom synthesis, suggesting mRNA deadenylation, general endonucleolytic RNA degradation and selective degradation of high molecular weight message components.
Animals↗
The size of fibroin messenger RNA and its polyadenylic acid content.
The molecular weight of fibroin messenger RNA has been measured by its contour length in the electron microscope and its electrophoretic mobility under conditions of complete denaturation. These methods give values of 5.6 times 10-6 and 6.0 times 10-6 daltons, respectively. The average molecular weight of 5.8 times 10-6 (1.6 times 10-4 base residues) would be expected to code for a protein of 414,000 daltons, a value about 12 percent larger than the most recently published size for fibroin. When the mRNA is prepared carefully to avoid shear breakage, it is as homogeneous in size as ribosomal RNA. Between 43 percent and 81 percent of the mRNA molecules bind to oligo(dT)-cellulose and are presumed to contain a poly(A) sequence. This sequence, isolated after ribonuclease digestion, comprises about 0.6 percent of the molecule, or an estimated 100 nucleotides. The poly(A) has been localized at the end of the molecule by hybridizing with a small poly (dT:dA) piece and subsequent visualization by electron microscopy. This observation, along with the ability of reverse transcriptase to transcribe fibroin mRNA with an oligo(dT) primer, demonstrates that the poly(A) is at the 3' end of the mRNA.
Adenine Nucleotides↗
Species-specific expression of cholecystokinin messenger RNA in rodent dorsal root ganglia.
The expression of cholecystokinin (CCK) messenger RNA (mRNA) was examined in dorsal root ganglia of rat and guinea pig using in situ hybridization histochemistry and RNA (Northern) blot hybridization with synthetic oligodeoxyribonucleotide (oligomer) probes. In guinea pig, CCK mRNA was detected in small and medium-sized neuronal perikarya comprising approximately 10-15% of the total dorsal root ganglia cell population. In contrast, in neurons of rat dorsal root ganglia, CCK mRNA was not detectable. Northern blot analyses revealed a single CCK mRNA species of expected size (0.8 kb) in guinea pig, but not rat, dorsal root ganglia. A 0.8 kb CCK mRNA was, however, detected in cortex of both rat and guinea pig. These data suggest that CCK is normally not synthesized in neurons of rat dorsal root ganglia and that there are species differences in CCK gene expression in mammalian sensory ganglia.
Animals↗
Repression of hspA2 messenger RNA in human testes with abnormal spermatogenesis.
OBJECTIVE: To evaluate the messenger RNA (mRNA) expression of hspA2 in testes of infertile men with azoospermia. DESIGN: Prospective study. SETTING: Center for Reproduction and Genetics, Pundang Je-Saeng General Hospital, Dae-Jin Medical Center, Korea. PATIENT(S): Azoospermic patients (n = 15) undergoing testicular biopsy for pathologic evaluation were selected. INTERVENTION(S): After pathologic evaluation, testicular biopsy specimens were subdivided into three groups: group 1, normal spermatogenesis (n = 5); group 2, spermatocyte arrest (n = 5); and group 3, Sertoli cell-only syndrome (n = 5). The levels of hspA2 mRNA expression were compared in testes of group 1, group 2, and group 3 with the use of a competitive reverse transcription polymerase chain reaction (RT-PCR) technique. MAIN OUTCOME MEASURE(S): Comparison of hspA2 mRNA levels in testes. RESULT(S): On competitive RT-PCR analyses for hspA2 mRNA, significant hspA2 expression was observed in group 1, whereas a very low level of hspA2 was expressed in groups 2 and 3. CONCLUSION(S): This study demonstrates that hspA2 gene expression is down-regulated in human testes with abnormal spermatogenesis, which in turn suggests that the hspA2 gene might play a specific role during meiosis in human testes.
Cell Cycle↗
Rapid generation of homologous internal standards and evaluation of data for quantitation of messenger RNA by competitive polymerase chain reaction.
Sensitive and quantitative measurement of messenger RNA (mRNA) is important for accurate assessment of gene expression. Conventional methods of mRNA measurement frequently lack the sensitivity required to detect mRNA expressed at low level, such as mRNA encoding receptors and intracellar signaling molecules. Thus, the extremely sensitive RT-PCR has become the method of choice for examination of gene expression. However, quantitation of mRNA by PCR is difficult because small variations in amplification efficiencies among sample tubes can lead to substantial differences in product yield, thereby rendering direct comparisons between samples invalid. Development of protocols for quantitative RT-PCR has relied on internal standards to monitor the efficiency of the RT-PCR in different reaction tubes. Technically, the two most serious limitations to routine successful application of competitive quantitative PCR is ready access to competitive internal standards and efficient methods for accurate quantitative analysis of the data. In the present manuscript, application and validation of a simple approach to generate homologous internal competitive standards and to quantitate data for rapid, accurate determination of the expression level of genes by quantitative PCR is described. Generation of the competitive standard from a previously amplified PCR product by the methods described requires only one additional primer pair, and an additional two-step reaction; it can be completed in 1-2 days. Analyzing the results of the competitive PCR reaction via phosphoimager analysis provides a simple, rapid method for accurate quantitation of results. Data presented here clearly illustrate that the methods described have been successfully applied, and that they should have wide application for competitive quantitative PCR analysis of gene expression.
Aorta↗
Coordinate hormonal and synaptic regulation of vasopressin messenger RNA.
Previous studies have shown that adrenalectomy augments arginine vasopressin (AVP) messenger RNA levels in the adult paraventricular nucleus. It is now demonstrated that unilateral lesions in the lateral septal nucleus enhance the adrenalectomy-induced expression of AVP mRNA. This effect was entirely ipsilateral to the lesion and most prominent in the rostral paraventricular nucleus and related nuclei. Moreover, AVP and AVP mRNA were found to be colocalized with oxytocin in a few neurons. These results indicate that mRNA expression is modulated by synaptic influences and raise the possibility that synaptically mediated selection of neuronal phenotypes is a dynamic feature of the mature central nervous system.
Adrenalectomy↗
Messenger RNA decay in mammalian cells: the exonuclease perspective.
The majority of messenger RNA (mRNA) decay in mammalian cells appears to be the work of a series of RNA exoribonucleases. A set of multiple poly(A)-specific deadenylases has been identified, some, if not most, of which are likely to play a role in the key first step of mRNA turnover--the regulated shortening of the poly(A) tail. After deadenylation, the transcript likely gets degraded by either a 5'-to-3' or a 3'-to-5' exonucleolytic pathway. Interestingly, multiple exonucleases have been identified for both of these pathways that appear to form multicomponent complexes with diverse roles in cellular biology. Therefore these enzymes appear not only to be important components of the mRNA turnover machinery, but also may function in a networked fashion in the post-transcriptional control of gene expression.
Animals↗
Effects of a single intranasal dose of modified-live bovine respiratory syncytial virus vaccine on cytokine messenger RNA expression following viral challenge in calves.
OBJECTIVE: To characterize cytokine messenger RNA (mRNA) expression in intranasally vaccinated calves after bovine respiratory syncytial virus (BRSV) challenge. ANIMALS: Twelve 8- to 12-week-old calves. PROCEDURES: Calves received modified-live BRSV vaccine (vaccinated) or spent tissue culture medium (mock-vaccinated) intranasally, followed by challenge 30 days later with BRSV, or mock challenge with spent tissue culture medium (mock-challenge controls). Interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) mRNA was measured in lungs, bronchoalveolar lavage (BAL) fluid cells, pharyngeal tonsils, and tracheobronchial lymph nodes, and tumor necrosis factor-alpha (TNF-alpha) mRNA was measured in lungs and BAL fluid cells by reverse transcriptase-competitive polymerase chain reaction assay. RESULTS: Resistance to clinical signs of disease was conferred in vaccinated calves. Expression of TNF-alpha mRNA in lungs and BAL fluid cells was higher in mock-vaccinated calves than control or vaccinated calves. In the lung, IL-4 mRNA expression was higher in vaccinated calves than control or mock-vaccinated calves. In pharyngeal tonsils, expression of mRNA for IL-4 and IFN-gamma was higher in mock-vaccinated calves than control calves. In tracheobronchial lymph nodes, IFN-gamma mRNA expression was higher in mock-vaccinated calves than vaccinated calves. CONCLUSIONS AND CLINICAL RELEVANCE: Although vaccinated calves had decreased clinical signs of disease after BRSV challenge, compared with mock-vaccinated calves, this difference was not related to a T helper type 1 bias, as determined by increased expression of interferon-gamma mRNA relative to interleukin-4 mRNA in lungs, BAL fluid cells, or tracheobronchial lymph nodes of vaccinated calves. Pulmonary inflammation was decreased in vaccinated calves as determined by decreased expression of TNF-alpha mRNA.
Administration, Intranasal↗