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At least 667 records · Page 37Linked to original sources
PubMed1991

Co-expression of cholecystokinin mRNA and tyrosine hydroxylase mRNA in populations of rat substantia nigra cells; a study using a combined radioactive and non-radioactive in situ hybridization procedure.

Improvements in the sensitivity of non-radioactive in situ hybridization histochemistry methods for detection of mRNA now make it feasible to combine the use of non-radioactive and radioactive in situ methods to visualize two mRNAs on the same tissue section. The method reported here allows the simultaneous detection of two mRNAs in one cell and therefore is ideally suited to the studies of co-expression. Here we demonstrate the co-expression of tyrosine hydroxylase (TH) mRNA and cholecystokinin (CCK) mRNA in the ventral mesencephalic dopaminergic neurones of the rat. The distribution of dopaminergic neurones containing both TH and CCK transcripts suggests, on the basis of earlier anatomical studies that these CCK/TH-containing doubled-labelled cells project mainly to the striatal matrix. Dopamine neurones believed to project to the patch compartment did not contain CCK mRNA.

Animals↗
PubMed1991

In situ hybridization with single-stranded RNA probes to demonstrate infrequently elevated gli mRNA and no increased ras mRNA levels in meningiomas and astrocytomas.

Using computer-assisted digital image-analysis, the level of ras and gli messenger RNA (mRNA) in neoplasms of meningeal and glial origin was determined in an attempt to correlate these parameters with histological tumor severity. We used single-stranded, asymmetric, radiolabelled RNA probes to detect the amount of ras and gli mRNA present in formalin-fixed, paraffin-embedded tumor material. Such archival material can provide an immediate, larger sample base than with fresh samples. The extent of ras mRNA expression in 25 brain tumors was not significantly different than normal cerebellum. However, five of 74 astrocytomas of relatively high malignant potential demonstrated gli mRNA levels above normal cerebellum.

Analysis of Variance↗
PubMed1995

Lens epithelial cell mRNA, II. Expression of a mRNA encoding a lipid-binding protein in rat lens epithelial cells.

A lens epithelial (LE) cell cDNA clone, designated pLELBP, was isolated by subtraction-hybridization methods between a 4-week-old rat LE cell and rat lens fiber cell lambda ZAP cDNA libraries. The cDNA contained 683 bp, an ATG at bp 42, and an open reading frame (ORF) encoding a protein of 135 amino acids (aa), and a poly(A) signal at bp 641 (GenBank/EMBL accession No. U13253). Northern blot analysis showed that the lens mRNA was at a concentration that exceeded 100-fold that found in non-ocular tissues examined, except in skin it was found at levels equal to about 1/15 of the lens levels. In the retina, it was also found at about 1/15 of that present in the lens. By in situ hybridization analysis, the sense RNA was localized to the LE cells and to the glial cells of the retina, and negligibly to the lens fiber cells. Search of GenBank, EMBL and SwissProt data bases revealed that the LE cell mRNA and its aa encoded protein showed extensive sequence homology to members of the cytosolic lipid-binding protein (LBP) family. It matched to 91% in aa sequence homology to the protein encoded by ORF of mal1 cDNA, isolated from mouse skin squamous cell carcinoma [Krieg et al., J. Biol. Chem. 268 (1993) 17362-17369], and 99% in aa sequence homology to the protein encoded by ORF of rat skin LBP mRNA [Watanabe et al., Biochem. Biophys. Res. Commun. 200 (1994) 253-259]. Occurrence of high levels of mRNA for a specific LBP in the LE cells relative to other non-ocular tissues is consistent with a hypothesis that the encoded protein may be involved in several lens epithelial cell-specific mechanisms, possibly including differentiation, protective and nutritional processes.

Animals↗
PubMed1994

An alpha 1 adrenergic mechanism mediates estradiol stimulation of LHRH mRNA synthesis and estradiol inhibition of POMC mRNA synthesis in the hypothalamus of the prepubertal female rat.

We showed previously that the surge of luteinizing hormone-releasing hormone (LHRH) induced by estradiol-17 beta (E2) in the female rat can be blocked by an alpha 1 adrenergic antagonist. The aim of the present study was to determine whether this was due to a direct action of E2 on noradrenergic projections to LHRH neurons or whether it also involved other systems such as the arcuate pro-opiomelanocortin (POMC) neurons which are thought to inhibit LHRH biosynthesis and release. The experimental preparation was the prepubertal female rat in which an LHRH surge is induced by pregnant mare serum gonadotropin. Prazosin was used as a specific alpha 1 adrenergic antagonist and LHRH and POMC mRNA concentrations and cell numbers, in the medial preoptic area and rostral arcuate nucleus, respectively, were determined by in situ hybridization. Prazosin significantly reduced the total number of LHRH mRNA expressing cells, and increased the total number of POMC mRNA expressing cells and the concentration of POMC mRNA per cell. These results suggest that the inhibition of E2-stimulated LHRH biosynthesis and release by alpha 1 adrenergic blockade may be mediated by two mechanisms; (i) increased POMC synthesis leading to inhibition of LHRH neurons and (ii) direct inhibition of a stimulatory alpha 1 adrenergic/LHRH mechanism.

Animals↗
PubMed2004

Effects of 1,25(OH)2D3, 25OHD3, and EB1089 on cell growth and Vitamin D receptor mRNA and 1alpha-hydroxylase mRNA expression in primary cultures of the canine prostate.

The aim of this study was to investigate effects of 1,25(OH)(2)D(3) (calcitriol), 25OHD(3), and EB1089 on cell growth and on Vitamin D receptor (VDR) mRNA and 1alpha-hydroxylase (1alpha-OHase) mRNA expression in normal canine prostatic primary cultures. Canine prostatic epithelial cells were isolated, cultured, and treated with vehicle (ethanol), calcitriol, 25OHD(3), and EB1089 at 10(-9) and 10(-7)M. The VDR was present in epithelial and stromal cells of the canine prostate gland. 1,25(OH)(2)D(3), 25OHD(3), and EB1089 inhibited epithelial cell growth at 10(-7)M compared to vehicle-treated controls [calcitriol (P < 0.01), EB1089 (P < 0.01), and 25OHD(3) (P < 0.05)]. Epithelial cells treated with calcitriol and EB1089 at 10(-7)M had slightly increased VDR mRNA expression (0.2-0.3-fold) at 6 and 12h compared to controls. There was no difference in 1alpha-OHase mRNA expression in epithelial cells treated with these three compounds. 1,25(OH)(2)D(3) and its analogs may be effective antiproliferative agents of epithelial cells in certain types of prostate cancer.

Animals↗
PubMed2004

SMG7 acts as a molecular link between mRNA surveillance and mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that eliminates mRNAs containing premature termination codons (PTCs). The proteins UPF1, SMG5, SMG6, and SMG7 are essential NMD factors in metazoa. SMG5 and SMG7 form a complex with UPF1 and interact with each other via their N-terminal domains. Here we show that SMG5 and SMG7 colocalize in cytoplasmic mRNA decay bodies, while SMG6 forms separate cytoplasmic foci. When SMG7 is tethered to a reporter transcript, it elicits its degradation, bypassing the requirement for a PTC, UPF1, SMG5, or SMG6. This activity is mediated by the C-terminal domain of SMG7. In contrast, SMG5 requires SMG7 to trigger mRNA decay and to localize to decay bodies. Our findings indicate that SMG7 provides a link between the NMD and the mRNA degradation machinery by interacting with SMG5 and UPF1 via its N-terminal domain and targeting bound transcripts for decay via its C-terminal domain.

Blotting, Western↗
PubMed2005

Iron-mediated stability of PAI-1 mRNA in adenocarcinoma cells-involvement of a mRNA-binding nuclear protein.

This study reports the stability of mRNA of type-1 plasminogen activator inhibitor (PAI-1), the major physiologic inhibitor of plasminogen activation, by deferoxamine-aided iron deprivation, in PC3 adenocarcinoma cells. ELISA and Northern analyses studies revealed dose-dependent increase in PAI-1 expression by deferoxamine-treated cells. Co-treatment with ferric citrate quenched the effect of deferoxamine, confirming the role of iron in PAI-1 regulation. DRB-based RNA chase experiments suggested that post-transcriptional mechanism was involved in PAI-1 regulation. De-novo protein synthesis was necessary for this regulation. Electrophoretic mobility shift assay revealed the presence of a nuclear protein, binding to the 3'-UTR of PAI-1 mRNA in an iron-mediated manner. This is the first report of iron-mediated mRNA-protein interaction in PAI-1, involved in mRNA stability.

3' Untranslated Regions↗
PubMed1999

Tyrosine hydroxylase mRNA is increased in old age and norepinephrine uptake transporter mRNA is decreased in middle age in locus coeruleus of Brown-Norway rats.

In normal aging, cell loss occurs in the locus coeruleus (LC), the major noradrenergic nucleus in the brain. This study examined changes in the LC of aged rats by measuring mRNA expression for tyrosine hydroxylase (TH) and the norepinephrine uptake transporter (NET). TH and NET mRNA expression were measured by in situ hybridization in young, middle-aged and aged rats. It appears that in middle age, the transporter system responds initially to LC cell loss by decreasing NET mRNA expression. Then, with further aging and cell loss, TH mRNA expression increases which may potentially increase NE synthesis in the remaining neurons. These findings suggest that multiple regulatory components are used to maintain stable noradrenergic synaptic levels despite neuronal loss. Published by Elsevier Science B.V.

Aging↗
PubMed1998

Adrenal neuropeptide Y mRNA but not preproenkephalin mRNA induction by stress is impaired by aging in Fischer 344 rats.

Relatively few molecular markers of stress have been studied in aged individuals. Interactions of age and stress on adrenal neuropeptide Y (NPY) and preproenkephalin (ppENK) expression have not been reported. The purpose of these studies was to characterize the adrenal NPY and ppENK responses to stress using a common stressor, physical restraint for 2 h, in Fischer 344 rats at 7, 16 and 23 months of age. Northern blot techniques were used to evaluate induction by stress of adrenal NPY mRNA and adrenal ppENK mRNA. Two humoral responses to stress, serum glucose and corticosterone, were measured to corroborate that a stress response occurred. We observed that the induction by stress of adrenal NPY mRNA is impaired with age but the stress-induced elevation of adrenal ppENK mRNA, blood glucose, and corticosterone show no evidence of age-related impairments.

Adrenal Glands↗
PubMed2002

Differential regulation of glutamic acid decarboxylase mRNA and tyrosine hydroxylase mRNA expression in the aged manganese-treated rats.

Recent studies have implicated chronic elevated exposures to environmental agents, such as metals (e.g. manganese, Mn) and pesticides, as contributors to neurological disease. Eighteen-month-old rats received intraperitoneal injections of manganese chloride (6 mg Mn/kg/day) or equal volume of saline for 30 days in order to study the effect of manganese on the dopamine- and GABA-neurons. The structures studied were substantia nigra, striatum, ventral tegmental area, nucleus accumbens and globus pallidus. First, we studied the enzymatic activity of mitochondrial complex II succinate dehydrogenase (SDH). We found an overall decrease of SDH in the different brain areas analyzed. We then studied the mRNA levels for tyrosine hydroxylase (TH) and the dopamine transporter (DAT) by in situ hybridization. TH mRNA but not DAT mRNA was significantly induced in substantia nigra and ventral tegmental area following Mn treatment. Correspondingly, TH immunoreactivity was increased in substantia nigra and ventral tegmental area. Manganese treatment significantly decreased GAD mRNA levels in individual GABAergic neurons in globus pallidus but not in striatum. We also quantified the density of glial fibrillary acidic protein (GFAP)-labeled astrocytes and OX-42 positive cells. Reactive gliosis in response to Mn treatment occurred only in striatum and substantia nigra and the morphology of the astrocytes was different than in control animals. These results suggest that the nigrostriatal system could be specifically damaged by manganese toxicity. Thus, changes produced by manganese treatment on 18-month-old rats could play a role in the etiology of Parkinson's disease.

Aging↗
PubMed2002

Plasminogen mRNA induction in the mouse brain after kainate excitation: codistribution with plasminogen activator inhibitor-2 (PAI-2) mRNA.

Plasminogen (Plg), which can be converted to the active protease plasmin by plasminogen activators, has been previously implicated in brain plasticity and in toxicity inflicted in hippocampal pyramidal neurons by kainate. Here we have localized Plg. mRNA through in situ hybridization in brain cryosections derived from normal adult mice or after kainate injection (i.p.). The results indicated that Plg mRNA was undetectable in the normal brain, but after kainate injection it was induced in neuronal cells in multiple, but specific areas, including layers II-III of the neocortex; the olfactory bulb, anterior olfactory nucleus, and the piriform cortex; the caudate/putamen and accumbens nucleus shell; throughout the amygdaloid complex; and in the CAI/CA3 subfields of the hippocampus. Interestingly, this distribution pattern coincided with what we have recently described for the plasminogen activator inhibitor-2 (PAI-2) mRNA, however differing from that of the plasminogen activator inhibitor-1 (PAI-1) mRNA, as also shown here. These results suggest that enhanced Plg gene expression could be involved in events associated with olfactory, striatal, and limbic structures. Furthermore, because PAI-2 is thought to intracellularly counteract cytotoxic events, our results raise the possibility that PAI-2 can act in the brain as an intracellular neuroprotector against potential plasmin-mediated toxicity.

Animals↗
PubMed1997

Co-localization of preprosomatostatin mRNA and preprotachykinin A mRNA in neurons of the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN), a circadian oscillator, contains various peptides arranged in the compartment-specific manner. Somatostatin (SS) and substance P (SP), a peptide derived from preprotachykinin A (PPT-A), are expressed in neurons in the intermediate zone, a narrow area between the major dorsomedial and ventrolateral subdivisions. In the present study, we examined the possibility of co-localization of SS and SP in the SCN by a double-labeling in situ hybridization method using 35S-labeled and digoxigenin-labeled cRNA probes. In the SCN, most of preprosomatostatin (PPSS) mRNA-containing neurons expressed PPT-A mRNA (86%) and, in turn, almost all preprotachykinin A (PPT-A) mRNA-expressing neurons expressed PPSS mRNA signals (97%). Both PPSS and PPT-A mRNAs were also detected in the cerebral cortex and the caudate-putamen, however, their co-existence was extremely rare (< 4%) in these regions. Since the pharmacological effects of SS and SP are similar to that of the light pulses exposed on animals under constant darkness, the co-release of peptides might be an important process for entraining the circadian clock in the SCN.

Animals↗
PubMed1999

Quercetin inhibited DNA synthesis and induced apoptosis associated with increase in c-fos mRNA level and the upregulation of p21WAF1CIP1 mRNA and protein expression during liver regeneration after partial hepatectomy.

Quercetin, a widely distributed bioflavonoid, inhibited DNA synthesis in regenerating liver after partial hepatectomy. This inhibition was accompanied by apoptosis, evidenced by in situ end-labeling and gel electrophoresis of DNA fragmentation. Characteristic DNA fragmentation was detected as early as 2 h after injection. Northern blot analysis revealed that quercetin induced the increases in c-fos and p21WAF1CIP1 mRNA levels within 2 h. The expression of p21 protein was also enhanced, while p53 mRNA and protein levels were not affected by quercetin. These results suggest that quercetin-induced apoptosis is associated with the increase in c-fos mRNA level and the upregulation of p21 mRNA and protein expression, probably in a p53-independent pathway.

Animals↗
PubMed2001

Corticotropin-releasing hormone mRNA levels in response to chronic mild stress rise in male but not in female rats while tyrosine hydroxylase mRNA levels decrease in both sexes.

Corticotropin-releasing hormone (CRH) and catecholamines are suggested to play a significant role in the pathophysiology of depression. In the present study we investigated gene expression of CRH in the hypothalamic paraventricular nucleus (PVN) and tyrosine hydroxylase (TH) in the locus coeruleus (LC) in an experimental model of depression. A chronic mild stress model was applied in rats of both genders for a three-week period. Anhedonic behaviour, a typical sign of depression-like state, was measured by a sucrose preference test. The chronic mild stress induced a decrease in sucrose preference in both genders. The body weight gain was reduced in males only. The total activity in the open field test was unchanged, however, male rats exposed to chronic mild stress showed enhanced locomotor activity during the first minute of the session, suggesting increased anxiety. Basal plasma corticosterone levels, thymus and adrenal weights measured on the third day after cessation of the stress regimen, were not affected by the stress procedure. Evaluation of CRH mRNA levels in the PVN by in situ hybridisation revealed a significant rise in response to chronic mild stress in males. In females, the basal CRH mRNA levels were higher compared to those in males, but the stress-induced rise was absent. Chronic mild stress resulted in a decrease in TH mRNA levels in the LC. These data demonstrate that chronic mild stress model of depression induces a specific stress response with a reduction of TH gene expression in the LC and clear gender differences in gain of body weight, anxiety-like behaviour, and CRH mRNA levels in the PVN.

Adrenal Cortex↗
PubMed1999

Down-regulation of L-type calcium channel and sarcoplasmic reticular Ca(2+)-ATPase mRNA in human atrial fibrillation without significant change in the mRNA of ryanodine receptor, calsequestrin and phospholamban: an insight into the mechanism of atrial electrical remodeling.

OBJECTIVES: We investigated the gene expression of calcium-handling genes including L-type calcium channel, sarcoplasmic reticular calcium adenosine triphosphatase (Ca(2+)-ATPase), ryanodine receptor, calsequestrin and phospholamban in human atrial fibrillation. BACKGROUND: Recent studies have demonstrated that atrial electrical remodeling in atrial fibrillation is associated with intracellular calcium overload. However, the changes of calcium-handling proteins remain unclear. METHODS: A total of 34 patients undergoing open heart surgery were included. Atrial tissue was obtained from the right atrial free wall, right atrial appendage, left atrial free wall and left atrial appendage, respectively. The messenger ribonucleic acid (mRNA) amount of the genes was measured by reverse transcription-polymerase chain reaction and normalized to the mRNA levels of glyceraldehyde 3-phosphate dehydrogenase. RESULTS: The mRNA of L-type calcium channel and of Ca(2+)-ATPase was significantly decreased in patients with persistent atrial fibrillation for more than 3 months (0.36+/-0.26 vs. 0.90+/-0.88 for L-type calcium channel; 0.69+/-0.42 vs. 1.21+/-0.68 for Ca(2+)-ATPase; both p < 0.05, all data in arbitrary unit). We further demonstrated that there was no spatial dispersion of the gene expression among the four atrial tissue sampling sites. Age, gender and underlying cardiac disease had no significant effects on the gene expression. In contrast, the mRNA levels of ryanodine receptor, calsequestrin and phospholamban showed no significant change in atrial fibrillation. CONCLUSIONS: L-type calcium channel and the sarcoplasmic reticular Ca(2+)-ATPase gene were down-regulated in atrial fibrillation. These changes may be a consequence of, as well as a contributory factor for, atrial fibrillation.

Adolescent↗
PubMed2000

Long-term repetitive transcranial magnetic stimulation increases the expression of brain-derived neurotrophic factor and cholecystokinin mRNA, but not neuropeptide tyrosine mRNA in specific areas of rat brain.

Repetitive transcranial magnetic stimulation (rTMS) is increasingly used as a therapeutic tool in various neurological and psychiatric disorders, and we recently found that it has a neuroprotective effect both in vitro and in vivo. However, the neurochemical mechanisms underlying the therapeutic effects are still unknown. We investigated the effects of long-term rTMS on the expression of brain-derived neurotrophic factor (BDNF), cholecystokinin (CCK), and neuropeptide tyrosine (NPY) mRNA in rat brain. In situ hybridization revealed a significant increase in BDNF mRNA in the hippocampal areas CA3 and CA3c, the granule cell layer, as well as in the parietal and the piriform cortex after rTMS. BDNF-like immunoreactivity was markedly increased in the same areas. A significant increase in CCK mRNA was observed in all brain regions examined. NPY mRNA expression, in contrast, was not altered. The present results suggest that BDNF may contribute to the neuroprotective effects of rTMS. Furthermore, the rTMS-induced changes in BDNF and CCK expression are similar to those reported after antidepressant drug treatment and electroconvulsive seizures, suggesting that a common molecular mechanism may underlie different antidepressant treatment strategies.

Animals↗
PubMedDate not supplied

Expression of mRNA and proteins for testicular steroidogenic enzymes and brain and pituitary mRNA for glutamate receptors in rats exposed to immobilization stress.

The objectives of this study were to determine whether stress attenuates the pituitary LH response to excitatory amino acids by altering expression of glutamate receptor 1 (GluR1) and N-methyl-D-aspartic acid (NMDA) receptor mRNA levels in the hypothalamus or pituitary, and assess whether stress influences testicular levels of mRNA or protein for steroidogenic enzymes. Three hours (h) of immobilization stress was associated with a greater than 7-fold increase in serum corticosterone, and a marked reduction in serum testosterone (T) concentrations. Stress did not significantly alter hypothalamic or pituitary GluR1 and NMDA receptor mRNA levels. Although transcript levels for P450SCC and P45017alpha mRNA in the testis were unchanged in stressed rats, western blotting of testicular fractions revealed reduced amounts of P450SCC and 3beta-HSD, but not P45017alpha. The data suggest that immobilization stress reduces T production by suppressing the translation of transcripts for P450SCC and 3beta-HSD, but the attenuated LH response of stressed animals to NMDA is not mediated by altered hypothalamic or pituitary expression of GluR1 and NMDA receptor levels.

3-Hydroxysteroid Dehydrogenases↗
PubMed2003

EP3-2 receptor mRNA expression is reduced and EP3-6 receptor mRNA expression is increased in gravid human myometrium.

OBJECTIVE: To assess relative expression levels of mRNA for EP3 isoforms EP3-2, EP3-3, and EP3-6 in human myometrium in various physiologic states. METHODS: Using semiquantitative reverse transcriptase-polymerase chain reaction, we analyzed myometrial samples from 46 women (ten menopausal, ten nongravid premenopausal, 19 gravidas, seven premenopausal misoprostol-treated nongravid women) for the specific expression of mRNA messages for the EP3 receptor isoforms EP3-2, EP3-3, and EP3-6. RESULTS: We found that the expression of EP3-2 was significantly lower in gravid than nongravid myometrium (P < .0001). Levels of the EP3-6 message were higher in gravid samples than in nongravid specimens (P < .02). EP3-3 mRNA was present in higher levels in menopausal samples compared with the other groups. CONCLUSIONS: The selective fluctuation of mRNA expression of the different EP3 isoforms suggests the possibility of an important regulatory role for these receptors in myometrial contractility. Additional studies are necessary to evaluate the exact mechanism of action of EP3 receptor isoforms in human myometrium.

Adult↗