hnRNP A2 selectively binds the cytoplasmic transport sequence of myelin basic protein mRNA

Segregation of mRNAs in the cytoplasm of polar cells has been demonstrated for proteins involved in Xenopus and Drosophila oogenesis, and for some proteins in somatic cells. It is assumed that vectorial transport of the messages is generally responsible for this localization. The mRNA encoding the basic protein of central nervous system myelin is selectively transported to the distal ends of the processes of oligodendrocytes, where it is anchored to the myelin membrane and translated. This transport is dependent on a 21-nucleotide cis-acting segment of the 3'-untranslated region (RTS). Proteins that bind to this cis-acting segment have now been isolated from extracts of rat brain. A group of six 35-42-kDa proteins bind to a 35-base oligoribonucleotide incorporating the RTS, but not to several oligoribonucleotides with the same composition but randomized sequences, thus establishing specificity for the base sequence in the RTS. The most abundant of these proteins has been identified, by Edman sequencing of tryptic peptides and mass spectroscopy, as heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a 36-kDa member of a family of proteins that are primarily, but not solely, intranuclear. This protein was most abundant in samples from rat brain and testis, with lower amounts in other tissues. It was separated from the other polypeptides by using reverse-phase HPLC and shown to retain preferential association with the RTS. In cultured oligodendrocytes, hnRNP A2 was demonstrated by confocal microscopy to be distributed throughout the nucleus, cell soma, and processes.

Acute intermittent porphyria: alternative splicing of hydroxymethylbilane synthase mRNA excludes exons 3 and 12

The hydroxymethylbilane synthase (HMBS) mRNAs from 44 control individuals and 30 patients suffering from acute intermittent porphyria (AIP), were screened for length differences by reverse transcriptase polymerase chain reaction (RT-PCR) and any abnormalities were characterized by direct sequencing. Examination of the mRNAs extracted from the peripheral blood lymphocytes of the samples revealed varying degrees of alternative splicing, involving the removal of exons 3 and 12. Approximately 10-50% of the mRNA molecules were affected, despite the absence of genomic splice site mutations or any major deviance from consensus splice sequence values. The preliminary data obtained from this study suggest that this event is a normal occurrence in peripheral blood lymphocytes, and may not be associated with the molecular pathology responsible for AIP. (C) 1998 Academic Press Limited.

Chemokine and cell adhesion molecule mRNA expression and neutrophil infiltration in lipopolysaccharide-induced hepatitis in ethanol-fed rats

Neutrophil infiltration is a feature of alcoholic hepatitis (AH), and although the mechanism by which this occurs is unclear, it may involve a chemotactic gradient. We used lipopolysaccharide (LPS) to induce, in ethanol-fed rats, liver damage similar to that seen in AH. To our knowledge, this study is the first to examine the effect of ethanol on LPS-stimulated chemokine mRNA expression in this model. Hepatic cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1 beta, MIP-2, and eotaxin mRNA levels were elevated 1 to 3 hr post-LPS in both groups. Maximal expression of MIP-2 and MCP-1 mRNA was higher in ethanol-fed rats 1 hr post-LPS, whereas CINC-2 mRNA expression was elevated above controls at 12 to 24 hr. Hepatic intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 mRNA levels were elevated in both groups at 1 hr, whereas L-selectin expression in ethanol-fed rats was elevated above controls at 12 to 24 hr. Hepatic neutrophil infiltration was highest during maximal hepatocyte necrosis. These data suggest that cell adhesion molecules, in conjunction with elevated cytokines and the subsequently induced chemokines, may assist in the formation of a chemotactic gradient within the liver, causing the neutrophil infiltration seen both in this model and possibly in AH.

mRNA differential display of 2-amino-1-methyl-6-phenylinlidazo[4,5-b]pyridine-induced rat mammary gland tumors

The mRNA differential display technique was used to compare mRNAs between normal mammary gland and turner-derived epithelial cells from female Sprague-Dawley rat mammary gland tumors induced by the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and promoted by a high-fat diet (23.5% corn oil). Two genes, beta-casein and transferrin, were identified as differentially expressed. The expression of these genes was examined across a bank of rat mammary gland tumors derived from animals on a low-fat diet (5% corn oil) or the high-fat diet. Carcinomas had over a 10- and 50-fold lower expression of beta-casein and transferrin, respectively than normal mammary gland. In addition, carcinomas from animals on the high-fat diet showed on average a 5-fold higher expression of beta-casein, and transferrin than carcinomas from animals on the low-fat diet. The results indicate the process of mammary gland tumorigenesis alters the expression of certain genes in the mammary gland, and that the level of dietary fat further modulates the expression of these genes.

Chronic ethanol exposure and withdrawal influence NMDA receptor subunit and splice variant mRNA expression in the rat cerebral cortex

Chronic ethanol exposure and subsequent withdrawal are known to change NMDA receptor activity. This study examined the effects of chronic ethanol administration and withdrawal on the expression of several NMDA receptor subunit and splice variant mRNAs in the rat cerebral cortex. Ethanol dependence was induced by ethanol vapour exposure. To delineate between seizure-induced changes in expression during withdrawal and those due to withdrawal per se, another group of naive rats was treated with pentylenetetrazol (PTZ) injection (30 mg/kg, i.p.). RNA samples from the cortices of chronically treated and withdrawing animals were compared to those from pairfed controls. Changes in NMDA receptor mRNA expression were determined using ribonuclease protection assays targetting the NR2A, -2B, -2C and NR1-pan subunits as well as the three alternatively spliced NR1 inserts (NR1-pan describes all the known NR1 splice variants generated from the 5' insert and the two 3' inserts). The ratio of NR1 mRNA incorporating the 5' insert vs, that lacking it was decreased during ethanol exposure and up to 48 h after withdrawal. NR2B mRNA expression was elevated during exposure, but returned to control levels 18 h after withdrawal. Levels of NR2A, NR2C, NR1-pan and both 3' NR1 insert mRNAs from the ethanol-treated groups did not alter compared with the pair-fed control group. No changes in the level of any NMDA receptor subunit mRNA was detected in the PTZ-treated animals. These data support the hypothesis that changes in NMDA receptor subunit composition may underlie a neuronal adaptation to the chronic ethanol-inhibition and may therefore be important in the precipitation of withdrawal hyperactivity. (C) 1999 Elsevier Science B.V. All rights reserved.

PMCA1 mRNA expression in rat aortic myocytes: A real-time RT-PCR study

The plasmalemmal Ca2+ adenosine triphosphatase (PMCA) is a key regulator of Ca2+ efflux in vascular smooth muscle. In these studies are developed a realtime reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay for assessing PMCA1 mRNA levels in rat primary cultured aortic myocytes. This assay detected fetal bovine serum-induced increases in PMCA1 mRNA (relative to 18S rRNA) 4, 8, and 24 h after stimulation. Early fetal bovine serum-induced increases in PMCA1 mRNA were insensitive to the Ca2+ channel blockers nifedipine, flunarizine, and SKF-96365. These studies demonstrate the feasibility of real-time RT-PCR to assess mRNA levels of PMCA1 and illustrate dynamic regulation of this Ca2+ pump isoform in rat primary cultured aortic myocytes, (C) 2000 Academic Press.

Human cyclophilin 40 is a heat shock protein that exhibits altered intracellular localization following heat shock

The unactivated steroid receptors are chaperoned into a conformation that is optimal for binding hormone by a number of heat shock proteins, including Hsp90, Hsp70, Hsp40, and the immunophilin, FKBP52 (Hsp56). Together with its partner cochaperones, cyclophilin 40 (CyP40) and FKBP51, FKBP52 belongs to a distinct group of structurally related immunophilins that modulate steroid receptor function through their association with Hsp90. Due to the structural similarity between the component immunophilins, FKBP52 and cyclophilin 40, we decided to investigate whether CyP40 is also a heat shock protein. Exposure of MCF-7 breast cancer cells to elevated temperatures (42 degreesC for 3 hours) resulted in a 75-fold increase in CyP40 mRNA levels, but no corresponding increase in CyP40 protein expression, even after 7 hours of heat stress. The use of cycloheximide to inhibit protein synthesis revealed that in comparison to MCF-7 cells cultured at 37 degreesC, those exposed to heat stress (42 degreesC for 3 hours) displayed an elevated rate of degradation of both CyP40 and FKBP52 proteins. Concomitantly, the half-life of the CyP40 protein was reduced from more than 24 hours to just over 8 hours following heat shock. As no alteration in CyP40 protein levels occurred in cells exposed to heat shock, an elevated rate of degradation would imply that CyP40 protein was synthesized at an increased rate. hence the designation of human CyP40 as a heat shock protein. Application of heat stress elicited a marked redistribution of CyP40 protein in MCF-7 cells from a predominantly nucleolar localization, with some nuclear and cytoplasmic staining, to a pattern characterized by a pronounced nuclear accumulation of CyP40, with no distinguishable nucleolar staining. This increase in nuclear CyP40 possibly resulted from a redistribution of cytoplasmic and nucleolar CyP40, as no net increase in CyP40 expression levels occurred in response to stress. Exposure of MCF-7 cells to actinomycin D for 4 hours resulted in the translocation of the nucleolar marker protein, B23, from the nucleolus, with only a small reduction in nucleolar CyP40 levels. Under normal growth conditions, MCF-7 cells exhibited an apparent colocalization of CyP40 and FKBP52 within the nucleolus.

Quantification of prolactin-releasing peptide (PrRP) mRNA expression in specific brain regions of the rat during the oestrous cycle and in lactation

Real-time Taqman(TM) RT-PCR was used to make quantitative comparisons of the levels of PrRP mRNA expression in micropunch brain samples from rats at different stages of the oestrous cycle and in lactation. The nucleus of the solitary tract and ventrolateral reticular nuclei of the medulla oblongata contained significantly (P < 0.05) greater levels of PrRP mRNA than any hypothalamic region. Within the hypothalamus, the highest level of PrRP expression was localised to the dorsomedial aspect of the ventromedial hypothalamus. All other hypothalamic regions exhibited significantly (P < 0.05) lower levels of expression, including the rostral and caudal dorsomedial hypothalamus. Very low levels of PrRP expression were observed in the arcuate nucleus, paraventricular nucleus, medial preoptic nucleus and ventrolateral aspect of the ventromedial hypothalamus. No significant changes in PrRP expression were noted in any sampled region between proestrus, oestrus or dioestrus. Similarly, PrRP expression in hypothalamic regions did not differ between lactating and non-lactating (dioestrous) animals. During validation of RT-PCR techniques we cloned and sequenced a novel splice variant of PrRP from the hypothalamus. This variant arises from alternative splicing of the donor site within exon 2, resulting in an insert of 64 base pairs and shift in the-codon:reading frame with the introduction of an early stop codon. In the hypothalamus and brainstem, mRNA expression of the variant was restricted to regions that expressed PrRP. These results suggest that PrRP expression in the hypothalamus may be more Widespread than previously reported. However, the relatively low level of PrRP in the hypothalamus and the lack of significant changes in expression during the oestrous cycle and lactation provides further evidence that PrRP is unlikely to be involved in the regulation of prolactin, secretion. (C) 2003 Elsevier Science B.V. All rights reserved.

Elevated plasma membrane and sarcoplasmic reticulum Ca2+ pump mRNA levels in cultured aortic smooth muscle cells from spontaneously hypertensive rats

We have observed previously that Ca2+ pump-mediated Ca2+ efflux is elevated in cultured aortic smooth muscle cells from spontaneously hypertensive rats compared to those from Wistar-Kyoto rat controls. The objective of this work was to determine if these strains differ in mRNA levels for the PMCA1 isoform of the plasma membrane Ca2+-ATPase and the SERCA2 isoform of the sarcoplasmic reticulum Ca2+-ATPase. mRNA levels were compared in cultured aortic smooth muscle cells from 10-week-old male rats. PMCA1 and SERCA2 mRNA levels were elevated in SHR compared to WKY. Angiotensin II increased the level of PMCA1 and SERCA2 mRNA in both strains. These studies provide further evidence for alterered Ca2+ homeostasis in hypertension at the level of Ca2+ transporting ATPases in the spontaneously hypertensive rat model. These data are also consistent with the hypothesis that the expression of these two Ca2+ pumps may be linked. (C) 1997 Academic Press

Expression of recombinant human follicle stimulating hormone mRNA in Chinese hamster ovary cells under different promoters

Levels of recombinant human follicle stimulating hormone (r-hFSH) mRNA expressed under butyrate and zinc treatment were compared in two CHO-K1 derived cell lines. In King cells under the metallothionein promoter, butyrate induced the increase in both r-hFSH productivity (q(FSH)) and mRNA levels proportionally. In the presence of 1 mM butyrate and 40 mu M zinc, a 4-fold increase in q(FSH) and mRNA levels was achieved as compared to zinc (40) alone; this wasa approximately 6 times higher than in serum free medium. In Darren cells under the beta-actin promotor butyrate induced an increase in q(SFH) but not in mRNA levels.

Glutamate(NMDA) receptor NR1 subunit mRNA expression in Alzheimer's disease

We analyzed the expression profile of two NMDAR1 mRNA isoform subsets. NR1(0xx) and NR1(1xx), in discrete regions of human cerebral cortex. The subsets are characterized by the absence or presence of a 21-amino acid N-terminal cassette. Reverse transcription polymerase chain reaction for NR1 isoforms was performed on total RNA preparations from spared and susceptible regions from 10 pathologically confirmed Alzheimer's disease (AD) cases and 10 matched controls. Primers spanning the splice insert yielded two bands, 342 bp (NR1(0xx)) and 405 bp (NR1(1xx)), on agarose gel electrophoresis. The bands were visualized with ethidium and quantified by densitometry. NR1(1xx) transcript expression was calculated as a proportion of the NR1(1xx) + NR1(0xx) total. Values were significantly lower in AD cases than in controls in mid-cingulate cortex, p < 0.01, superior temporal cortex, p < 0.01 and hippocampus, p similar to 0.05. Cortical proportionate NR1(1xx) transcript expression was invariant over the range of ages acid areas of controls tested, at similar to 50%. This was also true for AD motor and occipital cortex. Proportionate NR1(1xx) expression in AD cingulate and temporal cortex was lower at younger ages and increased with age: this regression was significantly different from that in the homotropic areas of controls. Variations in NR1 N-terminal cassette expression may underlie the local vulnerability to excitotoxic damage of some areas in the AD brain. Alternatively, changes in NR1 mRNA expression may arise as a consequence of the AD disease process.

Development of a real-time RT-PCR assay for plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA levels in a human breast epithelial cell line.

The plasma membrane Ca2+ pump is a key regulator of cytosolic free Ca2+. Recent studies have demonstrated the dynamic expression of the plasma membrane Ca2+ pump in a variety of cell types. Furthermore, alterations in plasma membrane calcium pump activity have now been implicated in human disease. In this study, the development of a technique to quantitatively assess mRNA expression of the human plasma membrane Ca2+ ATPase (PMCA1) isoform of the plasma membrane Ca2+ pump, using a real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay in a human breast epithelial cell line (MCF-7) is described. The sequences of the PMCA1 primers and probe for real-time RT-PCR are presented. The results also indicate that PMCA1 mRNA can be normalized to both 18S ribosomal RNA (18S rRNA) and human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) in MCF-7 cells. Real-time RT-PCR will be most useful in assessing PMCA1 mRNA expression in cases where only low amounts of RNA are available and/or when numerous samples must be assessed simultaneously. (C) 2001 Elsevier Science Inc. All rights reserved.