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PubMed1994

Leukaemia inhibitory factor mRNA concentration peaks in human endometrium at the time of implantation and the blastocyst contains mRNA for the receptor at this time.

Maternal endometrial leukaemia inhibitory factor (LIF) is required for successful implantation in mice. Mice with homozygous deletions in this gene fail to support implantation. The localization of immunoreactive LIF and the concentration of the mRNA encoding human LIF in normal endometrium during the menstrual cycle were investigated. The amount of RNA was low or undetectable in the proliferative phase but increased by approximately six times in the mid- to late secretory phase. The protein can only be detected by immunocytochemistry in glandular epithelium in the mid- or late secretory phase. To investigate the possible target for the endometrial LIF, we undertook reverse transcription-PCR analysis of early human embryos to determine whether they contain the mRNA encoding the LIF receptor. This study indicated that at the time of implantation in humans, the maternal endometrium produces LIF and that the blastocyst expresses LIF receptor mRNA and therefore may be capable of responding to this signal.

Base Sequence↗
PubMed2004

Chorionic gonadotrophin beta subunit mRNA but not luteinising hormone beta subunit mRNA is expressed in the pituitary of the common marmoset (Callithrix jacchus).

The pituitary gonadotrophins LH and FSH are responsible for regulation of gametogenesis in the testis and ovary. Chorionic gonadotrophin (CG), a third closely related glycoprotein hormone derived by gene duplication of the LHbeta gene and secreted by the placenta in primates, is essential for the rescue of the corpus luteum and maintenance of pregnancy. We have recently shown that marmoset (m) CGbeta mRNA is highly expressed in the pituitary of the common marmoset (Callithrix jacchus) and that LH is less active than human CG in activating the human LH receptor lacking exon 10. To investigate further which gonadotrophin is the actual ligand of the LH receptor (LHR) of the marmoset monkey that naturally lacks exon 10, we identified and characterised the genomic organisation of the mLHbeta gene and its expression. Intergenic PCR amplification of the region encompassing the mLHbeta and the mCGbeta genes revealed that, surprisingly, mCGbeta is located 20 kbp upstream of the LHbeta gene, whereas in other species the intergenic distance is approximately 2-3 kbp. Sequence analysis of the mLHbeta coding region showed 70% identity to mCGbeta and 90% identity to human LHbeta at the amino acid level. Both gonadotrophin beta subunits are present at the genomic level, but RT-PCR of pituitary and placental total RNA using specific oligonucleotides for mCGbeta and mLHbeta showed high expression of mCGbeta mRNA in both tissues, whereas LHbeta was expressed neither in the pituitary nor in the placenta. Thus mLHbeta mRNA is lacking in the marmoset pituitary. Immunohistochemistry of marmoset pituitaries showed that mCG was confined to the gonadotrophes, and partly co-localised in cells stained positively for FSH. Western blot analysis confirmed the presence of mCG in the pituitary. Northern blot analysis using mCGbeta as a probe displayed one transcript of 0.7 kb in the pituitary and detected two transcripts of 1.1 kb and 2 kb in the marmoset placenta. Our results suggest that, in the common marmoset, CG is the only gonadotrophin with luteinising function that is present in the pituitary. We postulate that, owing to an unknown mutational event in evolution, expression of mLH was completely abolished, and CG - which, unlike LH, acts normally even when exon 10 is missing from the LHR - took over its function.

Amino Acid Sequence↗
PubMed1998

Alternative splicing of follicle-stimulating hormone receptor pre-mRNA: cloning and characterization of two alternatively spliced mRNA transcripts.

Glycoprotein hormone receptors contain a large extracellular domain that is encoded by multiple exons, facilitating the possibility of expressing alternatively spliced transcripts. We have cloned two new splice variants of the rat follicle-stimulating hormone (FSH) receptor gene: FSH-R1 and FSH-R2. The splice variant FSH-R1 differs from the full-length FSH receptor mRNA by the inclusion of a small extra exon between exons 9 and 10. FSH-R2 lacks the first three base pairs of exon 4, contains an extra exon between exons 4 and 5, and has an extended 3'-untranslated region. According to the predicted open reading frames, both mRNAs encode truncated FSH receptor proteins, consisting of the entire extracellular domain (FSH-R1) or the amino-terminal half of the extracellular domain (FSH-R2), and are expressed at a low level in testes and ovaries. The levels of expression of the FSH-R1 and FSH-R2 mRNAs in the gonads show a constant ratio to the expression level of the full-length FSH receptor mRNA. Furthermore, in vitro co-expression of either one of the truncated proteins with the full-length FSH receptor in COS1 cells did not affect signal transduction through the full-length FSH receptor. The absence of a function of the truncated FSH receptors in FSH signal transduction in vitro, and the lack of differential regulation of the alternative transcripts, indicate that there is no clear function for alternative splicing of the FSH receptor pre-mRNA in the postnatal testis and the cycling adult ovary.

Alternative Splicing↗
PubMed1997

Transforming growth factor-alpha mRNA and epidermal growth factor receptor mRNA expression in normal and neoplastic mammary glands of four strains of mice with different mammary tumor potentials.

Transforming growth factor-alpha (TGF alpha) and epidermal growth factor receptor (EGF-R) mRNAs were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) in the normal and neoplastic mammary glands of four strains of mice with different mammary tumor potentials (from highest to lowest potential): SHN, GR/A, SLN and C3H/He. At 2 months of age, when the mammary glands of these strains consisted mostly of normal tissue, the samples examined showed the positive expressions of both TGF alpha and EGF-R mRNAs in all strains (4-6 mice per group), except for EGF-R mRNA in the SLN mice, expressed in only 2 of 4 samples associated with no end-bud formation in the mammary glands. At 10 months, all of the samples from all four strains had a positive expression of TGF alpha mRNA. The EGF-R mRNA expression paralleled the degree of the formation of preneoplastic hyperplastic alveolar nodules (HAN) in all strains. These findings indicate that TGF alpha and EGF-R participate in the growth of the mammary glands, and that EGF-R especially contributes to the formation of end-buds at younger ages and to that of preneoplastic HAN at later ages. All of the samples of mammary tumors from four strains had positive expressions of both TGF alpha and EGF-R mRNAs.

Animals↗
PubMed2007

Direct modification of mRNA by ferrocenyl carbodiimide and its application to electrochemical detection of mRNA.

Ferrocenyl carbodiimide (1) could be used for the direct labeling of synthetic RNA and expressed mRNA in vitro with the electrochemically active ferrocene moieties. These RNAs modified by 1 could be detected electrochemically coupled with a DNA probe-immobilized electrode. After hybridization of 1.1 Kb mRNA modified by 1 with the DNA probe-immobilized electrode, the peak charge observed by an Osteryoung square wave voltammetry (SWV) measurement correlated well with the concentration of mRNA, having a detection limit at the sub nanogram level.

Carbodiimides↗
PubMed2000

Xplore mRNA assays for the quantification of IL-1 beta and TNF-alpha mRNA in lipopolysaccharide-induced mouse macrophages.

Because the accurate measurement of a number of cytokine mRNA transcripts provides valuable knowledge about cytokine gene regulation, we have developed the Xplore assay for the quantification of cytokine mRNA. This microplate-based assay is rapid (under four hours), quantitative over three orders of magnitude and carries no risk of false-positive values from contamination with amplified target. Here, we describe the use of Xplore assays to measure the steady-state mRNA levels of TNF-alpha and IL-1 beta produced by mouse WEHI and J774 macrophage-like cell lines.

Animals↗
PubMed2003

c-jun mRNA expression and profilin mRNA amplification in rat alveolar macrophages exposed to volcanic ash and sulfur dioxide.

Local residents exposed to heavy falls of ash discharged by Mt. Sakurajima, an active volcano, have been reported to develop acute and chronic inflammation of the respiratory tract. The present study aimed to determine the primary cause of this inflammation using an experimental model. Wistar rats were exposed for 5 days (4 h/d) to air containing 100 mg/m3 volcanic ash (mass median aerodynamic diameter, 4.3 microm; geometric standard deviation, 1.7) with or without 1.5 ppm sulfur dioxide (SO2). The lungs were then lavaged, and mRNA was extracted from alveolar macrophages and assessed by reverse transcription-polymerase chain reaction (RT-PCR). In the lavage fluid, no change in cellularity or increase in the content of tumor necrosis factor (TNF)-alpha was detected. However, at 1 h following exposure, 80% of macrophages were seen to have phagocytosed the volcanic ash. This percentage was unchanged at 24 h after exposure. Profilin mRNA content of the macrophages was elevated, and c-jun mRNA was expressed. Alveolar macrophages exposed to volcanic ash and SO2, therefore, are likely to have some inflammatory and fibrogenic potential.

Animals↗
PubMed1998

Expression of ornithine decarboxylase mRNA and c-myc mRNA in breast tumours.

We examined the expression of ornithine decarboxylase (ODC) mRNA in 53 female cases of breast cancer by a reverse transcriptase-polymerase chain reaction (RT-PCR) assay to determine the clinicopathologic significance of its expression. A significantly higher expression of ODC mRNA was, observed in younger patients than in older patients. The patients with a larger sized tumour possessed a significantly higher expression of ODC mRNA. In addition, the cases with a poor prognosis showed significantly higher expression of ODC. Previous studies have reported in vivo and in vitro correlation between the expression of ODC and c-myc genes in human carcinomas. We disclosed a significant correlation between these genes in primary breast carcinomas. We conclude that the expression of ODC may potentially be a new biological marker for breast carcinoma.

Adolescent↗
PubMed1997

[Study on platelet-derived growth factor mRNA and copper-zinc superoxide dismutase mRNA expression changes of aortic artery endothelium with restenosis after aortic artery dilatation treated with buyang huanwu decoction].

OBJECTIVE: To study on dynamic mechanism of restenosis after PTCA treated with Buyang Huanwu Decoction (BYHWD). METHODS: Twenty-eight male New Zealand rabbits with mimic restenosis after aortic artery dilatation were detected by prime in situ labeling in aortic endothelium. The items included gene expression of copper zinc superoxide dismutase (SOD-1) and platelet-derived growth factor receptor (PDGFR) mRNA to analyse dynamic mechanism of restenosis. RESULTS: Seven days after aortic artery dilatation, PDGFR mRNA of aortic endothelium decreased clearly. The comparison between experimental and the control group showed significant difference (P < 0.05). By the same condition, the gene expression of SOD-1 was increasing, which showed a significant difference with the control group (P < 0.05). CONCLUSIONS: BYHWD increased SOD-1 gene expression of artery endothelium, to improve oxygen feed for ischemia reperfusion in the same way, BYHWD suppressed PDGFR mRNA expression to control cellular pathological proliferation.

Angioplasty, Balloon, Coronary↗
PubMed2000

Cell-cell contact of human T cells with fibroblasts changes lymphocytic mRNA expression: increased mRNA expression of interleukin-17 and interleukin-17 receptor.

Using random arbitrarily primed-reverse transcribed-PCR and sequence analysis, we investigated changes in lymphocytic molecules after cell-cell contact with fibroblasts. An mRNA species which was upregulated in Jurkat T cells by cell-cell contact with MRHF cells (a human foreskin fibroblast line) was identified as coding for the human interleukin-17 receptor. This finding was confirmed by quantitative RT-PCR for the HUT78 and Jurkat T cell lines, for peripheral blood lymphocytes, and for tonsillar T cells. Furthermore, the interleukin-17 mRNA, coding for a proinflammatory cytokine, was also upregulated in peripheral blood lymphocytes and tonsillar T cells after cell-cell contact with fibroblasts. Supernatants obtained from cell-cell contact-stimulated peripheral blood lymphocytes enhance the production of interleukin-6 and interleukin-8 by fibroblast-like synoviocytes and this effect could be blocked by interleukin-17 antibodies. Changes in the mRNA levels of Jurkat T cells induced by cell-cell contact with adherent cells were also found for M-type pyruvate kinase, for tropomyosin TM30 and for the p54nrb gene product.

Cell Communication↗
PubMedDate not supplied

Telomerase activity is down regulated via decreases in hTERT mRNA but not TEP1 mRNA or hTERC during the differentiation of leukemic cells.

BACKGROUND: Recent studies have determined several telomerase-associated molecules, but the precise mechanisms regulating telomerase activity by those molecules has not been fully understood. MATERIALS AND METHODS: The telomerase activity was determined by TRAP assay. Using TaqMan RT-PCR, the quantitative and kinetic values of mRNA expression of the three telomerase-associated molecules were examined in HL60 cells differentiated with tumor necrosis factor mutant and all-transretinoic acid. RESULTS: The levels of telomerase activity in leukemic cell lines, leukemic cells from patients, and normal peripheral blood cells were distributed over a very wide range. Human telomerase catalytic subunit (hTERT) mRNA expression declined to nearly undetectable levels more rapidly than the inhibition of telomerase activity after treatment with these reagents in HL60 cells. Telomerase-associated protein (TEP1) mRNA increased approximately 6-fold over its level in untreated cells. The levels of human telomerase RNA component (hTERC) also increased approximately 2.7-fold at 5 days after treatment. CONCLUSIONS: These results suggest that telomerase activity is down-regulated mainly via decreases in hTERT, but not TEP1 and hTERC expression during the differentiation of leukemic cells.

Adolescent↗
PubMed2000

[Effects of shuanglong pill on mRNA expression of platelet-derived growth factor A and monocyte chemoattractant protein-1 mRNA].

OBJECTIVE: To explore the molecular mechanism of Shuanglong Pill (SLP) in intervening atherosclerosis, mainly the effect of SLP on the mRNA expression of the platelet-derived growth factor-A (PDGF-A) and the monocyte chemoattractant protein-1 (MCP-1). METHODS: Using the hyperlipoprotein serum (HLS) of cultured rabbits' aortic smooth muscle cell (SMC) to build the atherosclerotic cell model, and medicated serum acting on the cell model by serum pharmacologic method. The effect of SLP medicated serum on the level of PDGF-A, MCP-1 mRNA expression were observed and analysed by means of reverse transcriptase-polymerase chain reaction assay. RESULTS: It was found that HLS stimulated mRNA expression of the PDGF-A and the MCP-1, but the medicated serum of SLP lowered their expression level. CONCLUSION: SLP could inhibit the function of cellular auto-secreting growth factor, it is one of the possible pharmacologic mechanism in intervening atherosclerosis.

Animals↗
PubMed2000

[The significance of the expressions of lipopolysaccharide binding protein mRNA and lipopolysaccharide receptor CD14 mRNA in the liver of burned rat].

OBJECTIVE: To explore the significance and the correlation between enteric endotoxin translocation and hepatic endotoxin -- sensitivity -- enhancing system--lipopolysaccharide -- binding protein (LBP)/lipopolysaccharide receptor CD14 (LBP/CD14) after burn. METHODS: Wistar rats subjected to 35% III degree burn were employed as the model. The rats were randomly divided into three groups: i.e. normal control (C, n = 8) group, thermal injury (T, n = 10) group and recombinant bactericidal/permeability -- increasing protein (rBPI(21)) treatment (R, n = 6) group. The rats in T and R groups were sacrificed at 12 postburn hour (PBH) and the liver tissue was collected for the detection of the mRNA expressions of LBP, CD14 and tumor necrosis factor (TNFalpha) and blood samples collected for hepatic functional indices. RESULTS: The hepatic endotoxin content increased significantly postburn (P < 0.01), and the expressions of LBP/CD14 and TNFalpha mRNA in liver tissue increased obviously. However the use of rBPI(21) could evidently lower hepatic endotoxin content and inhibit the expression of tissue LBP/CD14 and TNFalpha. In addition, rBPI(21) could also significantly decrease serum level of glutamic -- pyruvic transaminase (ALT) (P < 0.01). CONCLUSION: Aggregation of endotoxin in liver due to postburn translocation might obviously stimulate the expression of LBP/CD14 mRNA locally. The up -- regulation of the expression of LBP/CD14 might be the principle molecular basis enhandced activity of translocated endotoxin on inflammatory cells.

Acute-Phase Proteins↗
PubMedDate not supplied

[The structure of nuclear pre-mRNA. VII. The hybridization and renaturation properties of double-stranded sequences of nuclear pre-mRNA].

Some properties of the double-stranded regions of pre-mRNA are discribed. 1. The double-stranded regions contain approximately 80 base pairs. 2. The material contains the heterogeneous populations of sequences and some homogenous material which renatures with a COT1/2 value of (1.5-3) X 10(-4). 3. Identical sequences of fast-renaturing "hairpins" may be found in various tissues. 4. Double-stranded RNA and mRNA have some sequences complementary to each other. These results consistent with the view that the hairpin sequences may act as specific recognition sites for ribonucleases involved in processing of pre-mRNA.

Base Sequence↗
PubMed2004

Upregulation of mRNA in MAPK signaling: transcriptional activation or mRNA stabilization?

Mitogen-activated protein kinases (MAPKs), found in all eukaryotes, are signal-transducing enzymes that play a central role in a variety of biological processes. MAPK phosphatase has dual catalytic activity toward phosphotyrosine- and phosphothreonine-containing proteins, and is known to inactivate ERKs and JNKs/SAPKs, thus playing a crucial role in MAPK regulation. Although MAPK phosphatase has been implicated in a feedback loop that inactivates MAPKs after stimulation by mitogens and during the cellular response to stress, signaling pathways leading to MAPK phosphatase gene expression have not been fully elucidated. Recently, we have shown that a novel RNA-binding protein Rnc1 plays a crucial role in negative feedback regulation of MAPK signaling by stabilizing the mRNA of a MAPK phosphatase at the post-transcriptional level. One important aspect of our findings is that the increase in mRNA levels involves not only the transcriptional upregulation, but also the post-transcriptional gene regulation, especially the regulation of mRNA stability. Our discovery highlights a potential role and an emerging view of RNA-binding protein as a regulator of cell signaling and as a future target of drug discovery.

Animals↗
PubMed1991

Preexposure of macrophages to low doses of lipopolysaccharide inhibits the expression of tumor necrosis factor-alpha mRNA but not of IL-1 beta mRNA.

LPS is known to be a potent activator of macrophages and induces the production of TNF-alpha and IL-1. However, the signaling events and regulatory mechanisms required for the activation of macrophages by LPS have not been resolved precisely. We show that LPS modulates its own response in macrophages. Proteose peptone-induced murine peritoneal macrophages (P-PEM) produce significant amount of TNF-alpha and IL-1 after stimulation with LPS. However, preexposure of macrophages to low doses (less than 1 ng/ml) of LPS renders them refractory to stimulation by a second round of LPS, as evaluated by production of TNF-alpha. The loss of sensitivity to a second round of LPS was selective for TNF-alpha production as the LPS-primed macrophages retained the ability to produce IL-1. Northern blot analysis was performed with total RNA obtained from control and LPS- (1 ng/ml) primed P-PEM after 3-h stimulation with a second round of LPS. The expression of TNF-alpha mRNA was inhibited in LPS-primed P-PEM, whereas the expression of IL-1 beta mRNA was the same in control and LPS-primed P-PEM, consistent with the data of biologic activities of these two cytokines. Zymosan-induced TNF-alpha production was the same in control and LPS-primed macrophages, indicating that not all of the pathways required for TNF-alpha production were affected by LPS priming. Monokines such as human (h) rIL-1 alpha, hrTNF-alpha, hrIL-6, and murine rIFN-beta could not substitute for the action of low doses of LPS, and addition of indomethacin could not restore TNF-alpha production. These results suggest that exposure of macrophages to low doses of LPS suppresses the production of TNF-alpha, but not of IL-1, by inhibiting the expression of mRNA through a noncyclooxygenase-dependent mechanism. Thus, LPS-induced production of TNF-alpha and IL-1 in macrophages are differently regulated.

Animals↗
PubMed1989

Construction of mRNA genes for the synthesis and translation of duck alpha globin mRNA.

In studies of the effects of changes in mRNA structure and sequence on the initiation of protein synthesis, we used a generally applicable approach to transcribe reconstructed genes for duck alpha A globin by the bacteriophage SP6 RNA polymerase promoter in a pGEM-2 plasmid vector. The genes were reconstructed such that the first nucleotide to be transcribed, the 5' adenosine, was placed directly adjacent to the SP6 promoter sequence. The 3' ends of the genes were constructed such that cleavage with Ssp 1 endonuclease yielded a template that directed the synthesis of mRNA terminating in a poly A tail containing 56 adenosines and a single 3' uridine. Special conditions using a Mn++ buffer were developed to enable the SP6 RNA polymerase to initiate at the 5' adenosine and synthesize the A-start transcription product. The mRNA could be capped and was subsequently used as an effective template for in vitro translation and synthesis of duck alpha A globin.

Animals↗
PubMed1987

Identification of a new type of mammalian myosin heavy chain by molecular cloning. Overlap of its mRNA with preprotachykinin B mRNA.

In this paper, we report the polypeptide sequence, the mRNA sequence, and gene organization of a new category of mammalian myosin heavy chain, termed myosin I heavy chain-like protein (MIHC), on the basis of sequence analyses of cDNA clones isolated from a bovine intestinal cDNA library and a genomic DNA clone containing the MIHC gene. The MIHC mRNA shares its 3' sequence with the 5' sequence of the preprotachykinin B mRNA encoding the precursor for the neuromedin K neuropeptide. An overlap of these functionally unrelated mRNAs results from overlapping transcription of the same strand of the genomic DNA sequence. The deduced amino acid sequence comprising the amino-terminal two-thirds of the 119-kDa MIHC polypeptide is highly homologous to that portion of conventional myosin heavy chains which forms the globular head domain. The homology is particularly strong in the regions of sequence thought to be involved in forming the nucleotide- and actin-binding sites. By contrast, the deduced sequence comprising the carboxyl-terminal one-third of MIHC shows no significant homology to that portion of conventional myosin heavy chains which forms the alpha-helical coiled-coil rod-like tail. In this regard, MIHC more closely resembles the monomolecular, non-filamentous, low molecular weight myosin I heavy chain from Acanthamoeba castellanii. Furthermore, the size of the MIHC polypeptide, together with its preferential expression in intestinal tissue, leads us to speculate that MIHC and the 110-kDa intestinal brush-border protein (Collins, J. H., and Borysenko, C. W. (1984) J. Biol. Chem. 259, 14128-14135), which binds actin and has myosin-like ATPase activity, are the same or closely related proteins.

Acanthamoeba↗