72 A comparison of 2 PCR-based methods for the quantitation of mRNA levels of putative membrane receptors in transfected mammallan cell clones

1998 ◽  
Vol 26 (4) ◽  
pp. S362-S362
Author(s):  
Paul R. Murdock ◽  
Nicole C. Herrity ◽  
Owen Jenkins
Keyword(s):  
Membrane Receptors ◽  
Mrna Levels ◽  
Cell Clones ◽  
Quantitation Of Mrna

Multiplex TaqMan® combined RT-PCR: a novel method for the quantitation of mRNA levels in receptor-transfected mammalian cell clones

1999 ◽  
Vol 27 (5) ◽  
pp. A150-A150
Author(s):  
Paul R Murdock ◽  
Kong B Tan ◽  
Nicole C Herrity ◽  
Gillian I Rennie ◽  
Owen Jenkins
Keyword(s):  
Mammalian Cell ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Cell Clones ◽  
Novel Method ◽  
Quantitation Of Mrna

P2863Regional specific remodeling of cGMP pathway in human atrial fibrillation

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
N I Bork ◽  
N G Pavlidou ◽  
B Reiter ◽  
H Reichenspurner ◽  
T Christ ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Sinus Rhythm ◽  
Ryanodine Receptors ◽  
Membrane Receptors ◽  
Mrna Levels ◽  
Atrial Myocytes ◽  
Human Atrial Myocytes ◽  
Cgmp Pathway ◽  
The Right

Abstract Background Atrial fibrillation (AF) is accompanied by a profound remodeling of membrane receptors and alterations in cyclic nucleotides-dependent regulation of Ca2+-handling. Thus, while basal ryanodine receptors activity is upregulated, L-type calcium current (ICa,L) density is diminish in AF, due to local microdomain-specific cAMP dynamics. The same seems true for cGMP regulation in AF. In AF cGMP-mediated increase in ICa,L is blunted but NO-mediated attenuation of β-adrenoceptors stimulation-mediated increase is preserved. However, although the role of cGMP in controling atrial function and pathophysiology is controversial, no study has been ever performed in human myocytes to measure cGMP directly. Methods We isolated myocytes from the right and/or left atrium of 27 patients in sinus rhythm (SR), and with AF. Cells were then transfected with adenovirus to express the cytosolic FRET-based cGMP sensor red-cGES-DE5 and cultured for 48 hours. Förster resonance energy transfer (FRET) was used to measure cGMP in 61 living human atrial myocytes. We stimulated cells with the C-type natriuretic peptide CNP (100 nM and 1 μM), and the non-selective phosphodiesterases (PDEs) inhibitor IBMX (100 μM). Additionally, PDE specific inhibitors for PDE2 (Bay 60–7550, 100 nM) and PDE3 (Cilostamide, 10 μM) as well as inhibitor of the soluble guanylyl cyclase (ODQ, 50 μM) were used. We also measured PDE2 and PDE3 mRNA levels in atrial tissue samples from both groups of patients using RT-qPCR. Results We could show that stimulation with CNP increased cGMP levels in human atrial myocytes. However, in myocytes from patients with AF global cGMP responses to CNP and to IBMX was reduced compared to SR. Additionally, there was a difference in response to CNP and IBMX in patients with AF between the right and the left atria. Whereas in the right atria IBMX could further increase cGMP levels in the cell, in the left atria leaded to a reduction in cGMP levels. RT-qPCR showed a tendency of PDE3 to be reduced in AF. On the other hand, PDE2A gene expression was upregulated in the left atria. Conclusions We have shown that PDEs contributes cGMP signaling in the human atria and that they are involved in atrial pathophysiology. Now our data clearly show differences in cGMP regulation in cardiomyocytes isolated from left and right atrium from patients in atrial fibrillation and sinus rhythm. We observe a major role of PDEs, regulating cGMP pathway promoted by the reduced responses in AF, especially PDE2 in the left atria.

BACH2 Is Required for Pre-B Cell Receptor Checkpoint Control and p53-Dependent Tumor Surveillance

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1300-1300
Author(s):  
Srividya Swaminathan ◽  
Huining Kang ◽  
Richard C. Harvey ◽  
Chuanxin Huang ◽  
Maike Buchner ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
Clinical Outcome ◽  
B Cell ◽  
Leukemia Cells ◽  
Mrna Levels ◽  
B Cell Differentiation ◽  
Childhood All ◽  
Cell Clones ◽  
Favorable Clinical Outcome

Abstract Abstract 1300 Background: BACH2 (BTB and CNC homology 1, basic leucine zipper transcription factor 2) is required for class-switch recombination and somatic hypermutation of immunoglobulin genes during affinity maturation of mature germinal center B cells. We and others found that BACH2 is strongly upregulated in BCR-ABL1-transformed acute lymphoblastic leukemia (Ph+ ALL) cells upon treatment with tyrosine kinase inhibitors (TKI). Results: Bach2 mRNA levels are significantly lower in Ph+ ALL (n=72) compared to normal human bone marrow pre-B cells (n=10). Studying gene expression in a clinical trial for children with high risk ALL (Children's Oncology Group, P9906; n=207), we found in a multivariate analysis that high Bach2 levels at the time of diagnosis represents an independent predictor of favorable clinical outcome (negative MRD at day and higher overall and relapse-free survival; p<0.0001). We next studied 49 sample pairs from patients with childhood ALL at diagnosis and relapse. In 44 of these sample pairs, the relapse sample showed drastically reduced mRNA levels of Bach2 (p=0.019), suggesting that loss of BACH2 expression is associated with relapse of childhood ALL. A comparison of the methylation status of BACH2 promoter of normal pre-B cells (n=5), with Ph+ ALL cells (n=70) revealed that CpG islands in the BACH2 promoter were heavily hypermethylated in the leukemia samples. These findings are also consistent with genomic analyses on patient derived samples and the identification of small deletions at 6q15 in 4 of 11 cases of childhood ALL cases that all span the BACH2 locus. To study the role of Bach2 in pre-B ALL in a genetic experiment, we transformed pre-B cells from Bach2−/− mice with BCR-ABL1. We observed that Bach2−/− normal pre-B cells lack the ability to counterselect pre-B cell clones that failed to undergo successful V(D)J rearrangement. In the absence of Bach2, a significant number of B cells survive even though they failed to rearrange immunoglobulin heavy chain genes. Besides this unexpected role in early B cell differentiation, quantitative RT-PCR and Western blot confirmed that BACH2 is also required for expression of the tumor suppressors Cdkn2a (Arf), p53 and Btg2. Consistent with extremely low protein levels of Arf and p53 in Bach2−/− leukemia cells, Bach2−/− ALL cells are more resistant to TKI-treatment, more actively proliferating (increased S-phase; p=0.02) and exhibit a ∼90-fold increased ability to form colonies in methyl cellulose (p=0.001). Studying Cre-mediated inducible deletion of p53 in p53-fl/fl leukemia cells, we found that Bach2-induced tumor suppression is largely dependent on p53 function. Forced overexpression of Myc results in oncogene-induced senescence (OIS) followed by apoptosis. Whereas Bach2+/+ leukemia cells are non-permissive to forced Myc expression and die within four days after Myc induction, Bach2−/− ALL cells tolerate forced expression of Myc and evade OIS and subsequent cell death. Similarly, overexpression of Myc alone fails to transform Bach2+/+ pre-B cells. By contrast, retroviral overexpression of Myc results in rapid transformation and growth factor-independence of Bach2−/− pre-B cells. Bach2−/− Myc-high pre-B cells cause fatal leukemia in 100% of recipient mice within 22 days, whereas all mice that received Bach2+/+ Myc-high pre-B cells survived without signs of disease until day 67, when all mice were sacrificed and analyzed for MRD by flow cytometry and PCR. No evidence of MRD was detected in most mice injected with Bach2+/+ Myc-high pre-B cells. Three mice had positive MRD PCR findings, however, at 4 log orders below findings in mice injected with Bach2−/−Myc-high pre-B cells. Conclusions: Our findings identify Bach2 as a novel tumor suppressor upstream of p53 in pre-B ALL. Bach2 is a regulator of negative selection during normal pre-B cell differentiation but also limits excessive proliferation of pre-B cell clones by induction of oncogene-induced senescence and activation of p53. In addition, our multivariate analyses identify high expression levels of Bach2 as powerful predictor of favorable clinical outcome in children, which may be useful in future approaches for risk stratification. Disclosures: No relevant conflicts of interest to declare.

Comparison of Expression of Human Globin Genes Transferred Into Mouse Erythroleukemia Cells and in Transgenic Mice

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3416-3421 ◽  
Author(s):  
E. Skarpidi ◽  
G. Vassilopoulos ◽  
G. Stamatoyannopoulos ◽  
Q. Li
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Globin Gene ◽  
Mrna Levels ◽  
Globin Genes ◽  
Position Effects ◽  
Erythroleukemia Cells ◽  
Cell Clones ◽  
Globin Gene Expression ◽  
Mouse Erythroleukemia

To examine whether transfer of γ globin genes into mouse erythroleukemia cells can be used for the analysis of regulatory elements of γ globin gene promoter, Aγ gene constructs carrying promoter truncations that have been previously analyzed in transgenic mice were used for production of stably transfected mouse erythroleukemia (MEL) cell clones and pools. We found that constructs, which contain a microlocus control region (μLCR) that efficiently protects globin gene expression from the effects of the position of integration in transgenic mice, display position-dependent globin gene expression in MEL cell clones. Aγ globin gene expression among MEL cell clones carrying the μLCR(−201)Aγ and μLCR(−382)Aγ gene constructs ranged 15.5-fold and 17.6-fold, respectively, and there was no correlation between theAγ mRNA levels and the copies of the transgene (r= .28, P = .18). There was significant variation in per copy Aγ globin gene expression among MEL cell pools composed of 10 clones, but not among pools composed of 50 clones, indicating that position effects are averaged in pools composed by large numbers of clones. The overall pattern of Aγ globin gene expression in MEL cell pools resembled that observed in transgenic mice indicating that MEL cell transfections can be used in the study ofcis elements controlling γ globin gene expression. MEL cell transfections, however, are not appropriate for investigation of cis elements, which either sensitize or protect the globin transgenes from position effects. © 1998 by The American Society of Hematology.

Comparison of Expression of Human Globin Genes Transferred Into Mouse Erythroleukemia Cells and in Transgenic Mice

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3416-3421 ◽  
Author(s):  
E. Skarpidi ◽  
G. Vassilopoulos ◽  
G. Stamatoyannopoulos ◽  
Q. Li
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Globin Gene ◽  
Mrna Levels ◽  
Globin Genes ◽  
Position Effects ◽  
Erythroleukemia Cells ◽  
Cell Clones ◽  
Globin Gene Expression ◽  
Mouse Erythroleukemia

Abstract To examine whether transfer of γ globin genes into mouse erythroleukemia cells can be used for the analysis of regulatory elements of γ globin gene promoter, Aγ gene constructs carrying promoter truncations that have been previously analyzed in transgenic mice were used for production of stably transfected mouse erythroleukemia (MEL) cell clones and pools. We found that constructs, which contain a microlocus control region (μLCR) that efficiently protects globin gene expression from the effects of the position of integration in transgenic mice, display position-dependent globin gene expression in MEL cell clones. Aγ globin gene expression among MEL cell clones carrying the μLCR(−201)Aγ and μLCR(−382)Aγ gene constructs ranged 15.5-fold and 17.6-fold, respectively, and there was no correlation between theAγ mRNA levels and the copies of the transgene (r= .28, P = .18). There was significant variation in per copy Aγ globin gene expression among MEL cell pools composed of 10 clones, but not among pools composed of 50 clones, indicating that position effects are averaged in pools composed by large numbers of clones. The overall pattern of Aγ globin gene expression in MEL cell pools resembled that observed in transgenic mice indicating that MEL cell transfections can be used in the study ofcis elements controlling γ globin gene expression. MEL cell transfections, however, are not appropriate for investigation of cis elements, which either sensitize or protect the globin transgenes from position effects. © 1998 by The American Society of Hematology.

Progesterone-induced sphingosine kinase-1 expression in the rat uterus during pregnancy and signaling consequences

2007 ◽  
Vol 292 (4) ◽  
pp. E1110-E1121 ◽  
Author(s):  
Yow-Jiun Jeng ◽  
Victor R. Suarez ◽  
Michael G. Izban ◽  
Hui-Qun Wang ◽  
Melvyn S. Soloff
Keyword(s):  
Cyclin D1 ◽  
Ectopic Expression ◽  
Sphingosine Kinase ◽  
Membrane Receptors ◽  
Target Cells ◽  
Mrna Levels ◽  
Sphingosine Kinase 1 ◽  
Rat Uterus ◽  
Serum Progesterone ◽  
Pregnant Rat

Sphingosine 1-phosphate (Sph-1- P), a product of sphingomyelin metabolism, can act via a family of cognate G protein-coupled receptors or as an intracellular second messenger for agonists acting through their membrane receptors. In view of the general growth promoting and developmental effects of Sph-1- P on target cells, we hypothesized that it plays a role in adaptation of the uterus to pregnancy. We analyzed its potential role and that of the related lysophospholipid lysophosphatidic acid in the pregnant rat uterus by examining changes in mRNA levels of cognate receptors and enzymes involved in their turnover. Of these, only sphingosine kinase-1 (SphK1) was markedly changed (∼30-fold increase), being localized in the glandular epithelium, vasculature, and the myometrium. Uterine SphK1 mRNA and protein levels paralleled those of serum progesterone, and treatment with progesterone or an antagonist elevated or reduced SphK1 mRNA expression, respectively. Progesterone also increased SphK1 mRNA steady-state levels in a rat myometrial/leiomyoma cell line (ELT3). Overexpressing human SphK1 in these cells resulted in increased levels of the cell cycle regulator cyclin D1 and increased myosin light-chain phosphorylation. Ectopic expression of SphK1 also resulted in increased proliferation rates, possibly in conjunction with increased cyclin D1 expression. These studies suggest that the uterine expression of SphK1 mediates processes involved in growth and differentiation of uterine tissues during pregnancy.

Studies of hprt mutation, hprt mRNA levels and karyotypes in human T-cell clones

1989 ◽  
Vol 216 (1) ◽  
pp. 71-72
Author(s):  
B. Andersson ◽  
S.-H. He ◽  
D. Hellgren ◽  
K. Holmberg ◽  
S. Marcus ◽  
...  
Keyword(s):  
T Cell ◽  
Mrna Levels ◽  
T Cell Clones ◽  
Human T Cell ◽  
Cell Clones
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