Northern blot analysis of human placenta and cultured human trophoblast cells for cystic fibrosis transmembrane regulator (CFTR) messenger RNA

1994 ◽  
Vol 39 (2) ◽  
pp. 147-148
Keyword(s):  
Cystic Fibrosis ◽  
Human Placenta ◽  
Northern Blot ◽  
Blot Analysis ◽  
Messenger Rna ◽  
Northern Blot Analysis ◽  
Cystic Fibrosis Transmembrane Regulator ◽  
Human Trophoblast ◽  
Cystic Fibrosis Transmembrane ◽  
Human Trophoblast Cells

scholarly journals Induction of hepatic metallothioneins determined at isoprotein and messenger RNA levels in glucocorticoid-treated rats

1988 ◽  
Vol 249 (2) ◽  
pp. 429-433 ◽  
Author(s):  
L D Lehman-McKeeman ◽  
G K Andrews ◽  
C D Klaassen
Keyword(s):  
Detection Limit ◽  
Northern Blot ◽  
Blot Analysis ◽  
Messenger Rna ◽  
Time Course ◽  
Northern Blot Analysis ◽  
Blot Hybridization ◽  
Northern Blot Hybridization ◽  
Single Dosage ◽  
Rna Levels

Induction of metallothionein-I (MT-I) and metallothionein-II (MT-II) by glucocorticoids was determined by h.p.l.c. analysis of proteins and Northern-blot analysis of MT mRNAs. Rats were injected with dexamethasone (0.03-10 mumol/kg) and hepatic concentrations of MTs were determined 24 h later. In control rats, only MT-II was detected (9.4 +/- 2.5 micrograms/g of liver), whereas the hepatic concentration of MT-I was below the detection limit (5 micrograms of MT/g). Dexamethasone did not increase MT-I above the detection limit at any dosage tested, but MT-II increased to 2.5 times control values at dosages of 0.30 mumol/kg and higher. Time-course experiments indicated that MT-II reached a maximum at 24 h after a single dosage of dexamethasone and returned to control values by 48 h. To determine whether dexamethasone increased MT-I in liver, samples were saturated with 109Cd, after which the amount of 109Cd in MT-I and MT-II was determined. Results indicated that, by this approach, MT-I and MT-II could be detected in control rats, and there was approx. 1.8 times more 109Cd in MT-II than in MT-I. At 24 h after administration of dexamethasone (1 mumol/kg), there was a small increase in the amount of 109Cd bound to MT-I, whereas the amount of 109Cd bound to MT-II increased to more than 2 times control values. Northern-blot hybridization with mouse cRNA probes indicated that MT-I and MT-II mRNAs increased co-ordinately after administration of dexamethasone. Thus, although glucocorticoids increase both MT-I and MT-II mRNAs, MT-II preferentially accumulates after administration of dexamethasone.

scholarly journals Physiologic regulation and tissue localization of renal erythropoietin messenger RNA

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 316-318 ◽  
Author(s):  
SJ Schuster ◽  
JH Wilson ◽  
AJ Erslev ◽  
J Caro
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Messenger Rna ◽  
Northern Blot Analysis ◽  
De Novo ◽  
Rat Kidney ◽  
Acute Hypoxia ◽  
Mrna Levels ◽  
Mrna Content ◽  
Lag Times

Abstract Although erythropoietin (Epo) is produced primarily by the kidneys in response to hypoxia, the precise cell type(s) and mechanisms by which these cells regulate production are poorly understood. In the experiments we report, the kinetics of renal Epo production in response to acute hypoxia and the intrarenal localization of cellular Epo synthesis were studied at the level of Epo mRNA. Erythropoietin mRNA expression was determined by Northern blot analysis of rat kidney RNAs using a probe derived from the mouse Epo gene. Renal Epo mRNA content increased as early as 1 hour after initiation of hypoxia and continued to accumulate during 4 hours of stimulation. Discontinuation of the hypoxic stimulus resulted in rapid decay of mRNA levels. Kidney and plasma Epo levels measured by radioimmunoassay paralleled, with respective lag times, the changes in renal Epo mRNA content, suggesting that Epo production in response to acute hypoxia represents de novo synthesis and is regulated by changes in Epo mRNA. Northern blot analysis of RNAs extracted from separated glomerular and tubular tissue fractions revealed Epo mRNA in the tubular fraction, whereas glomerular tissue did not contain Epo mRNA. Thus, the site of cellular Epo synthesis is located in the renal tubule or its interstitium and not in the glomerular tuft.

scholarly journals Expression and Regulation of P2U-Purinergic Receptor in Human Granulosa-Luteal Cells1

2000 ◽  
Vol 85 (4) ◽  
pp. 1591-1597 ◽  
Author(s):  
Chen-Jei Tai ◽  
Sung Keun Kang ◽  
Kwai Wa Cheng ◽  
Kyung-Chul Choi ◽  
Parimal S. Nathwani ◽  
...  
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Messenger Rna ◽  
Northern Blot Analysis ◽  
Purinergic Receptor ◽  
Cytosolic Calcium ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Complementary Dna ◽  
Pcr Product

The P2U purinoceptor (P2UR) has been identified pharmacologically in the ovary. However, the expression and regulation of the P2UR messenger RNA (mRNA) in human ovarian cells are still poorly characterized. The present study was designed to examine the expression and regulation of the P2UR in human granulosa-luteal cells (hGLCs) by RT-PCR and Northern blot analysis. A PCR product corresponding to the expected 599-bp P2UR complementary DNA was obtained from hGLCs. Molecular cloning and sequencing of the PCR product revealed an identical sequence to the reported P2UR complementary DNA. Two mRNA transcripts of 2.0 kb and 4.6 kb were identified in hGLCs using Northern blot analysis. The expression of the P2UR mRNA was down-regulated by human CG in a dose- and time-dependent manner. Treatment with 8-bromo-cAMP and forskolin also attenuated P2UR mRNA levels. Calcium signaling following the activation of the P2UR in single hGLCs was studied using microspectrofluorimetry. It revealed that, like ATP, uridine triphosphate (UTP) also induced cytosolic calcium mobilization in a dose-dependent manner. These results demonstrate for the first time that the P2UR mRNA is expressed in hGLCs and that P2UR mRNA is regulated by human CG, cAMP, and forskolin. The P2UR expressed in hGLCs functional because activation of the P2UR by ATP or UTP resulted in rapid and transient mobilization of cytosolic calcium at the single cell level. These findings further support a potential role of this neurotransmitter receptor in the human ovary.

scholarly journals Effects of colony stimulating factor-1 on human extravillous trophoblast growth and invasion

1998 ◽  
Vol 159 (1) ◽  
pp. 69-77 ◽  
Author(s):  
GS Hamilton ◽  
JJ Lysiak ◽  
AJ Watson ◽  
PK Lala
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Messenger Rna ◽  
Northern Blot Analysis ◽  
Tritiated Thymidine ◽  
Extravillous Trophoblast ◽  
Colony Stimulating Factor ◽  
Matrigel Invasion Assay ◽  
Matrigel Invasion ◽  
Stimulating Factor

Colony stimulating factor (CSF)-1 has been localized in a variety of tissues and shown to influence proliferation and differentiation of numerous cell types. Messenger RNA and protein products of CSF-1 and its receptor (c-fms) have been identified in the human placenta and decidua. We examined whether CSF-1 and c-fms mRNA and protein are expressed by normal human first trimester invasive extravillous trophoblast (EVT) cells propagated in culture and whether CSF-1 influences proliferation and/or invasion of these cells. CSF-1 mRNA and protein expression was determined by RT-PCR and immunofluorescence microscopy. Proliferation was assessed by the cellular uptake of tritiated thymidine and invasion was evaluated by Matrigel invasion assay as well as Northern blot analysis of mRNA expression for invasion-associated enzymes and their inhibitors. Results revealed that normal invasive EVT cells in culture express both CSF-1 and c-fms mRNA and protein. Under serum-free conditions, exogenous CSF-1 greatly stimulated the proliferation of these cells. CSF-1 neutralizing and c-fms receptor blocking antibody (Ab) each abolished the growth stimulatory effects of CSF-1, indicating that CSF-1 and c-fms interaction was responsible for these effects. In fact, c-fms Ab alone reduced proliferation to below background levels. While exogenous CSF-1 failed to influence EVT cell invasiveness, Northern blot analysis of mRNA indicated a slight upregulation of the invasion-associated enzyme 72 kDa type IV collagenase as well as its natural inhibitor tissue inhibitor of metalloprotease (TIMP)-1, so that the balance between the two remained unaltered. These findings suggest that CSF-1 may represent an autocrine (and possibly paracrine) growth stimulatory factor for the invasive trophoblast cells in situ with no net effect on their invasiveness.

scholarly journals Physiologic regulation and tissue localization of renal erythropoietin messenger RNA

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 316-318 ◽  
Author(s):  
SJ Schuster ◽  
JH Wilson ◽  
AJ Erslev ◽  
J Caro
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Messenger Rna ◽  
Northern Blot Analysis ◽  
De Novo ◽  
Rat Kidney ◽  
Acute Hypoxia ◽  
Mrna Levels ◽  
Mrna Content ◽  
Lag Times

Although erythropoietin (Epo) is produced primarily by the kidneys in response to hypoxia, the precise cell type(s) and mechanisms by which these cells regulate production are poorly understood. In the experiments we report, the kinetics of renal Epo production in response to acute hypoxia and the intrarenal localization of cellular Epo synthesis were studied at the level of Epo mRNA. Erythropoietin mRNA expression was determined by Northern blot analysis of rat kidney RNAs using a probe derived from the mouse Epo gene. Renal Epo mRNA content increased as early as 1 hour after initiation of hypoxia and continued to accumulate during 4 hours of stimulation. Discontinuation of the hypoxic stimulus resulted in rapid decay of mRNA levels. Kidney and plasma Epo levels measured by radioimmunoassay paralleled, with respective lag times, the changes in renal Epo mRNA content, suggesting that Epo production in response to acute hypoxia represents de novo synthesis and is regulated by changes in Epo mRNA. Northern blot analysis of RNAs extracted from separated glomerular and tubular tissue fractions revealed Epo mRNA in the tubular fraction, whereas glomerular tissue did not contain Epo mRNA. Thus, the site of cellular Epo synthesis is located in the renal tubule or its interstitium and not in the glomerular tuft.

CPX, a selective A1-adenosine-receptor antagonist, regulates intracellular pH in cystic fibrosis cells

1995 ◽  
Vol 269 (1) ◽  
pp. C226-C233 ◽  
Author(s):  
V. Casavola ◽  
R. J. Turner ◽  
C. Guay-Broder ◽  
K. A. Jacobson ◽  
O. Eidelman ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Receptor Antagonist ◽  
Intracellular Ph ◽  
Adenosine Receptor ◽  
Ph Regulation ◽  
Cystic Fibrosis Transmembrane Regulator ◽  
Wild Type ◽  
A1 Adenosine Receptor ◽  
Adenosine Receptor Antagonist ◽  
Cystic Fibrosis Transmembrane

The selective A1-adenosine-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPX), has been reported to activate Cl- efflux from cystic fibrosis cells, such as pancreatic CFPAC-1 and lung IB3 cells bearing the cystic fibrosis transmembrane regulator(delta F508) mutation, but has little effect on the same process in cells repaired by transfection with wild-type cystic fibrosis transmembrane regulator (O. Eidelman, C. Guay-Broder, P. J. M. van Galen, K. A. Jacobson, C. Fox, R. J. Turner, Z. I. Cabantchik, and H. B. Pollard. Proc. Natl. Acad. Sci. USA 89: 5562-5566, 1992). We report here that CPX downregulates Na+/H+ exchange activity in CFPAC-1 cells but has a much smaller effect on cells repaired with the wild-type gene. CPX also mildly decreases resting intracellular pH. In CFPAC-1 cells, this downregulation is dependent on the presence of adenosine, since pretreatment of the cells with adenosine deaminase blocks the CPX effect. We also show that, by contrast, CPX action on these cells does not lead to alterations in intracellular free Ca2+ concentration. We conclude that CPX affects pH regulation in CFPAC-1 cells, probably by antagonizing the tonic action of endogenous adenosine.

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