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Activin-A up-regulates type I activin receptor mRNA levels in human immortalized extravillous trophoblast cells.

Chen VT, Peng C, Leung PC - Reprod. Biol. Endocrinol. (2003)

Bottom Line: ActRI mRNA levels were increased in a dose-dependent manner by activin-A with the maximal effect observed at the dose of 10 ng/ml.Time course studies revealed that activin-A had maximal effects on ActRI mRNA levels at 6 hours after treatment.In addition, inhibin-A inhibited basal, as well as activin-A-induced ActRI mRNA levels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, BC, Canada. victorchen@shaw.ca

ABSTRACT
Activin is known to play an important regulatory role in reproduction, including pregnancy. To further examine the role and signaling mechanism of activin in regulating placental function, the steady-state level of activin type I receptor (ActRI) mRNA in immortalized extravillous trophoblasts (IEVT) cells was measured using competitive PCR (cPCR). An internal standard of ActRI cDNA for cPCR was constructed for the quantification of ActRI mRNA levels in IEVT cells. ActRI mRNA levels were increased in a dose-dependent manner by activin-A with the maximal effect observed at the dose of 10 ng/ml. Time course studies revealed that activin-A had maximal effects on ActRI mRNA levels at 6 hours after treatment. The effects of activin-A on ActRI mRNA levels was blocked by follistatin, an activin binding protein, in a dose-dependent manner. In addition, inhibin-A inhibited basal, as well as activin-A-induced ActRI mRNA levels. These findings provide evidence, for the first time, that activin-A modulates ActRI mRNA levels in human trophoblast cells.

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Schematic structure of the ActRI cDNA and primers used in PCR or cPCR. An 156 bp internal deletion has been done between the restriction sites Sty I and Eco47 III. Co-amplification of the ActRI native cDNA and mutant cDNA template using primer ActRI-1 and primer ActRI-2 resulted in 700 bp and 544 bp PCR product, respectively. Co-amplification of the ActRI native cDNA and mutant cDNA template using primer ActRI-3 and primer ActRI-4 resulted in 650 bp and 494 bp PCR product, respectively.
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Figure 1: Schematic structure of the ActRI cDNA and primers used in PCR or cPCR. An 156 bp internal deletion has been done between the restriction sites Sty I and Eco47 III. Co-amplification of the ActRI native cDNA and mutant cDNA template using primer ActRI-1 and primer ActRI-2 resulted in 700 bp and 544 bp PCR product, respectively. Co-amplification of the ActRI native cDNA and mutant cDNA template using primer ActRI-3 and primer ActRI-4 resulted in 650 bp and 494 bp PCR product, respectively.

Mentions: Cloning of native ActRI PCR product using primers ActRI-1 and ActRI-2 was performed by ligating the PCR product into a pDirect (BD Biosciences Clontech, Palo Alto, CA), followed by sequencing analysis to confirm the identity. To construct an internal standard for comparative PCR, a 156 base pair segment was removed from the cloned native ActRI PCR product by restriction digestion using StyI and Eco47III (Fig. 1). With the same pair of primers is used in PCR, the subcloned mutant ActRI template yielded a PCR product 156 base pairs smaller than that from the native cDNA template.


Activin-A up-regulates type I activin receptor mRNA levels in human immortalized extravillous trophoblast cells.

Chen VT, Peng C, Leung PC - Reprod. Biol. Endocrinol. (2003)

Schematic structure of the ActRI cDNA and primers used in PCR or cPCR. An 156 bp internal deletion has been done between the restriction sites Sty I and Eco47 III. Co-amplification of the ActRI native cDNA and mutant cDNA template using primer ActRI-1 and primer ActRI-2 resulted in 700 bp and 544 bp PCR product, respectively. Co-amplification of the ActRI native cDNA and mutant cDNA template using primer ActRI-3 and primer ActRI-4 resulted in 650 bp and 494 bp PCR product, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC153493&req=5

Figure 1: Schematic structure of the ActRI cDNA and primers used in PCR or cPCR. An 156 bp internal deletion has been done between the restriction sites Sty I and Eco47 III. Co-amplification of the ActRI native cDNA and mutant cDNA template using primer ActRI-1 and primer ActRI-2 resulted in 700 bp and 544 bp PCR product, respectively. Co-amplification of the ActRI native cDNA and mutant cDNA template using primer ActRI-3 and primer ActRI-4 resulted in 650 bp and 494 bp PCR product, respectively.
Mentions: Cloning of native ActRI PCR product using primers ActRI-1 and ActRI-2 was performed by ligating the PCR product into a pDirect (BD Biosciences Clontech, Palo Alto, CA), followed by sequencing analysis to confirm the identity. To construct an internal standard for comparative PCR, a 156 base pair segment was removed from the cloned native ActRI PCR product by restriction digestion using StyI and Eco47III (Fig. 1). With the same pair of primers is used in PCR, the subcloned mutant ActRI template yielded a PCR product 156 base pairs smaller than that from the native cDNA template.

Bottom Line: ActRI mRNA levels were increased in a dose-dependent manner by activin-A with the maximal effect observed at the dose of 10 ng/ml.Time course studies revealed that activin-A had maximal effects on ActRI mRNA levels at 6 hours after treatment.In addition, inhibin-A inhibited basal, as well as activin-A-induced ActRI mRNA levels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, BC, Canada. victorchen@shaw.ca

ABSTRACT
Activin is known to play an important regulatory role in reproduction, including pregnancy. To further examine the role and signaling mechanism of activin in regulating placental function, the steady-state level of activin type I receptor (ActRI) mRNA in immortalized extravillous trophoblasts (IEVT) cells was measured using competitive PCR (cPCR). An internal standard of ActRI cDNA for cPCR was constructed for the quantification of ActRI mRNA levels in IEVT cells. ActRI mRNA levels were increased in a dose-dependent manner by activin-A with the maximal effect observed at the dose of 10 ng/ml. Time course studies revealed that activin-A had maximal effects on ActRI mRNA levels at 6 hours after treatment. The effects of activin-A on ActRI mRNA levels was blocked by follistatin, an activin binding protein, in a dose-dependent manner. In addition, inhibin-A inhibited basal, as well as activin-A-induced ActRI mRNA levels. These findings provide evidence, for the first time, that activin-A modulates ActRI mRNA levels in human trophoblast cells.

Show MeSH
Related in: MedlinePlus