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A calmodulin inhibitor, W-7 influences the effect of cyclic adenosine 3', 5'-monophosphate signaling on ligninolytic enzyme gene expression in Phanerochaete chrysosporium.

Sakamoto T, Yao Y, Hida Y, Honda Y, Watanabe T, Hashigaya W, Suzuki K, Irie T - AMB Express (2012)

Bottom Line: Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that transcription of most LiP and MnP isozyme genes was statistically significantly upregulated in the presence of the cAMP and IBMX compared to the untreated condition.However, 100 μM calmodulin (CaM) inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which had insignificant effects on fungal growth and intracellular cAMP concentration, not only offset the increased activity and transcription induced by the drugs, but also decreased them to below basal levels.Like the isozyme genes, transcription of the CaM gene (cam) was also upregulated by cAMP and IBMX.

View Article: PubMed Central - HTML - PubMed

Affiliation: Environmental Science Graduate School, The University of Shiga Prefecture, 2500 Hassaka-cho, Hikone City, Shiga, 522-8533, Japan. tirie@ses.usp.ac.jp.

ABSTRACT
The capacity of white-rot fungi to degrade wood lignin may be highly applicable to the development of novel bioreactor systems, but the mechanisms underlying this function are not yet fully understood. Lignin peroxidase (LiP) and manganese peroxidase (MnP), which are thought to be very important for the ligninolytic property, demonstrated increased activity in Phanerochaete chrysosporium RP-78 (FGSC #9002, ATCC MYA-4764™) cultures following exposure to 5 mM cyclic adenosine 3', 5'-monophosphate (cAMP) and 500 μM 3'-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that transcription of most LiP and MnP isozyme genes was statistically significantly upregulated in the presence of the cAMP and IBMX compared to the untreated condition. However, 100 μM calmodulin (CaM) inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which had insignificant effects on fungal growth and intracellular cAMP concentration, not only offset the increased activity and transcription induced by the drugs, but also decreased them to below basal levels. Like the isozyme genes, transcription of the CaM gene (cam) was also upregulated by cAMP and IBMX. These results suggest that cAMP signaling functions to increase the transcription of LiP and MnP through the induction of cam transcription.

No MeSH data available.


Related in: MedlinePlus

Relative quantity of transcripts of the 25S rRNA (transcribed by RNA polymerase I), act (encoding actin), and gpd (encoding GAPDH) genes (transcribed by RNA polymerase II) under various conditions for determination of the internal standard (Figure 4). Drugs were added into 48 h culture, and total RNA was extracted from each culture at 24 h after the drug addition. Each real-time RT-PCRs was performed using 3 ng total RNA. Error bars show the SD for 4 biological repetitions. A common letter indicates cases where values were insignificantly different between drug groups (P < 0.05), estimated by Turkey's HSD test following one-way factorial ANOVA. Primers 5'-CGTCAACGACCCCTTCATTG-3' and 5'-CGACATAGAGCTTGCCGTCCT-3' were used for the gpd gene. The other primers are listed in Sakamoto et al. (2010).
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Figure 1: Relative quantity of transcripts of the 25S rRNA (transcribed by RNA polymerase I), act (encoding actin), and gpd (encoding GAPDH) genes (transcribed by RNA polymerase II) under various conditions for determination of the internal standard (Figure 4). Drugs were added into 48 h culture, and total RNA was extracted from each culture at 24 h after the drug addition. Each real-time RT-PCRs was performed using 3 ng total RNA. Error bars show the SD for 4 biological repetitions. A common letter indicates cases where values were insignificantly different between drug groups (P < 0.05), estimated by Turkey's HSD test following one-way factorial ANOVA. Primers 5'-CGTCAACGACCCCTTCATTG-3' and 5'-CGACATAGAGCTTGCCGTCCT-3' were used for the gpd gene. The other primers are listed in Sakamoto et al. (2010).

Mentions: Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) analysis was conducted as previously described (Sakamoto et al. 2010). Total RNA was isolated using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer's protocol. After treatment with RNase-free DNase (TaKaRa, Shiga, Japan), mRNA was reverse transcribed using the PrimeScript RT Regent Kit (TaKaRa, Shiga, Japan) according to the manufacturer's instructions and used for analysis. Quantitative real-time RT-PCR amplification was carried out for all isozyme genes of ligninolytic peroxidase, i.e. 10 lip isozyme genes (protein_id 10957, 121822, 131738, 6811, 11110, 122202, 8895, 121806, 131707, 131709), 5 mnp isozyme genes (protein_id 140708, 3589, 878, 8191, 4636), and cam (protein_id 10767). An actin gene (protein_id 139298) was used as endogenous reference gene, which was not valuable in quantity of its transcript among the culture conditions used in this study (Figure 1). The genes were predicted using data from the P. chrysosporium v2.0 genome database (Martinez et al. 2004) available at DOE Joint Genome Institute (JGI; http://genome.jgi-psf.org/Phchr1/Phchr1.home.html). The amplification was performed using gene-specific primers (Sakamoto et al. 2010) and SYBR® Premix Ex TaqTM II (TaKaRa, Shiga, Japan). The experiment was repeated 4 times. PCR amplifications using a Thermal Cycler Dice TM real-time system (TaKaRa, Shiga, Japan) were performed as follows: (i) an initial denaturation step at 95°C for 10 s and (ii) 40 cycles, with each cycle consisting of denaturation at 95°C for 5 s and annealing and elongation at 60°C for 30 s. The standard curve of each gene was constructed from real-time PCR results using dilution series of the PCR product made by the same primer pair template as for real-time RT-PCR. Transcription of each gene was quantified using the standard curve. For comparisons between different culture conditions, the total amount of complementary DNA (cDNA) was normalized against that of actin.


A calmodulin inhibitor, W-7 influences the effect of cyclic adenosine 3', 5'-monophosphate signaling on ligninolytic enzyme gene expression in Phanerochaete chrysosporium.

Sakamoto T, Yao Y, Hida Y, Honda Y, Watanabe T, Hashigaya W, Suzuki K, Irie T - AMB Express (2012)

Relative quantity of transcripts of the 25S rRNA (transcribed by RNA polymerase I), act (encoding actin), and gpd (encoding GAPDH) genes (transcribed by RNA polymerase II) under various conditions for determination of the internal standard (Figure 4). Drugs were added into 48 h culture, and total RNA was extracted from each culture at 24 h after the drug addition. Each real-time RT-PCRs was performed using 3 ng total RNA. Error bars show the SD for 4 biological repetitions. A common letter indicates cases where values were insignificantly different between drug groups (P < 0.05), estimated by Turkey's HSD test following one-way factorial ANOVA. Primers 5'-CGTCAACGACCCCTTCATTG-3' and 5'-CGACATAGAGCTTGCCGTCCT-3' were used for the gpd gene. The other primers are listed in Sakamoto et al. (2010).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3275468&req=5

Figure 1: Relative quantity of transcripts of the 25S rRNA (transcribed by RNA polymerase I), act (encoding actin), and gpd (encoding GAPDH) genes (transcribed by RNA polymerase II) under various conditions for determination of the internal standard (Figure 4). Drugs were added into 48 h culture, and total RNA was extracted from each culture at 24 h after the drug addition. Each real-time RT-PCRs was performed using 3 ng total RNA. Error bars show the SD for 4 biological repetitions. A common letter indicates cases where values were insignificantly different between drug groups (P < 0.05), estimated by Turkey's HSD test following one-way factorial ANOVA. Primers 5'-CGTCAACGACCCCTTCATTG-3' and 5'-CGACATAGAGCTTGCCGTCCT-3' were used for the gpd gene. The other primers are listed in Sakamoto et al. (2010).
Mentions: Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) analysis was conducted as previously described (Sakamoto et al. 2010). Total RNA was isolated using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer's protocol. After treatment with RNase-free DNase (TaKaRa, Shiga, Japan), mRNA was reverse transcribed using the PrimeScript RT Regent Kit (TaKaRa, Shiga, Japan) according to the manufacturer's instructions and used for analysis. Quantitative real-time RT-PCR amplification was carried out for all isozyme genes of ligninolytic peroxidase, i.e. 10 lip isozyme genes (protein_id 10957, 121822, 131738, 6811, 11110, 122202, 8895, 121806, 131707, 131709), 5 mnp isozyme genes (protein_id 140708, 3589, 878, 8191, 4636), and cam (protein_id 10767). An actin gene (protein_id 139298) was used as endogenous reference gene, which was not valuable in quantity of its transcript among the culture conditions used in this study (Figure 1). The genes were predicted using data from the P. chrysosporium v2.0 genome database (Martinez et al. 2004) available at DOE Joint Genome Institute (JGI; http://genome.jgi-psf.org/Phchr1/Phchr1.home.html). The amplification was performed using gene-specific primers (Sakamoto et al. 2010) and SYBR® Premix Ex TaqTM II (TaKaRa, Shiga, Japan). The experiment was repeated 4 times. PCR amplifications using a Thermal Cycler Dice TM real-time system (TaKaRa, Shiga, Japan) were performed as follows: (i) an initial denaturation step at 95°C for 10 s and (ii) 40 cycles, with each cycle consisting of denaturation at 95°C for 5 s and annealing and elongation at 60°C for 30 s. The standard curve of each gene was constructed from real-time PCR results using dilution series of the PCR product made by the same primer pair template as for real-time RT-PCR. Transcription of each gene was quantified using the standard curve. For comparisons between different culture conditions, the total amount of complementary DNA (cDNA) was normalized against that of actin.

Bottom Line: Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that transcription of most LiP and MnP isozyme genes was statistically significantly upregulated in the presence of the cAMP and IBMX compared to the untreated condition.However, 100 μM calmodulin (CaM) inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which had insignificant effects on fungal growth and intracellular cAMP concentration, not only offset the increased activity and transcription induced by the drugs, but also decreased them to below basal levels.Like the isozyme genes, transcription of the CaM gene (cam) was also upregulated by cAMP and IBMX.

View Article: PubMed Central - HTML - PubMed

Affiliation: Environmental Science Graduate School, The University of Shiga Prefecture, 2500 Hassaka-cho, Hikone City, Shiga, 522-8533, Japan. tirie@ses.usp.ac.jp.

ABSTRACT
The capacity of white-rot fungi to degrade wood lignin may be highly applicable to the development of novel bioreactor systems, but the mechanisms underlying this function are not yet fully understood. Lignin peroxidase (LiP) and manganese peroxidase (MnP), which are thought to be very important for the ligninolytic property, demonstrated increased activity in Phanerochaete chrysosporium RP-78 (FGSC #9002, ATCC MYA-4764™) cultures following exposure to 5 mM cyclic adenosine 3', 5'-monophosphate (cAMP) and 500 μM 3'-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that transcription of most LiP and MnP isozyme genes was statistically significantly upregulated in the presence of the cAMP and IBMX compared to the untreated condition. However, 100 μM calmodulin (CaM) inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which had insignificant effects on fungal growth and intracellular cAMP concentration, not only offset the increased activity and transcription induced by the drugs, but also decreased them to below basal levels. Like the isozyme genes, transcription of the CaM gene (cam) was also upregulated by cAMP and IBMX. These results suggest that cAMP signaling functions to increase the transcription of LiP and MnP through the induction of cam transcription.

No MeSH data available.


Related in: MedlinePlus