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Increases in apoptosis, caspase activity and expression of p53 and bax, and the transition between two types of mitochondrion-rich cells, in the gills of the climbing perch, Anabas testudineus, during a progressive acclimation from freshwater to seawater.

Ching B, Chen XL, Yong JH, Wilson JM, Hiong KC, Sim EW, Wong WP, Lam SH, Chew SF, Ip YK - Front Physiol (2013)

Bottom Line: Seawater acclimation apparently activated both the extrinsic and intrinsic pathways, as reflected by significant increases in branchial caspase-8 and caspase-9 activities.The involvement of the intrinsic pathway was confirmed by the significant increase in branchial mRNA expression of bax between day 4 (salinity 20) and day 6 (seawater).Western blotting results revealed the presence of a freshwater Na(+)/K(+)-ATPase (Nka) α-isoform, Nka α1a, and a seawater isoform, Nka α1b, the protein abundance of which decreased and increased, respectively, during seawater acclimation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, National University of Singapore Kent Ridge, Singapore, Singapore.

ABSTRACT
This study aimed to test the hypothesis that branchial osmoregulatory acclimation involved increased apoptosis and replacement of mitochdonrion-rich cells (MRCs) in the climbing perch, Anabas testudineus, during a progressive acclimation from freshwater to seawater. A significant increase in branchial caspase-3/-7 activity was observed on day 4 (salinity 20), and an extensive TUNEL-positive apoptosis was detected on day 5 (salinity 25), indicating salinity-induced apoptosis had occurred. This was further supported by an up-regulation of branchial mRNA expression of p53, a key regulator of cell cycle arrest and apoptosis, between day 2 (salinity 10) and day 6 (seawater), and an increase in branchial p53 protein abundance on day 6. Seawater acclimation apparently activated both the extrinsic and intrinsic pathways, as reflected by significant increases in branchial caspase-8 and caspase-9 activities. The involvement of the intrinsic pathway was confirmed by the significant increase in branchial mRNA expression of bax between day 4 (salinity 20) and day 6 (seawater). Western blotting results revealed the presence of a freshwater Na(+)/K(+)-ATPase (Nka) α-isoform, Nka α1a, and a seawater isoform, Nka α1b, the protein abundance of which decreased and increased, respectively, during seawater acclimation. Immunofluorescence microscopy revealed the presence of two types of MRCs distinctly different in sizes, and confirmed that the reduction in Nka α1a expression, and the prominent increases in expression of Nka α1b, Na(+):K(+):2Cl(-) cotransporter 1, and cystic fibrosis transmembrane conductance regulator Cl(-) channel coincided with the salinity-induced apoptotic event. Since modulation of existing MRCs alone could not have led to extensive salinity-induced apoptosis, it is probable that some, if not all, freshwater-type MRCs could have been removed through increased apoptosis and subsequently replaced by seawater-type MRCs in the gills of A. testudineus during seawater acclimation.

No MeSH data available.


Related in: MedlinePlus

A multiple alignment of the deduced amino acid sequence of p53 from gills of Anabas testudineus (Genbank accession number KC513732) with those from Epinephelus coioides (grouper; ADN04912.1), Oreochromis niloticus (tilapia; ADE21938.1), Xiphophorus maculatus (platyfish; AAC31134.1), Salmo salar (salmon; ACN10490.1), Ictalurus punctatus (catfish; NP_001187005.1), Danio rerio (zebrafish; NP_571402.1), Xenopus laevis (frog; CAA54672.1), Gallus gallus (chicken; NP_990595.1), Mus musculus (mouse; BAA82344.1), and Homo sapiens (human; BAC16799.1). Identical amino acids are indicated by shaded black residues and similar amino acids (threshold value 50%) are indicated by shaded grey residues. Domains present, as indicated by line, are transactivation domain (TAD), proline-rich region (PR), DNA binding domain (DNA-BD), oligomerization domain (OD) and carboxy-terminal regulatory domain (CTD). Important residues are in boxes. Within the TAD, key residues are F19, W23, and L26 which make crucial Murine Double Minute 2 (MDM2) contact, and S15, T18, and S20 which are important phosphorylation sites. Within the DNA-BD, key residues that make direct contact with DNA are K120, S241, R248, R273, A276, C277, and R280. Within the OD, there is conserved R333, G334, E349, and highly conserved intermolecular salt bridge between R337 and D352. L348 has also been found to be one of the key hydrophobic residues in this region, while L344 is part of a leucine-rich nuclear export signal. In the CTD, K381, K382, K386, and S392 are important regions for posttranslational modification.
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Figure 3: A multiple alignment of the deduced amino acid sequence of p53 from gills of Anabas testudineus (Genbank accession number KC513732) with those from Epinephelus coioides (grouper; ADN04912.1), Oreochromis niloticus (tilapia; ADE21938.1), Xiphophorus maculatus (platyfish; AAC31134.1), Salmo salar (salmon; ACN10490.1), Ictalurus punctatus (catfish; NP_001187005.1), Danio rerio (zebrafish; NP_571402.1), Xenopus laevis (frog; CAA54672.1), Gallus gallus (chicken; NP_990595.1), Mus musculus (mouse; BAA82344.1), and Homo sapiens (human; BAC16799.1). Identical amino acids are indicated by shaded black residues and similar amino acids (threshold value 50%) are indicated by shaded grey residues. Domains present, as indicated by line, are transactivation domain (TAD), proline-rich region (PR), DNA binding domain (DNA-BD), oligomerization domain (OD) and carboxy-terminal regulatory domain (CTD). Important residues are in boxes. Within the TAD, key residues are F19, W23, and L26 which make crucial Murine Double Minute 2 (MDM2) contact, and S15, T18, and S20 which are important phosphorylation sites. Within the DNA-BD, key residues that make direct contact with DNA are K120, S241, R248, R273, A276, C277, and R280. Within the OD, there is conserved R333, G334, E349, and highly conserved intermolecular salt bridge between R337 and D352. L348 has also been found to be one of the key hydrophobic residues in this region, while L344 is part of a leucine-rich nuclear export signal. In the CTD, K381, K382, K386, and S392 are important regions for posttranslational modification.

Mentions: The complete coding cDNA sequence of p53 (1143 bp) obtained from the gills of A. testudineus (Genbank accession number KC513732) putatively coded for 380 amino acids with an estimated molecular mass of 42.4 kDa (Figure 3). p53 of A. testudineus has a DNA-binding domain (DNA-BD) and a oligomerization domain flanked by intrinsically disordered regions at the C-terminal and N-terminal regions (Figure 3). The C-terminal region is a natively unfolded region consisting of regulatory domains while the N-terminal region comprises of an intrinsically disordered transactivation domain and a proline-rich region. The mRNA expression of p53 in the gills of A. testudineus kept in freshwater for 1 day was comparable to that of fish kept in freshwater for 11 days (Figure 4). However, during seawater acclimation, there were significant increases in the mRNA expression of p53 in the gills of fish exposed to salinity 10 (day 2), salinity 20 (day 4), salinity 25 (day 5), and salinity 30 (seawater; day 6; Figure 4). After 6 days of acclimation to seawater (day 11), the mRNA expression of p53 in the gills returned to a level comparable to that of the day 1 freshwater control (Figure 4). There was a general trend of increase in the protein abundance of p53 in the gills of fish during seawater acclimation, and the branchial p53 protein abundance of fish exposed to seawater on day 6 was significantly greater than that of the freshwater control (Figure 5).


Increases in apoptosis, caspase activity and expression of p53 and bax, and the transition between two types of mitochondrion-rich cells, in the gills of the climbing perch, Anabas testudineus, during a progressive acclimation from freshwater to seawater.

Ching B, Chen XL, Yong JH, Wilson JM, Hiong KC, Sim EW, Wong WP, Lam SH, Chew SF, Ip YK - Front Physiol (2013)

A multiple alignment of the deduced amino acid sequence of p53 from gills of Anabas testudineus (Genbank accession number KC513732) with those from Epinephelus coioides (grouper; ADN04912.1), Oreochromis niloticus (tilapia; ADE21938.1), Xiphophorus maculatus (platyfish; AAC31134.1), Salmo salar (salmon; ACN10490.1), Ictalurus punctatus (catfish; NP_001187005.1), Danio rerio (zebrafish; NP_571402.1), Xenopus laevis (frog; CAA54672.1), Gallus gallus (chicken; NP_990595.1), Mus musculus (mouse; BAA82344.1), and Homo sapiens (human; BAC16799.1). Identical amino acids are indicated by shaded black residues and similar amino acids (threshold value 50%) are indicated by shaded grey residues. Domains present, as indicated by line, are transactivation domain (TAD), proline-rich region (PR), DNA binding domain (DNA-BD), oligomerization domain (OD) and carboxy-terminal regulatory domain (CTD). Important residues are in boxes. Within the TAD, key residues are F19, W23, and L26 which make crucial Murine Double Minute 2 (MDM2) contact, and S15, T18, and S20 which are important phosphorylation sites. Within the DNA-BD, key residues that make direct contact with DNA are K120, S241, R248, R273, A276, C277, and R280. Within the OD, there is conserved R333, G334, E349, and highly conserved intermolecular salt bridge between R337 and D352. L348 has also been found to be one of the key hydrophobic residues in this region, while L344 is part of a leucine-rich nuclear export signal. In the CTD, K381, K382, K386, and S392 are important regions for posttranslational modification.
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Related In: Results  -  Collection

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Figure 3: A multiple alignment of the deduced amino acid sequence of p53 from gills of Anabas testudineus (Genbank accession number KC513732) with those from Epinephelus coioides (grouper; ADN04912.1), Oreochromis niloticus (tilapia; ADE21938.1), Xiphophorus maculatus (platyfish; AAC31134.1), Salmo salar (salmon; ACN10490.1), Ictalurus punctatus (catfish; NP_001187005.1), Danio rerio (zebrafish; NP_571402.1), Xenopus laevis (frog; CAA54672.1), Gallus gallus (chicken; NP_990595.1), Mus musculus (mouse; BAA82344.1), and Homo sapiens (human; BAC16799.1). Identical amino acids are indicated by shaded black residues and similar amino acids (threshold value 50%) are indicated by shaded grey residues. Domains present, as indicated by line, are transactivation domain (TAD), proline-rich region (PR), DNA binding domain (DNA-BD), oligomerization domain (OD) and carboxy-terminal regulatory domain (CTD). Important residues are in boxes. Within the TAD, key residues are F19, W23, and L26 which make crucial Murine Double Minute 2 (MDM2) contact, and S15, T18, and S20 which are important phosphorylation sites. Within the DNA-BD, key residues that make direct contact with DNA are K120, S241, R248, R273, A276, C277, and R280. Within the OD, there is conserved R333, G334, E349, and highly conserved intermolecular salt bridge between R337 and D352. L348 has also been found to be one of the key hydrophobic residues in this region, while L344 is part of a leucine-rich nuclear export signal. In the CTD, K381, K382, K386, and S392 are important regions for posttranslational modification.
Mentions: The complete coding cDNA sequence of p53 (1143 bp) obtained from the gills of A. testudineus (Genbank accession number KC513732) putatively coded for 380 amino acids with an estimated molecular mass of 42.4 kDa (Figure 3). p53 of A. testudineus has a DNA-binding domain (DNA-BD) and a oligomerization domain flanked by intrinsically disordered regions at the C-terminal and N-terminal regions (Figure 3). The C-terminal region is a natively unfolded region consisting of regulatory domains while the N-terminal region comprises of an intrinsically disordered transactivation domain and a proline-rich region. The mRNA expression of p53 in the gills of A. testudineus kept in freshwater for 1 day was comparable to that of fish kept in freshwater for 11 days (Figure 4). However, during seawater acclimation, there were significant increases in the mRNA expression of p53 in the gills of fish exposed to salinity 10 (day 2), salinity 20 (day 4), salinity 25 (day 5), and salinity 30 (seawater; day 6; Figure 4). After 6 days of acclimation to seawater (day 11), the mRNA expression of p53 in the gills returned to a level comparable to that of the day 1 freshwater control (Figure 4). There was a general trend of increase in the protein abundance of p53 in the gills of fish during seawater acclimation, and the branchial p53 protein abundance of fish exposed to seawater on day 6 was significantly greater than that of the freshwater control (Figure 5).

Bottom Line: Seawater acclimation apparently activated both the extrinsic and intrinsic pathways, as reflected by significant increases in branchial caspase-8 and caspase-9 activities.The involvement of the intrinsic pathway was confirmed by the significant increase in branchial mRNA expression of bax between day 4 (salinity 20) and day 6 (seawater).Western blotting results revealed the presence of a freshwater Na(+)/K(+)-ATPase (Nka) α-isoform, Nka α1a, and a seawater isoform, Nka α1b, the protein abundance of which decreased and increased, respectively, during seawater acclimation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, National University of Singapore Kent Ridge, Singapore, Singapore.

ABSTRACT
This study aimed to test the hypothesis that branchial osmoregulatory acclimation involved increased apoptosis and replacement of mitochdonrion-rich cells (MRCs) in the climbing perch, Anabas testudineus, during a progressive acclimation from freshwater to seawater. A significant increase in branchial caspase-3/-7 activity was observed on day 4 (salinity 20), and an extensive TUNEL-positive apoptosis was detected on day 5 (salinity 25), indicating salinity-induced apoptosis had occurred. This was further supported by an up-regulation of branchial mRNA expression of p53, a key regulator of cell cycle arrest and apoptosis, between day 2 (salinity 10) and day 6 (seawater), and an increase in branchial p53 protein abundance on day 6. Seawater acclimation apparently activated both the extrinsic and intrinsic pathways, as reflected by significant increases in branchial caspase-8 and caspase-9 activities. The involvement of the intrinsic pathway was confirmed by the significant increase in branchial mRNA expression of bax between day 4 (salinity 20) and day 6 (seawater). Western blotting results revealed the presence of a freshwater Na(+)/K(+)-ATPase (Nka) α-isoform, Nka α1a, and a seawater isoform, Nka α1b, the protein abundance of which decreased and increased, respectively, during seawater acclimation. Immunofluorescence microscopy revealed the presence of two types of MRCs distinctly different in sizes, and confirmed that the reduction in Nka α1a expression, and the prominent increases in expression of Nka α1b, Na(+):K(+):2Cl(-) cotransporter 1, and cystic fibrosis transmembrane conductance regulator Cl(-) channel coincided with the salinity-induced apoptotic event. Since modulation of existing MRCs alone could not have led to extensive salinity-induced apoptosis, it is probable that some, if not all, freshwater-type MRCs could have been removed through increased apoptosis and subsequently replaced by seawater-type MRCs in the gills of A. testudineus during seawater acclimation.

No MeSH data available.


Related in: MedlinePlus