Occludin immunolocalization and protein expression in goldfish

Abstract

SUMMARYTight junctions (TJs) are an integral component of models illustrating ion transport mechanisms across fish epithelia; however, little is known about TJ proteins in fishes. Using immunohistochemical methods and Western blot analysis, we examined the localization and expression of occludin, a transmembrane TJ protein, in goldfish tissues. In goldfish gills, discontinuous occludin immunostaining was detected along the edges of secondary gill lamellae and within parts of the interlamellar region that line the lateral walls of the central venous sinus. In the goldfish intestine, occludin immunolocalized in a TJ-specific distribution pattern to apical regions of columnar epithelial cells lining the intestinal lumen. In the goldfish kidney, occludin was differentially expressed in discrete regions of the nephron. Occludin immunostaining was strongest in the distal segment of the nephron, moderate in the collecting duct and absent in the proximal segment. To investigate a potential role for occludin in the maintenance of the hydromineral balance of fishes, we subjected goldfish to 1, 2 and 4 weeks of food deprivation, and then examined the endpoints of hydromineral status, Na + ,K + -ATPase activity and occludin protein expression in the gills, intestine and kidney. Occludin expression altered in response to hydromineral imbalance in a tissue-specific manner suggesting a dynamic role for this TJ protein in the regulation of epithelial permeability in fishes.

Effect of β-Glucans in Diets on Growth, Survival, Digestive Enzyme Activity, and Immune System and Intestinal Barrier Gene Expression for Tropical Gar (Atractosteus tropicus) Juveniles

“…Therefore, expression of OCC in the intestine can be modified via feed formulations to improve the function of the cellular barrier against possible pathogens. In studies conducted on goldfish (Carassius auratus, Linnaeus 1758) [59], it has been reported that a low expression of OCC can affect the permeability of transmembrane junctions, causing a decrease in the intestinal epithelium that potentially lowers the resistance to pathogens by the redistribution of tight junction proteins, thereby altering the correct functioning of the intestinal barrier. However, the concentrations used in this study did not show an apparent negative effect on the health of the fish.…”

An animal homolog of plant Mep/Amt transporters promotes ammonia excretion by the anal papillae of the disease vector mosquito,Aedes aegypti

“…Samples (5-7 µg protein) were then electrophoretically separated by SDS-PAGE and western blot analysis of AeAmt1 was conducted according to a previously described protocol (Chasiotis and Kelly, 2008), while analysis of NHE3 was carried out by overnight transfer of protein to Immobilon-P polyvinylidene-difluoride (PVDF) membrane (EMD Millipore, USA) at 4°C. A custom-made polyclonal antibody that was produced in rabbit against a custom-made synthetic peptide (CPLWEREVELSDGLM) corresponding to a 14-amino acid region of AeAmt1 (GenScript USA Inc., Piscataway, NJ, USA) was used at 1:500 dilution.…”

“…Immunolocalization of AeAmt1, NKA, VA and NHE3 in paraffin-embedded sections of anal papillae was conducted according to previously described protocols (Pullikuth et al, 2006;Chasiotis and Kelly, 2008) using a 1:40 dilution of the custom-made anti-AeAmt1 antibody described above, a 1:10 dilution of a mouse monoclonal anti-α5 antibody for NKA (Douglas Fambrough, Developmental Studies Hybridoma Bank, IA, USA), a 1:100 dilution of a mouse polyclonal anti-ATP6V0A1 antibody for VA (Abnova, Taipei, Taiwan) or a 1:250 dilution of the NHE3 antibody described above. A sheep anti-mouse antibody conjugated to Cy2 or a goat anti-mouse antibody conjugated to Cy5 (Jackson Immunoresearch) was applied at 1:500 and 1:1000, respectively, to visualize NKA.…”

Aedes aegypti Rhesus glycoproteins contribute to ammonia excretion by larval anal papillae

“…Samples were then electrophoretically separated by SDS-PAGE and western blot analysis of AeRh50 was conducted according to procedures outlined by Chasiotis and Kelly (2008). A custom-synthesized polyclonal antibody (1:2000 dilution) was raised in rabbit against the epitope HHKDDAYWETPAES corresponding to a 14-amino acid region of AeRh50-1 (GenScript USA, Piscataway, NJ, USA).…”