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Muc5b is the major polymeric mucin in mucus from thoroughbred horses with and without airway mucus accumulation.

Rousseau K, Cardwell JM, Humphrey E, Newton R, Knight D, Clegg P, Thornton DJ - PLoS ONE (2011)

Bottom Line: Immunohistochemical staining of normal equine trachea showed that Muc5ac and Muc5b are produced by cells in the submucosal glands, as well as surface epithelial goblet cells.The change in amount of Muc5b and Muc5ac indicates that Muc5b remains the most abundant mucin in mucus.In summary, we have developed mucin specific polyclonal antibodies, which have allowed us to show that there is a significant increase in Muc5b and Muc5ac in mucus accumulated in equine airways and these increases correlated with the numbers of bacteria.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

ABSTRACT
Mucus accumulation is a feature of inflammatory airway disease in the horse and has been associated with reduced performance in racehorses. In this study, we have analysed the two major airways gel-forming mucins Muc5b and Muc5ac in respect of their site of synthesis, their biochemical properties, and their amounts in mucus from healthy horses and from horses with signs of airway mucus accumulation. Polyclonal antisera directed against equine Muc5b and Muc5ac were raised and characterised. Immunohistochemical staining of normal equine trachea showed that Muc5ac and Muc5b are produced by cells in the submucosal glands, as well as surface epithelial goblet cells. Western blotting after agarose gel electrophoresis of airway mucus from healthy horses, and horses with mucus accumulation, was used to determine the amounts of these two mucins in tracheal wash samples. The results showed that in healthy horses Muc5b was the predominant mucin with small amounts of Muc5ac. The amounts of Muc5b and Muc5ac were both dramatically increased in samples collected from horses with high mucus scores as determined visually at the time of endoscopy and that this increase also correlated with increase number of bacteria present in the sample. The change in amount of Muc5b and Muc5ac indicates that Muc5b remains the most abundant mucin in mucus. In summary, we have developed mucin specific polyclonal antibodies, which have allowed us to show that there is a significant increase in Muc5b and Muc5ac in mucus accumulated in equine airways and these increases correlated with the numbers of bacteria.

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Related in: MedlinePlus

Purification of Muc5ac and Muc5b by caesium chloride density gradient centrifugation.Tracheal washes were dissolved in equal volume of 8 M guanidinium chloride and the mucins separated from other proteins by CsCl density gradient centrifugation. Panel A: Fractions were analysed for optical density at 280 nm (open squares) and the mucin containing fractions were determined by PAS staining (black triangles). The density of each fraction was determined by weighing (dashed line). Panel B: MANeq5b-I and MANeq5ac-I antisera were used to probe a Western blot after agarose gel electrophoresis of the mucins in fraction 5.
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pone-0019678-g002: Purification of Muc5ac and Muc5b by caesium chloride density gradient centrifugation.Tracheal washes were dissolved in equal volume of 8 M guanidinium chloride and the mucins separated from other proteins by CsCl density gradient centrifugation. Panel A: Fractions were analysed for optical density at 280 nm (open squares) and the mucin containing fractions were determined by PAS staining (black triangles). The density of each fraction was determined by weighing (dashed line). Panel B: MANeq5b-I and MANeq5ac-I antisera were used to probe a Western blot after agarose gel electrophoresis of the mucins in fraction 5.

Mentions: In order to characterise Muc5ac and Muc5b, four tracheal wash samples from horses showing signs of mucus hypersecretion were pooled, solubilised in 4M guanidinium chloride and analysed by caesium chloride density gradient centrifugation (buoyant density), anion exchange chromatography (charge distribution) and rate-zonal sedimentation (size distribution). PAS staining of fractions across the density distribution (Fig. 2A) showed that mucins were enriched in the high buoyant density fractions (1.37–1.55 g/ml). The mucins in fraction 5 were reduced and carboxymethylated and subjected to agarose gel electrophoresis (Fig. 2B). A Western blot of the gel probed with MANeq5b-I showed three bands. In contrast, MANeq5ac-I detected a single, broad band that migrated in a similar position to the fastest migrating Muc5b-band.


Muc5b is the major polymeric mucin in mucus from thoroughbred horses with and without airway mucus accumulation.

Rousseau K, Cardwell JM, Humphrey E, Newton R, Knight D, Clegg P, Thornton DJ - PLoS ONE (2011)

Purification of Muc5ac and Muc5b by caesium chloride density gradient centrifugation.Tracheal washes were dissolved in equal volume of 8 M guanidinium chloride and the mucins separated from other proteins by CsCl density gradient centrifugation. Panel A: Fractions were analysed for optical density at 280 nm (open squares) and the mucin containing fractions were determined by PAS staining (black triangles). The density of each fraction was determined by weighing (dashed line). Panel B: MANeq5b-I and MANeq5ac-I antisera were used to probe a Western blot after agarose gel electrophoresis of the mucins in fraction 5.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3094342&req=5

pone-0019678-g002: Purification of Muc5ac and Muc5b by caesium chloride density gradient centrifugation.Tracheal washes were dissolved in equal volume of 8 M guanidinium chloride and the mucins separated from other proteins by CsCl density gradient centrifugation. Panel A: Fractions were analysed for optical density at 280 nm (open squares) and the mucin containing fractions were determined by PAS staining (black triangles). The density of each fraction was determined by weighing (dashed line). Panel B: MANeq5b-I and MANeq5ac-I antisera were used to probe a Western blot after agarose gel electrophoresis of the mucins in fraction 5.
Mentions: In order to characterise Muc5ac and Muc5b, four tracheal wash samples from horses showing signs of mucus hypersecretion were pooled, solubilised in 4M guanidinium chloride and analysed by caesium chloride density gradient centrifugation (buoyant density), anion exchange chromatography (charge distribution) and rate-zonal sedimentation (size distribution). PAS staining of fractions across the density distribution (Fig. 2A) showed that mucins were enriched in the high buoyant density fractions (1.37–1.55 g/ml). The mucins in fraction 5 were reduced and carboxymethylated and subjected to agarose gel electrophoresis (Fig. 2B). A Western blot of the gel probed with MANeq5b-I showed three bands. In contrast, MANeq5ac-I detected a single, broad band that migrated in a similar position to the fastest migrating Muc5b-band.

Bottom Line: Immunohistochemical staining of normal equine trachea showed that Muc5ac and Muc5b are produced by cells in the submucosal glands, as well as surface epithelial goblet cells.The change in amount of Muc5b and Muc5ac indicates that Muc5b remains the most abundant mucin in mucus.In summary, we have developed mucin specific polyclonal antibodies, which have allowed us to show that there is a significant increase in Muc5b and Muc5ac in mucus accumulated in equine airways and these increases correlated with the numbers of bacteria.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

ABSTRACT
Mucus accumulation is a feature of inflammatory airway disease in the horse and has been associated with reduced performance in racehorses. In this study, we have analysed the two major airways gel-forming mucins Muc5b and Muc5ac in respect of their site of synthesis, their biochemical properties, and their amounts in mucus from healthy horses and from horses with signs of airway mucus accumulation. Polyclonal antisera directed against equine Muc5b and Muc5ac were raised and characterised. Immunohistochemical staining of normal equine trachea showed that Muc5ac and Muc5b are produced by cells in the submucosal glands, as well as surface epithelial goblet cells. Western blotting after agarose gel electrophoresis of airway mucus from healthy horses, and horses with mucus accumulation, was used to determine the amounts of these two mucins in tracheal wash samples. The results showed that in healthy horses Muc5b was the predominant mucin with small amounts of Muc5ac. The amounts of Muc5b and Muc5ac were both dramatically increased in samples collected from horses with high mucus scores as determined visually at the time of endoscopy and that this increase also correlated with increase number of bacteria present in the sample. The change in amount of Muc5b and Muc5ac indicates that Muc5b remains the most abundant mucin in mucus. In summary, we have developed mucin specific polyclonal antibodies, which have allowed us to show that there is a significant increase in Muc5b and Muc5ac in mucus accumulated in equine airways and these increases correlated with the numbers of bacteria.

Show MeSH
Related in: MedlinePlus