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. 2014 Sep 5;10:204.
doi: 10.1186/s12917-014-0204-9.

Alkaline Phosphatase in Nasal Secretion of Cattle: Biochemical and Molecular Characterisation

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Free PMC article

Alkaline Phosphatase in Nasal Secretion of Cattle: Biochemical and Molecular Characterisation

M Faizal Ghazali et al. BMC Vet Res. .
Free PMC article

Abstract

Background: Nasal secretion (NS) was investigated as a source of information regarding the mucosal and systemic immune status of cattle challenged by respiratory disease. A method for the collection of substantial volumes (~12 ml) of NS from cattle was developed to establish a reference range of analytes that are present in the NS of healthy cattle. Biochemical profiles of NS from a group of 38 healthy Holstein-Friesian cows revealed high alkaline phosphatase (AP) activity of up to 2392 IU/L. The character and source of the high activity of AP in bovine NS was investigated.

Results: Histochemical analysis confirmed the localization of the AP enzyme activity to epithelial cells and serous glands of the nasal respiratory mucosa. Analysis of mRNA levels from nasal mucosa by end point RT-PCR and PCR product sequencing confirmed that the AP was locally produced and is identical at the nucleotide level to the non-specific AP splice variant found in bovine liver, bone and kidney. Analysis by isoelectric focussing confirmed that AP was produced locally at a high level in nasal epithelium demonstrating that AP from nasal secretion and nasal mucosa had similar pI bands, though differing from those of the liver, kidney, bone and intestine, suggesting different post-translational modification (PTM) of AP in these tissues.

Conclusions: A nasal isozyme of AP has been identified that is present at a high activity in NS, resulting from local production and showing distinctive PTM and may be active in NS as an anti-endotoxin mediator.

Figures

Figure 1
Figure 1
Schematic representation of a collecting tube for nasal secretion. The saturated tampon from nostril was inserted into a modified collecting tube consisted of a 30 ml universal tube with four × 2 mm holes drilled into the bottom, inserted in a 50 ml Falcon tube.
Figure 2
Figure 2
Histology and alkaline phosphatase activity histochemistry of nasal mucosa. Routine staining of nasal mucosa with Haematoxilin and Eosin (left column) and histochemical staining of AP activity using Vector Red substrate (central column) showed strong AP activity in the mucus at the luminal surface and in the basal cell layers of the epithelium (yellow arrow); as well as in serous glands (green arrow). Levamisole, a non-specific AP inhibitor, abolished the histochemical staining of these cells (right column). Image captures at x10, x20 and x40 magnifications are shown with appropriate scale bars.
Figure 3
Figure 3
Alkaline phosphatase mRNA analysis from bovine tissues by RT- PCR. Bovine tissues examined by RT-PCR to determine expression of genes for AP. The presence of a single 500 bp DNA fragment corresponding to the predicted product size was visualised when using (a) non-specific AP primers and (b) intestinal AP primers. PCR products were present in nasal mucosa only when non-specific AP primers were used. M = Marker (100 bp DNA ladder); MQ = high purity water; B = blank; N = nasal mucosa; L = liver; I = intestine.
Figure 4
Figure 4
Isoelectric focussing of alkaline phosphatase in bovine tissue and nasal secretion. Bovine tissue extracts and nasal secretion separated on isoelectric focusing gels over a pH 3–7 range and stained with 5-Bromo-4-chloro-3-indolyl phosphate with nitro blue tetrazolium chloride (BCIP/NBT) substrate. M = IEF standard protein marker (stained with Coomassie blue); −ve = cathode; +ve = anode. Two different samples of NS were run in duplicate. Samples of extracts from nasal mucosa, bone and intestine were run in duplicate with liver and kidney extract being run in single tracks. Annotated boxes around isoforms bands as discussed in the text.

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