, 2003), Hagfish (Myxine glutinosa L.) ( Subramanian et al., 2009) and catfish (Pelteobagrus fulvidraco) ( Su, 2011). These observations suggest that mucus is a good source of novel molecules for fish and human health-related applications. We also recently reported that Selleckchem GSK126 the mucus of the stingray Potamotrygon. cf. henlei shows antimicrobial effects and a pro-inflammatory response ( Monteiro-Dos-Santos et al., 2011). The aim of the present study was therefore to identify and characterize the major component(s) with antimicrobial activity in the mucus of

P. cf. henlei, which is a very common stingray found in northern and central-western rivers from Brazil ( Carvalho et al., 2003). Their spines are hard, sharp, bilaterally retroserrated and covered by an integumentary sheath with a ventrolateral glandular groove containing venom glands along both edges ( Halstead, 1970) and the mucus of biological importance that covers the entire body of these animals.

This study employed a screening approach on mucus components that were purified by RP-HPLC, and characterized by ESI-MS and Edman degradation. By this approach, several compounds including peptides were obtained and a protein similar to Hemoglobin β-chain was identified, isolated and characterized. Following antimicrobial and hemolytic assays, intravital microscopy was used to image the effects of the protein on the microcirculation. Specimens Romidepsin datasheet of adult female and male (n = 15) P. cf. henlei fish were collected from the Manoel Alves River in the state of Tocantins, Brazil. Mucus dispersed all over the body was collected by scraping the skin with a glass slide, and immediately stored on ice, then diluted in 0.15 M phosphate-buffered sterile saline, pH 7.4, homogenized, and centrifuged (5000 × g for 20 min at 4 °C) for collection of the supernatant. The supernatant was collected and stored at −20 °C.

Protein content was determined by the method of Bradford (1976) using bovine serum albumin (Sigma Chemical Co., St Louis, MO) as standard protein. The supernatants were loaded onto solid phase extraction cartridges Sep-Pak, C18 (Waters Corporation, Taunton, MA, USA) equilibrated in acidified water (0.1% trifluoroacetic Montelukast Sodium acid (TFA)). A single aliquot of 3 mg diluted in 3 mL of 0.1% TFA was loaded and the elution was performed sequentially with 40 and 80% acetonitrile. These fractions were further concentrated by a vacuum centrifugation. Aliquots of 1 mg of the samples were dissolved in 1 mL of deionized water in 0.1% TFA and centrifuged at 5000 × g for 20 min (10 °C). The supernatants were applied to a system of RP – HPLC (Äkta basic, Amersham Biosciences – Sweden) for the sample separation. The sample was loaded in a Jupiter C18 column (4.6 mm × 150 mm, 5 μm, Phenomenex, USA) in a two-solvent system: (A) TFA/H2O (1:1000) and (B) TFA/Acetonitrile (ACN)/H2O (1:900:100). The column was eluted at a flow rate of 1.