MW: molecular weight markers

MW: molecular weight markers. weight markers. Each lane was loaded with 75 g of protein.(TIF) pone.0066689.s003.tif (1.3M) GUID:?2139B90D-4FA2-44E0-B6FA-CFEB4D0B90F1 Abstract Digestive proteases of the NOTCH1 Mulberroside A digestive tract of the apple snail were studied. Luminal protease activity was found in the crop, the style sac and the coiled gut and was significantly higher in the coiled gut. Several protease bands and their apparent molecular weights were identified in both tissue extracts and luminal contents by gel zymography: (1) a 125 kDa protease in salivary gland extracts and in the crop content; (2) a 30 kDa protease throughout all studied luminal contents and in extracts of the midgut gland and of the endosymbionts isolated from this gland; (3) two proteases of 145 and 198 kDa in the coiled gut content. All these proteases were inhibited by aprotinin, a serine-protease inhibitor, and showed maximum activity between 30C and 35C and pH between 8.5 and 9.5. Tissue L-alanine-N-aminopeptidase activity was determined in the wall of the crop, the style sac and the coiled gut and was significantly higher in the coiled gut. Our findings show that protein digestion in is carried out through a battery of diverse proteases originated from the salivary glands and the endosymbionts lodged in the midgut gland and by proteases of uncertain origin that occur in the coiled gut lumen. Introduction (Lamarck 1822) (Caenogastropoda, Ampullariidae) is a highly invasive apple snail original from Central and Northern Argentina, Southern Brazil and Uruguay, and that has spread to Southeast Asia, North America and Europe where it has become a plague for rice and other crops [1], [2], [3]. Knowledge on the digestive tract of this polyphagous snail is essentially morphological and several specializations have been found [4] (Figure 1): (a) the buccal cavity receives the openings of a pair of salivary glands, (b) the esophagus has a pair of ventro-lateral pouches and an expanded crop in its medial portion that retains food during digestion, (c) a three-chambered stomach, which comprises a muscular gizzard, a vestibule that receives the openings of the midgut gland and the style sac, (d) a thin gut, (e) a coiled gut, and (f) the rectum with an anal gland. Open in a separate window Figure 1 Schematic view of the digestive system of host two types of endosymbiotic pigmented corpuscles which are considered morphotypes of the same organism and are identified as C and K corpuscles [5], [6], [7], [8]. The possible role of this endosymbiont in protein digestion was suggested by the unexpected finding of protease activity in extracts of C corpuscles isolated from the midgut gland of this snail (Vega, unpublished). Proteolytic enzymes have been studied in vetigastropods (genera and and zymography. Materials and Methods Animals and Culture Conditions Adult snails (shell length 35C40 mm) from a cultured strain of were used [8]. Room temperature was regulated (23C25C) and artificial lighting was provided 14 h per day. The animals were maintained in aquaria containing 2 L of tap water and the aquarium water was changed thrice weekly. Unless otherwise indicated, animals were fed with lettuce from Monday through Friday and this was supplemented with high protein fish food pellets (40% total protein content; Peishe Car Shulet?, Argentina) on Thursday and with excess toilet paper on Friday. Luminal Protease Activity Snail acclimation Animals were acclimated to feed exclusively on fish food pellets for 48 h, after which they were fasted for 24 h. After fasting, each animal was isolated in a vessel containing 70 mL water and 3 food pellets; 90 min after the first pellet was swallowed, each animal was immersed in an ice bath during 10 min to minimize pain and then the shell was cracked and the samples were obtained. The fish food pellets were approximately cubical (2.5 mm wide) and each one could be swallowed at once by the snails, without any visible fragmentation or spilling. The snails consumed all the offered food pellets during the 90 min period preceding ice-bathing. Sampling Immediately, after shell removal, an autostatic forceps was fixed on the posterior esophagus to prevent any passage of contents between the crop and the stomach during sampling. The crop and the style sac contents were collected with a 1 mL syringe by puncturing the walls with an 18-gauge needle. The coiled gut content was collected by gentle squeezing the sectioned gut. For protein extraction,.MW?=?molecular weight markers. snails. MW?=?molecular weight markers. Each lane was loaded with 75 g of protein.(TIF) pone.0066689.s002.tif (190K) GUID:?B2CB1F6F-0278-4103-9015-8A036122D57A Figure S3: Zymograms of gut contents in presence of E64, pepstatin A or EDTA. Zymograms (10% polyacrylamide gels copolymerized with 1 mg/mL gelatin) of contents of the crop, style sac and coiled gut contents that were incubated with or without E64 (10 M), or pepstatin A (1.45 M), or EDTA (1 mM). MW: molecular weight markers. Each lane was loaded with 75 g of protein.(TIF) pone.0066689.s003.tif (1.3M) GUID:?2139B90D-4FA2-44E0-B6FA-CFEB4D0B90F1 Abstract Digestive proteases of the digestive tract of the apple snail were studied. Luminal protease activity was found in the crop, the style sac and the coiled gut and was significantly higher in the coiled gut. Several protease bands and their apparent molecular weights were identified in both tissue extracts and luminal contents by gel zymography: (1) a 125 kDa protease in salivary gland extracts and in the crop content; (2) a 30 kDa protease throughout all studied luminal contents and in extracts of the midgut gland and of the endosymbionts isolated from this gland; (3) two proteases of 145 and 198 kDa in the coiled gut content. All these proteases were inhibited by aprotinin, a serine-protease inhibitor, and showed maximum activity between 30C and 35C and pH between 8.5 and 9.5. Tissue L-alanine-N-aminopeptidase activity was determined in the wall of the crop, the style sac and the coiled gut and was significantly higher in the coiled gut. Our findings show that protein digestion in is carried out through a battery of diverse proteases originated from the salivary glands and the endosymbionts lodged in the midgut gland and by proteases of uncertain origin that occur in the coiled gut lumen. Introduction (Lamarck 1822) (Caenogastropoda, Ampullariidae) is a highly invasive apple snail original from Central and Northern Argentina, Southern Brazil and Uruguay, and that has spread to Southeast Asia, North America and Europe where it has become a plague for rice and other crops [1], [2], [3]. Knowledge on the Mulberroside A digestive tract of this polyphagous snail is essentially morphological Mulberroside A and several specializations have been found [4] (Number 1): (a) the buccal cavity receives the openings of a pair of salivary glands, (b) the esophagus has a pair of ventro-lateral pouches and an expanded crop in its medial portion that retains food during digestion, (c) a three-chambered belly, which comprises a muscular gizzard, a vestibule that receives the openings of the midgut gland and the style sac, (d) a thin gut, (e) a coiled gut, and (f) the rectum with an anal gland. Open in a separate window Number 1 Schematic look at of the digestive system of sponsor two types of endosymbiotic pigmented corpuscles which are considered morphotypes of the same organism and are identified as C and K corpuscles [5], [6], [7], [8]. The possible role of this endosymbiont in protein digestion was suggested by the unpredicted getting of protease activity in components of C corpuscles isolated from your midgut gland of this snail (Vega, unpublished). Proteolytic enzymes have been analyzed in vetigastropods (genera and and zymography. Materials and Methods Animals and Culture Conditions Adult snails (shell size 35C40 mm) from a cultured strain of were used [8]. Space temperature was regulated (23C25C) and artificial lighting was offered 14 h per day. The animals were managed in aquaria comprising 2 L of tap water and the aquarium water was changed thrice weekly. Unless normally indicated, animals were fed with lettuce from Monday through Friday and this was supplemented with high protein fish food pellets (40% total protein content material; Peishe Car Shulet?, Argentina) on Thursday and with extra toilet paper on Friday. Luminal Protease Activity Snail acclimation Animals were acclimated to feed exclusively on fish food pellets for 48 h, after which they were fasted for 24 h. After fasting, each animal was isolated inside a vessel comprising 70 mL water and 3 food pellets; 90 min after the 1st pellet was swallowed, each animal was immersed in an snow bath during 10 min to minimize pain and then the shell was cracked and the samples were obtained. The fish food pellets were approximately cubical (2.5 mm wide) and each one could be swallowed at once from the snails, without any visible fragmentation or spilling. The snails.