Cloning, Expression, Sequence Analysis and Homology Modeling of the Prolyl Endoprotease from Eurygaster integriceps Puton

Eurygaster integriceps Puton, commonly known as sunn pest, is a major pest of wheat in Northern Africa, the Middle East and Eastern Europe. This insect injects a prolyl endoprotease into the wheat, destroying the gluten. The purpose of this study was to clone the full length cDNA of the sunn pest prolyl endoprotease (spPEP) for expression in E. coli and to compare the amino acid sequence of the enzyme to other known PEPs in both phylogeny and potential tertiary structure. Sequence analysis shows that the 5ꞌ UTR contains several putative transcription factor binding sites for transcription factors known to be expressed in Drosophila that might be useful targets for inhibition of the enzyme. The spPEP was first identified as a prolyl endoprotease by Darkoh et al., 2010. The enzyme is a unique serine protease of the S9A family by way of its substrate recognition of the gluten proteins, which are greater than 30 kD in size. At 51% maximum identity to known PEPs, homology modeling using SWISS-MODEL, the porcine brain PEP (PDB: 2XWD) was selected in the database of known PEP structures, resulting in a predicted tertiary structure 99% identical to the porcine brain PEP structure. A Km for the recombinant spPEP was determined to be 210 ± 53 µM for the zGly-Pro-pNA substrate in 0.025 M ethanolamine, pH 8.5, containing 0.1 M NaCl at 37 °C with a turnover rate of 172 ± 47 µM Gly-Pro-pNA/s/µM of enzyme

Enzymes as Feed Additive to Aid in Responses Against Eimeria Species in Coccidia-Vaccinated Broilers Fed Corn-Soybean Meal Diets with Different Protein Levels

This research aimed to evaluate the effects of adding a combination of exogenous enzymes to starter diets varying in protein content and fed to broilers vaccinated at day of hatch with live oocysts and then challenged with mixed Eimeria spp. Five hundred four 1-d-old male Cobb-500 chickens were distributed in 72 cages. The design consisted of 12 treatments. Three anticoccidial control programs [ionophore (IO), coccidian vaccine (COV), and coccidia-vaccine + enzymes (COV + EC)] were evaluated under 3 CP levels (19, 21, and 23%), and 3 unmedicated-uninfected (UU) negative controls were included for each one of the protein levels. All chickens except those in unmedicated-uninfected negative controls were infected at 17 d of age with a mixed oral inoculum of Eimeria acervulina, Eimeria maxima, and Eimeria tenella. Live performance, lesion scores, oocyst counts, and samples for gut microflora profiles were evaluated 7 d postinfection. Ileal digestibility of amino acids (IDAA) was determined 8 d postinfection. Microbial communities (MC) were analyzed by G + C%, microbial numbers were counted by flow cytometry, and IgA concentrations were measured by ELISA. The lowest CP diets had poorer (P ≤ 0.001) BW gain and feed conversion ratio in the preinfection period. Coccidia-vaccinated broilers had lower performance than the ones fed ionophore diets during pre- and postchallenge periods. Intestinal lesion scores were affected (P ≤ 0.05) by anticoccidial control programs, but responses changed according to gut section. Feed additives or vaccination had no effect (P ≥ 0.05) on IDAA, and diets with 23% CP had the lowest (P ≤ 0.001) IDAA. Coccidial infection had no effect on MC numbers in the ileum but reduced MC numbers in ceca and suppressed ileal IgA production. The COV + EC treatment modulated MC during mixed coccidiosis infection but did not significantly improve chicken performance. Results indicated that feed enzymes may be used to modulate the gut microflora of cocci-vaccinated broiler chickens

The Addition of Arachidin 1 or Arachidin 3 to Human Rotavirus-infected Cells Inhibits Viral Replication and Alters the Apoptotic Cell Death Pathway

Rotavirus (RV) infections are a leading cause of severe gastroenteritis in infants and children under the age of five. There are two vaccines available in the United States and one in India that can be administered early in childhood, however they only protect against specific strains1. From our previous work, both arachidin-1 (A1) and arachidin-3 (A3) from peanut (Arachis hypogaea) hairy root cultures significantly inhibit simian RV replication2,3,4. The purpose of this study was to determine if a human intestinal cell line, HT29.f8, infected with a human RV, Wa, was affected by A1 and A3. Cell viability assays were utilized to determine if A1 and A3 affect the HT29.f8 cells with/without RV infections. At eighteen hours post infection (hpi), supernatants from the RV-infected HT29.f8 cells with/without the arachidins were used in plaque forming assays to quantify and compare the amount of infectious RV particles that are produced during an infection. Transmission electron microscopy (TEM) was used to visualize cell ultrastructure and individual RV particles. Additionally, tunable resistive pulse sensing technology (TRPS) using the qNano system by IZON was employed to quantify and measure virus particle sizes, and display the size distribution of RV particles. Likewise, quantitative real time polymerase chain reactions (qRT-PCR) were performed to determine if A1 and A3 regulated cell death pathways in the HT29.f8 cell line. This data will guide our future studies to determine the antiviral mechanism(s) of action of A1 and A3

Cloning, Expression, Sequence Analysis and Homology Modeling of the Prolyl Endoprotease from Eurygaster integriceps Puton

eurygaster integriceps Puton, commonly known as sunn pest, is a major pest of wheat in Northern Africa, the Middle East and Eastern Europe. This insect injects a prolyl endoprotease into the wheat, destroying the gluten. The purpose of this study was to clone the full length cDNA of the sunn pest prolyl endoprotease (spPEP) for expression in E. coli and to compare the amino acid sequence of the enzyme to other known PEPs in both phylogeny and potential tertiary structure. Sequence analysis shows that the 5ꞌ UTR contains several putative transcription factor binding sites for transcription factors known to be expressed in Drosophila that might be useful targets for inhibition of the enzyme. The spPEP was first identified as a prolyl endoprotease by Darkoh et al., 2010. The enzyme is a unique serine protease of the S9A family by way of its substrate recognition of the gluten proteins, which are greater than 30 kD in size. At 51% maximum identity to known PEPs, homology modeling using SWISS-MODEL, the porcine brain PEP (PDB: 2XWD) was selected in the database of known PEP structures, resulting in a predicted tertiary structure 99% identical to the porcine brain PEP structure. A Km for the recombinant spPEP was determined to be 210 ± 53 µM for the zGly-Pro-pNA substrate in 0.025 M ethanolamine, pH 8.5, containing 0.1 M NaCl at 37 °C with a turnover rate of 172 ± 47 µM Gly-Pro-pNA/s/µM of enzyme