Anti-Proliferative and Apoptotic Activities of Rumex crispus.

Colorectal cancer is the fourth leading cause of cancer death and the third most common cancer diagnosed in the United States. Several anticancer compounds from natural products have been of great interest in cancer chemotherapy and are currently in clinical trials. Natural products that present the targeted killing of cancerous cell and are soluble in water with minimal side effects are ideal candidates. In this study, water-soluble compounds from Rumex crispus plants were screened for anti-proliferative and apoptotic activity against human colorectal adenocarcinoma (DLD-1) cells. The most potent fraction with the highest cell killing and caspase fold change rates was selected for further experiments. The observed changes were further validated by measuring the caspase fold change using RT-qPCR. Furthermore, gene transcript levels were evaluated using an RT2 Profiler assay and a microarray experiment. Our results showed that the most potent L19 fraction exhibits anti-proliferative activity in a dose-dependent manner. The L19 fraction was found to induce apoptotic pathways by triggering different caspases and inflammatory pathways through the activation of non-apoptotic genes. Our study identified and validated the anticancer property of the L19 fraction, which can serve as a strong lead compound for the synthesis of other novel potent analogues.

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.

Structure and properties of the exopolysaccharides produced by Pseudomonas mutabilis T6 and P. mutabilis ATCC 31014.

This paper presents a study on the purification, primary structure, and rheological properties of exopolysaccharides isolated from cultures of Pseudomonas mutabilis T6 and P. mutabilis ATCC 31014. Both polymers are exopolysaccharides of D-mannose. The mannan isolated from P. mutabilis T6 contains on average about 5% of residual ß-D-glucose, in contrast to the mannan from P. mutabilis ATCC 31014, which contained only trace amounts of residual ß-D-glucose (less than 1%). Based on the (13)C NMR spectra, all of the remaining carbohydrates in the exopolysaccharides occur in the form of pyranose rings. All of the mannose residues have the α configuration at the anomeric carbon atom while the glucose adopts the ß configuration. The reaction of both polysaccharide hydrolysates with an optically active alcohol indicates that all of the sugar residues have the D configuration. We found that the main chain of the exopolysaccharide is composed of mannose residues connected through α-(1→6) linkages, of which a large number are substituted on O2 with D-mannose and the remaining are substituted with di- to pentasaccharide fragments. The rheological properties of the exopolysaccharide isolated from P. mutabilis T6 show that its viscosity is over 30 times greater than that of P. mutabilis ATCC 31014.

Characterization of a prolyl endoprotease from Eurygaster integriceps Puton (Sunn pest) infested wheat.

Sunn pest, Eurygaster integriceps, Puton, infested and uninfested wheat seeds were obtained from the International Center for Agriculture Research in the Dry Areas (ICARDA), Aleppo, Syria, with the primary objective to identify the type of enzyme deposited by the Sunn pest on the wheat responsible for the gluten degradation. Enzyme levels were extremely low due to the enzyme being secreted by the insect in localized areas on the seed. Only extract from the infested wheat contained glutenase activity. Anion exchange, Cu(2+) sepharose, and gel filtration chromatography were used to partially purify and enrich protein samples from both infested wheat and uninfested wheat. An SDS-gluten assay was used to show gluten specificity while a commercially available chromogenic proline peptide, benzyloxycarbonyl-Gly-Pro-p-nitroanalide (ZGPpNA), was utilized to identify fractions containing the active proline specific enzyme activity and to determine Michaelis-Menten kinetics. Despite low levels of enzyme on the infested wheat, the enzyme was partially purified and enriched exhibiting a specific activity of 4.5 U/mg of total protein for gluten in a SDS gluten assay (1 U of enzyme activity was defined as the decrease in gel height in millimeters in 1 h) and exhibited a high-affinity Km of 65 microM for ZGPpNA, cleaving at the carboxy terminus of the proline residue. The enzyme exhibited optimal activity between pH 8 and 10.0 at temperatures between 20 degrees and 35 degrees C. The enzyme was identified to be a prolyl endoprotease.