ABSTRACT
The coffee berry borer, Hypothenemus hampei (Ferrari), is an important devastating coffee pest worldwide. Both trypsin and chymotrypsin enzyme activities from H. hampei larval midgut can be inactivated by proteinaceous enzyme-inhibitors. A serine proteinase inhibitor belonging to the Bowman-Birk class was purified from a wild accession of Phaseolus coccineus L. seeds. The inhibitor (PcBBI1) is a cysteine-rich protein that is heat-stable at alkaline pH. MALDI-TOF/MS analysis showed that PcBBI1 occurs in seeds as a monomer (8689 Da) or dimer (17,378 Da). Using in vitro inhibition assays, it was found that PcBBI1 has a high inhibitory activity against H. hampei trypsin-like enzymes, bovine pancreatic chymotrypsin, and trypsin. According to this, PcBBI1 could be a promising tool to make genetically modified coffee with resistance to coffee berry borer.
Subject(s)
Coleoptera/enzymology , Peptide Hydrolases/metabolism , Phaseolus/chemistry , Serine Proteinase Inhibitors/pharmacology , Amino Acid Sequence , Animals , Larva/enzymology , Molecular Sequence Data , Molecular Weight , Seeds/chemistry , Serine Proteinase Inhibitors/chemistryABSTRACT
The laticifer fluid of Calotropis procera is rich in proteins and there is evidence that they are involved in the pharmacological properties of the latex. However, not much is known about how the latex-containing proteins are produced or their functions. In this study, laticifer proteins of C. procera were pooled and examined by 1D and 2D electrophoresis, masses spectrometry (MALDI-TOF) and characterized in respect of proteolytic activity and oxidative enzymes. Soluble laticifer proteins were predominantly composed of basic proteins (PI>6.0) with molecular masses varying between 5 and 95 kDa. Proteins with a molecular mass of approximately 26,000 Da were more evident. Strong anti-oxidative activity of superoxide dismutase (EC 1.15.1.1) (1007.74+/-91.89 Ug(-1)DM) and, to a lesser extent ascorbate peroxidase (EC 1.11.1.1) (0.117(d)+/-0.013 microMol H(2)O(2)g(-1)min(-1)), were detected. However, catalase (EC 1.11.1.6) was absent. The strong proteolytic activities of laticifer proteins from C. procera were shown to be shared by at least four distinct cysteine proteinases (EC 3.4.22.16) that were isolated by gel filtration chromatography. Serine and metaloproteinases were not detected and aspartic proteinase activities were barely visible. Chitinases (EC 3.2.1.14) were also isolated in a chitin column and their activities quantified. The presence of these enzymatic activities in latex from C. procera may confirm their involvement in resistance to phytopathogens and insects, mainly in its leaves where the latex circulates abundantly.
Subject(s)
Calotropis/metabolism , Latex/metabolism , Plant Proteins/metabolism , Ascorbate Peroxidases , Chitin/chemistry , Chitin/metabolism , Chitinases/chemistry , Chitinases/metabolism , Chromatography, Affinity , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Latex/chemistry , Molecular Weight , Peroxidases/chemistry , Peroxidases/metabolism , Plant Proteins/chemistry , Protons , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Superoxide Dismutase/chemistry , Superoxide Dismutase/metabolism , TemperatureABSTRACT
Fibronectin (FN), a large family of plasma and extracellular matrix glycoproteins, plays an important role in wound healing. PURPOSE: To evaluate the effect of fibronectin on the healing of sutured duodenal wounds, correlating with the serum and tissue level of the substance. METHODS: An experimental study was done in 30 adult Wistar rats divided into two group. In the control group (n=15) a duodenal suture was treated with saline solution 0,9 percent and in the test group the duodenal wounds were treated with 1 percent FN. The duodenal wound healing process was studied in the 5th, 7tn and 10th postoperative days, by histological sections stained by hematoxylin-eosin, Masson trichromic and immunohistochemical reaction for FN. A digital histological grading system was used to obtain a score for each group and to observe the healing process. RESULTS: the FN was present in the several layers of the duodenum and the cellular and plasmatic FN increased with the evolution of healing. In the test group the FN enhanced the wound healing within 5, 7 and 10 days after injury, when compared with the control group. CONCLUSION: The topical use of FN in duodenal sutured wounds in rats enhances healing by stimulating the appearence of fibroblasts into the wound site and development of granulation tissue. This acceleration of the repair process may have an important application in the healing of duodenal wounds.
