RESUMEN
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.
Asunto(s)
Escarabajos/enzimología , Péptido Hidrolasas/metabolismo , Phaseolus/química , Inhibidores de Serina Proteinasa/farmacología , Secuencia de Aminoácidos , Animales , Larva/enzimología , Datos de Secuencia Molecular , Peso Molecular , Semillas/química , Inhibidores de Serina Proteinasa/químicaRESUMEN
Toxicity and toxin identification in Colomesus asellus, an Amazonian (Brazil) freshwater puffer fish. By using four different techniques--mouse bioassay, ELISA, HPLC and mass spectrometry-we evaluated the toxicity in the extracts of C. asellus, a freshwater puffer fish from the rivers of the Amazon, and identified for the first time the components responsible for its toxicity. The T20G10 monoclonal antibody raised against TTX, and employed in an indirect competitive enzyme immunoassay, showed very low affinity for the C. asellus extracts, indicating that TTX and its analogs are not the main toxic components of the extracts. This antibody was efficient in detecting presence of TTX in a total extract of Sphoeroides spengleri, which is one of the most toxic puffer fish found in the Atlantic coast. Extracts of C. asellus were toxic when administered intraperitonially into mice with an average toxicity of 38.6+/-12 mouse unit (MU)/g, while HPLC analysis indicated a lower toxin content (7.6+/-0 5MU/g). The HPLC profile showed no traces of TTX, but only the presence of PSPs (STX, GTX 2 and GTX 3). These toxins were also confirmed by electrospray ionization mass spectrometry.