Orally hypoglycemic activity of an insulin mimetic glycoprotein isolated from Cnidoscolus quercifolius Pohl. (Euphorbiaceae) seeds, Cq-IMP.

The genus Cnidoscolus (Euphorbiaceae) is widely distributed in tropical areas. In the Northeast of Brazil, the species C. quercifolius is endemic and has been used in traditional medicine. In this study, a novel protein was isolated from C. quercifolius seeds and characterized by its molecular weight, primary structure, isoelectric point (pI), and carbohydrate content. The hypoglycemic activity of this protein was investigated by in vitro assay with the RIN-5F glucose-responsive cell line and in vivo test using alloxan-induced diabetic mice models. In addition, safe use of the protein was also investigated by cytotoxicity, hemagglutinating, and immunogenicity assays. The protein which was named Cq-IMP (Cnidoscolus quercifolius - Insulin Mimetic Protein) showed a single 11.18 KDa glycopolypeptide chain (16.4% of carbohydrates, m/m), pI of 8.0 and N-terminal sequence (TKDPELKQcKKQQKKqQQYDDDDKK) with similarity around 46-62% to sucrose binding protein-like and vicilin-like protein that was confirmed by mass spectrometry tryptic peptides analysis. Besides that, Cq-IMP presented anti-insulin antibody cross-reactivity as hypoglycemic activity in both in vitro and in vivo models. Additionally, it did not present any toxicity by methods tested. In conclusion, Cq-IMP is an insulin-mimetic protein, with a potent hypoglycemic activity and no toxicity showing great potential for therapeutic applications and drug development.

Identification of a novel antimicrobial peptide from Brazilian coast coral Phyllogorgia dilatata.

The marine ecosystem is able to provide enormous biomolecule diversity that could be used for treatment of various diseases. In this highly competitive environment, organisms need chemical barriers to reduce or avoid microorganism contamination. Among the molecules that protect these animals the antimicrobial peptides (AMPs) are included. In the present study, crude extracts of coral coral specimens Carijoa riisei, Muriceopsis sulphurea, Neospongodes atlantica, Palythoa caribeorum, Phyllogorgia dilatata and Plexaurella grandiflora were challenged against multiple Grampositive and -negative bacteria showing different activities. P. dilatata crude extract showed the antibacterial activity, and was ammonium-sulfate (0-40%) fractionated, being able to control the growth of K. pneumoniae, S. flexineri and S. aureus. Rich-fraction was further purified by using Amicon® Ultra Centrifugal 10 kDa associated with reversed-phase HPLC chromatography (C18), producing the peptide named Pd-AMP1. Pd-AMP1 was able to inhibit S. aureus development. Mass spectrometry analyses showed a monoisotopic mass of 5372.66 Da and N-terminal sequence showed no significant match with databank. In this view, the prospecting of protein biomolecules and biotechnological potential from marine animals is still little explored and may serve as an alternative to common antibiotics.

Purification of a chitin-binding protein from Moringa oleifera seeds with potential to relieve pain and inflammation.

Moringa oleifera Lam. is a perennial multipurpose tree that has been successfully used in folk medicine to cure several inflammatory processes. The aim of this study was to purify and characterize a chitin-binding protein from Moringa oleifera seeds, named Mo-CBP4, and evaluate its antinociceptive and anti-inflammatory effects in vivo. The protein was purified by affinity chromatography on chitin followed by ion exchange chromatography. Acetic acid-induced abdominal constrictions assay was used for the antinociceptive and anti-inflammatory activity assessments. Mo-CBP4 is a glycoprotein (2.9% neutral carbohydrate) composed of two protein subunits with apparent molecular masses of 28 and 18 kDa (9 kDa in the presence of reducing agent). The intraperitoneal injection of Mo-CBP4 (3.5 and 10 mg/kg) into mice 30 min before acetic acid administration potently and significantly reduced the occurrence of abdominal writhing in a dose dependent manner by 44.7% and 100%, respectively. In addition, the oral administration of the protein (10 mg/kg) resulted in 18% and 52.8% reductions in abdominal writhing when given 30 and 60 min prior to acetic acid administration, respectively. Mo-CBP4, when administered by intraperitoneal route, also caused a significant and dose-dependent inhibition of peritoneal capillary permeability induced by acid acetic and significantly inhibited leukocyte accumulation in the peritoneal cavity. In conclusion, this pioneering study describes that the chitin-binding protein Mo-CBP4, from M. oleifera seeds, exhibits anti-inflammatory and antinociceptive properties and scientifically supports the use of this multipurpose tree in folk medicine.

Effects of a novel pathogenesis-related class 10 (PR-10) protein from Crotalaria pallida Roots with papain inhibitory activity against root-knot nematode Meloidogyne incognita.

A novel pathogenesis-related class 10 (PR-10) protein with papain inhibitory activity, named CpPRI, was purified from Crotalaria pallida roots by ammonium sulfate precipitation followed by three reverse-phase high-performance liquid chromatographies (HPLCs). CpPRI is made up of a single polypeptide chain with a M(r) of 15 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This protein exhibited a K(i) value of 1.8 x 10(-9) M and operates via a noncompetitive inhibition mechanism. The alignment of the N-terminal amino acid sequence of CpPRI with other proteins revealed its identity with PR-10 proteins. CpPRI acts against digestive proteinase from root-knot nematode Meloidogyne incognita and demonstrated nematostatic and nematicide effects on this parasite in bioassays. In a localization study, fluorescein-5-isothiocyanate (FITC)-CpPRI was observed to internalize and diffuse over the entire J2 body after 6 h of incubation. This fact could explain the natural tolerance of this plant species to nematodes.

Expression of the Canavalia brasiliensis lectin (ConBr) in tobacco plants.

Tobacco plants were transformed with gene constructs encoding prepro-ConBr (Canavalia brasiliensis lectin). Transgenic plants confirmed by PCR expressed the recombinant protein as revealed by Western blot. However, the apparent molecular mass of the recombinant polypeptide (ca. 34 kDa) was higher than the native lectin (about 30 kDa), showing that further proteolytic processing of pro-ConBr was not detected.