Inhibition mechanism of human salivary α-amylase by lipid transfer protein from Vigna unguiculata.

VuLTP1.1, a LTP1 from Vigna unguiculata, inhibits 78.1 % of the human salivary α-amylase (HSA) activity at 20 µM. We had performed a correlation study between VuLTP1.1 structure and HSA inhibitory activity and showed that two VuLTP1.1 regions are responsible for HSA inhibition. In one of them we had characterized the crucial importance of an Arg39 for inhibition. In this work, we analyzed the VuLTP1.1-HSA interaction by protein-protein docking to understand the most probable interaction model and the mechanism of HSA inhibition by VuLTP1.1. The VuLTP1.1 tertiary structure quality and refinement as well as the docking assay between VuLTP1.1 and HSA were done by bioinformatic programs. HSA inhibition occurs by direct interaction of the VuLTP1.1 with the HSA causing the obstruction of the carbohydrate biding cleft with Gibbs free energy of -18.5 Kcal/mol and the dissociation constant of 2.6E-14 M. The previously identified Arg39 of VuLTP1.1 is burrowed into the active site of the HSA and there it interacts with the Asp300 of HSA catalytic site by a hydrogen bond. We had confirmed the importance of the Arg39 of VuLTP1.1 for the HSA inhibition which interacts with the Asp300 at the HSA active site. I-2, a LTP-like peptide, presents the same HSA inhibition pattern that VuLTP1.1, which indicates that the inhibition mechanism of the LTPs towards α-amylase is very similar. For the best of our knowledge, it is the first time that the HSA inhibition mechanism was understood and described for the LTP1s using VuLTP1.1 and I-2 as prototype inhibitors.

Anti-Neuroblastoma Properties of a Recombinant Sunflower Lectin.

According to their sugar recognition specificity, plant lectins are proposed as bioactive proteins with potential in cancer treatment and diagnosis. Helja is a mannose-specific jacalin-like lectin from sunflower which was shown to inhibit the growth of certain fungi. Here, we report its recombinant expression in a prokaryotic system and its activity in neurobalstoma cells. Helja coding sequence was fused to the pET-32 EK/LIC, the enterokinase/Ligation-independent cloning vector and a 35 kDa protein was obtained in Escherichia coli representing Helja coupled to thioredoxin (Trx). The identity of this protein was verified using anti-Helja antibodies. This chimera, named Trx-rHelja, was enriched in the soluble bacterial extracts and was purified using Ni+2-Sepharose and d-mannose-agarose chromatography. Trx-rHelja and the enterokinase-released recombinant Helja (rHelja) both displayed toxicity on human SH-SY5Y neuroblastomas. rHelja decreased the viability of these tumor cells by 75% according to the tetrazolium reduction assay, and microscopic analyses revealed that the cell morphology was disturbed. Thus, the stellate cells of the monolayer became spheroids and were isolated. Our results indicate that rHelja is a promising tool for the development of diagnostic or therapeutic methods for neuroblastoma cells, the most common solid tumors in childhood.

The structure of colleters in several species of Simira (Rubiaceae).

BACKGROUND AND AIMS: Colleters are secretory structures consisting of a parenchymatic middle axis surrounded by a layer of palisade-like epidermal cells. Colleters occur in a large number of rubiaceous species. Their function is to protect the developing shoot apex. They are also taxonomically useful in the Rubiaceae. This study characterized the structure of the colleters of Simira glaziovii, S. pikia and S. rubra and the biochemistry of secretions in S. glaziovii. METHODS: Stipules of the shoot apices of the three species studied were collected at Barragem de Saracuruna, in Rio de Janeiro state, Brazil. The samples were fixed according to the usual methods for light and electron microscopy. Secretion stipules of S. glaziovii were washed with 0.1 m Tris-HCl plus 0.1 %Triton X-100 to extract proteins and carbohydrates. KEY RESULTS: Colleters in these species are located at the base of the stipule. Each species shows a different pattern of distribution. They form as emergentia from the stipules. Simira glaziovii was different from the other two species because it exhibited vascular traces. The epidermal cells of colleters have dense cytoplasm, nuclei, small vacuoles, endoplasmic reticulum, Golgi apparatus, mitochondria and extraplasmic spaces if they are secretory. The outer cell wall of the mature colleters differs from the outer cell wall of stipule cells and immature colleters. Both carbohydrates and proteins were found in secretions from the stipules of S. glaziovii. CONCLUSIONS: Few ultrastructural differences were noted among the three species. These secretory structures not only protect the shoot apex, but also have taxonomic importance below the genus level.