Structure-Activity relationship in mutated pyrazinamidases from Mycobacterium tuberculosis

The pncA gene codes the pyrazinamidase of Mycobacterium tuberculosis, which converts pyrazinamide to ammonia and pyrazinoic-acid, the active antituberculous compound. Pyrazinamidase mutations are associated to pyrazinamide-resistant phenotype, however how mutations affect the structure of the pyrazinamidase, and how structural changes affect the enzymatic function and the level of pyrazinamide-resistance is unknown. The structures of mutated pyrazinamidases from twelve Mycobacterium tuberculosis strains and the pyrazinamide-susceptible H37Rv reference strain were modelled using homology modelling and single amino acid replacement. Physical-chemical and structural parameters of each pyrazinamidase were calculated. These parameters were: The change of electrical charge of the mutated amino acid, the change of volume of the mutated amino acid, the change of a special amino acid, the distance of the mutated amino acid to the active site, the distance of the mutated amino acid to the metal-coordination site, and the orientation of the side-chain of the mutated amino acid. The variability of the enzymatic activity of the recombinant pyrazinamidases, and the microbiological susceptibility to pyrazinamide determined by BACTEC 460TB, were modelled in multiple linear regressions. Physical-chemical and structural parameters of the mutated pyrazinamidases were tested as predictors. Structural and physical-chemical variations of the pyrazinamidase explained 75% of the variability of the enzymatic activity, 87% of the variability of the kinetic constant and 40% of the variability of the pyrazinamide-resistance level. Based on computer models of mutated pyrazinamidases, the structural parameters explained a high variability of the enzymatic function, and to a lesser extent the resistance level.     

The natural compound trans-chalcone induces programmed cell death in Arabidopsis thaliana roots

Chalcone (1,3-diphenyl-2-propen-1-one) is an aromatic ketone precursor of important molecules in plants such as flavonoids or anthocyanins. Its phytotoxicity has been demonstrated on different plant species, but to date little is known about the mechanisms of action of this secondary metabolite at plant cellular level. Detailed analysis by light and transmission electron microscopy (TEM) was conducted to examine the root meristems' ultrastructure of control and chalcone-treated Arabidopsis seedlings. Mitochondrial dysfunction was analysed by measuring mitochondrial membrane potential with JC-1 fluorochrome. Finally, acridine orange/ethidium bromide staining was used for the detection of programmed cell death. Microscopy revealed tissue alterations, inhibition of root hair formation and important changes after 7 and 14 d at the chalcone IC(50) value. Chalcone-treated cells showed signs of programmed cell death such as mitochondrial condensation, disruption of organelles and chromatin fragmentation. Acridine orange/ethidium bromide staining confirmed the programmed cell death, which could be induced by the reduction of mitochondrial transmembrane potential (ΔΨ(m)) that was detected after chalcone treatment. These results confirm the phytotoxic activity of chalcone on Arabidopsis seedlings, the alteration of mitochondrial membrane potential and the induction of programmed cell death.     

Detoxifying Antitumoral Drugs via Nanoconjugation: The Case of Gold Nanoparticles and Cisplatin

Nanoparticles (NPs) have emerged as a potential tool to improve cancer treatment. Among the proposed uses in imaging and therapy, their use as a drug delivery scaffold has been extensively highlighted. However, there are still some controversial points which need a deeper understanding before clinical application can occur. Here the use of gold nanoparticles (AuNPs) to detoxify the antitumoral agent cisplatin, linked to a nanoparticle via a pH-sensitive coordination bond for endosomal release, is presented. The NP conjugate design has important effects on pharmacokinetics, conjugate evolution and biodistribution and results in an absence of observed toxicity. Besides, AuNPs present unique opportunities as drug delivery scaffolds due to their size and surface tunability. Here we show that cisplatin-induced toxicity is clearly reduced without affecting the therapeutic benefits in mice models. The NPs not only act as carriers, but also protect the drug from deactivation by plasma proteins until conjugates are internalized in cells and cisplatin is released. Additionally, the possibility to track the drug (Pt) and vehicle (Au) separately as a function of organ and time enables a better understanding of how nanocarriers are processed by the organism.     

Sublethal effects of antibiosis resistance on the reproductive biology of two spittlebug (Hemiptera: Cercopidae) species affecting Brachiaria spp

Several greenhouse experiments were used to measure how high levels of antibiosis resistance to nymphs in two interspecific Brachiaria (brachiariagrass) hybrids affect life history parameters of the spittlebugs Aeneolamia varia (F.) and Zulia carbonaria (Lallemand), two of the most important spittlebug (Hemiptera: Cercopidae) species affecting Brachiaria production in Colombia. The A. varia-resistant hybrid CIAT 36062, the Z. carbonaria-resistant hybrid SX01NO/0102, and the susceptible accession CIAT 0654 were used to compare the effect of all possible combinations of food sources for nymphs and adults. Calculation of growth indexes showed a significant impact of antibiosis resistance on the biology of immature stages of both species. Median survival times of adults feeding on resistant genotypes did not differ from those recorded on the susceptible genotype, suggesting that factors responsible for high mortality of nymphs in the resistant hybrids did not affect adult survival. Rearing nymphs of A. varia on CIAT 36062 and of Z. carbonaria on SX01NO/0102 had deleterious sublethal effects on the reproductive biology of resulting adult females. It is concluded that high nymphal mortality and subsequent sublethal effects of nymphal antibiosis on adults should have a major impact on the demography of the two spittlebug species studied.     

Slit2-Mediated chemorepulsion and collapse of developing forebrain axons

Diffusible chemorepellents play a major role in guiding developing axons toward their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a novel repulsive guidance system that prevents inappropriate axons from crossing the CNS midline; this repulsive system is mediated by the Roundabout (Robo) receptor and its secreted ligand Slit. In rodents, Robo and Slit are expressed in the spinal cord and Slit can repel spinal motor axons in vitro. Here, we extend these findings into higher brain centers by showing that Robo1 and Robo2, as well as Slit1 and Slit2, are often expressed in complementary patterns in the developing forebrain. Furthermore, we show that human Slit2 can repel olfactory and hippocampal axons and collapse their growth cones.     

Inhibitory effect of Thymus vulgaris and Origanum vulgare essential oils on virulence factors of phytopathogenic Pseudomonas syringae strains

• Pseudomonas syringae is a phytopathogenic bacterium that causes lesions in leaves during the colonisation process. The damage is associated with production of many virulence factors, such as biofilm and phytotoxins. The essential oils of Thymus vulgaris (thyme) and Origanum vulgare (oregano) have been demonstrated to inhibit P. syringae. The aim of this study was to investigate the effects of T. vulgaris and O. vulgare essential oils on production of virulence factors of phytopathogenic P. syringae strains, including anti‐biofilm and anti‐toxins activities.
• The broth microdilution method was used for determination of MIC and biofilm inhibition assays. Coronatine, syringomycin and tabtoxin were pheno‐ and genotypically evaluated.
• Both oils showed good inhibitory activity against P. syringae, with MIC values from 1.43 to 11.5 mg·ml^?1 for thyme and 5.8 to 11.6 mg·ml^?1 for oregano. Biofilm formation, production of coronatine, syringomycin and tabtoxin were inhibited by thyme and oregano essential oil in most strains.
• The results presented here are promising, demonstrating the bactericidal activity and reduction of virulence factor production after treatment with thyme and oregano oil, providing insight into how they exert their antibacterial activity. These natural products could be considered in the future for the control of diseases caused by P. syringae.

     

Design, synthesis, and biological evaluation of glycine-based molecular tongs as inhibitors of Abeta1-40 aggregation in vitro

A series of N-terminus benzamides of glycine-based symmetric peptides, linked to m-xylylenediamine and 3,4′-oxydianiline spacers, were prepared and tested as inhibitors of β-amyloid peptide Aβ_1–40 aggregation in vitro. Compounds with good anti-aggregating activity were detected. Polyphenolic amides showed the highest anti-aggregating activity, with IC₅₀ values in the micromolar range. Structure–activity relationships suggested that π–π stacking and hydrogen-bonding interactions play a key role in the inhibition of Aβ_1–40 self-assembly leading to amyloid fibrils.     

An electron microscope study of the regenerating nerve fibers

Summary Guinea pig sciatic nerves were severed in order to obtain a regenerative process of the nerve fibers. The animals were killed at different periods of time after the severing (24 hours, 6 days and 12 days) and the specimens obtained were prepared for electronmicroscopic study.The nerve fiber growing extremities (growing cones) were specially studied. The growing cones showed the following components: a) microvesicles; b) mitochondria; c) multivesicular bodies.The microvesicles are hollow elements of about 200 to 700 Å. They constitute the main component of the growing cone. The mitochondria were seen as elongated bodies of 80 m. They were seen in many cases changing to round dense bodies which appear to break-up in irregularly-shaped fragments.The multivesicular bodies were found present in most of the growing cones. Protoneurofibrils do not exist in the growing cone but a close relationship between microvesicles and protoneurofibrils was found in the segments next to the growing cone.The above-mentioned components were found in all growing cones, disregarding the time elapsed after the nerve severing.Director of the Institute.Assistant of the Department of Neurohistology and Experimental Histology.Head of the Department of Cell Ultrastructure.