Antimicrobial peptides from chili pepper seeds causes yeast plasma membrane permeabilization and inhibits the acidification of the medium by yeast cells

During the last few years, a growing number of cysteine-rich antimicrobial peptides has been isolated from plants and particularly from seeds. It has become increasingly clear that these peptides play an important role in the protection of plants against microbial infection. In this work, proteins from chili pepper (Capsicum annuum L.) seeds were extracted in phosphate buffer, pH 5.4 and peptides purification were performed by employing ion-exchange chromatographies on DEAE, CM-Sepharose, Sephacryl S-100 and reverse phase in HPLC. Three peptide enriched fractions, namely F1, F2 and F3, were obtained after the CM-Sepharose chromatography. The F1 fraction, mainly composed of three peptides ranging from 6 to 10 kDa, was submitted to N-terminal amino acid sequencing. The closer to 10 kDa peptide showed high sequence homology to lipid transfer proteins (LTPs) previously isolated from others seeds. F1 fraction exhibited strong fungicidal activity against Candida albicans, Saccharomyces cerevisiae and Schizosaccharomyces pombe and also promoted several morphological changes to C. albicans, including the formation of pseudohyphae, as revealed by scanning electron micrography. F1 fraction also reduced the glucose stimulated acidification of the medium mediated by H(+)-ATPase of S. cerevisiae cells in a dose-dependent manner and caused the permeabilization of yeast plasma membrane to the dye SYTOX Green, as verified by confocal laser microscopy.     

Mitochondrial GPX1 silencing triggers differential photosynthesis impairment in response to salinity in rice plants

The physiological role of plant mitochondrial glutathione peroxidases is scarcely known. This study attempted to elucidate the role of a rice mitochondrial isoform(GPX1) in photosynthesis under normal growth and salinity conditions. GPX1 knockdown rice lines(GPX1s) were tested in absence and presence of 100 mM NaCl for 6 d.Growth reduction of GPX1 s line under non-stressful conditions, compared with non-transformed(NT) plants occurred in parallel to increased H_2O_2 and decreased GSH contents. These changes occurred concurrently with photosynthesis impairment, particularly in Calvin cycle's reactions, since photochemical efficiency did not change.Thus, GPX1 silencing and downstream molecular/metabolic changes modulated photosynthesis differentially. In contrast, salinity induced reduction in both phases of photosynthesis, which were more impaired in silenced plants.These changes were associated with root morphology alterations but not shoot growth. Both studied lines displayed increased GPX activity but H_2O_2 content did not change in response to salinity. Transformed plants exhibited lower photorespiration, water use efficiency and root growth, indicating that GPX1 could be important to salt tolerance. Growth reduction of GPX1 s line might be related to photosynthesis impairment, which in turn could have involved a cross talk mechanism between mitochondria and chloroplast originated from redox changes due to GPX1 deficiency.     

Polymorphism Detection by Microsatellite Markers in a Magnaporthe oryzae Population From Different Geographical Areas of Brazil

This study aimed to examine Brazilian M. oryzae populations using 18 microsatellites. Fifty cultivars were sown in plastic trays for the pathotyping of 847 isolates. The DNA of 494 isolates was extracted and purified using the modified Doyle and Doyle method, the genetic structure was determined by the software Structure, and the actual number was selected from the prediction method based on the K values. Nei's genetic distance among the subpopulations was determined with the aid of the program Genetix, and the amova was performed with the program Arlequin. Out of 847 inoculated monosporic isolates, 528 infected their respective cultivars; of the 528 isolates pathotyped, there was a prevalence of group IA and pathotype IF‐1, which was the most frequent pathotype in the rice production areas of Brazil. The Bayesian clustering analysis indicated that 19 was the optimal value of K; this value was the lowest standard deviation and log (ln K) closest to zero, which predicted the 494 isolates of M. oryzae that were selected for molecular studies to be grouped into 19 subpopulations. The AMOVA detected a 37.13% variability within the 19 subpopulations and 62.87% variability among the subpopulations. The polymorphic information content (PIC) ranged from 0 to 0.756. Thirty three rare alleles were found distributed among 15 out of 19 subpopulations. The Margalef index ranged from 38.69 to 79.21 for all 18 analysed locus. The results indicated that the identification of different blast resistance genes must consider the composition of each subpopulation and that the identification is most effective when performed within a subpopulation and then between subpopulations.     

Identification of potential allosteric binding sites in cathepsin K based on intramolecular communication

Network theory methods and molecular dynamics (MD) simulations are accepted tools to study allosteric regulation. Indeed, dynamic networks built upon correlation analysis of MD trajectories provide detailed information about communication paths between distant sites. In this context, we aimed to understand whether the efficiency of intramolecular communication could be used to predict the allosteric potential of a given site. To this end, we performed MD simulations and network theory analyses in cathepsin K (catK), whose allosteric sites are well defined. To obtain a quantitative measure of the efficiency of communication, we designed a new protocol that enables the comparison between properties related to ensembles of communication paths obtained from different sites. Further, we applied our strategy to evaluate the allosteric potential of different catK cavities not yet considered for drug design. Our predictions of the allosteric potential based on intramolecular communication correlate well with previous catK experimental and theoretical data. We also discuss the possibility of applying our approach to other proteins from the same family.     

The cysteine-rich protein thimet oligopeptidase as a model of the structural requirements for S-glutathiolation and oxidative oligomerization

Thimet oligopeptidase (EP24.15) is a cysteine-rich metallopeptidase containing fifteen Cys residues and no intra-protein disulfide bonds. Previous work on this enzyme revealed that the oxidative oligomerization of EP24.15 is triggered by S-glutathiolation at physiological GSSG levels (10-50 μM) via a mechanism based on thiol-disulfide exchange. In the present work, our aim was to identify EP24.15 Cys residues that are prone to S-glutathiolation and to determine which structural features in the cysteinyl bulk are responsible for the formation of mixed disulfides through the reaction with GSSG and, in this particular case, the Cys residues within EP24.15 that favor either S-glutathiolation or inter-protein thiol-disulfide exchange. These studies were conducted by in silico structural analyses and simulations as well as site-specific mutation. S-glutathiolation was determined by mass spectrometric analyses and western blotting with anti-glutathione antibody. The results indicated that the stabilization of a thiolate sulfhydryl and the solvent accessibility of the cysteines are necessary for S-thiolation. The Solvent Access Surface analysis of the Cys residues prone to glutathione modification showed that the S-glutathiolated Cys residues are located inside pockets where the sulfur atom comes into contact with the solvent and that the positively charged amino acids are directed toward these Cys residues. The simulation of a covalent glutathione docking onto the same Cys residues allowed for perfect glutathione posing. A mutation of the Arg residue 263 that forms a saline bridge to the Cys residue 175 significantly decreased the overall S-glutathiolation and oligomerization of EP24.15. The present results show for the first time the structural requirements for protein S-glutathiolation by GSSG and are consistent with our previous hypothesis that EP24.15 oligomerization is dependent on the electron transfer from specific protonated Cys residues of one molecule to previously S-glutathionylated Cys residues of another one.     

Field and laboratory studies provide insights into the meaning of day-time activity in a subterranean rodent (Ctenomys aff. knighti), the tuco-tuco

South American subterranean rodents (Ctenomys aff. knighti), commonly known as tuco-tucos, display nocturnal, wheel-running behavior under light-dark (LD) conditions, and free-running periods >24 h in constant darkness (DD). However, several reports in the field suggested that a substantial amount of activity occurs during daylight hours, leading us to question whether circadian entrainment in the laboratory accurately reflects behavior in natural conditions. We compared circadian patterns of locomotor activity in DD of animals previously entrained to full laboratory LD cycles (LD12:12) with those of animals that were trapped directly from the field. In both cases, activity onsets in DD immediately reflected the previous dark onset or sundown. Furthermore, freerunning periods upon release into DD were close to 24 h indicating aftereffects of prior entrainment, similarly in both conditions. No difference was detected in the phase of activity measured with and without access to a running wheel. However, when individuals were observed continuously during daylight hours in a semi-natural enclosure, they emerged above-ground on a daily basis. These day-time activities consisted of foraging and burrow maintenance, suggesting that the designation of this species as nocturnal might be inaccurate in the field. Our study of a solitary subterranean species suggests that the circadian clock is entrained similarly under field and laboratory conditions and that day-time activity expressed only in the field is required for foraging and may not be time-dictated by the circadian pacemaker.     

Synthesis and vasodilatory activity of new N-acylhydrazone derivatives, designed as LASSBio-294 analogues

Conventional therapy to treat hypertension often involves arterial vasodilation. Decrease of blood pressure by vasodilators is normally associated with adverse effects because of their low vascular selectivity. This is of interest to develop new molecules with potential for clinical use and fewer side effects. Recently, a new bioactive compound of the N-acylhydrazone class, LASSBio-294, was shown to produce a cardioinotropic effect and vasodilation. In this report, new derivatives of LASSBio-294 were designed and tested on the contractile response of vascular smooth muscle from Wistar rats. Phenylephrine-induced contracture in the aorta was inhibited by the derivatives LASSBio-785 and LASSBio-788. The concentrations necessary to cause 50% reduction of the maximal vascular response (IC50) were 10.2 +/- 0.5 and 67.9 +/- 6.5 microM. Vasodilation induced by both derivatives is likely to be mediated by a direct effect on smooth muscle because it was not dependent on the integrity of vascular endothelium. LASSBio-785 was seven times more potent than the reference compound LASSBio-294 (IC50 = 74 microM) in producing an endothelium-independent vasodilator effect.