Kinetic mechanism determination and analysis of metal requirement of dehydroquinate synthase from Mycobacterium tuberculosis H37Rv: an essential step in the function-based rational design of anti-TB drugs

The number of new cases of tuberculosis (TB) arising each year is increasing globally. Migration, socio-economic deprivation, HIV co-infection and the emergence of drug-resistant strains of Mycobacterium tuberculosis, the main causative agent of TB in humans, have all contributed to the increasing number of TB cases worldwide. Proteins that are essential to the pathogen survival and absent in the host, such as enzymes of the shikimate pathway, are attractive targets to the development of new anti-TB drugs. Here we describe the metal requirement and kinetic mechanism determination of M. tuberculosis dehydroquinate synthase (MtDHQS). True steady-state kinetic parameters determination and ligand binding data suggested that the MtDHQS-catalyzed chemical reaction follows a rapid-equilibrium random mechanism. Treatment with EDTA abolished completely the activity of MtDHQS, and addition of Co(2+) and Zn(2+) led to, respectively, full and partial recovery of the enzyme activity. Excess Zn(2+) inhibited the MtDHQS activity, and isotitration microcalorimetry data revealed two sequential binding sites, which is consistent with the existence of a secondary inhibitory site. We also report measurements of metal concentrations by inductively coupled plasma atomic emission spectrometry. The constants of the cyclic reduction and oxidation of NAD(+) and NADH, respectively, during the reaction of MtDHQS was monitored by a stopped-flow instrument, under single-turnover experimental conditions. These results provide a better understanding of the mode of action of MtDHQS that should be useful to guide the rational (function-based) design of inhibitors of this enzyme that can be further evaluated as anti-TB drugs.     

A Mycobacterium leprae Hsp65 mutant as a candidate for mitigating lupus aggravation in mice

Hsp60 is an abundant and highly conserved family of intracellular molecules. Increased levels of this family of proteins have been observed in the extracellular compartment in chronic inflammation. Administration of M. leprae Hsp65 [WT] in [NZBxNZW]F(1) mice accelerates the Systemic Lupus Erythematosus [SLE] progression whereas the point mutated K(409)A Hsp65 protein delays the disease. Here, the biological effects of M. leprae Hsp65 Leader pep and K(409)A pep synthetic peptides, which cover residues 352-371, are presented. Peptides had immunomodulatory effects similar to that observed with their respective proteins on survival and the combined administration of K(409)A+Leader pep or K(409)A pep+WT showed that the mutant forms were able to inhibit the deleterious effect of WT on mortality, indicating the neutralizing potential of the mutant molecules in SLE progression. Molecular modeling showed that replacing Lysine by Alanine affects the electrostatic potential of the 352-371 region. The number of interactions observed for WT is much higher than for Hsp65 K(409)A and mouse Hsp60. The immunomodulatory effects of the point-mutated protein and peptide occurred regardless of the catalytic activity. These findings may be related to the lack of effect on survival when F(1) mice were inoculated with Hsp60 or K(409)A pep. Our findings indicate the use of point-mutated Hsp65 molecules, such as the K(409)A protein and its corresponding peptide, that may minimize or delay the onset of SLE, representing a new approach to the treatment of autoimmune diseases.     

Purification and enzymatic properties of a peroxidase from leaves of Phytolacca dioica L. (Ombú tree)

A peroxidase (PD-cP; 0.47 mg/100 g leaves) was purified from autumn leaves of Phytolacca dioica L. and characterized. PD-cP was obtained by acid precipitation followed by gel-filtration and cation exchange chromatography. Amino acid composition and N-terminal sequence of PD-cP up to residue 15 were similar to that of Spinacia oleracea (N-terminal pairwise comparison showing four amino acid differences). PD-cP showed a molecular mass of approx. 36 kDa by SDS-PAGE, pH and temperature optima at 3.0 and 50.0°C, respectively and seasonal variation. The Michaelis-Menten constant (K(M)) for H(2)O(2) was 5.27 mM, and the velocity maximum (V(max)) 1.31 nmol min(-1), while the enzyme turnover was 0.148 s(-1). Finally, the presence of Ca(2+) and Mg(2+) enhanced the PD-cP activity, with Mg(2+) 1.4-fold more effective than Ca(2+)     

Is the quantity of orbicules released by Dactylis glomerata and Cynosurus echinatus (Poaceae) big enough to play an allergenic role?

Orbicule characteristics of Dactylis glomerata and Cynosurus echinatus (Poaceae) were investigated using light (LM), scanning electron (SEM) and transmission electron microscopy (TEM). Based on SEM micrographs, the number of orbicules per 100?µm² of the locule wall surface was determined in both dehisced and undehisced anthers and was further compared statistically. A total of 100 pollen grains were examined using SEM in search for orbicules attached to the pollen exine. Orbicules were not found distributed freely in the anther locules. They were attached to the locule wall surface through sporopolleninous fibrils, the orbicule wall being firmly embedded in, and often in continuity with the thin layer of sporopollenin lining each locule. The orbicule density on the locule wall surface of both the dehisced and undehisced anthers did not differ significantly. Only a few orbicules were seen attached to the pollen exine in both species. It is concluded that orbicules are not easily removed from the surface of the locule wall and, consequently, that the number of orbicules emitted from the two grass species is too low to play a significant role in triggering allergic diseases.     

Temporal and spatial distributions of the fish larval assemblages of the Ivinheima River sub-basin (Brazil)

Fish, like other animals, choose environments for reproduction that are favorable for their offspring’s initial development. For example, these environments may be chosen to provide shelter and food. This study examined the fish larvae inhabiting the Ivinheima River sub-basin. The study aimed to (i) characterize the taxonomic composition of the larval fish fauna, (ii) analyze the structure of the larval assemblage, and (iii) verify the relationship between the larval assemblages and environmental variables. The sampling areas included three environments: the Ivinheima River, the Finado Raimundo Lagoon and the Patos Lagoon. Sampling was performed between October 2002 and March 2006 during four reproductive periods (RPs). The data were analyzed with correspondence analysis (CA) and canonical correlation analysis (CCA). A total of 120 619 larvae were collected. These larvae belonged to several species characterized by different reproductive strategies. Our results showed differences between the composition of the Ivinheima River’s assemblages, where there were more larvae of migratory fish, and the lagoons, where larvae of sedentary species were predominant. In addition, differences were found among the RPs evaluated in the Ivinheima River: the first and second RPs were characterized by milder floods and by a greater abundance of migratory piscivore larvae, whereas the third and fourth RPs had more outflow and a greater abundance of larvae of detritivore and omnivore species. We conclude that the Ivinheima River plays a significant role in the maintenance of native migratory fish stocks in the upper Paraná River basin and that the environment shows a segregation of the spatial and temporal composition and abundance of larvae related to running and flooding/standing water.     

Circulation of Different Lineages of Dengue Virus 2, Genotype American/Asian in Brazil: Dynamics and Molecular and Phylogenetic Characterization

The American/Asian genotype of Dengue virus type 2 (DENV-2) was introduced into the Americas in the 80′s. Although there is no data showing when this genotype was first introduced into Brazil, it was first detected in Brazil in 1990. After which the virus spread throughout the country and major epidemics occurred in 1998, 2007/08 and 2010. In this study we sequenced 12 DENV-2 genomes obtained from serum samples of patients with dengue fever residing in São José do Rio Preto, São Paulo (SJRP/SP), Brazil, in 2008. The whole open reading frame or envelope sequences were used to perform phylogenetic, phylogeographic and evolutionary analyses. Isolates from SJRP/SP were grouped within one lineage (BR3) close to isolates from Rio de Janeiro, Brazil. Isolates from SJRP were probably introduced there at least in 2007, prior to its detection in the 2008 outbreak. DENV-2 circulation in Brazil is characterized by the introduction, displacement and circulation of three well-defined lineages in different times, most probably from the Caribbean. Thirty-seven unique amino acid substitutions were observed among the lineages, including seven amino acid differences in domains I to III of the envelope protein. Moreover, we dated here, for the first time, the introduction of American/Asian genotype into Brazil (lineage BR1) to 1988/89, followed by the introduction of lineages BR2 (1998–2000) and BR3 (2003–05). Our results show a delay between the introduction and detection of DENV-2 lineages in Brazil, reinforcing the importance and need for surveillance programs to detect and trace the evolution of these viruses. Additionally, Brazilian DENV-2 differed in genetic diversity, date of introduction and geographic origin and distribution in Brazil, and these are important factors for the evolution, dynamics and control of dengue.     

Corneal Absorption of a New Riboflavin-Nanostructured System for Transepithelial Collagen Cross-Linking

Corneal collagen cross-linking (CXL) has been described as a promising therapy for keratoconus. According to standard CXL protocol, epithelium should be debrided before treatment to allow penetration of riboflavin into the corneal stroma. However, removal of the epithelium can increase procedure risks. In this study we aim to evaluate stromal penetration of a biocompatible riboflavin-based nanoemulsion system (riboflavin-5-phosphate and riboflavin-base) in rabbit corneas with intact epithelium. Two riboflavin nanoemulsions were developed. Transmittance and absorption coefficient were measured on corneas with intact epithelia after 30, 60, 120, 180, and 240 minutes following exposure to either the nanoemulsions or standard 0.1% or 1% riboflavin-dextran solutions. For the nanoemulsions, the epithelium was removed after measurements to assure that the riboflavin had passed through the hydrophobic epithelium and retained within the stroma. Results were compared to de-epithelialized corneas exposed to 0.1% riboflavin solution and to the same riboflavin nanoemulsions for 30 minutes (standard protocol). Mean transmittance and absorption measured in epithelialized corneas receiving the standard 0.1% riboflavin solution did not reach the levels found on the debrided corneas using the standard technique. Neither increasing the time of exposure nor the concentration of the riboflavin solution from 0.1% to 1% improved riboflavin penetration through the epithelium. When using riboflavin-5-phosphate nanoemulsion for 240 minutes, we found no difference between the mean absorption coefficients to the standard cross-linking protocol (p = 0.54). Riboflavin nanoemulsion was able to penetrate the corneal epithelium, achieving, after 240 minutes, greater stromal concentration when compared to debrided corneas with the standard protocol (p = 0.002). The riboflavin-5-phosphate nanoemulsion diffused better into the stroma than the riboflavin-base nanoemulsion.