Bioactivity quantification of crude bacteriocin solutions

Bacteriocin activity can be quantified by a simple parallel line model based on an agar diffusion assay. The quotient between the inhibition zone area and the sensitivity of the indicator bacterium can be used to compare different bacteriocins. Deferred calculations of relative potencies are possible, once purified compounds are obtained.     

Inhibition of the proton pumping ATPases of yeast and oat root plasma membranes by dicyclohexylcarbodiimide

The inhibition of the proton-pumping ATPases of yeast and oat root plasma membranes by dicyclohexylcarbodiimide (DCCD) can be correlated with the covalent incorporation of the inhibitor. Full inhibition of the yeast enzyme required the incorporation of about 1 mol DCCD/mol of the ATPase polypeptide of 100 kDa. A kinetic study of the interaction of DCCD with the yeast and oat ATPases indicates a second-order rate constant of about 500 M-1 min-1 and a stoichiometry of 1 mol DCCD/mol of enzyme, in agreement with the amount of DCCD incorporated by the yeast enzyme. It is proposed that DCCD reacts with a single carboxylic group present in a hydrophobic region of these proton-pumping ATPases and which could participate in proton binding and transport.     

A high-throughput fluorescence-based assay for Plasmodium dihydroorotate dehydrogenase inhibitor screening

Plasmodium dihydroorotate dehydrogenase (DHODH) is a mitochondrial membrane-associated flavoenzyme that catalyzes the rate-limiting step of de novo pyrimidine biosynthesis. DHODH is a validated target for malaria, and DSM265, a potent inhibitor, is currently in clinical trials. The enzyme catalyzes the oxidation of dihydroorotate to orotate using flavin mononucleotide (FMN) as cofactor in the first half of the reaction. Reoxidation of FMN to regenerate the active enzyme is mediated by ubiquinone (CoQ_D), which is the physiological final electron acceptor and second substrate of the reaction. We have developed a fluorescence-based high-throughput enzymatic assay to find DHODH inhibitors. In this assay, the CoQ_D has been replaced by a redox-sensitive fluorogenic dye, resazurin, which changes to a fluorescent state on reduction to resorufin. Remarkably, the assay sensitivity to find competitive inhibitors of the second substrate is higher than that reported for the standard colorimetric assay. It is amenable to 1536-well plates with Z′ values close to 0.8. The fact that the human enzyme can also be assayed in the same format opens additional applications of this assay to the discovery of inhibitors to treat cancer, transplant rejection, autoimmune diseases, and other diseases mediated by rapid cellular growth.     

Lopesia indaiensis (Diptera,Cecidomyiidae), a new species of gall midge feeding on Andira fraxinifolia Benth (Fabaceae), an endemic plant in Brazil

Lopesia indaiensis (Diptera, Cecidomyiidae), a new species of gall midge found causing galls on Andira fraxinifolia (Fabaceae), an endemic plant species in Brazil, is described based on larva, pupa, male and female. L. indaiensis galls were collected in Dores do Indaiá, State of Minas Gerais, Southeastern Brazil. Larvae were removed from the galls and pupae and adults were obtained by rearing. The specimens were mounted on slides and the most important morphological characters were illustrated. The new species was compared to the other species of Lopesia.     

Retrophosphorylation of Mkk1 and Mkk2 MAPKKs by the Slt2 MAPK in the yeast cell integrity pathway

In Saccharomyces cerevisiae, a variety of stresses and aggressions to the cell wall stimulate the activation of the cell wall integrity MAPK pathway, which triggers the expression of a series of genes important for the maintenance of cell wall homeostasis. This MAPK module lies downstream of the Rho1 small GTPase and protein kinase C Pkc1 and consists of MAPKKK Bck1, MAPKKs Mkk1 and Mkk2, and the Slt2 MAPK. In agreement with previous reports suggesting that Mkk1 and Mkk2 were functionally redundant, we show here that both Mkk1 and Mkk2 alone or even chimerical proteins constructed by interchanging their catalytic and regulatory domains are able to efficiently maintain signal transduction through the pathway. Both Mkk1 and Mkk2 are phosphorylated in vivo concomitant to activation of the cell integrity pathway. Interestingly, hyperphosphorylation of the MEKs required not only the upstream components of the pathway, but also a catalytically competent Slt2 MAPK downstream. Active Slt2 purified from yeast extracts was able to phosphorylate Mkk1 and Mkk2 in vitro. We have mapped Ser(50) as a direct phosphorylation target for Slt2 in Mkk2. However, substitution of all (Ser/Thr)-Pro canonical MAPK target sites with alanine did not totally abrogate Slt2-dependent Mkk2 phosphorylation. Mutation or deletion of a conserved MAPK-docking site at the N-terminal extension of Mkk2 precluded its interaction with Slt2 and negatively affected retrophosphorylation. Our data show that the cell wall integrity MAPKKs are targets for their downstream MAPK, suggesting the existence of complex feedback regulatory mechanisms at this level.     

Lopesia leandrae (Diptera,Cecidomyiidae), a new species of gall midge associated with Leandra ionopogon (Mart.) Cogn. (Melastomataceae), a native plant to Brazil

Lopesia leandrae (Diptera, Cecidomyiidae), a new galling species associated with Leandra ionopogon (Melastomataceae), a native plant species to Brazil, is described based on larva, pupal exuvia, male and female. L. leandrae galls were collected in an area of Atlantic Forest in Bertioga, state of São Paulo, Southeastern Brazil. The new species was compared to the other species of Lopesia.     

A new species of Youngomyia Felt from Brazil and new morphological data on Youngomyia pouteriae Maia (Insecta,Diptera, Cecidomyiidae)

Youngomyia matogrossensis Proença & Maia a new species of Cecidomyiidae (Insecta: Diptera) that induces cylindrical hairy galls on leaves of Pouteria torta (Mart.) Radlk. (Sapotaceae) is herein described and illustrated (larvae, pupal exuviae, male and female). The galler, gall and host plant were collected at Parque Nacional da Chapada dos Guimarães, in the state of Mato Grosso (Brazil). New morphological data and photographs of pupal exuviae, male and female of Youngomyia pouteriae Maia, 2001 are also provided.     

Charge effect on the photoinactivation of Gram-negative and Gram-positive bacteria by cationic meso-substituted porphyrins

Background  

In recent times photodynamic antimicrobial therapy has been used to efficiently destroy Gram (+) and Gram (-) bacteria using cationic porphyrins as photosensitizers. There is an increasing interest in this approach, namely in the search of photosensitizers with adequate structural features for an efficient photoinactivation process. In this study we propose to compare the efficiency of seven cationic porphyrins differing in meso-substituent groups, charge number and charge distribution, on the photodynamic inactivation of a Gram (+) bacterium (Enterococcus faecalis) and of a Gram (-) bacterium (Escherichia coli). The present study complements our previous work on the search for photosensitizers that might be considered good candidates for the photoinactivation of a large spectrum of environmental microorganisms.