Crystallographic and pre-steady-state kinetics studies on binding of NADH to wild-type and isoniazid-resistant enoyl-ACP(CoA) reductase enzymes from Mycobacterium tuberculosis

An understanding of isoniazid (INH) drug resistance mechanism in Mycobacterium tuberculosis should provide significant insight for the development of newer anti-tubercular agents able to control INH-resistant tuberculosis (TB). The inhA-encoded 2-trans enoyl-acyl carrier protein reductase enzyme (InhA) has been shown through biochemical and genetic studies to be the primary target for INH. In agreement with these results, mutations in the inhA structural gene have been found in INH-resistant clinical isolates of M. tuberculosis, the causative agent of TB. In addition, the InhA mutants were shown to have higher dissociation constant values for NADH and lower values for the apparent first-order rate constant for INH inactivation as compared to wild-type InhA. Here, in trying to identify structural changes between wild-type and INH-resistant InhA enzymes, we have solved the crystal structures of wild-type and of S94A, I47T and I21V InhA proteins in complex with NADH to resolutions of, respectively, 2.3 angstrom, 2.2 angstrom, 2.0 angstrom, and 1.9 angstrom. The more prominent structural differences are located in, and appear to indirectly affect, the dinucleotide binding loop structure. Moreover, studies on pre-steady-state kinetics of NADH binding have been carried out. The results showed that the limiting rate constant values for NADH dissociation from the InhA-NADH binary complexes (k(off)) were eleven, five, and tenfold higher for, respectively, I21V, I47T and S94A INH-resistant mutants of InhA as compared to INH-sensitive wildtype InhA. Accordingly, these results are proposed to be able to account for the reduction in affinity for NADH for the INH-resistant InhA enzymes. (c) 2006 Elsevier Ltd. All rights reserved.

THERMOTOLERANCE BEHAVIOR IN SUGAR-CANE SYRUP FERMENTATIONS OF WILD-TYPE YEAST STRAINS SELECTED UNDER PRESSURES OF TEMPERATURE, HIGH SUGAR AND ADDED ETHANOL

The selected yeast strains were examined for their ability lo grow, to retain cell viability and to ferment diluted sugar cane juice (15% total sugar, w/v) to ethanol at 40-degrees-C. The degree of agitation (aeration) affects the thermotolerance while the method used for isolation of the strains appears to have no significant effect. The yeast isolated are aerobically fermentative with increased levels of fermentation and growth resulting from agitation (aeration), the exact level of these increases being dependent on the strain used.

Crystallographic studies on the binding of isonicotinyl-NAD adduct to wild-type and isoniazid resistant 2-trans-enoyl-ACP (CoA) reductase from Mycobacterium tuberculosis

The resumption of tuberculosis led to an increased need to understand the molecular mechanisms of drug action and drug resistance, which should provide significant insight into the development of newer compounds. Isoniazid (INH), the most prescribed drug to treat TB, inhibits an NADH-dependent enoyl-acyl carrier protein reductase (InhA) that provides precursors of mycolic acids, which are components of the mycobacterial cell wall. InhA is the major target of the mode of action of isoniazid. INH is a pro-drug that needs activation to form the inhibitory INH-NAD adduct. Missense mutations in the inhA structural gene have been identified in clinical isolates of Mycobacterium tuberculosis resistant to INH. To understand the mechanism of resistance to INH, we have solved the structure of two InhA mutants (121V and S94A), identified in INH-resistant clinical isolates, and compare them to INH-sensitive WT InhA structure in complex with the INH-NAD adduct. We also solved the structure of unliganded INH-resistant S94A protein, which is the first report on apo form of InhA. The salient features of these structures are discussed and should provide structural information to improve our understanding of the mechanism of action of, and resistance to, INH in M. tuberculosis. The unliganded structure of InhA allows identification of conformational changes upon ligand binding and should help structure-based drug design of more potent antimycobacterial agents. (c) 2007 Elsevier B.V. All rights reserved.

Study of lobation and vascularization of the lungs of wild boar (Sus scrofa)

The purpose of this study was to describe the anatomy of the lungs of wild boars for comparison with those of domestic swine. It was found that the right lung of the wild boar is divided into four lobes: cranial, median, caudal and accessory, whereas the left lung is divided into two lobes: cranial and caudal. In 93.4% of the cases, right pulmonary artery separates into the ascendant, descendant, median, accessory and caudal branches. In 73.3% of the cases, left pulmonary artery separates most frequently to form three branches to the cranial lobe, whereas the median lobe is generally supplied by only one arterial branch. There is a single pattern of bronchial distribution: in the right lung a tracheal bronchus leads to the cranial lobe, where it separates into the cranial and caudal bronchi and there are also bronchi to the median, caudal and accessory lobes. In the left lung, the large bronchus separates to form two branches, one of which further separates to form two branches to the cranial lobe whereas the other forms a single branch to the caudal lobe.

Anxiety levels and wild running susceptibility in rats: assessment with elevated plus maze test and predator odor exposure

It is reported in the literature that nearly 20% of rats are susceptible to displays of wild running (WR) behavior when submitted to high intensity acoustic stimulation. Some characteristics of WR suggest that it can be viewed as a panic-like reaction. This work aimed to test whether WR-sensitive rats show higher levels of anxiety in elevated-plus-maze (EPM) and predator-odor exposure paradigms in comparison with WR-resistant ones. Male adult Wistar rats were submitted to two trials of acoustic stimulation (104 dB, 60 s) in order to assess WR susceptibility. Seven WR-sensitive and 15 WR-resistant rats were evaluated by the EPM test. Other 13 WR-sensitive and 18 WR-resistant animals were submitted to the predator-odor exposure test which consisted of a 10 min-session of free exploration in a specific apparatus containing two odoriferous stimuli: cotton swab imbedded with snake cloacal gland secretion or with iguana feces (control). WR-sensitive rats presented a significantly higher closed-to open-ann-entry ratio in the EPM test. All rats responded with anxiety-like behaviors to the predator odor exposure, although the WR-sensitive ones showed a marked behavioral inhibition regardless of the odor condition. We conclude that WR-sensitive rats present elevated levels of anxiety manifested by means of passive behavioral strategies. (C) 2004 Elsevier B.V. All rights reserved.

HERITABILITY OF THE NUMBER OF OVARIOLES IN HONEY-BEE WORKERS (APIS-MELLIFERA L) (HYM, APIDAE)

Narrow and broad sense heritabilities (h(2)) of the number of ovarioles in Apis mellifera L. workers were estimated using two distinct data sets, based on groups of half (queens inseminated by several drones) and super-sisters (single drone insemination). The values found are in the usual range for economically important characters (0.25-0.38).

Species diversity and seasonality of free-living ticks (Acari : Ixodidae) in the natural habitat of wild Marsh deer (Blastocerus dichotomus) in Southeastern Brazil

This study evaluated the presence and seasonal activity of free-living ticks in remaining marsh areas by the Parana river, in Brazil. Eight field trips (once per season) for collection of ticks were performed during 2 years. Using CO2 traps, dragging, and visual inspection of vegetation, five free-living tick species were collected, in the following order of abundance: Amblyomma cajennense, Amblyom\ma dubitatum, Amblyomma triste, Amblyomma coelebs, and Amblyomma nodosum. The seasonal pattern of A. cajennense was characterized by the highest peaks for adult ticks in the summer/spring months, for nymphs in the winter and for larvae in the autumn and winter. A. dubitatum and A. triste presented similar seasonal patterns characterized by peaks of adult ticks in the autumn. Nymphs of A. dubitatum peaked in the winter of the first year and in the winter/spring of the second year. A. triste was the only species to be collected in significantly higher numbers in the marsh than in surrounding drier areas such as forest patches. Among domestic animals living close the marsh areas, horses were infested by Anocentor nitens, A. cajennense, and Boophilus microplus, bovines were infested solely by B. microplus, and dogs were infested by Rhipicephalus sanguineus. Adults of A. triste showed to be well adapted to the marsh environment. This result, at least partially, explains local previous observations on the association of A. triste with marsh deer, as this vertebrate host inhabits mainly the marsh area. (c) 2006 Published by Elsevier B.V.

COMPARATIVE STUDIES ON PIGMENT GRANULES AND PIGMENT DISTRIBUTION IN THE COMPOUND EYES OF WILD-TYPE AND MUTANT WORKER HONEYBEES .2. AN ULTRASTRUCTURAL ANALYSIS

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Hepatozoon canis infection of wild carnivores in Brazil

Hepatozoon canis was diagnosed in a crab-eating fox (Cerdocyon thous) found on a highway in the region of Botucatu, São Paulo, Brazil, after being hit by a car. The fox had bilateral fractures of the olecranon, which was corrected by osteosynthesis. Hematologic findings included a neutrophilia, eosinophilia, monocytosis and mild anemia. In the Leishman-stained blood film, gametocytes of Hepatozoon canis in neutrophils were identified measuring 9.1+/-0.54x5.3+/-0.46 mu m. (C) 1997 Elsevier B.V. B.V.

DEVELOPMENT of MICROSATELLITE MARKERS FOR PIMENTA PSEUDOCARYOPHYLLUS (MYRTACEAE), A WILD SOUTH AMERICAN SPECIES

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Differentiation of the honey bee (Apis mellifera L.) antennal lobes during metamorphosis: a comparative study among castes and sexes

In insects the antennal lobes (AL) constitute the brain deutocerebrum. In bees they consist of two neuropil regions, each associated with one antenna, delimited by a layer of glial cells and somata of neurons. The neuropil is organized in distinct globular structures of dense synaptic axons coming from the olfactory organs of the antennae, known as glomeruli. In Apis mellifera, as in other eusocial species of bees, queens, workers, and drones perform different functions in the colony and consequently the organs associated with these functions undergo a differential development. In this paper we analyzed the structure and size of the differentiating AL of queens, workers, and drones during metamorphosis using light microscopy. During metamorphosis the neuropil enlarge and differentiates into concentric structures known as glomeruli. The results showed size, structural and temporal differences in the glomeruli development among the classes of individuals of the colony. The neuropil differentiation starts early and is faster in drones and newly emerged worker is the colony individual class with greater neuropil area in AL. These results are discussed taking in account the functions of the individuals in the colony. (C) Koninklijke Brill NV, Leiden, 2011.

Effects of sex and locality on the abundance of lice on the wild rodent Oligoryzomys nigripes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)