Fumarate hydratase isoforms of Leishmania major: Subcellular localization, structural and kinetic properties

Fumarate hydratases (FHs; EC 4.2.1.2) are enzymes that catalyze the reversible hydration of fumarate to S-malate. Parasitic protists that belong to the genus Leishmania and are responsible for a complex of vector-borne diseases named leishmaniases possess two genes that encode distinct putative FH enzymes. Genome sequence analysis of Leishmania major Friedlin reveals the existence of genes LmjF24.0320 and LmjF29.1960 encoding the putative enzymes LmFH-1 and LmFH-2, respectively. In the present work, the FH activity of both L. major enzymes has been confirmed. Circular dichroism studies suggest important differences in terms of secondary structure content when comparing LmFH isoforms and even larger differences when comparing them to the homologous human enzyme. CD melting experiments revealed that both LmFH isoforms are thermolabile enzymes. The catalytic efficiency under aerobic and anaerobic environments suggests that they are both highly sensitive to oxidation and damaged by oxygen. Intracellular localization studies located LmFH-1 in the mitochondrion, whereas LmFH-2 was found predominantly in the cytosol with possibly also some in glycosomes. The high degree of sequence conservation in different Leishmania species, together with the relevance of FH activity for the energy metabolism in these parasites suggest that FHs might be exploited as targets for broad-spectrum antileishmanial drugs. (c) 2012 Elsevier B.V. All rights reserved.

Low-Resolution Molecular Models Reveal the Oligomeric State of the PPAR and the Conformational Organization of Its Domains in Solution

The peroxisome proliferator-activated receptors (PPARs) regulate genes involved in lipid and carbohydrate metabolism, and are targets of drugs approved for human use. Whereas the crystallographic structure of the complex of full length PPAR gamma and RXR alpha is known, structural alterations induced by heterodimer formation and DNA contacts are not well understood. Herein, we report a small-angle X-ray scattering analysis of the oligomeric state of hPPAR gamma alone and in the presence of retinoid X receptor (RXR). The results reveal that, in contrast with other studied nuclear receptors, which predominantly form dimers in solution, hPPAR gamma remains in the monomeric form by itself but forms heterodimers with hRXR alpha. The low-resolution models of hPPAR gamma/RXR alpha complexes predict significant changes in opening angle between heterodimerization partners (LBD) and extended and asymmetric shape of the dimer (LBD-DBD) as compared with X-ray structure of the full-length receptor bound to DNA. These differences between our SAXS models and the high-resolution crystallographic structure might suggest that there are different conformations of functional heterodimer complex in solution. Accordingly, hydrogen/deuterium exchange experiments reveal that the heterodimer binding to DNA promotes more compact and less solvent-accessible conformation of the receptor complex.

Synthesis and characterization of a diruthenium(II,III)-ketoprofen compound and study of the in vitro effects on CRC cells in comparison to the naproxen and ibuprofen derivatives

A new diruthenium(II,III) complex, of formula [Ru2Cl(ket)(4)], Ruket, containing the non-steroidal anti-inflammatory drug ketoprofen was synthesized and mainly characterized by electrospray ionization mass spectrometry (ESI-MS), UV-Vis-IR electronic spectroscopy and FTIR and Raman vibrational spectroscopies. The four drug-carboxylato bridging ligands stabilize a Ru-2(II,III) mixed valent core in a paddlewheel type structure as confirmed by ESI mass spectra, electronic and vibrational spectroscopies and magnetic measurements. Ruket and the analogous compounds containing ibuprofen, Ruibp, and naproxen, Runpx, were tested for the biological effects in the human colon carcinoma cells HT-29 and Caco-2 expressing high and low levels of COX-2 respectively. All compounds only weakly affected the proliferation of the colorectal cancer cells HT-29 and Caco-2, and similarly only partially inhibited the production/activity of MMP-2 and MMP-9 by HT-29 cells, suggesting that COX-2 inhibition by these drugs can only partially be involved in the pharmacological effects of these derivatives. (c) 2012 Elsevier Ltd. All rights reserved.

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD369↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.

In situ effect of chewing gum containing CPP–ACP on the mineral precipitation of eroded bovine enamel

Objectives: Stimulation of salivary flow is considered a preventive strategy for dental erosion. Alternatively, products containing calcium phosphate, such as a complex of casein phosphopeptide–amorphous calcium phosphate (CPP–ACP), have also been tested against dental erosion. Therefore, this in situ study analyzed the effect of chewing gum containing CPP–ACP on the mineral precipitation of initial bovine enamel erosion lesions. Methods: Twelve healthy adult subjects wore palatal appliances with two eroded bovine enamel samples. The erosion lesions were produced by immersion in 0.1% citric acid (pH 2.5) for 7 min. During three experimental crossover in situ phases (1 day each), the subjects chewed a type of gum, 3 times for 30 min, in each phase: with CPP–ACP (trident total), without CPP–ACP (trident), and no chewing gum (control). The Knoop surface microhardness was measured at baseline, after erosion in vitro and the mineral precipitation in situ. The differences in the degree of mineral precipitation were analyzed using repeated measures (RM-) ANOVA and post hoc Tukey’s test ( p < 0.05). Results: Significant differences were found among the remineralizing treatments ( p < 0.0001). Chewing gum (19% of microhardness recovery) improved the mineral precipitation compared to control (10%) and the addition of CPP–ACP into the gum promoted the best mineral precipitation effect (30%). Conclusions: Under this protocol, CPP–ACP chewing gum improved the mineral precipitation of eroded enamel. Clinical significance: Since the prevalence of dental erosion is steadily increasing, CPP–ACP chewing gum might be an important strategy to reduce th eprogression of initial erosion lesions.

Spatio-temporal and functional structure of the amphipod communities off Santos, Southwestern Atlantic

The amphipod fauna was employed to investigate a bottom environmental gradient in the continental shelf adjacent to Santos Bay. The constant flow of less saline water from the estuarine complex of the Santos and São Vicente rivers besides the seasonal intrusion of the cold saline South Atlantic Central Water (SACW) bring a highly dynamic water regime to the area. Density, distribution, diversity and functional structure of the communities were studied on a depth gradient from 10 to 100 m on two cruises in contrasting seasons, winter 2005 and summer 2006. Twenty-one sediment samples were taken with a 0.09m² box corer. Temperature and salinity were measured at each station and an additional surface sediment sample was obtained with the box corer for granulometric and chemical analyses. Sixty species were collected on each survey and higher density values were found in summer. A priori one-way Analysis of Similarities (ANOSIM) indicated the existence of three different groups of amphipods related to the depth gradient: the Coastal group, the Mixed Zone group and the Deep Zone group. The Coastal Zone in both cruises was inhabited by a community presenting low diversity and density, besides high dominance of the infaunal tube-dweller Ampelisca paria; the area around 30 m presented the highest values of all the ecological indicators and the species showed several life styles; the outer area, situated between 50 and 100 m depth in the SACW domain, presented a community characterized by lower diversity and high biomass and density values. A season-depth ANOSIM showed the influence of depth and season for the Coastal and Mixed Zone groups whereas no seasonal difference was obtained for the Deep Zone group. The synergistic effect of the SACW and depth in the first place, followed by physical changes in substrate, seem to be the main factors controlling the fauna's distribution. In addition, the estuarine waters from Santos Bay apparently had no effect on the establishment of the environmental gradient observed on the adjacent shelf. Diversity, distribution, functional groups and trophic conditions of superficial sediments are discussed in the light of the main oceanographic processes present on the southern Brazilian shelf.

Role of the amygdala in the reinforcement omission effect

The reinforcement omission effect (ROE) has been attributed to both motivational and attentional consequences of surprising reinforcement omission. Recent evidence suggests that the basolateral complex of the amygdala is involved in motivational components related to reinforcement value, whereas the central nucleus of the amygdala is involved in the processing of the attentional consequences of surprise. This study was designed to verify whether the mechanisms involved in the ROE depend on the integrity of either the basolateral amygdala complex or central nucleus of the amygdala. The ROE was evaluated in rats with lesions of either the central nucleus or basolateral complex of the amygdala and trained on a fixed-interval schedule procedure (Experiment 1) and fixed-interval with limited hold signaled schedule procedure (Experiment 2). The results of Experiment 1 showed that sham-operated rats and rats with lesions of either the central nucleus or basolateral area displayed the ROE. In contrast, in Experiment 2, subjects with lesions of the central nucleus or basolateral complex of the amygdala exhibited a smaller ROE compared with sham-operated subjects. Thus, the effects of selective lesions of amygdala subregions on the ROE in rats depended on the training procedure. Furthermore, the absence of differences between the lesioned groups in either experiment did not allow the dissociation of attentional or motivational components of the ROE with functions of specific areas of the amygdala. Thus, results did not show a functional double-dissociation between the central nucleus and basolateral area in the ROE.

Cryptic species in the cosmopolitan Bugulan eritina complex (Bryozoa,Cheilostomata)

Previous analyses of the mitochondrial gene cytochrome c oxidase subunit 1 (COI) and γ-proteobacterial endosymbiont diversity have suggested that the marine bryozoan Bugula neritina is a complex of three cryptic species, namely Types S, D and N. Types D and N were previously reported to have restricted distributions along California (western USA) and Delaware and Connecticut (eastern USA), respectively, whereas Type S is considered widespread in tropical, subtropical and temperate regions due to anthropogenic transport. Here, Bayesian species delimitation analysis of a data set composed of two mitochondrial (COI and large ribosomal RNA subunit [16S]) and two nuclear genes (dynein light chain roadblock type-2 protein [DYN] and voltage-dependent anion-selective channel protein [VDAC]) demonstrated that Types S, D and N correspond to three biological species. This finding was significantly supported, in spite of the combinations of priors applied for ancestral population size and root age. Furthermore, COI sequences were used to assess the introduction patterns of the cosmopolitan Type S species. Two COI haplotypes of Type S (S1a and S1d) were found occurring at a global scale. Mantel tests showed correlation between these haplotypes and local sea surface temperature tolerance. Accordingly, the distributions of Type S haplotypes may reflect intraspecific temperature tolerance variation, in addition to the role of introduction vectors. Finally, we show that the Type N may also have been introduced widely, as this species was found for the first time in Central California and north-eastern Australia.

Redox regulation of the proteasome via S-glutathionylation

The proteasome is a multimeric and multicatalytic intracellular protease responsible for the degradation of proteins involved in cell cycle control, various signaling processes, antigen presentation, and control of protein synthesis. The central catalytic complex of the proteasome is called the 20S core particle. The majority of these are flanked on one or both sides by regulatory units. Most common among these units is the 19S regulatory unit. When coupled to the 19S unit, the complex is termed the asymmetric or symmetric 26S proteasome depending on whether one or both sides are coupled to the 19S unit, respectively. The 26S proteasome recognizes poly-ubiquitinylated substrates targeted for proteolysis. Targeted proteins interact with the 19S unit where they are deubiquitinylated, unfolded, and translocated to the 20S catalytic chamber for degradation. The 26S proteasome is responsible for the degradation of major proteins involved in the regulation of the cellular cycle, antigen presentation and control of protein synthesis. Alternatively, the proteasome is also active when dissociated from regulatory units. This free pool of 20S proteasome is described in yeast to mammalian cells. The free 20S proteasome degrades proteins by a process independent of poly-ubiquitinylation and ATP consumption. Oxidatively modified proteins and other substrates are degraded in this manner. The 20S proteasome comprises two central heptamers (β-rings) where the catalytic sites are located and two external heptamers (α-rings) that are responsible for proteasomal gating. Because the 20S proteasome lacks regulatory units, it is unclear what mechanisms regulate the gating of α-rings between open and closed forms. In the present review, we discuss 20S proteasomal gating modulation through a redox mechanism, namely, S-glutathionylation of cysteine residues located in the α-rings, and the consequence of this post-translational modification on 20S proteasomal function.

Anthropogenic influences in a lagoonal environment: a multiproxy approach at the valo grande mouth, Cananéia-Iguape system (SE Brazil)

The Cananeia-Iguape system, SE Brazil, consists of a complex of lagoonal channels, located in a United Nations Educational, Scientific and Cultural Organization (UNESCO) Biosphere Reserve. Nevertheless, important environmental changes have occurred in approximately the last 150 yrs due to the opening of an artificial channel, the Valo Grande, connecting the Ribeira de Iguape River to the lagoonal system. Our objective is to assess the historical record of the uppermost layers of the sedimentary column of the lagoonal system in order to determine the history of environmental changes caused by the opening of the artificial channel. In this sense, an integrated geochemical-faunal approach is used. The environmental changes led significant modifications in salinity, in changes of the depositional patterns of sediments and foraminiferal assemblages (including periods of defaunation), and, more drastically, in the input of heavy metals to the coastal environment. The concentrations Pb in the core analyzed here were up to two times higher than the values measured in contaminated sediments from the Santos estuary, the most industrialized coastal zone in Brazil.

Cryptic species in Iphisa elegans Gray, 1851 (Squamata: Gymnophthalmidae) revealed by hemipenial morphology and molecular data

Iphisa elegans Gray, 1851 is a ground-dwelling lizard widespread over Amazonia that displays a broadly conserved external morphology over its range. This wide geographical distribution and conservation of body form contrasts with the expected poor dispersal ability of the species, the tumultuous past of Amazonia, and the previously documented prevalence of cryptic species in widespread terrestrial organisms in this region. Here we investigate this homogeneity by examining hemipenial morphology and conducting phylogenetic analyses of mitochondrial (CYTB) and nuclear (C-MOS) DNA sequence data from 49 individuals sampled across Amazonia. We detected remarkable variation in hemipenial morphology within this species, with multiple cases of sympatric occurrence of distinct hemipenial morphotypes. Phylogenetic analyses revealed highly divergent lineages corroborating the patterns suggested by the hemipenial morphotypes, including co-occurrence of different lineages. The degrees of genetic and morphological distinctness, as well as instances of sympatry among mtDNA lineages/morphotypes without nuDNA allele sharing, suggest that I. elegans is a complex of cryptic species. An extensive and integrative taxonomic revision of the I. elegans complex throughout its wide geographical range is needed. (c) 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 361376.

Low polymorphism revealed in new microsatellite markers for Bemisia tabaci (Hemiptera: Aleyrodidae)

The silverleaf whitefly (Bemisia tabaci) is actually a complex of morphologically indistinct species that vary in their capacity to transmit plant viruses and to induce physiological disorders in plants of economic importance. The worldwide impact of this whitefly has increased greatly, as it is a vector of various types of phytovirus, especially geminiviruses, in plants of economic importance. The adaptability of B. tabaci to many regions of the world has fostered the appearance of various biotypes that attack a broad spectrum of host plants. We developed microsatellite markers to study genetic variability and population structure of this whitefly in Brazil. Thirteen polymorphic microsatellite markers were isolated and characterized in 20 individuals from a natural population that were collected in soybean in Campinas (SP). The number of alleles per locus ranged from one to two, and the expected heterozygosity ranged from 0.000 to 0.505. These microsatellite markers will be useful for studies and management of B. tabaci. The low polymorphism found in these molecular markers is probably associated with homology of genes expressed in these markers.

Streptococcus pneumoniae: susceptibility to penicillin and moxifloxacin

Objective: To determine the minimum inhibitory concentrations (MICs) of parenteral penicillin and moxifloxacin against Streptococcus pneumoniae strains isolated at a hospital center. Methods: In-vitro, prospective study involving 100 S. pneumoniae isolates collected from patients who had been treated, between October of 2008 and July of 2010, at the Hospital das Clinicas complex of the University of Sao Paulo School of Medicine, located in the city of Sao Paulo, Brazil. The isolates were obtained from respiratory tract cultures or blood samples unrelated to meningeal infections, and they were tested for penicillin and moxifloxacin susceptibility by E-test. The MIC category interpretations were based on updated standards. Results: All isolates were fully susceptible to parenteral penicillin (MIC <= 2 mu g/mL), and, consequently, they were also susceptible to amoxicillin, ampicillin, third/fourth generation cephalosporins, and ertapenem. Of the S. pneumoniae strains, 99% were also susceptible to moxifloxacin, and only one strain showed an MIC = 1.5 mu g/mL (intermediate). Conclusions: Our results showed high susceptibility rates to parenteral penicillin and moxifloxacin among S. pneumoniae isolates unrelated to meningitis, which differs from international reports. Reports on penicillin resistance should be based on updated breakpoints for non-meningitis isolates in order to guide the selection of an antimicrobial therapy and to improve the prediction of the clinical outcomes.

Non-functionant urinary bladder paraganglioma

Introduction: Paragangliomas are rare tumors origined on chromaffin-cells of neural crest and can be located from skull base to pelvis, on sympathetic or parasympathetic paraganglia. They account on less than 0,06% of all urinary bladder tumors, with only few hundreds of cases reported in literature since the first record by Zimmermmann in 1953. Case Report: A 63 year-old woman referring irritative urinary symptoms was submitted to an ultrassonography that disclosed an irregular-shaped nodulation on her bladder. CT confirmed the existence of a nodulation on bladder's anterior wall. Patient had normal levels of urinary catecholamins and Vanilmandelic acid. Tumor was excised and posterior immunohistochemical study revealed it was a paraganglioma. Nowadays, ten months after surgery, patient stills healthy and disease-free. Discussion: Paragangliomas can be classified as functionant or non-functionant, according to its production of cathecolamins, which can cause the same symptom complex of pheocromocytomas. About 10-15% of bladder paragangliomas are malignant, and potential metastasis are more common to lymph nodes, lungs and bones. 131-MIBG iodine cyntilography is the most sensitive method for diagnosis and surgery (transurethral resection or cystectomy) is the best choice for treatment.

Estudo anatômico e funcional do complexo ligamentar colateral medial do cotovelo

OBJETIVO: Realizar um estudo anatômico do ligamento colateral medial, um importante estabilizador do cotovelo, em diferentes graus de flexo-extensão do cotovelo. MÉTODOS: Foram dissecados 40 cotovelos para analisar o comportamento funcional das bandas anterior, posterior e transversa do ligamento nas manobras de estresse em valgo do cotovelo durante seu movimento de flexão e extensão em diferentes graus. Determinou-se dois grupos: no GPA foi seccionado, a banda posterior do ligamento, depois a cápsula articular e, por fim, a banda anterior; no GAP, a ordem de dissecação foi inversa. RESULTADOS: No GPA observou-se instabilidade somente na terceira etapa e a média de abertura foi maior entre 50&º e 70&º de flexão de cotovelo; no GAP, a instabilidade apareceu desde a primeira etapa e os graus de flexão com maior instabilidade foram nos mesmos do grupo A. CONCLUSÃO: A banda anterior do ligamento colateral medial do cotovelo é o mais importante estabilizador na instabilidade em valgo do cotovelo e sua atuação principal acontece entre 50º e 70º de flexão do cotovelo. Nível de evidência III, Estudo Diagnóstico - Investigação de um exame para diagnóstico.