Caracterização de interações entre subunidades do complexo de iniciação da tradução EIF4F e homólogos da proteína de ligação ao poli-A (PABP) de Leishmania sp

Os tripanossomatídeos são caracterizados por processos moleculares diferenciados como a transcrição policistrônica e regulação pós-transcricional da expressão gênica. Em mamíferos, a tradução se inicia com a ligação do complexo eIF4F (formado pelos eIF4A, eIF4E e eIF4G) a extremidade 5' dos mRNAs, o que facilita seu reconhecimento pelo ribossomo. A atividade do eIF4F é reforçada pela proteína de ligação a cauda poli-A (PABP), na extremidade 3' dos mRNAs, que interage com o eIF4G. Dois complexos do tipo eIF4F foram identificados em tripanossomatídeos: o primeiro formado pelos EIF4G3, EIF4E4 e EIF4AI com a PABP1; e um outro baseado na interação do EIF4G4 com o EIF4E3 e o EIF4A1. Este trabalho buscou caracterizar as interações entre as subunidades destes complexos e sua associação com PABPs de Leishmania, avaliando o efeito de mutações em motivos específicos. Proteínas recombinantes foram geradas fusionadas a GST e avaliadas quanto a sua habilidade de interagir com parceiros marcados radioativamente em ensaios do tipo pull-down. Para o EIF4G3, mutações individuais em dois resíduos vizinhos (I8A e R9A), afetaram a interação com o EIF4E4 e a mutação de ambos os resíduos equivalentes do EIF4G4 (IL25-26AA) também impediu sua ligação ao EIF4E3, sugerindo um motivo comum para a ligação aos seus parceiros. As proteínas EIF4E3 e EIF4E4 foram avaliadas quanto à capacidade de interagir com a PABP2 e PABP1 respectivamente, e mutações em motivos conservados nas regiões N-terminais dos EIF4E (Boxes A, B e C) aboliram sua interação com os homólogos da PABP. Para identificar que regiões da PABP1 estão relacionadas às interações com o parceiro EIF4E4, foram obtidas proteínas PABP1 mutantes em motivos conservados e observou-se que a mutação no motivo TGM, C-terminal, aboliu sua interação com o EIF4E4. Com estas abordagens conseguiu-se avançar na definição das interações entre as referidas subunidades do eIF4F e PABP, identificando-se diferenças relevantes em relação a outros eucariotos

Identificação de motivos relevantes para a função do fator de iniciação da tradução EIF4E3 de Leishmania sp

Os tripanossomatídeos são organismos caracterizados pelo controle póstranscricional da expressão gênica, principalmente em nível de tradução. Na tradução em eucariotos, tem grande destaque o complexo eIF4F, sendo um de seus principais componentes o fator de iniciação da tradução eIF4E. Já foram descritos em tripanossomatídeos seis homólogos para o eIF4E, nomeados EIF4E1 a 6. Em um estudo com Leishmania amazonensis, focado no EIF4E3, percebeu-se que seu perfil de expressão se alterava rapidamente numa curva de crescimento, com este apresentando ao menos duas bandas. As mudanças observadas sugeriam modificações pós-traducionais do tipo fosforilação, algo posteriormente confirmado. Analisando-se a sequência do EIF4E3 de Leishmania, foi possível identificar a presença de possíveis sítios de fosforilação e de ligação a parceiros funcionais como homólogos do eIF4G, outro componente do complexo eIF4F, e da proteína de ligação á cauda poli-A (PABP). No presente estudo foi analisado o perfil de expressão e a capacidade de ligação a parceiros funcionais do EIF4E3 de Leishmania superexpresso em células transfectadas e no qual foram introduzidas mutações em motivos específicos. Os resultados mostraram um perfil de expressão de ao menos três bandas para o EIF4E3 de L. amazonensis e duas para L. infantum, com o sítio S75, presente apenas na primeira, sendo o responsável por esta diferença. Em ensaios de imunoprecipitação, foi identificado um motivo que, quando mutado, aboliu a ligação do EIF4E3 com a PABP3, sugerindo este como o sítio de interação entre os fatores. Com a análise do efeito de mutações no EIF4E3 de L. amazonensis, foi percebido que ao se mutar três motivos implicados na à PABP e o possível sítio de ligação ao EIF4G, sua fosforilação diminuiu drasticamente, sugerindo a necessidade destas interações para que a fosforilação ocorra. Estes resultados indicam um complexo mecanismo de modificações pós-traducionais responsáveis pela regulação do EIF4E3 e contribuem a para a caracterização da sua função em Leishmania

Corynebacterium uterequi sp. nov., a non-lipophilic bacterium isolated from urogenital samples from horses

Three strains of a Gram-positive, catalase-positive, fermentative, non-lipophilic, previously unknown bacterium were isolated from urogenital samples taken from mares in Scotland (M401624/00/1) and Sweden (VM 2074 and VM 2298T). All were deposited with the CCUG with tentative identifications as Corynebacterium spp. The strains were characterized using a polyphasic taxonomic approach. Biochemically, the strains were very similar to each other, but phylogenetically distinct from Corynebacterium species with validly published names (≤95% sequence similarity). rpoB gene sequence data confirmed the strains belonged to the same species (>99% sequence similarity) and were distinct from species with validly published names (>13% sequence divergence). On the basis of phenotypic and sequence data, the strains represent a novel species within the genus Corynebacterium, for which the name Corynebacterium uterequi is proposed. The type strain is VM 2298T (=CCUG 61235T = DSM 45634T), isolated from equine uterus.

Sponge layer feedbacks in middle-atmosphere models

Middle-atmosphere models commonly employ a sponge layer in the upper portion of their domain. It is shown that the relaxational nature of the sponge allows it to couple to the dynamics at lower levels in an artificial manner. In particular, the long-term zonally symmetric response to an imposed extratropical local force or diabatic heating is shown to induce a drag force in the sponge that modifies the response expected from the “downward control” arguments of Haynes et al. [1991]. In the case of an imposed local force the sponge acts to divert a fraction of the mean meridional mass flux upward, which for realistic parameter values is approximately equal to exp(−Δz/H), where Δz is the distance between the forcing region and the sponge layer and H is the density scale height. This sponge-induced upper cell causes temperature changes that, just below the sponge layer, are of comparable magnitude to those just below the forcing region. In the case of an imposed local diabatic heating, the sponge induces a meridional circulation extending through the entire depth of the atmosphere. This circulation causes temperature changes that, just below the sponge layer, are of opposite sign and comparable in magnitude to those at the heating region. In both cases, the sponge-induced temperature changes are essentially independent of the height of the imposed force or diabatic heating, provided the latter is located outside the sponge, but decrease exponentially as one moves down from the sponge. Thus the effect of the sponge can be made arbitrarily small at a given altitude by placing the sponge sufficiently high; e.g., its effect on temperatures two scale heights below is roughly at the 10% level, provided the imposed force or diabatic heating is located outside the sponge. When, however, an imposed force is applied within the sponge layer (a highly plausible situation for parameterized mesospheric gravity-wave drag), its effect is almost entirely nullified by the sponge-layer feedback and its expected impact on temperatures below largely fails to materialize. Simulations using a middle-atmosphere general circulation model are described, which demonstrate that this sponge-layer feedback can be a significant effect in parameter regimes of physical interest. Zonally symmetric (two dimensional) middle-atmosphere models commonly employ a Rayleigh drag throughout the model domain. It is shown that the long-term zonally symmetric response to an imposed extratropical local force or diabatic heating, in this case, is noticeably modified from that expected from downward control, even for a very weak drag coefficient

Optimization of a sponge cake formulation with inulin as fat replacer: structure, physicochemical, and sensory properties

The effects of several fat replacement levels (0%, 35%, 50%, 70%, and 100%) by inulin in sponge cake microstructure and physicochemical properties were studied. Oil substitution for inulin decreased significantly (P < 0.05) batter viscosity, giving heterogeneous bubbles size distributions as it was observed by light microscopy. Using confocal laser scanning microscopy the fat was observed to be located at the bubbles’ interface, enabling an optimum crumb cake structure development during baking. Cryo-SEM micrographs of cake crumbs showed a continuous matrix with embedded starch granules and coated with oil; when fat replacement levels increased, starch granules appeared as detached structures. Cakes with fat replacement up to 70% had a high crumb air cell values; they were softer and rated as acceptable by an untrained sensory panel (n = 51). So, the reformulation of a standard sponge cake recipe to obtain a new product with additional health benefits and accepted by consumers is achieved.

Optimizing mixing during the sponge cake manufacturing process

Sponge cakes have traditionally been manufactured using multistage mixing methods to enhance potential foam formation by the eggs. Today, use of all-in (single-stage) mixing methods is superseding multistage methods for large-scale batter preparation to reduce costs and production time. In this study, multistage and all-in mixing procedures and three final high-speed mixing times (3, 5, and 15 min) for sponge cake production were tested to optimize a mixing method for pilot-scale research. Mixing for 3 min produced batters with higher relative density values than did longer mixing times. These batters generated well-aerated cakes with high volume and low hardness. In contrast, after 5 and 15 min of high-speed mixing, batters with lower relative density and higher viscosity values were produced. Although higher bubble incorporation and retention were observed, longer mixing times produced better developed gluten networks, which stiffened the batters and inhibited bubble expansion during mixing. As a result, these batters did not expand properly and produced cakes with low volume, dense crumb, and high hardness values. Results for all-in mixing were similar to those for the multistage mixing procedure in terms of the physical properties of batters and cakes (i.e., relative density, elastic moduli, volume, total cell area, hardness, etc.). These results suggest the all-in mixing procedure with a final high-speed mixing time of 3 min is an appropriate mixing method for pilot-scale sponge cake production. The advantages of this method are reduced energy costs and production time.

Bone Formation Following Implantation of Titanium Sponge Rods into Humeral Osteotomies in Dogs: A Histological and Histometrical Study

Background: Titanium (Ti) is widely proven to enhance bone contact and growth on its surface. It is expected that bone defects could benefit from Ti to promote healing and to increase strength of the implanted area. Purpose: The present study aimed at comparing the potential of porous Ti sponge rods with synthetic hydroxyapatite (HA) for the healing of bone defects in a canine model. Material and Methods: Six mongrel dogs were submitted to three trephined osteotomies of 6.0 x 4.0 mm in one humerus and after 2 months another three osteotomies were performed in the contralateral humerus. A total of 36 defects were randomly filled either with Ti foam, particulate HA, or coagulum (control). The six animals were killed 4 months after the first surgery for histological and histometrical analysis. Results: The Ti-foam surface was frequently found in intimate contact with new bone especially at the defect walls. Control sites showed higher amounts of newly formed bone at 2 months - Ti (p = 0.000) and HA (p = 0.009) - and 4 months when compared with Ti (p = 0.001). Differently from HA, the Ti foam was densely distributed across the defect area which rendered less space for bone growth in the latter`s sites. The use of Ti foams or HA resulted in similar amounts of bone formation in both time intervals. Nevertheless, the presence of a Ti-foam rod preserved defect`s marginal bone height as compared with control groups. Also, the Ti-foam group showed a more mature bone pattern at 4 months than HA sites. Conclusion: The Ti foam exhibited good biocompatibility, and its application resulted in improved maintenance of bone height compared with control sites. The Ti foam in a rod design exhibited bone ingrowth properties suitable for further exploration in other experimental situations.

Henneguya pseudoplatystoma n. sp causing reduction in epithelial area of gills in the farmed pintado, a South American catfish: Histopathology and ultrastructure

The present study is part of an ongoing investigation into the characteristics of Myxozoan parasites of Brazilian freshwater fish and was carried out using morphology, histopathology and electron microscopy analysis. A new Myxosporea species (Henneguya pseudoplatystoma) is described causing an important reduction in gill function in the farmed pintado (a hybrid fish from a cross between Pseudoplatystoma corruscans and Pseudoplatystoma fasciatum), which is a commercially important South American catfish. From a total of 98 pintado juveniles from fish farms in the states of Sao Paulo and Mato Grosso do Sul (Brazil), 36 samples (36.7%) exhibited infection of the gill filaments. infection was intense, with several plasmodia occurring on a same gill filament. The plasmodia were white and measured up to 0.5 mm in length; mature spores were ellipsoidal in the frontal view, measuring 33.2 +/- 1.9 mu m in total length, 10.4 +/- 0.6 mu m in body length, 3.4 +/- 0.4 mu m in width and 22.7 +/- 1.7 mu m in the caudal process. The polar capsules were elongated, measuring 3.3 +/- 0.4 mu m in length and 1.0 +/- 0.1 mu m in width and the polar filaments had six to seven turns. Histopathological analysis revealed the parasite in the connective tissue of the gill filaments and lamella. No inflammatory process was observed, but the development of the plasmodia reduced the area of functional epithelium. Ultrastructural analyses revealed a single plasmodial wall, which was in direct contact with the host cells and had numerous projections in direction of the host cells as well as extensive pinocytotic canals. A thick layer (2-6 mu m) of fibrous material and numerous mitochondria were found in the ectoplasm. Generative cells and the earliest stage of sporogenesis were seen more internally. Advanced spore developmental stages and mature spores were found in the central portion of the plasmodia. (C) 2009 Elsevier B.V. All rights reserved.

Light, electron microscopy and histopathology of Myxobolus salminus n. sp., a parasite of Salminus brasiliensis from the Brazilian Pantanal

In this report, we describe the morphology and histopathology of Myxobolus salminus n. sp., a parasite of the gill filaments of wild Salminus brasiliensis (dourado) from the Brazilian Pantanal. The small polysporic plasmodia were similar to 100 mu m in diameter and the development was asynchronous. The mature spores were oval to pear shaped and had a smooth wall. The spore measurements were (mean +/- S.D., with range in parentheses): length 10.1 +/- 0.4 mu m (9.6-10.5), width 6.1 +/- 0.4 mu m (5.8-6.6) and thickness 5.0 +/- 0.6 mu m (4.7-5.3). The polar capsules were elongated and of equal size: length 4.6 +/- 0.2 mu m (4.3-4.8) and width 1.7 +/- 0.1 mu m (1.5-1.9). The histological analysis revealed numerous plasmodia in the blood vessels of the gill filaments. The site of parasite development was the wall of the large-caliber blood vessel of the gill filament, with progressive growth towards the lumen, resulting in the obstruction of blood flow, congestion and perivascular edema. The ultrastructural study revealed that the plasmodial wall was composed of two membranes, had numerous pinocytic canals and was in direct contact with the basement membrane of the vessel. The development of the parasite was asynchronous, with mature spores, immature spores and young developmental stages randomly distributed throughout the plasmodium. The prevalence of the parasite was 4.4%. with male and female fish being infected. (C) 2009 Elsevier B.V. All rights reserved.

Myxobolus cordeiroi n. sp., a parasite of Zungaro jahu (Siluriformes: Pimelodiade) from Brazilian Pantanal: Morphology, phylogeny and histopathology

This work is part of an ongoing investigation into the characteristics of Myxozoan parasites of freshwater fish in Brazil and was carried out using morphology, histopathology and molecular analysis. A new Myxosporea species (Myxobolus cordeiroi) is described infecting the jau catfish (Zungaro jahu). Fifty jau specimens were examined and 78% exhibited plasmodia of the parasite. The plasmodia were white and round, measuring 0.3-2.0 mm in diameter and the development occurred in the gill arch, skin, serosa of the body cavity, urinary bladder and eye. The spores had an oval body and the spore wall was smooth. Partial sequencing of the 18S rDNA gene resulted in a total of 505 bp and the alignment of the sequences obtained from samples in different organs revealed 100% identity. In the phylogenetic analysis, the Myxobolus species clustered into two clades-one primarily parasites of freshwater fish and the other primarily parasites of marine fish. M. cordeiroi n. sp. was clustered in a basal position in the freshwater fish species clade. The histological analysis revealed the parasite in the connective tissue of the different infected sites, thereby exhibiting affinity to this tissue. The plasmodium was surrounded by an outer collagen capsule of fibers with distinct orientation from the adjacent connective tissue and an inner layer composed of delicate collagen fibrils-more precisely reticular fibers. The development of the parasite in the cornea and urinary bladder caused considerable stretching of the epithelium. (C) 2009 Elsevier B.V. All rights reserved.

Host-parasite-environment relationship, morphology and molecular analyses of Henneguya eirasi n. sp parasite of two wild Pseudoplatystoma spp. in Pantanal Wetland, Brazil

A new myxosporean species, Henneguya eirasi n. sp., is described parasitizing the gill filaments of Pseudoplatystoma corruscans and Pseudoplatystoma fasciatum (Siluriformes: Pimelodidae) caught in the Patanal Wetland of the state of Mato Grosso, Brazil. The parasite formed white, elongated plasmodia measuring up to 3 mm. Mature spores were ellipsoidal in the frontal view, measuring 37.1 +/- 1.8 mu m in total length, 12.9 +/- 0.8 mu m in body length, 3.4 +/- 0.3 mu m in width, 3.1 +/- 0.1 mu m in thickness and 24.6 +/- 2.2 mu m in the caudal process. Polar capsules were elongated and equal in size, measuring 5.4 +/- 0.5 mu m in length and 0.7 +/- 0.1 mu m in width. Polar filaments had 12-13 coils. Histopathological analysis revealed that the parasite developed in the sub-epithelial connective tissue of the gill filaments and the plasmodia were surrounded by a capsule of host connective tissue. The plasmodia caused slight compression of the adjacent tissues, but no inflammatory response was observed in the infection site. Ultrastructure analysis revealed a single plasmodial wall connected to the ectoplasmic zone through numerous pinocytotic canals. The plasmodial wall exhibited numerous projections and slightly electron-dense material was found in the ectoplasm next to the plasmodial wall, forming a line just below the wall. Partial sequencing of the 18S rDNA gene of H. eirasi n. sp. obtained from P. fasciatum resulted in a total of 1066 bp and this sequence did not match any of the Myxozoa available in the GenBank. Phylogenetic analysis revealed the Henneguya species clustering into clades following the order and family of the host fishes. H. eirasi n. sp. clustered alone in one clade, which was the basal unit for the clade composed of Henneguya species parasites of siluriform ictalurids. The prevalence of the parasite was 17.1% in both fish species examined. Parasite prevalence was not influenced by season, host sex or host size. (C) 2011 Elsevier B.V. All rights reserved.

Light and scanning electron microscopy of Henneguya arapaima n. sp (Myxozoa: Myxobolidae) and histology of infected sites in pirarucu (Arapaima gigas: Pisces: Arapaimidae) from the Araguaia River, Brazil

In this report, we describe Henneguya arapaima n. sp., a parasite of the gill arch and gall bladder of Arapaima gigas (pirarucu) collected in the Araguaia River, in the municipality of Nova Crixas, Goias State, central Brazil. The plasmodia were white, round or ellipsoidal and measured 200-600 mu m. Parasite development was asynchronous and the mature spores were fusifonn and had smooth wall. The spores measurements were (range, with means +/- S.D. in parentheses): total length-48.4-53.1 mu m (51.6 +/- 3.4 mu m), body length-13.5-15.2 mu m (14.2 +/- 0.8 mu m), body width-5.1-6.1 mu m (5.7 +/- 0.5 mu m), body thickness-4.7-5.3 mu m (4.9 +/- 0.2 mu m) and caudal process length-38.0-41.2 mu m (38.3 +/- 2.9 mu m). The polar capsules were elongated and of unequal size, with lengths of 6.3-6.8 mu m (6.5 +/- 0.2) and 6.2-6.6 mu m (6.3 +/- 0.1) for the longest and shortest axes, respectively. Capsule width was 1.4-1.6 mu m (1.5 +/- 0.1). Histological analysis showed that the plasmodia occurred in the tunica adventitia of the gall bladder and were delimited by a thin capsule of connective tissue. In the gill arch, the plasmodia were also surrounded by connective tissue similar to the endomesium, of striated skeletal muscle cells. Sixty-five juvenile specimens of A. gigas weighing 1.0-25.0 kg were examined, 17 (26.1%) of which were infected. Of these, 14 (82.3%) had cysts in the gall bladder, two (11.7%) had cysts in the gill arch and only one (5.9%) had cysts in both organs. When the fish were grouped by weight, the prevalence of infection in fish weighing up to 10.0 kg (20.7%) was significantly lower than in fish weighing 10.1-25.0 kg (50%) (G = 3.93; d.f. = 1; p < 0.05). (C) 2008 Elsevier B.V. All rights reserved.

Phylogenetic relationships in Kumanoa (Batrachospermales, Rhodophyta) species in Brazil with the proposal of Kumanoa amazonensis sp nov.

This Study evaluated the species-level taxonomy and phylogenetic relationship among Kumanoa species from Brazil with other regions of the world based on the plastid-encoded RUBISCO large Subunit gene (rhcL). Partial rbcL sequences were obtained for 11 Kulnanoa specimens. Eight species are recognised from Brazil on the basis of molecular and morphological data: seven previously described (K abilii, K ambignia, K. breviarticulata, K. cipoensis, K. equisetoidea, K. globospora and K procarpa) and a new species here proposed (K. amazonensis sp. nov. Necchi & Vis). The new species has reduced and dense whorls but differs from the two closest related species in lacking secondary fascicles. Previously proposed infrageneric categories were not supported by the molecular data. Species described and endemic (K. breviarticulata, K. cipoensis, K equiseloidea and K. procarpa) to Brazil are not grouped together but are variously related to other species from North America, Europe and Australasia. With the species recognised in this study using molecular and morphological data and those previously distinguished by morphology, 13 species of Kumanoa are Currently documented from Brazil.