Fine structure of the branchial epithelium in the teleost Oreochromis niloticus

We have studied the gill epithelium of Oreochromis niloticus using transmission electron microscopy with the particular interested relationship between cell morphology and osmotic, immunoregulatory, or other non‐regulatory functions of the gill. Pavement cells covered the filament epithelium and lamellae of gills, with filament pavement cells showing distinct features from lamellar pavement cells. The superficial layer of the filament epithelium was formed by osmoregulatory elements, the columnar mitochondria‐rich, mucous and support cells, as well as by their precursors. Light mitochondria‐rich cells were located next to lamellae. They exhibited an apical crypt with microvilli and horizontal small dense rod‐like vesicles, sealed by tight junctions to pavement cells. Dark mitochondria‐rich cells had long dense rod‐like vesicles and a small apical opening sealed by tight junctions to pavement cells. The deep layer of the filament epithelium was formed by a network of undifferentiated cells, containing neuroepithelial and myoepithelial cells, macrophage and eosinophil‐like cells and their precursors, as well as precursors of mucous cells. The lateral‐basal surface was coated by myoepithelial cells and a basal lamina. The lamellar blood lacunae was lined by pillar cells and surrounded by a basal lamina and pericytes. The data presented here support the existence of two distinct types of pavement cells, mitochondria‐rich cells, and mitochondria‐rich cells precursors, a structural role for support cells, a common origin for pavement cells and support cells, a paracrine function for neuroepithelial cells in the superficial layer, and the control of the lamellar capillary base by endocrine and contractile cells. Data further suggest that the filament superficial layer is involved in gill osmoregulation, that may interact, through pale mitochondria‐rich cells, with the deep layer and lamellae, whereas the deep layer, through immune and neuroendocrine systems, acts in the regeneration and defense of the tissue. J. Morphol. 2010. © 2010 Wiley‐Liss, Inc.     

Invasion of MDCK epithelial cells with altered expression of Rho GTPases by Trypanosoma cruzi amastigotes and metacyclic trypomastigotes of strains from the two major phylogenetic lineages

In order to invade mammalian cells, Trypanosoma cruzi infective forms cause distinct rearrangements of membrane and host cell cytoskeletal components. Rho GTPases have been shown to regulate three separate signal transduction pathways, linking plasma membrane receptors to the assembly of distinct actin filament structures. Here, we examined the role of Rho GTPases on the interaction between different T. cruzi infective forms of strains from the two major phylogenetic lineages with nonpolarized MDCK cells transfected with different Rho GTPase constructs. We compared the infectivity of amastigotes isolated from infected cells (intracellular amastigotes) with forms generated from the axenic differentiation of trypomastigotes (extracellular amastigotes), and also with metacyclic trypomastigotes. No detectable effect of GTPase expression was observed on metacyclic trypomastigote invasion and parasites of Y and CL (T. cruzi II) strains invaded to similar degrees all MDCK transfectants, and were more infective than either G or Tulahuen (T. cruzi I) strains. Intracellular amastigotes were complement sensitive and showed very low infectivity towards the different transfectants regardless of the parasite strain. Complement-resistant T. cruzi I extracellular amastigotes, especially of the G strain, were more infective than T. cruzi II parasites, particularly for the Rac1V12 constitutively active GTPase transfectant. The fact that in Rac1N17 dominant-negative cells, the invasion of G strain extracellular amastigotes was specifically inhibited suggested an important role for Rac1 in this process.     

Volatile oil from Guarea macrophylla ssp. tuberculata: seasonal variation and electroantennographic detection by Hypsipyla grandella

GC and GC-MS analyses of the volatile oils from Guarea macrophylla (Meliaceae) collected during three different periods in one year (February, June and October) indicated a seasonal variation in chemical composition. Whilst sesquiterpenes were the predominant class of components present in the leaf oil, a seasonal dependent variation in the degree of oxygenation of these compounds was detected, which seemed to be associated with phenological factors. The leaf oil, and fractions thereof, were subjected to GC coupled with electroantennographic detection employing antennae of females of Hypsipyla grandella, an insect pest that attacks several meliaceous species. Five compounds elicited significant responses and these were identified as ledol, 1-cubenol, guai-6-en-10beta-ol, 1-epi-cubenol, and tau-muurolol. The results suggest that these components could be responsible for the attraction of H. grandella to G. macrophylla.     

Evaluation of sample preparation methods for the analysis of papaya leaf proteins through two‐dimensional gel electrophoresis

Introduction – A variety of sample preparation protocols for plant proteomic analysis using two‐dimensional gel electrophoresis (2‐DE) have been reported. However, they usually have to be adapted and further optimised for the analysis of plant species not previously studied. Objective – This work aimed to evaluate different sample preparation protocols for analysing Carica papaya L. leaf proteins through 2‐DE. Methodology – Four sample preparation methods were tested: (1) phenol extraction and methanol–ammonium acetate precipitation; (2) no precipitation fractionation; and the traditional trichloroacetic acid–acetone precipitation either (3) with or (4) without protein fractionation. The samples were analysed for their compatibility with SDS–PAGE (1‐DE) and 2‐DE. Fifteen selected protein spots were trypsinised and analysed by matrix‐assisted laser desorption/ionisation time‐of‐flight tandem mass spectrometry (MALDI‐TOF‐MS/MS), followed by a protein search using the NCBInr database to accurately identify all proteins. Results – Methods number 3 and 4 resulted in large quantities of protein with good 1‐DE separation and were chosen for 2‐DE analysis. However, only the TCA method without fractionation (no. 4) proved to be useful. Spot number and resolution advances were achieved, which included having an additional solubilisation step in the conventional TCA method. Moreover, most of the theoretical and experimental protein molecular weight and pI data had similar values, suggesting good focusing and, most importantly, limited protein degradation. Conclusion – The described sample preparation method allows the proteomic analysis of papaya leaves by 2‐DE and mass spectrometry (MALDI‐TOF‐MS/MS). The methods presented can be a starting point for the optimisation of sample preparation protocols for other plant species. Copyright © 2009 John Wiley & Sons, Ltd.     

Elevated temperature may intensify the positive effects of nutrients on microbial decomposition in streams

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Enantioselective behavior of lipases from <Emphasis Type="Italic">Aspergillus niger</Emphasis> immobilized in different supports

Considering the extraordinary microbial diversity and importance of fungi as enzyme producers, the search for new biocatalysts with special characteristics and possible applications in biocatalysis is of great interest. Here, we report the performance in the resolution of racemic ibuprofen of a native enantioselective lipase from Aspergillus niger, free and immobilized in five types of support (Accurel EP-100, Amberlite MB-1, Celite, Montmorillonite K10 and Silica gel). Amberlite MB-1 was found to be the best support, with a conversion of 38.2%, enantiomeric excess of 50.7% and enantiomeric ratio (E value) of 19 in 72 h of reaction. After a thorough optimization of several parameters, the E value of the immobilized Aspergillus niger lipase was increased (E = 23) in a shorter reaction period (48 h) at 35°C. Moreover, the immobilized Aspergillus niger lipase maintained an esterification activity of at least 80% after 8 months of storage at 4°C and could be reused at least six times.     

Plant growth promoting bacteria in Brachiaria brizantha

Brachiaria brizantha is considered one of the preferred fodders among farmers for having high forage yield and large production of root mass. The association of beneficial bacteria with these grasses can be very valuable in the recovery of the pasture areas with nutritional deficiency. With the aim of studying this possibility, we carried out the sampling of soil and roots of B. brizantha in three areas (Nova Odessa-SP, S?o Carlos-SP and Campo Verde-MT, Brazil). Seventy-two bacterial strains were isolated and used in tests to evaluate their biotechnological potential. Almost all isolates presented at least one positive feature. Sixty-eight isolates produced analogues of indole-3-acetic acid, ten showed nitrogenase activity when subjected to the method of increasing the concentration of total nitrogen (total N) in the culture medium and sixty-five isolates showed nitrogenase activity when subjected to acetylene reduction technique. The partial sequencing of 16S rRNA of these isolates allowed the identification of seven main groups, with the prevalence of those affiliated to the genus Stenotrophomonas (69?%). At the end, this work elected the strains C4 (Pseudomonadaceae) and C7 (Rhodospirillaceae) as promising organisms for the development of inoculants due to their higher nitrogenase activity.     

DNA Barcoding of Neotropical Sand Flies (Diptera,Psychodidae, Phlebotominae): Species Identification and Discovery within Brazil

DNA barcoding has been an effective tool for species identification in several animal groups. Here, we used DNA barcoding to discriminate between 47 morphologically distinct species of Brazilian sand flies. DNA barcodes correctly identified approximately 90% of the sampled taxa (42 morphologically distinct species) using clustering based on neighbor-joining distance, of which four species showed comparatively higher maximum values of divergence (range 4.23–19.04%), indicating cryptic diversity. The DNA barcodes also corroborated the resurrection of two species within the shannoni complex and provided an efficient tool to differentiate between morphologically indistinguishable females of closely related species. Taken together, our results validate the effectiveness of DNA barcoding for species identification and the discovery of cryptic diversity in sand flies from Brazil.     

Azilsartan Reduced TNF-α and IL-1β Levels,Increased IL-10 Levels and Upregulated VEGF,FGF, KGF,and TGF-α in an Oral Mucositis Model

Oral mucositis (OM) is a common complication of treatments for head and neck cancer, particularly radiotherapy with or without chemotherapy. OM is characterised by oral erythema, ulceration, and pain. The aim of this study was to evaluate the effect of azilsartan (AZT), an angiotensin II receptor antagonist, on 5-fluorouracil (5-FU)-induced oral mucositis (OM) in Syrian hamsters. OM was induced by the intraperitoneal administration of 5-FU on experimental days 1 (60mg/Kg) and 2 (40mg/Kg). Animals were pretreated with oral AZT (1, 5, or 10 mg/kg) or vehicle 30 min before 5-FU injection and daily until day 10. Experimental treatment protocols were approved by the Animal Ethics Committee Use/CEUA (Number 28/2012) of the UFRN. Macroscopic analysis and cheek pouch samples were removed for histopathologic analysis. Myeloperoxidase (MPO), Malonyldialdehyde (MDA), interleukin-1 beta (IL-1β), interleukin-10 (IL-10), and tumour necrosis factor-alpha (TNF-α) were analysed by Enzyme Linked Immuno Sorbent Assay (ELISA). Vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), and transforming growth factor (TGF)-α were measured by immunohistochemistry. Analysis of variance followed by Bonferroni’s test was used to calculate the means of intergroup differences (p ≤ 0.05). Treatment with 1 mg/kg AZT reduced levels MPO (p<0.01), MDA (p<0.5) and histological inflammatory cell infiltration, and increased the presence of granulation tissue. AZT treatment at 1 mg/kg reduced the TNF-α (p<0.05) and IL-1β (p<0.05) levels, increased the cheek pouch levels of IL-10 (p<0.01), and upregulated VEGF, FGF, KGF, and TGF-α. Administration of AZT at higher doses (5 and 10 mg/kg) did not significantly reverse the OM. AZT at a dose of 1 mg/kg prevented the mucosal damage and inflammation associated with 5-FU-induced OM, increasing granulation and tissue repair.