Spermatogenesis of Zaprionus indianus and Zaprionus sepsoides (Diptera, Drosophilidae): Cytochemical, structural and ultrastructural characterization

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Fermion localization on branes with generalized dynamics

In this letter we consider a specific model of braneworld with nonstandard dynamics diffused in the literature, specifically we focus our attention on the matter energy density, the energy of system, the Ricci scalar and the thin-brane limit. As the model is classically stable and capable of localize gravity, as a natural extension we address the issue of fermion localization of fermions on a thick brane constructed out from one scalar field with nonstandard kinetic terms coupled with gravity. The contribution of the nonstandard kinetic terms to the problem of fermion localization is analyzed. It is found that the simplest Yukawa coupling η ωφ ω supports the localization of fermions on the thick brane. It is shown that the zero mode for left-handed fermions can be localized on the thick brane depending on the values for the coupling constant η. Copyright © EPLA, 2013.

Paracoccidoides brasiliensis 30 kDa Adhesin: Identification as a 14-3-3 Protein, Cloning and Subcellular Localization in Infection Models

Paracoccidoides brasiliensis adhesion to lung epithelial cells is considered an essential event for the establishment of infection and different proteins participate in this process. One of these proteins is a 30 kDa adhesin, pI 4.9 that was described as a laminin ligand in previous studies, and it was more highly expressed in more virulent P. brasiliensis isolates. This protein may contribute to the virulence of this important fungal pathogen. Using Edman degradation and mass spectrometry analysis, this 30 kDa adhesin was identified as a 14-3-3 protein. These proteins are a conserved group of small acidic proteins involved in a variety of processes in eukaryotic organisms. However, the exact function of these proteins in some processes remains unknown. Thus, the goal of the present study was to characterize the role of this protein during the interaction between the fungus and its host. To achieve this goal, we cloned, expressed the 14-3-3 protein in a heterologous system and determined its subcellular localization in in vitro and in vivo infection models. Immunocytochemical analysis revealed the ubiquitous distribution of this protein in the yeast form of P. brasiliensis, with some concentration in the cytoplasm. Additionally, this 14-3-3 protein was also present in P. brasiliensis cells at the sites of infection in C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells for 72 h (acute infections) and 30 days (chronic infection). An apparent increase in the levels of the 14-3-3 protein in the cell wall of the fungus was also noted during the interaction between P. brasiliensis and A549 cells, suggesting that this protein may be involved in host-parasite interactions, since inhibition assays with the protein and this antibody decreased P. brasiliensis adhesion to A549 epithelial cells. Our data may lead to a better understanding of P. brasiliensis interactions with host tissues and paracoccidioidomycosis pathogenesis. © 2013 Silva et al.

Immunophenotypic, immunocytochemistry, ultrastructural, and cytogenetic characterization of mesenchymal stem cells from equine bone marrow

The aim of this study was to isolate, culture, and characterize mesenchymal stem cells (MSCs) from horse bone marrow (BM) using the techniques of flow cytometry, immunocytochemistry, cytogenetics, and electron microscopy. Immunophenotypic analysis revealed the presence of MSCs with high expression of the CD90 marker, lower expression of the CD44 marker, and absent expression of the CD34 marker. In assays of differentiation, the positive response to osteogenic (OST), chondrogenic (CDG), and adipogenic (ADP) differentiation signals was observed and characterized by deposition of calcium-rich extracellular matrix (OST), proteoglycans and collagen II (CDG) and intracellular deposition of fat drops (ADP). In immunocytochemical characterization, MSCs were immunopositive for CD44, vimentin, and PCNA, and they were negative for CD13. In the ultrastructural analysis of MSCs, the most outstanding characteristic was the presence of rough endoplasmic reticulum with very dilated cisterns filled with a low electrodensity material. Additionally, MSCs had normal karyotypes (2n=64) as evidenced by cytogenetic analysis, and aneuploidy in metaphase was not observed. The protocols for isolating, culturing, and characterizing equine MSCs used in this study were shown to be appropriate for the production of a cell population with a good potential for differentiation and without aneuploidy that can be used to study future cellular therapies. © 2013 Wiley Periodicals, Inc.

Subcellular localization and kinetic characterization of a gill (Na +, K+)-ATPase from the giant freshwater prawn macrobrachium rosenbergii

The stimulation by Mg2+, Na+, K+, NH 4 +, and ATP of (Na+, K+)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na +, K+)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M r. Under saturating Mg2+, Na+, and K+ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V M = 115.0 ± 2.3 U mg-1, K 0.5 = 0.10 ± 0.01 mmol L-1. Stimulation by Na+ (V M = 110.0 ± 3.3 U mg-1, K 0.5 = 1.30 ± 0.03 mmol L -1), Mg2+ (V M = 115.0 ± 4.6 U mg -1, K 0.5 = 0.96 ± 0.03 mmol L-1), NH4 + (V M = 141.0 ± 5.6 U mg -1, K 0.5 = 1.90 ± 0.04 mmol L-1), and K+ (V M = 120.0 ± 2.4 U mg-1, K M = 2.74 ± 0.08 mmol L-1) followed single saturation curves and, except for K+, exhibited site-site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K I = 12.4 ± 1.3 mol L-1. Complementary inhibition studies suggest the presence of F0F1-, Na+-, or K +-ATPases, but not V(H+)- or Ca2+-ATPases, in the gill microsomal preparation. K+ and NH4 + synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH4 +, and K+ of the gill enzyme. © 2013 Springer Science+Business Media New York.

Localization patterns of steroid and luteinizing hormone receptors in the corpus luteum of Nelore (Bos taurus indicus) cows throughout the estrous cycle

The aim of the present study was to detect progesterone receptors (A and B isoforms), α and β estrogen receptors, luteinizing hormone receptors and aromatase cytochrome P450 enzymes in the corpus luteum of Nelore (Bos taurus indicus) cows using immunohistochemistry. The estrous cycles of 16 Nelore cows were synchronized, and luteal samples were collected via an incision into the vaginal vault. Samples were collected during specific days of the estrous cycle (days 6, 10, 15 and 18) and 24. h after circulating progesterone dropped, after luteolysis had occurred. After each biopsy was taken, all animals were resynchronized so that each biopsy was performed during a different estrous cycle. Our results showed that the concentration of studied proteins vary throughout the bovine estrous cycle. The highest concentration of α and β estrogen receptors and the highest concentration of plasma progesterone were both observed on days 10 and 15 of the estrous cycle. The highest concentration of progesterone receptors was observed on days 6 and 10 of the estrous cycle, and the most intense immunostaining for cytochrome P450 aromatase enzymes was observed on day 10 of the estrous cycle. The highest score of cells with plasma membrane immunostaining for LH receptors was observed on day 15 of the estrous cycle. In conclusion, this study demonstrates the varying concentrations of specific proteins within the corpus luteum of Nelore cows during the estrous cycle. This finding suggests that these receptors and enzymes, and their interactions, are important in regulating luteal viability. © 2013 Elsevier B.V.

Chromosomal organization of repetitive DNA sequences in Astyanax bockmanni (Teleostei, Characiformes): dispersive location, association and co-localization in the genome

Repetitive DNA sequences constitute a great portion of the genome of eukaryotes and are considered key components to comprehend evolutionary mechanisms and karyotypic differentiation. Aiming to contribute to the knowledge of chromosome structure and organization of some repetitive DNA classes in the fish genome, chromosomes of two allopatric populations of Astyanax bockmanni were analyzed using classic cytogenetics techniques and fluorescent in situ hybridization, with probes for ribosomal DNA sequences, histone DNA and transposable elements. These Astyanax populations showed the same diploid number (2n = 50), however with differences in chromosome morphology, distribution of constitutive heterochromatin, and location of 18S rDNA and retroelement Rex3 sites. In contrast, sites for 5S rDNA and H1, H3 and H4 histones showed to be co-located and highly conserved. Our results indicate that dispersion and variability of 18S rDNA and heterochromatin sites are not associated with macro rearrangements in the chromosome structure of these populations. Similarly, distinct evolutionary mechanisms would act upon histone genes and 5S rDNA, contributing to chromosomal association and co-location of these sequences. Data obtained indicate that distinct mechanisms drive the spreading of repetitive DNAs in the genome of A. bockmanni. Also, mobile elements may account for the polymorphism of the major rDNA sites and heterochromatin in this genus. © 2013 Springer Science+Business Media Dordrecht.

Ultrastructural characteristics of the spermatogenesis during the four phases of the annual reproductive cycle of the black myotis bat, Myotis nigricans (Chiroptera: Vespertilionidae)

Myotis nigricans is an endemic species of vespertilionid bat, from the Neotropical region, that resembles temperate zone bats in their reproductive cycle; presenting an annual reproductive cycle with two periods of testicular regression, which are not linked to the apoptotic process and seems to be not directly linked to any seasonal abiotic variation. Thus, this study aimed to ultrastructurally evaluate their reproductive cycle. The process of testicular regression could be divided into four periods: active; regressing; regressed and recrudescence; with all presenting distinct characteristics. The active period was similar to that of other bats, presenting the complete occurrence of spermatogenesis, with three main types of spermatogonia (Ad, Ap, and B) and 12 steps in spermatid differentiation; however, it differed in having the outer dense fibers 1, 5, 6, and 9 larger than the others. These three types of spermatogonia undergo considerable morphologic changes from regressing to the regressed period, and in the recrudescence, they return to the basic morphology, which reactivates spermatogenesis. In conclusion, our study described the process of spermatogenesis, the ultrastructure of the spermatozoa and the distinct morphologic variations in the ultrastructure of the testicular cells of M. nigricans during the four different periods of its annual reproductive cycle. Microsc. Res. Tech., 76:1035-1049, 2013. © 2013 Wiley Periodicals, Inc.

Gene expression and protein localization of TLR-1, -2, -4 and -6 in amniochorion membranes of pregnancies complicated by histologic chorioamnionitis

Objectives: To determine whether histologic chorioamnionitis is associated with changes in gene expression of TLR-1, -2, -4 and -6, and to describe the localization of these receptors in fetal membranes. Study design: A total of 135 amniochorion membranes with or without histologic chorioamnionitis from preterm or term deliveries were included. Fragments of membranes were submitted to total RNA extraction. RNA was reverse transcribed and the quantification of TLRs expression measured by real time PCR. Results: All amniochorion membranes expressed TLR-1 and TLR-4, whereas 99.1% of membranes expressed TLR-2 and 77.4% expressed TLR-6. TLR-1 and TLR-2 expressions were significantly higher in membranes with histologic chorioamnionitis as compared to membranes without chorioamnionitis in preterm pregnancies (p = 0.003 and p < 0.001, respectively). Among the membranes of term pregnancies there were no differences in the expressions of such receptors regardless of inflammatory status. Regarding TLR-4 and TLR-6 expression, there was no difference among membranes with or without histologic chorioamnionitis, regardless gestational age at delivery. TLR-1, TLR-2, TLR-4 and TLR-6 expressions were observed in amniotic epithelial, chorionic and decidual cells. Conclusion: Amniochorion membranes express TLR-1, TLR-2, TLR-4 and TLR-6 and increased expression of TLR-1 and TLR-2 is related to the presence of histologic chorioamnionitis in preterm pregnancies. This study provides further evidence that amniochorion membranes act as a mechanical barrier to microorganisms and as components of the innate immune system. © 2013 Elsevier B.V. All rights reserved.

Estudo morfológico comparativo das glândulas salivares de fêmeas de carrapatos Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae) em dois estágios de alimentação

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Análise citogenética clássica, molecular e ultraestrutural em escorpiões da família Buthidae: um enfoque evolutivo

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

Caracterização da enolase de Paracoccidioides brasiliensis e identificação proteômica de novas moléculas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Ultrastructural description of Ceratomyxa microlepis sp. nov. (Phylum Myxozoa): a parasite infecting the gall bladder of Hemiodus microlepis, a freshwater teleost from the Amazon River

A new ceratomyxid parasite was examined for taxonomic identification, upon being found infecting the gall bladder of Hemiodus microlepis (Teleostei: Hemiodontidae), a freshwater teleost collected from the Amazon River, Brazil. Light and transmission electron microscopy revealed elongated crescent-shaped spores constituted by two asymmetrical shell valves united along a straight sutural line, each possessing a lateral projection. The spores body measured 5.2 ± 0.4 µm (n = 25) in length and 35.5 ± 0.9 µm (n = 25) in total thickness. The lateral projections were asymmetric, one measuring 18.1 ± 0.5 µm (n = 25) in thickness and the other measuring 17.5 ± 0.5 µm (n = 25) in thickness. Two equal-sized subspherical polar capsules measuring 2.2 ± 0.3 µm in diameter were located at the same level, each possessing a polar filament with 5-6 coils. The sporoplasm was binucleate. Considering the morphometric data analyzed from the microscopic observations, as well as the host species and its geographical location, this paper describes a new myxosporean species, herein named Ceratomyxa microlepis sp. nov.; therefore representing the first description of a freshwater ceratomyxid from the South American region.

Induction and Morpho-Ultrastructural Analysis of Organogenic Calli of a Wild Passionfruit

This work studied a new protocol for organogenic calli induction and characterization of the morphology and ultrastructure of callogenesis in leaf explants of Passiflora gibertii N. E. Brown, a native passion fruit species from Brazil. Calli induction was performed in different growth conditions (light and dark), different MS medium salt concentrations (MS and MS half strength) and the presence or absence of coconut water. The leaf explants maintained in the dark were more responsive to bud formation. In order to reduce spending on in vitro culture, the most suitable induction medium for P. gibertii organogenesis could, therefore be the MS half strength salt concentration medium maintained in the dark. The addition of coconut water to the culture medium was essential for both calli induction and bud formation. The morphological and ultrastructural features of the organogenic calli were isodiametric cells, characterized by an organized cellular system, nucleus with prominent nucleoli, presence of starch grains and dense cytoplasm rich in endoplasmic reticulum. The scanning electron microscopy demonstrated that buds were present on these calli.

Ultrastructural evaluation of the radioprotective effect of sodium selenite on submandibular glands in rats

The aim of this study was to evaluate the radioprotector effect of sodium selenite on the ultrastructure of submandibular glands in rats. Fifty-seven male albino Wistar rats were randomized to 4 groups: control, irradiated, sodium selenite and irradiated/sodium selenite. The animals in the sodium selenite and irradiated/sodium selenite groups received intraperitoneal injections of sodium selenite (0.5 mg/kg body weight) 24 h before irradiation. The animals belonging to the irradiated and irradiated/sodium selenite groups were submitted to 15 Gy of gamma radiation in the head and neck region. The submandibular glands were removed at 4, 8, 12, 24, 48 and 72 h after irradiation. The ionizing radiation induced damage to the secretory cells, especially the serous cells, right from the first period. Vacuolization, lysis of cytoplasmic inclusions and nuclear alterations occurred. The sodium selenite group also presented cellular alterations in the study periods, but with less damage compared to that caused by radiation. There was greater similarity between the irradiated/sodium selenite group and the control group than with the other groups treated in all study periods. Despite the alterations observed in the sodium selenite group, sodium selenite presented a radioprotective action on the secretory cells of submandibular glands.