Physical exercise on the rat ventral prostate: Steroid hormone receptors, apoptosis and cell proliferation

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

Viability and cell cycle analysis of equine fibroblasts cultured in vitro

This experiment aimed to study equine fibroblasts in culture analyzing and the cell cycle and viability of cells pre- and post-freezing. Skin fragments were obtained from 6 horses and cultured in DMEM high glucose + 10% FCS in 5% CO(2) until the beginning of confluence. Two passages were performed before freezing. Cells subjected to serum starvation (0.5% FCS) were analyzed for viability and cell cycle at 24, 48, 72, 96, 120, 144 and 168 h of culture. For the confluent groups, cells were analyzed at the moment they achieved confluence. Cellular viability was assisted with Hoescht 33342 and propidium iodide. The analysis of apoptosis/necrosis and cell cycle was performed using a flow cytometer (FACS Calibur BD(A (R))) after staining the cells with annexin V and propidium iodide. Both optical microscopy and flow cytometry confirmed that cellular viability was similar for serum starvation and confluent groups (average 84%). Similarly, both methods were efficient to synchronize the cell cycle before freezing. However, after thawing, serum starvation, for more than 24 h, was superior to culture for synchronizing cells in G0/G1 (69% x 90%). The results of this experiment indicate that equine fibroblasts can be efficiently cultured after thawing.

A marker-and-cell approach to free surface 2-D multiphase flows

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Population and reproductive biology of the crab Uca burgersi (Crustacea : Ocypodidae) in three subtropical mangrove forests

Population and reproductive biology were studied in three populations of the crab Uca burgersi Holthuis, 1967, in the Indaia, Cavalo and Ubatumirim mangrove forests (Ubatuba, São Paulo State, Brazil). Crabs were collected during low tide (August 2001 through July 2002), by digging the sediment, with a standard capture effort (two persons for 30 min.). Carapace width was measured, and gonad developmental stage was recorded from all specimens. U. burgersi was most abundant in the Cavalo mangrove, where the largest mate was found. Juvenile crabs were found year-round at all three sites. However, the ratio of ovigerous females was very low, even null in the Cavalo mangrove. The gonad development rate indicated that U. burgersi was reproducing continuously, but more intensively during spring and summer, with recruitment occurring in winter. The synchrony between the populational and reproductive biology in the three areas showed that local features were not the limiting factors. It is suggested that this species is a habitat generalist.

In situ localization of heat-shock proteins and cell death labelling in the salivary gland of acaricide-treated honeybee larvae

The effects of the acaricides, rotenone and oxalic acid (OA) on salivary glands of honeybee larvae were evaluated. Immunohistochemical methods were used to detect cell death and heat-shock protein (HSP70 and 90) localizations. Heat-shock proteins (HSP70 and 90) were localized in the cytoplasm and/or the nuclei of secretory gland cells, both under stress and in normal conditions. In rotenone-treated larvae, there were no changes in the normal level of cell death and also there were no morphological alterations in the secretory cells. In the larvae treated with oxalic acid, the salivary gland showed varying degrees of morphological cellular alteration and an increase in the cell death level. The present data suggest that stress-induced HSP70 might have an antiapoptotic effect while the stress-induced HSP90 might have a chaperone function in the larval salivary glands.

The neurotoxicological effects of mastoparan Polybia-MPII at the murine neuromuscular junction: an ultrastructural and immunocytochemical study

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Mitosis and cell death in the optic lobes of workers, queens and drones of the honey bee (Apis mellifera) during metamorphosis

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

Nectaries and reproductive biology of Croton sarcopetalus (Euphorbiaceae)

Flower morphology, nectary structure, nectar chemical composition, breeding system, floral visitors and pollination were analysed in Croton sarcopetalus, a diclinous-monoecious shrub from Argentina. Male flowers have five receptacular nectaries, with no special vascular bundles, that consist of a uniserial epidermis with stomata subtended by a secretory parenchyma. Female flowers bear two different types of nectaries: inner (IN) and outer (ON) floral nectaries. IN, five in all, are structurally similar to the nectaries of male flowers. The five ON are vascularized, stalked, and composed of secretory, column-shaped epidermal cells without stomata subtended by secretory and ground parenchyma. In addition, ON act as post-floral nectaries secreting nectar during fruit ripening. Extrafloral nectaries (EFN) are located on petioles, stipules and leaf margins. Petiolar EFN are patelliform, stalked and anatomically similar to the ON of the female flower. Nectar sampled from all nectary types is hexose dominant, except for the ON of the female flower at the post-floral stage that is sucrose dominant. The species is self-compatible, but geitonogamous fertilization is rarely possible because male and female flowers are not usually open at the same time in the same individual, i.e. there is temporal dioecism. Flowers are visited by 22 insect species, wasps being the most important group of pollinators. No significant differences were found in fruit and seed set between natural and hand pollinated flowers. This pattern indicates that fruit production in this species is not pollen/pollinator limited and is mediated by a wide array of pollinators. (C) 2001 the Linnean Society of London.

Ecology and reproductive biology of two species of Aplastodiscus (Anura : Hylidae) in the Atlantic forest, Brazil

The present study was conducted in the Serra do Japi in the State of São Paulo, Brazil. From March 2004 to January 2006 we studied the ecology and reproductive biology of two sympatric species of Aplastodiscus in three different habitats: lake, stream, and swamp. The majority of A. leucopygius males in calling activity were recorded during the rainy season and during sporadic rains in the dry season (April to September). The same was observed for A. arildae. Most courtship displays of A. leucopygius were observed mainly during the rainy period and only one courtship behaviour was observed for A. arildae during a rainy night, in December 2005. Aplastodiscus leucopygius males were found in activity in the three habitats surveyed. In contrast, males of A. arildae were found in only one habitat (stream). Spatial distribution seems to be the main mechanism of reproductive segregation between the studied species.

Localized matrix metalloproteinase (MMP)-2 and MMP-9 activity in the rat ventral prostate during the first week of postnatal development

The initial events in prostatic morphogenesis involve cell proliferation, epithelial canalization and outgrowth toward the stroma. We have hypothesized that stromal rearrangement takes place at the sites of epithelial growth and branching and that this rearrangement involves the action of gelatinases matrix metalloproteinase (MMP)-2 and MMP-9. Thus, the purpose of the present study was to characterize structural aspects of epithelial growth during the first week of postnatal development of the rat ventral prostate and to investigate the expression, localization and activity of MMP-2 and MMP-9 during this period by histological, ultrastructural and immunocytochemical analysis, in addition to gel zymography, in situ zymography and Western blotting. An increasing complexity of prostatic architeture was observed within the first postnatal week. Concurrently, the stroma became more organized and some cells differentiated into smooth muscle cells. Reticulin fibers appeared in a basket-like arrangement around both growing tips and epithelial sprouts, associated with a fainter staining for laminin. MMP-2 and MMP-9 activities were detected. MMP-2/MMP-9 expression decreased during the first week. Developing epithelial cords showed strong and difuse gelatinolytic activity. This activity coincided with the distribution of MMP-2 as determined by immunocytochemistry. on the other hand, MMP-9 was rather concentrated at the epithelial tips. These results suggest that gelatinolytic activity (with contribution of both MMP-2 and MMP-9) in the epithelium and at the epithelium-stroma interface are at least in part responsible for the tissue remodeling that allows epithelial growth and its projection into the surrounding stroma.

Characterization of mineral contents on androgen deficient and chronic ethanol consuming rats by biochemistry and non-destructive techniques

Ethanol can compromise the body mineral composition and affect bone, and when associated to hypogonadism is considered an important risk factor for osteoporosis in man. The aim of this study was to investigate the effect of androgen deficient and chronic ethanol consuming on mineral contents by biochemistry and non-destructive techniques. Wistar rat (n=54) were divided in orchiectomy (ORQ) or SHAM-operated and subdivided by diet. They were daily fed with a Lieber DeCarli diet model for 8 weeks long. The controls groups were free-diet and pair-fed. Ca and P were analyzed by biochemistry test in the blood and by nX-ray fluorescence and FT-Raman on the femur area. Serum analysis revealed hypocalcaemia and hypeiphosphataemia in ethanol groups more than pair-fed and free-diet. In similarity, spectroscopy indicated a decrease in bone Ca content in ORQ groups, mainly for ethanol groups. Phosphorus content and Ca/P molar ratio, otherwise, doesn't diverge in all 6 groups. Ethanol consumption impaired Ca and P homeostasis in ORQ rat more than SHAM. The relationships among ethanol consume and androgen deficit support the hypothesis that ethanol affects the mineral-regulating hormones and may mediate some effects on bone. These findings demonstrate that ethanol seemed to interfere with the normal compensatory response to these Ca and P levels and is more significant M androgen deficiency rats.

Yeast flotation viewed as the result of the interplay of supernatant composition and cell-wall hydrophobicity

Flotation is a process of cell separation based on the affinity of cells to air bubbles. In the present work, flotability and hydrophobicity were determined using cells from different yeasts (Hansenulla polymorpha, Saccharomyces cerevisiae, Candida albicans), which were propagated in different media and at different temperatures. Alterations to the supernatant of the cells were also carried out before the flotation assays. The results described here indicate that supernatants of the yeast cells can play a more important role on flotation than cell-wall hydrophobicity. For example, wall-hydrophobicity of strain FLT-01 of S. cerevisiae was high but flotation did not occur when their washed cells were resuspended in water. Additions of neopeptone to cultures of S. cerevisiae and H. polymorpha repressed flotation and increased the volume of foam. An additional task of the present work was to show that the relationship between cell-wall hydrophobicity and flotation performance was dependent on the method used for the measurement of hydrophobicity. Based on the assay procedure, two types of hydrophobicity were distinguished: (a) the apparent hydrophobicity for cells suspended in the medium and expressed by the degree of cell affinity to the organic solvent in the two-phase system supernatant/hexane; (b) the standard hydrophobicity, which was determined for cells suspended in a standard solution (acetate buffer, in the present work) within the acetate buffer/hexane system. Flotation of cells of S. cerevisiae and C albicans were best related to the degree of apparent hydrophobicity (varying with the supernatant composition at the cell/medium interface) rather than to the degree of standard hydrophobicity (varying with the alterations in the wall components, since the liquid phase was constant in the assay). However, depending on the yeast unpredictable results can be obtained. For example, cells of H. polymorpha exhibited good flotation associated to a high degree of standard hydrophobicity while having a lower degree of apparent hydrophobicity. Concerning growth temperature, flotation of cells of C albicans was strongly repressed when the temperature was raised from 30 to 38 degreesC while a similar effect was not observed in cultures of S. cerevisiae and H. polymorpha. It is difficult to understand and predict flotation of yeast cells but simple modifications made to the supernatant of cultures can activate or repress flotation. (C) 2003 Elsevier B.V. B.V. All rights reserved.

CYTOGENETIC EVIDENCE OF INVOLVEMENT OF CHROMOSOME REGION-15Q12 AND REGION-12Q15 IN CONDITIONS WITH ASSOCIATED OVERGROWTH

Syndromes with associated overgrowth are poorly understood. Besides their mode of inheritance, nothing is known regarding the basic genetic alterations that lead to their abnormal phenotypic manifestations. The chromosome localization of the genes involved remains unknown for this group of syndromes, with the only exception being the Wiedemann-Beckwith syndrome.

Fluorescence and confocal laser scanning microscopy imaging of elastic fibers in hematoxylin-eosin stained sections

We have studied the possibility of associating fluorescence microscopy and hematoxylin-eosin staining for the identification of elastic fibers in elastin-rich tissues. Elastic fibers and elastic laminae were consistently identified by the proposed procedure, which revealed itself to be easy and useful for the determination of such structures and their distribution. The fluorescence properties of stained elastic fibers are due to eosin staining as revealed by fluorescence analysis of the dye in solution, with no or only minor contribution by the elastin autofluorescence. The main advantage of this technique resides in the possibility of studying the distribution of elastic fibers in file material without further sectioning and staining. The use of the confocal laser scanning microscope greatly improved the resolution and selectivity of imaging elastic fibers in different tissues. The determination of the three-dimensional distribution and structure of elastic fiber and laminae using the confocal laser scanning microscope was evaluated and also produced excellent results.

Purification and partial characterization of cathepsin D from porcine (Sus scrofa) liver using affinity chromatography

Cathepsin D, a lysosomal aspartic protease, has been purified from porcine liver using a combination of pepstatin-A agarose and Affi-Gel Blue affinity chromatography, followed by size-exclusion chromatography. The purified protein consists of two polypeptide chains of 15 and 30 kDa, and has an isoelectric point of 6.8. Porcine liver cathepsin D has maximum activity at pH 2.5-3.0 as determined by its activity against hemoglobin, with a K-cat of 14.3 s(-1) and a k(cat)/K-M of 2.70 x 10(6) s(-1) M-1 as determined by the hydrolysis of a fluorogenic peptide substrate.