Differentiation of epithelial cells in the urinary tract

Uroplakins, cytokeratins and the apical plasma membrane were studied in the epithelia of mouse urinary tract. In the simple epithelium covering the inner medulla of the renal pelvis, no uroplakins or cytokeratin 20 were detected and cells had microvilli on their apical surface. The epithelium covering the inner band of the outer medulla became pseudostratified, with the upper layer consisting of large cells with stalks connecting them to the basal lamina. Uroplakins and cytokeratin 20 were not expressed in these cells. However, some superficial cells appeared without connections to the basal lamina; these cells expressed uroplakins Ia, Ib, II and III and cytokeratin 20, they contained sparse small uroplakin-positive cytoplasmic vesicles and their apical surface showed both microvilli and ridges. Cytokeratin 20 was seen as dots in the cytoplasm. This epithelium therefore showed partial urothelial differentiation. The epithelium covering the outer band of the outer medulla gradually changed from a two-layered to a three-layered urothelium with typical umbrella cells that contained all four uroplakins. Cytokeratin 20 was organized into a complex network. The epithelium possessed an asymmetric unit membrane at the apical cell surface and fusiform vesicles. Umbrella cells were also observed in the ureter and urinary bladder. In males and females, the urothelium ended in the bladder neck and was continued by a non-keratinized stratified epithelium in the urethra in which no urothelial cell differentiation markers were detected. We thus show here the expression, distribution and organization of specific proteins associated with the various cell types in the urinary tract epithelium.

Annexin A1 subcellular expression in laryngeal squamous cell carcinoma

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

Does alveolar soft-part sarcoma exhibit skeletal muscle differentiation? An immunocytochemical and biochemical study of myogenic regulatory protein expression

There has been persistent controversy regarding the nature of cell differentiation in alveolar soft-part sarcoma (ASPS) since its first description in 1952. Some studies suggest that ASPS might represent an unusual variant of skeletal muscle tumor, Given the availability of new monoclonal antibodies to probe for skeletal muscle differentiation and the rapid advance in immunocytochemical techniques for deparaffinized, formalin-fixed tissue sections, we wished to test the proposed hypothesis that ASPS might represent a new type of rhabdomyosarcoma Twelve archival samples of ASPS were retrieved, and we investigated the expression of two myogenic regulatory proteins, MyoD1 and myogenin, as rvell as other muscle-associated proteins, using sensitive immunocytochemical techniques. Despite the presence of desmin immunostaining in six ASPSs, no tumors were positive for either muscle actin or myoglobin Most importantly, no specimen showed nuclear expression of MyoD1 or myogenin, In 11 tumors, however, there was considerable granular immunostaining in the tumor cell cytoplasm with the anti-MyoD1 monoclonal antibody 5.8A, a phenomenon observed in various nonmuscle normal and neoplastic tissues with this antibody, To analyze the exact nature of immunostaining of MyoD1 and desmin in ASPS, biochemical analyses using available fresh frozen tumor tissue were performed, Although a 53-kDa band was noted with antidesmin antibody on Western blot analysis, no specific protein band that corresponds to the 45-kDa MyoD1 was detected with antibody 5.8A. These results confirm the presence of desmin in ASPS but argue against authentic expression of MyoD1, They also suggest that the cytoplasmic immunostaining observed with anti-MyoD1 antibody 5.8A most likely represents a nonspecific cross-reaction with an unknown cytoplasmic antigen, Considering the master role that MyoD1 and myogenin play in skeletal muscle commitment and differentiation and the lack of expression of these two proteins in ASPS as determined immunocytochemically and biochemically, we think that the histogenesis of ASPS remains unknown.

Mating influence in the ovary differentiation in adult queens of Apis mellifera L. (Hymenoptera, Apidae)

The present results show that in the ovarioles of a newly emerged (0 day) queen of A. mellifera only two regions may be distinguished: a proximal, short germarium and a very long distal, terminal filament. As the queen matures and gets ready for the nupcial flight, the germarium increases in lenght, advancing towered the distal end, as the terminal filament shortens. The ovarioles of queens ready to mate (6 to 8 days old) have, already one or two ovarian follicles, i.e. a very short proximal vitellarium, but a real vitellogenesis only starts after the fecundation. If the queen does not mate the ovarioles structure is disrupted (12-16 days old). In mated queen eggs the ovarioles present three differentiated regions, from the apice to the basis: a short terminal filament, a medium size germarium, and a very long basal vitellarium. As the eggs are laid, the emptied follicle collapses, degenerates and produces a corpus luteum.

Description of the nucleolar activity and karyotype in germinative cell lines of Rhodnius domesticus (Triatominae, Heteroptera)

The purpose of this work was to study the karyotype, spermatogenesis and nucleolar activity at meiosis, in the species Rhodnius domesticus (Heteroptera, Triatominae). The testicular tubules were cytologically prepared by the conventional method of cell crushing and subsequent application of cytogenetic staining techniques with lacto-acetic orcein and silver-ion impregnation. The species under study presented karyotype 2n= 20A+XY, the modal number of the subfamily Triatominae. The chromosomes presented no primary constriction and were therefore characterized as holocentric. It was observed that the sex chromosomes sometimes were located at the periphery, close to the ring formed by autosomes, at first meiotic division. At metaphases II, sex chromosomes were positioned in the center of the autosomal ring, thus evidencing a postreductional behavior. These same chromosomes showed late migration at anaphases and were clearly impregnated with silver-ions, suggesting they bore Nucleolar Organizer Regions. Dispersed nucleolar corpuscles in cytoplasm until telophase II and small dots in spermatids strongly impregnated with silver, could be seen. Thus, it may be inferred that, in triatomines, the nucleolus does not completely disappear but remains in the form of small corpuscles that have a role in cell differentiation.

Genomic and phenotypic profiles of two Brazilian breast cancer cell lines derived from primary human tumors

Breast cancer is the most common type of cancer among women worldwide. Research using breast cancer cell lines derived from primary tumors may provide valuable additional knowledge regarding this type of cancer. Therefore, the aim of this study was to investigate the phenotypic profiles of MACL-1 and MGSO-3, the only Brazilian breast cancer cell lines available for comparative studies. We evaluated the presence of hormone receptors, proliferation, differentiation and stem cell markers, using immunohistochemical staining of the primary tumor, cultured cells and xenografts implanted in immunodeficient mice. We also investigated the ability of the cell lines to form colonies and copy number alterations by array comparative genomic hybridization. Histopathological analysis showed that the invasive primary tumor from which the MACL-1 cell line was derived, was a luminal A subtype carcinoma, while the ductal carcinoma in situ (DCIS) that gave rise to the MGSO-3 cell line was a HER2 subtype tumor, both showing different proliferation levels. The cell lines and the tumor xenografts in mice preserved their high proliferative potential, but did not maintain the expression of the other markers assessed. This shift in expression may be due to the selection of an 'establishment' phenotype in vitro. Whole-genome DNA evaluation showed a large amount of copy number alterations (CNAs) in the two cell lines. These findings render MACL-1 and MGSO-3 the first characterized Brazilian breast cancer cell lines to be potentially used for comparative research. © 2013 Spandidos Publications Ltd. All rights reserved.

Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: An improved approach in cartilage tissue engineering

Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n=5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1×105) were than encapsulated inside 60μl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI. © 2013 Informa UK Ltd.

Isolation, Culture and Differentiation of Buffaloes Bone Marrow Mesenchymal Stem Cells Obtained from the Coxal Tuberosity

Currently, much attention has been devoted to the renewal of knowledge about Stem Cells and Cell Therapy in domestic species. In this sense, the present work aimed to develop a methodology for collecting, processing and cultivation of mesenchymal stem cells obtained from bone marrow of coxal tuberosity in buffaloes. The collection was performed using a Komiyashiki needle, which was introduced in the coxal tuberosity and the bone marrow aspirated into a heparinized syringe with the aid of negative pressure. Directly after collection samples were processed at the laboratory at FMVZ - UNESP. The samples took approximately 32 days to reach 80% confluence, when the first passage and differentiation was performed. To confirm the mesenchymal origin, cells were induced to differentiate into adipogenic and osteogenic lineages. Samples showed morphological changes during differentiation protocol, but not all presented production of extracellular deposits of calcium or intracellular fat droplets, observed after staining with Alizarin Red and Oil Red respectively. Compared with the material obtained from other species and processed in the same laboratory, the primary culture was longer. Therefore, more studies are needed to standardize the age of animals used and to test other inducers of cell differentiation.

Cissus quadrangularis Linn exerts dose-dependent biphasic effects: osteogenic and anti-proliferative, through modulating ROS, cell cycle and Runx2 gene expression in primary rat osteoblasts

Objectives: This report highlights phytoconstituents present in Cissus quadrangularis (CQ) extract and examines biphasic (proliferative and anti-proliferative) effects of its extract on bone cell proliferation, differentiation, mineralization, ROS generation, cell cycle progression and Runx2 gene expression in primary rat osteoblasts. Materials and methods: Phytoconstituents were identified using gas chromatography-mass spectroscopy (GC-MS). Osteoblasts were exposed to different concentrations (10-100g/ml) of CQ extract and cell proliferation and cell differentiation were investigated at different periods of time. Subsequently, intracellular ROS intensity, apoptosis and matrix mineralization of osteoblasts were evaluated. We performed flow cytometry for DNA content and real-time PCR for Runx2 gene expression analysis.Results: CQ extract's approximately 40 bioactive compounds of fatty acids, hydrocarbons, vitamins and steroidal derivatives were identified. Osteoblasts exposed to varying concentrations of extract exhibited biphasic variation in cell proliferation and differentiation as a function of dose and time. Moreover, lower concentrations (10-50g/ml) of extract slightly reduced ROS intensity, although they enhanced matrix mineralization, DNA content in S phase of the cell cycle, and levels of Runx2 expression. However, higher concentrations (75-100g/ml) considerably induced the ROS intensity and nuclear condensation in osteoblasts, while it reduced mineralization level, proportion of cells in S phase and Runx2 level of the osteogenic gene.Conclusions: These findings suggest that CQ extract revealed concentration-dependent biphasic effects, which would contribute notably to future assessment of pre-clinical efficacy and safety studies.

Efeitos da tricostatina A sobre a acetilação de histonas, proliferação celular e diferenciação de células tronco embrionárias murinas

Background: Embryonic stem cells are cells derived from early-stage embryos that are characterized by pluripotency and self-renewal capacity. The in vitro cultured murine embryonic stem cells can indefinitely propagate in an undifferentiated state in the presence of leukemia inhibitory factor (LIF). However, when stimulated, these cells can differentiate into cell lines derived from all three embryonic germ layers. The trichostatin A (TSA) is an epigenetic modifier agent and several studies have used the TSA to stimulate cellular differentiation. However, most of these studies only assessed one TSA concentration. Therefore, this study aimed to evaluate the effects of different TSA concentrations on histone hyperacetylation during in vitro cell differentiation of murine pluripotent embryonic stem cells, cultured with or without LIF, in the quest of to standardize their application on early cultures of embryonic stem cells.Materials, Methods & Results: Undifferentiated murine embryonic stem cells were plated in the presence of different TSA concentrations (0 nM, 15 nm, 50 nM and 100 nM) in the presence or absence of LIF. Thus, the treatments were evaluated in undifferentiated embryonic stem cells cultured in the presence of LIF (Control group: 0 nM LIF(+); Group 15 nM LIF+; Group 50 nM LIF+ and Group 100 nM LIF+), and in embryonic stem cells cultured in the absence of LIF (Control group: 0 nM LIF; Group 15 nM LIF(-); Group 50 nM LIF(-) and Group 100 nM LIF-). Treatment with TSA was performed for 24 h. After that the medium was replaced with fresh medium without TSA. Samples were collected at 0, 12, 24, 36 and 48 h after the beginning of the experiment. Three replicates were performed in each experimental group. The relative amount of Histone H3 lysine 9 acetylation was analyzed in all groups, as well as the cell proliferation in the embryonic stem cells cultured in the presence of LIF. In the control group (0 nM), the absence of LIF resulted in higher levels (P < 0.05) of H3lys9ac compared to the cultures supplemented with LIF. In the embryonic stem cells cultured in the presence of LIF, the 50 nM and 100 nM treatments resulted in higher levels (P < 0.05) of H3lys9ac when compared with 0 nM and 15 nM treatments. Evaluating the Hoechst area in the 0 nM group, it was observed that the number of cells increased (P < 0.05) according to the time of culture. Treatment with 15 nM also reflected a similar distribution, but the Hoechst area in 15 nM group was lower (P < 0.05) at 24 and 48h when compared to the observed in the control group. In the 100 nM treatment, was observed that the area of Hoechst was lower (P < 0.05) to that obtained in the control group at 12, 24 and 48h. In addition, it was observed that treatment with TSA induces greater cellular differentiation when compared to control groups in stem cells cultured in the presence of LIF as well as in the absence of LIF.Discussion: In the present study it was observed that TSA treatment increased the levels of histone acetylation in murine embryonic stem cells at a 50 nM concentration, making it possible to reduce the concentration recommended in the literature (100 nM). In addtion, it was concluded that the lower TSA concentrations utilized (15 nm and 50 nM) was less harmful to cellular proliferation than the 100 nM TSA concentration.

Regulation and action of fibroblast growth factor 17 in bovine follicles

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

Crystallization and preliminary x-ray analysis of Parkia pendula lectin

Lectins are proteins important in various biological processes such as infection, cell differentiation and metastasis. The Parkia pendula lectin has been crystallized using the hanging-drop vapour diffusion method. X-ray diffraction data were collected using a Rigaku RU300 rotating anode generator and R-AXIS IV diffractometer. The cell parameters for P. pendula lectin are a=93.7 Angstrom b=161.1 Angstrom, c=80.0 Angstrom and space group C222. The maximum resolution was of 2.98 Angstrom. These data showed a R-sym=12.8%.

The fibroblast growth factor family: involvement in the regulation of folliculogenesis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)