Expression of Pancreatic Endocrine Markers by Prolactin-Treated Rat Bone Marrow Mesenchymal Stem Cells

Background. Mesenchymal stem cells (MSCs) are an attractive source for generation of cells with beta-cell properties. Previous studies have demonstrated the ability of prolactin to induce an increase in beta-cell mass and maturation, which suggests beneficial effects of its use in MSC differentiation protocols. Objective. To evaluate the expression of endocrine differentiation markers in rat MSCs treated in vitro with prolactin. Methods. Mesenchymal stem cells from bone marrow of Wistar rats were isolated, expanded, and characterized. Differentiation of MSCs was induced in medium containing 23 mmol/L of glucose, and nicotinamide, 2-mercaptoethanol, and exendin-4, in the presence or absence of 500 ng/mL of rat recombinant prolactin. Expression of endocrine markers and prolactin receptor genes was evaluated using real-time polymerase chain reaction, and compared between culture stages and presence vs absence of prolactin in the culture medium. Expression of insulin, somatostatin, glucagon, and pancreatic and duodenal homeobox 1 was also evaluated at immunofluorescence microscopy. Results. Isolated cells were mostly MSCs, as confirmed at fluorescent-activated cell sorting and cytochemistry. Pax6, Ngn-3, Isl1, NeuroD1, Nkx2.2, and Nkx6.1 exhibited varied expression during culture stages. The long form of the prolactin receptor messenger RNA was induced in prolactin-treated cultures (P < .05). The somatostatin gene was induced in early stages of differentiation (P < .05), and its expression was induced by prolactin, as confirmed using immunofluorescence. Conclusion. Culture of rat bone marrow MSCs in differentiation medium induces expression of pancreatic endocrine-specific genes, and somatostatin and prolactin receptor expression was also induced by prolactin.

Islet neogenesis-associated protein signaling in neonatal pancreatic rat islets: involvement of the cholinergic pathway

Islet neogenesis associated protein (INGAP) increases islet mass and insulin secretion in neonatal and adult rat islets. lit the Present Study, we measured the short- and long-term effects of INGAP-PP (a pentadecapeptide having the 104-118 amino acid sequence of INGAP) upon islet protein expression and phosphorylation of components of the PI3K, MAPK and cholinergic pathways, and on insulin secretion. Short-term exposure of neonatal islets to INGAP-PP (90 s, 5, 15, and 30 min) significantly increased Akt1(-Ser473) and MAPK3/1(-Thr202/Tyr204) phosphorylation and INGAP-PP also acutely increased insulin secretion from islets perifused with 2 and 20 mM glucose. Islets cultured for 4 days in the presence of INGAP-PP showed an increased expression of Akt1, Frap1, and Mapk1 mRNAs as well as of the muscarinic M3 receptor subtype, and phospholipase C (PLC)-beta 2 proteins. These islets also showed increased Akt1 and MAPK3/1 protein phosphorylation. Brief exposure of INGAP-P-treated islets to carbachol (Cch) significantly increased P70S6K(-Thr389) and MAPK3/1 phosphorylation and these islets released more insulin when challenged with Cch that was prevented by the M3 receptor antagonist 4-DAMP in a concentration-dependent manner. In conclusion, these data indicate that short- and long-term exposure to INGAP-PP significantly affects the expression and the phosphorylation of proteins involved in islet PI3K and MAPK signaling pathways. The observations of INGAPP-PP-stimulated up-regulation of cholinergic M3 receptors and PLC-beta 2 proteins, enhanced P70S6K and MAIIK3/1 phosphorylation and Cch-induced insulin secretion suggest a participation of the cholinergic pathway in INGAP-PP-mediated effects.

Aspectos morfológicos, germinação e desenvolvimento inicial de plântulas de Archontophoenix alexandrae (F. Mueller) H. Wendl. e Drude (Arecaceae) em diferentes substratos

Este trabalho teve por objetivo descrever a morfologia dos diásporos e as suas fases de germinação, bem como determinar o substrato mais adequado para a germinação das sementes e o crescimento das plântulas de palmeira-da-rainha (Archontophoenix alexandrae (F. Mueller) H. Wendl. e Drude). Periodicamente, unidades representativas de cada fase de germinação foram retiradas para as descrições morfológicas. Os substratos utilizados foram Plantmax®, areia, terra (latossolo roxo) e outro com proporções iguais de terra, areia e esterco (TAE). Foi instalado um experimento em delineamento inteiramente casualizado. Avaliaram-se a porcentagem, o tempo médio de germinação e o crescimento inicial das plântulas aos 135 dias após a emergência, com base na altura, diâmetro do colo e número, comprimento e largura das folhas. As sementes são albuminosas, com endosperma ruminado e oleaginoso, e o embrião é lateral, periférico e relativamente indiferenciado. A germinação é do tipo criptocotiledonar hipógea, iniciando-se com a formação de uma massa de células indiferenciadas na depressão micropilar. Essa massa de células torna-se cilíndrica, com a diferenciação dos primórdios caulinares e radiculares. Concomitantemente, ocorre o desenvolvimento de raízes adventícias no eixo embrionário. O substrato mais indicado para a germinação de sementes de palmeira-da-rainha é o Plantmax®, porém, para o crescimento inicial, indica-se Plantmax® e TAE.

The effects of ovalbumin as a protein source during the in vitro production of bovine embryos

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

Aspectos anatômicos do olho e neuroquímicos da retina do mocó (Kerodon rupestris)

The visual system is an important link between the animal and the environment, com profound influences on the habits and lifestyle in various habitats. Adaptive mechanismsto the temporal niche are present in the visual system of many vertebrates, involving changins in ocular dimensios and design, retinal cell distribution and organization of neurochemical circuits related to the retinal resolution or sensitivity. The sensory system of the eye is represented by the retina, whose organization is responsible by receipty, initial analysis, and transmission of the information to the brain. The knowledge of the position of the eyes in the head and the distribution of retinal cells allow to identify adaptive aspects of each species to its visual field, which is characteristic to the ecological niche it occupies. In this research, we study eye anatomical characteristics and retina neurochemical features of the rock cavy (Kerodon rupestris), a tipical Brazilian rodent from the suborder Hystricomorpha, family Caviidae. The rock cavy has lateral eyes well constitute bony orbit and well differentiated extrinsic muscle. The study of the descriptive and morphometric anatomy of the showed mean values of axial diameter 10.7±0,5mm and equatorial diameter 11.6±0.7mm. The pupil is slit shaped and the lens has mean axial diameter 5.4±0.03 mm, corresponding to ~45% of the axial diameter of the eye. The posterior nodal distance and the retinal magnification factor were estimated at 6.74 mm e 118 μm/grau, respectively. Flat mounts were processed for Nissl stain, and the topographic distribution of ganglion cells showed a moderate visual band, just below the optic disc, with higher density in the ventral retina. Retinal vertical sections and flat mounts were processed for immunohistochemistry to visualize tyrosine hydroxilase (TH) and thus two types of TH+ cells were detected. Type 1 cells had strong TH-immunoreactivity, the body cell varied from 120.047 to 269.373 μm2 stratifying in the sublamina 1 of the IPL. Type 2 cells were weakly TH-imunoreactive, had cell body located mostly in the IPL, varying from 54.848 to 177.142 μm2, constituting ~10% of the TH+ cells. Both cell types exhibited similar topographic distribution with higher density found in a horizontal band along of the naso-temporal axis in the dorsal retina. The total population of dopaminergic cells was 2,156±469,4 cells, occupying an average area of 198,164 μm2. The presence of cones and rods was detected by immunohistochemistry in vertical sections and flat mounts. S cones density is around 10 times smaller than L cones, with different degree of spatial organization. Other retinal neuronal populations of the rock cavy were also detected in vertical sections with specific markers. Comparative analysis of the anatomical characteristics of the rock cavy eye 12 suggest that it was designed to acquire higher sensitivity to light, at expense of image sharpness, compatible with a vision at mesopic conditions. Additionally, the distribution of the 2 subtypes of dopaminergic cells in a naso-temporal band in the dorsal retina seems suitable to a gain in sensitivity, coherent with an animal with predominantly crepuscular activity pattern

Estudo das poliaminas na morfogênese in vitro de Hemerocallis sp.

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cultivation of Rubrivivax gelatinosus in fish industry effluent for depollution and biomass production

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

INGAP-PP up-regulates the expression of genes and proteins related to K-ATP(+) channels and ameliorates Ca2+ handling in cultured adult rat islets

Islet Neogenesis Associated Protein (INGAP) increases pancreatic beta-cell mass and potentiates glucose-induced insulin secretion. Here, we investigated the effects of the pentadecapeptide INGAP-PP in adult cultured rat islets upon the expression of proteins constitutive of the K-ATP(+) channel, Ca2+ handling, and insulin secretion. The islets were cultured in RPMI medium with or without INGAP-PP for four days. Thereafter, gene (RT-PCR) and protein expression (Western blotting) of Foxa2, SUR1 and Kir6.2, cytoplasmic Ca2+ ([Ca2+](i)), static and dynamic insulin secretion, and Rb-86 efflux were measured. INGAP-PP increased the expression levels of Kir6.2, SUR1 and Foxa2 genes, and SUR1 and Foxa2 proteins. INGAP-PP cultured islets released significantly more insulin in response to 40 mM KCl and 100 mu M tolbutamide. INGAP-PP shifted to the left the dose-response curve of insulin secretion to increasing concentrations of glucose (EC50 of 10.0 +/- 0.4 vs. 13.7 +/- 1.5 mM glucose of the controls). It also increased the first phase of insulin secretion elicited by either 22.2 mM glucose or 100 mu M tolbutamide and accelerated the velocity of glucose-induced reduction of Rb-86 efflux in perifused islets. These effects were accompanied by a significant increase in [Ca2+](i) and the maintenance of a considerable degree of [Ca2+](i) oscillations. These results confirm that the enhancing effect of INGAP-PP upon insulin release, elicited by different secretagogues, is due to an improvement of the secretory function in cultured islets. Such improvement is due, at least partly, to an increased K-ATP(+) channel protein expression and/or changing in the kinetic properties of these channels and augmented [Ca2+](i) response. Accordingly, INGAP-PP could potentially be used to maintain the functional integrity of cultured islets and eventually, for the prevention and treatment of diabetes. (C) 2008 Elsevier B.V. All rights reserved.

Experimental study of the centripetal force

We describe an experimental set-up for studying and determining the centripetal force acting on a body whose mass and radius of circular trajectory are known. The experimental behaviour is determined and compared with theoretical predictions by using a system that allows for the evaluation of the angular velocity by means of electric current measurements, and by assuming a potential dependence of the force on the body mass, radius and angular velocity. © 2007 IOP Publishing Ltd.

The aster mission: Exploring for the first time a triple system asteroid

2001 SN263 is a triple system asteroid. Although it was discovery in 2001, in 2008 astronomical observation carried out by Arecibo observatory revealed that it is actually a system with three bodies orbiting each other. The main central body is an irregular object with a diameter about 2.8 km, while the other two are small objects with less than 1 km across. This system presents an orbital eccentricity of 0.47, with perihelion of 1.04 and aphelion of 1.99, which means that it can be considered as a Near Earth Object. This interesting system was chosen as the target for the Aster mission - first Brazilian space exploration undertaking. A small spacecraft with 150 kg of total mass, 30 kg of payload with 110 W available for the instruments, is scheduled to be launched in 2015, and in 2018 it will approach and will be put in orbit of the triple system. This spacecraft will use electric propulsion and in its payload it will carry image camera, laser rangefinder, infrared spectrometer, mass spectrometer, and experiments to be performed in its way to the asteroid. This mission represents a great challenge for the Brazilian space program. It is being structured to allow the full engagement of the Brazilian universities and technological companies in all the necessary developments to be carried out. In this paper, we present some aspects of this mission, including the transfer trajectories to be used, and details of buss and payload subsystems that are being developed and will be used. Copyright ©2010 by the International Astronautical Federation. All rights reserved.

Canonical WNT signaling regulates development of bovine embryos to the blastocyst stage

Objectives were to evaluate the role of canonical WNT signaling in development of the preimplantation embryo. Signaling was activated with 2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) and inhibited with Dickkopf-related protein 1 (DKK1). Treatment of bovine embryos with AMBMP at day 5 after insemination decreased development to the blastocyst stage at day 7 and reduced numbers of trophectoderm and inner cell mass cells. At high concentrations, AMBMP caused disorganization of the inner cell mass. DKK1 blocked actions of AMBMP but did not affect development in the absence of AMBMP. Examination of gene expression in day 6 morulae by microarray revealed expression of 16 WNT genes and other genes involved in WNT signaling; differences in relative expression were confirmed by PCR for 7 genes. In conclusion, the preimplantation embryo possesses a functional WNT signaling system and activation of the canonical pathway can inhibit embryonic development.

Yeast proteins originated from the production of brazilian bioethanol quantification and content

The purpose of this work was to determine the levels of protein and the amino acid distribution in the cell mass of yeast strains (Saccharomyces sensu stricto) originated from Brazilian bioethanol industries. The protein was analyzed with the Kjeldahl method and the amino acids, by using high-performance liquid chromatography (HPLC). The percentages of the protein found ranged from 39 to 49%. The results show that in spite of some variation in numbers between the different yeast strains, all of them presented an amino acid profile similar to the one in the literature for S. cerevisae. The amino acids that have occurred in the largest amounts were: aspartic, glutamic acids and lysine, and those in the lowest amounts were: cysteine and methionine. Although the characteristics of the feedstock used and the process conditions are determinant of the protein values obtained in dry mass, this work elucidates that the intrinsic properties of the yeast strain influence these values.

Dynamics of DNA Methylation during Early Development of the Preimplantation Bovine Embryo

There is species divergence in control of DNA methylation during preimplantation development. The exact pattern of methylation in the bovine embryo has not been established nor has its regulation by gender or maternal signals that regulate development such as colony stimulating factor 2 (CSF2). Using immunofluorescent labeling with anti-5-methylcytosine and embryos produced with X-chromosome sorted sperm, it was demonstrated that methylation decreased from the 2-cell stage to the 6-8 cell stage and then increased thereafter up to the blastocyst stage. In a second experiment, embryos of specific genders were produced by fertilization with X- or Y-sorted sperm. The developmental pattern was similar to the first experiment, but there was stage × gender interaction. Methylation was greater for females at the 8-cell stage but greater for males at the blastocyst stage. Treatment with CSF2 had no effect on labeling for DNA methylation in blastocysts. Methylation was lower for inner cell mass cells (i.e., cells that did not label with anti-CDX2) than for trophectoderm (CDX2-positive). The possible role for DNMT3B in developmental changes in methylation was evaluated by determining gene expression and degree of methylation. Steady-state mRNA for DNMT3B decreased from the 2-cell stage to a nadir for D 5 embryos >16 cells and then increased at the blastocyst stage. High resolution melting analysis was used to assess methylation of a CpG rich region in an intronic region of DNMT3B. Methylation percent decreased between the 6-8 cell and the blastocyst stage but there was no difference in methylation between ICM and TE. Results indicate that DNA methylation undergoes dynamic changes during the preimplantation period in a manner that is dependent upon gender and cell lineage. Developmental changes in expression of DNMT3B are indicative of a possible role in changes in methylation. Moreover, DNMT3B itself appears to be under epigenetic control by methylation. © 2013 Dobbs et al.

Heparin and penicillamine-hypotaurine-epinephrine (PHE) solution during bovine in vitro fertilization procedures impair the quality of spermatozoa but improve normal oocyte fecundation and early embryonic development

The presence of heparin and a mixture of penicillamine, hypotaurine, and epinephrine (PHE) solution in the in vitro fertilization (IVF) media seem to be a prerequisite when bovine spermatozoa are capacitated in vitro, in order to stimulate sperm motility and acrosome reaction. The present study was designed to determine the effect of the addition of heparin and PHE during IVF on the quality and penetrability of spermatozoa into bovine oocytes and on subsequent embryo development. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes and mitochondrial function, was diminished (P < 0.05) in the presence of heparin and PHE. Oocyte penetration and normal pronuclear formation rates, as well as the percentage of zygotes presenting more than two pronuclei, was higher (P < 0.05) in the presence of heparin and PHE. No differences were observed in cleavage rates between treatment and control (P > 0.05). However, the developmental rate to the blastocyst stage was increased in the presence of heparin and PHE (P > 0.05). The quality of embryos that reached the blastocyst stage was evaluated by counting the inner cell mass (ICM) and trophectoderm (TE) cell numbers and total number of cells; the percentage of ICM and TE cells was unaffected (P > 0.05) in the presence of heparin and PHE (P < 0.05). In conclusion, this study demonstrated that while the supplementation of IVF media with heparin and PHE solution impairs spermatozoa quality, it plays an important role in sperm capacitation, improving pronuclear formation, and early embryonic development. © 2013 The Society for In Vitro Biology.

Cultivo e caracterização de células-tronco embrionárias de bovinos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)