A região anterior do intestino médio de Lepidoptera apresenta uma secreção microapócrina de enzimas digestivas com vesículas migrando pelo interior das microvilosidades. Essas vesículas brotam das microvilosidades intestinais como vesículas de membrana dupla e são descarregadas dentro do lúmen. O objetivo desse trabalho foi identificar as proteínas secretadas e aquelas envolvidas na maquinaria secretória microapócrina em Spodoptera frugiperda. Para isso, vesículas microapócrinas foram preparadas e usadas para a produção de anticorpo policlonal. Esse anticorpo foi utilizado para varrer uma biblioteca de expressão de cDNA do intestino médio de S. frugiperda. Também obtivemos um transcriptoma por pirosequenciamento de uma biblioteca de cDNA proveniente dos transcritos do intestino médio do mesmo inseto. Os clones positivos da varredura foram sequenciados, montados e submetidos a um BLASTN contra as sequências obtidas pelo pirosequenciamento, o que resultou na extensão dessas sequências. Usamos ainda as sequências geradas pelo pirosequenciamento para reanalisar sequências de proteínas presentes nas membranas microvilares, que tinham sido obtidas anteriormente em nosso laboratório (Ferreira et al., 2007). A reanálise das sequências de proteínas microvilares gerou 66 proteínas preditas. Dessas, 18 foram consideradas contaminantes de outros compartimentos celulares e 48 associadas às membranas microvilares. A análise das sequências obtidas das vesículas microapócrinas gerou 50 proteínas preditas que podem ser secretadas por essa rota. As sequências encontradas tanto em membrana microvilar quanto em vesículas microapócrinas podem ser classificadas em 8 grupos, de acordo com sua função: (1) enzimas digestivas; (2) proteínas da membrana peritrófica; (3) envolvidas com proteção; (4) transportadores; (5) receptores; (6) proteínas da maquinaria secretória; (7) proteínas de citoesqueleto; (8) com função desconhecida nesse local. Em ambas as preparações existem uma predominância de sequências de enzimas digestivas. Nas membranas microvilares a maioria das sequências são aminopeptidases, enquanto nas vesículas microapócrinas a maioria são lipases. Os cDNAs correspondentes as proteínas que poderiam estar envolvidas na maquinaria secretória foram clonados e sequenciados. São elas: fimbrina, cofilina, gelsolina-1 e miosina I. RT-PCRs semi-quantitativas dessas proteínas em diferentes tecidos do inseto (intestino, túbulos de Malpighi, corpo gorduroso e carcaça) mostraram que somente gelsolina-1 está presente exclusivamente no intestino. Os domínios G1-G3 da gelsolina característica do intestino (gelsolina-1) foram expressos e usados para produzir anticorpos em coelhos. Esses anticorpos reconhecem a proteína recombinante e uma proteína presente no epitélio intestinal com massa molecular compatível com a massa predita para gelsolina-1. Foi possível diminuir a expressão de gelsolina-1 utilizando RNA interferente. / Lepidoptera anterior midgut presents a microapocrine secretion of digestive enzymes with secretory vesicles migrating inside the microvilli. These vesicles bud from the midgut microvilli as double membrane vesicles and are discharged into the lumen. The aim of this work was to identify the proteins secreted and those involved in the microapocrine secretory machinery in Spodoptera frugiperda larvae. For this, microapocrine vesicles were prepared and used for polyclonal antibody production. This antibody was used to screen a cDNA expression library of S. frugiperda midgut. We also obtained a transcriptome by pyrosequencing a cDNA library derived from transcripts of the midgut. Positive clones from the screening were sequenced, assembled and N-blasted against S. frugiperda sequences obtained by pyrosequencing. This procedure led to the extension of the sequences previously obtained. We also used the sequences generated by pyrosequencing to reanalyze the sequences of microvillar membrane proteins obtained previously by Ferreira et al. (2007). This reanalysis generated 66 predicted proteins that are present in the microvillar membranes. Eighteen were considered to be contaminants from other compartments and 48 associated with the microvillar membranes. Analysing the sequences from microapocrine vesicles we found 50 predicted proteins that should be secreted by microapocrine vesicles. The sequences found in both microvillar membrane and microapocrine vesicles may be classified into 8 groups, according to their function: (1) digestive enzymes; (2) peritrophic membrane proteins; (3) protection; (4) transporters; (5) receptors; (6) secretory machinery; (7) cytoskeleton; (8) with unknown function. In both preparations there is a predominance of sequences of digestive enzymes. In microvillar membranes, there is a remarkable amount of aminopeptidases, while in microapocrine vesicles this is true for lipases. cDNAs coding for proteins that could be involved in the microapocrine secretory machinery were cloned and sequenced. They are: fimbrin, cofilin, gelsolin-1 and myosin I. Using RT-PCR, we showed that mRNAs coding for gelsolin-1 and myosin I are present only in the intestinal tissue. The mRNAs coding for other proteins were found in all tissues. The domains G1-G3 from gelsolin specific from intestinal midgut (gelsolin 1) were expressed and used to raise antibodies in rabbit. These antibodies were able to recognize the recombinant protein and a protein from the midgut epithelium that has a molecular weight similar to the one predicted from gelsolin-1 sequence. We succeed in decreasing the expression of gelsolin-1 by using interfering RNA

O fluido folicular (FF) é um líquido extracelular complexo que se acumula no antro dos folículos ovarianos durante o seu desenvolvimento. É o meio essencial para o crescimento e a maturação das células ovarianas somáticas e germinativas e contém substâncias envolvidas na diferenciação celular, maturação do oócito, qualidade do gameta, ruptura da parede folicular e luteinização. O estudo de seus componentes é fundamental para um melhor entendimento dos mecanismos que envolvem a dinâmica folicular na espécie equina. O objetivo deste trabalho foi comparar o perfil proteico do maior folículo, e entre o maior e o segundo maior folículo, em diferentes momentos do desenvolvimento folicular. Para este estudo, quarenta ovários, oriundos de vinte éguas Crioulas, não gestantes e cíclicas, foram coletados durante a estação reprodutiva, em um abatedouro. Antes do abate, as éguas foram divididas em quarto grupos de acordo com o diâmetro folicular, ecotextura uterina (EU) e presença de corpo lúteo (CL): G 15 (emergência) (n = 3) folículos até 15 mm, EU ≥ 1, CL ≥ 20 mm; G 20 (divergência) (n = 9) folículos entre 20 e 25 mm, EU 1-2, CL 15-20 mm; G 30 (dominância) (n = 4) folículos entre 30 e 35 mm, EU ≥ 2, CL ≤ 15 mm; G 40 (pré-ovulatória) (n = 4) folículos ≥ 40 mm, EU 2-3, CL ≤ 15 mm. Após o abate, os ovários foram coletados e o FF dos dois maiores folículos foi aspirado. A técnica de 2D-PAGE foi realizada, em duplicata, utilizando gel de acrilamida a 12%. Os géis foram corados com Comassie Brilliant Blue R-250, escaneados e analisados, utilizando o PDQuest software, para determinar a densidade óptica dos spots. A identificação proteica foi realizada através de espectometria de massa (MS). Um total de 43 spots foi observado. Sete spots, representando cinco proteínas (albumina, apolipoproteína A-1, gelsolina, transferrina e α-1-antiproteinase 2), apresentaram diferenças (P˂0,05) na expressão, no FF do maior folículo, nos diferentes grupos. Um spot, representado pela proteína POMZP3, demonstrou diferença (P=0,018) em sua expressão, entre o maior e o segundo maior folículo, nos diferentes grupos. E, por fim, um spot, identificado como a proteína α-1-antiproteinase 2, apresentou interação (P=0,047) entre o maior e o segundo maior folículo e as diferentes fases da foliculogênese. Os resultados deste trabalho demonstram que o perfil proteico do FF difere durante o desenvolvimento folicular e que, as maiores alterações, são observadas a partir da dominância. Além disso, provavelmente, algumas destas proteínas, bem como suas correlações, tenham grande importância nos eventos que ocorrem durante a foliculogênese. / The follicular fluid (FF) is a complex extracellular fluid that accumulates in the antrum follicles during the follicular development. It is the essential medium for the growth and maturation of somatic and germ ovarian cells and contains substances involved in cell differentiation, oocyte maturation, gamete quality, rupture of the follicle wall and luteinization. The study of its components is crucial for a better understanding of the mechanisms involved in follicular dynamics in mares. The objective of this study was to determine the protein profile of the largest follicle and among the largest and the second largest follicle at different stages of follicular development. In this study, 40 ovaries from 20 non pregnant Criollo cycling mares were collected during the breeding season in an abattoir. Before slaughter, the mares were divided into four groups according to follicular diameter, uterine ecotexture (UE) and the presence of corpus luteum (CL): G 15 (emergence) (n = 3), follicles up to 15mm, EU ≥ 1, CL ≥ 20 mm; G 20 (deviation) (n = 9), follicles between 20 and 25 mm, EU 1-2, CL 15-20 mm; G 30 (dominance) (n = 4), follicles between 30 e 35 mm, EU ≥ 2, CL ≥15 mm; G 40 (ovulation) (n = 4), follicles ≥ 40 mm, EU 2-3, CL ≥ 15 mm. After slaughter, the ovaries were collected and the FF of the two largest follicles was aspirated. The technique of 2D-PAGE was performed in duplicate using 12% acrylamide gel. Gels were stained with Comassie Brilliant Blue R-250, scanned and analyzed using the PDQuest software to determine the optical density of the spots. Protein identification was performed by mass spectrometry (MS). A total of 43 spots was observed. Seven spots representing five proteins (albumin, apolipoprotein A-1, gelsolin, transferrin e α-1-Antitrypsin 2) showed differences (P˂0.05) in expression, the FF of the largest follicle in the different groups. One spot, represented by POMZP3 protein showed a difference (P=0.018) in expression between the largest and second largest follicle in the different groups. Finally, one spot, identified as the protein α-1-antitrypsin 2, showed interaction (P=0.047) between the largest and second largest follicle. The results of this study demonstrated that the protein profile of FF differs during follicular development and that the largest changes are observed from the dominance. Also probably some of these proteins, as well as their correlations, have great importance in the events that occur during folliculogenesis.

O fluido folicular (FF) é um líquido extracelular complexo que se acumula no antro dos folículos ovarianos durante o seu desenvolvimento. É o meio essencial para o crescimento e a maturação das células ovarianas somáticas e germinativas e contém substâncias envolvidas na diferenciação celular, maturação do oócito, qualidade do gameta, ruptura da parede folicular e luteinização. O estudo de seus componentes é fundamental para um melhor entendimento dos mecanismos que envolvem a dinâmica folicular na espécie equina. O objetivo deste trabalho foi comparar o perfil proteico do maior folículo, e entre o maior e o segundo maior folículo, em diferentes momentos do desenvolvimento folicular. Para este estudo, quarenta ovários, oriundos de vinte éguas Crioulas, não gestantes e cíclicas, foram coletados durante a estação reprodutiva, em um abatedouro. Antes do abate, as éguas foram divididas em quarto grupos de acordo com o diâmetro folicular, ecotextura uterina (EU) e presença de corpo lúteo (CL): G 15 (emergência) (n = 3) folículos até 15 mm, EU ≥ 1, CL ≥ 20 mm; G 20 (divergência) (n = 9) folículos entre 20 e 25 mm, EU 1-2, CL 15-20 mm; G 30 (dominância) (n = 4) folículos entre 30 e 35 mm, EU ≥ 2, CL ≤ 15 mm; G 40 (pré-ovulatória) (n = 4) folículos ≥ 40 mm, EU 2-3, CL ≤ 15 mm. Após o abate, os ovários foram coletados e o FF dos dois maiores folículos foi aspirado. A técnica de 2D-PAGE foi realizada, em duplicata, utilizando gel de acrilamida a 12%. Os géis foram corados com Comassie Brilliant Blue R-250, escaneados e analisados, utilizando o PDQuest software, para determinar a densidade óptica dos spots. A identificação proteica foi realizada através de espectometria de massa (MS). Um total de 43 spots foi observado. Sete spots, representando cinco proteínas (albumina, apolipoproteína A-1, gelsolina, transferrina e α-1-antiproteinase 2), apresentaram diferenças (P˂0,05) na expressão, no FF do maior folículo, nos diferentes grupos. Um spot, representado pela proteína POMZP3, demonstrou diferença (P=0,018) em sua expressão, entre o maior e o segundo maior folículo, nos diferentes grupos. E, por fim, um spot, identificado como a proteína α-1-antiproteinase 2, apresentou interação (P=0,047) entre o maior e o segundo maior folículo e as diferentes fases da foliculogênese. Os resultados deste trabalho demonstram que o perfil proteico do FF difere durante o desenvolvimento folicular e que, as maiores alterações, são observadas a partir da dominância. Além disso, provavelmente, algumas destas proteínas, bem como suas correlações, tenham grande importância nos eventos que ocorrem durante a foliculogênese. / The follicular fluid (FF) is a complex extracellular fluid that accumulates in the antrum follicles during the follicular development. It is the essential medium for the growth and maturation of somatic and germ ovarian cells and contains substances involved in cell differentiation, oocyte maturation, gamete quality, rupture of the follicle wall and luteinization. The study of its components is crucial for a better understanding of the mechanisms involved in follicular dynamics in mares. The objective of this study was to determine the protein profile of the largest follicle and among the largest and the second largest follicle at different stages of follicular development. In this study, 40 ovaries from 20 non pregnant Criollo cycling mares were collected during the breeding season in an abattoir. Before slaughter, the mares were divided into four groups according to follicular diameter, uterine ecotexture (UE) and the presence of corpus luteum (CL): G 15 (emergence) (n = 3), follicles up to 15mm, EU ≥ 1, CL ≥ 20 mm; G 20 (deviation) (n = 9), follicles between 20 and 25 mm, EU 1-2, CL 15-20 mm; G 30 (dominance) (n = 4), follicles between 30 e 35 mm, EU ≥ 2, CL ≥15 mm; G 40 (ovulation) (n = 4), follicles ≥ 40 mm, EU 2-3, CL ≥ 15 mm. After slaughter, the ovaries were collected and the FF of the two largest follicles was aspirated. The technique of 2D-PAGE was performed in duplicate using 12% acrylamide gel. Gels were stained with Comassie Brilliant Blue R-250, scanned and analyzed using the PDQuest software to determine the optical density of the spots. Protein identification was performed by mass spectrometry (MS). A total of 43 spots was observed. Seven spots representing five proteins (albumin, apolipoprotein A-1, gelsolin, transferrin e α-1-Antitrypsin 2) showed differences (P˂0.05) in expression, the FF of the largest follicle in the different groups. One spot, represented by POMZP3 protein showed a difference (P=0.018) in expression between the largest and second largest follicle in the different groups. Finally, one spot, identified as the protein α-1-antitrypsin 2, showed interaction (P=0.047) between the largest and second largest follicle. The results of this study demonstrated that the protein profile of FF differs during follicular development and that the largest changes are observed from the dominance. Also probably some of these proteins, as well as their correlations, have great importance in the events that occur during folliculogenesis.

The mechanism of microparticle shedding from the plasma membrane of calcium-loaded cells has been investigated in erythrocytes and platelets. Recent studies have revealed the physiological and clinical importance of microparticle release from nucleated cells such as lymphocytes and endothelium. The experiments of this study were designed to address whether simple mechanisms discovered in platelets and erythrocytes also apply to the more complex nucleated cells. Four such mechanisms were addressed: potassium efflux, transbilayer phosphatidylserine migration, cytoskeleton degradation, and membrane lipid order. The rate and amount of microparticle release in the presence of a calcium ionophore, ionomycin, was assayed by light scatter at 500 nm. To inhibit the calcium-activated potassium current, cells were exposed to 1 mM quinine or a high-potassium buffer. Both interventions substantially attenuated microparticle shedding induced by ionomycin. Microparticle release was also greatly reduced in a lymphocyte cell line deficient in the expression of scramblase, the enzyme responsible for calcium-stimulated phosphatidylserine migration to the cell surface. This result indicated that such phosphatidylserine exposure is also required for microparticle shedding. The importance of cytoskeletal rearrangement was evaluated through the use of E64-d, a calpain inhibitor, which appeared to have no affect on release. Thus, if cytoskeleton degradation is important for microparticle release, a different enzyme or protein must be involved. Finally, the effect of membrane physical properties was addressed by varying the experimental temperature (32–42 °C). A significant positive trend in the rate of microparticle release as a function of temperature was observed. Fluorescence experiments with trimethylammoniumdiphenylhexatriene and patman revealed significant differences in the level of apparent membrane order along that temperature range. Ionomycin treatment appeared to cause further disordering of the membrane, although the magnitude of this change was minimally temperature-sensitive. Thus, it was concluded that microparticle release depends more on the initial level of membrane order than on the change imposed by calcium uptake. In general, mechanisms involved in particle release from platelets and erythrocytes appeared relevant tolymphocytes with the exception of the hydrolytic enzyme involved in cytoskeletal degradation.

Indiana University-Purdue University Indianapolis (IUPUI) / In response to glucose, pancreatic islet beta cells secrete insulin in a biphasic manner, and both phases are diminished in type 2 diabetes. In beta cells, cortical F-actin beneath the plasma membrane (PM) prevents insulin granule access to the PM and glucose stimulates remodeling of this cortical F-actin to allow trafficking of insulin granules to the PM. Glucose stimulation activates the small GTPase Cdc42, which then activates p21-activated kinase 1 (PAK1); both Cdc42 and PAK1 are required for insulin secretion. In conjunction with Cdc42-PAK1 signaling, the SNARE protein Syntaxin 4 dissociates from F-actin to allow SNARE complex formation and insulin exocytosis. My central hypothesis is that, in the pancreatic beta cell, glucose signals through a Cdc42-PAK1-mediated pathway to remodel the F-actin cytoskeleton to mobilize insulin granules to SNARE docking sites at the PM to evoke glucose stimulated second phase insulin secretion. To investigate this, PAK1 was inhibited in MIN6 beta cells with IPA3 followed by live-cell imaging of F-actin remodeling using the F-actin probe, Lifeact-GFP. PAK1 inhibition prevented normal glucose-induced F-actin remodeling. PAK1 inhibition also prevented insulin granule accumulation at the PM in response to glucose. The ERK pathway was implicated, as glucose-stimulated ERK activation was decreased under PAK1-depleted conditions. Further study showed that inhibition of ERK impaired insulin secretion and cortical F-actin remodeling. One of the final steps of insulin secretion is the fusion of insulin granules with the PM which is facilitated by the SNARE proteins Syntaxin 4 on the PM and VAMP2 on the insulin granule. PAK1 activation was also found to be critical for Syntaxin 4-F-actin complex dynamics in beta cells, linking the Cdc42-PAK1 signaling pathway to SNARE-mediated exocytosis. Syntaxin 4 interacts with the F-actin severing protein Gelsolin, and in response to glucose Gelsolin dissociates from Syntaxin 4 in a calcium-dependent manner to allow Syntaxin 4 activation. Disrupting the interaction between Syntaxin 4 and Gelsolin aberrantly activates endogenous Syntaxin 4, elevating basal insulin secretion. Taken together, these results illustrate that signaling to F-actin remodeling is important for insulin secretion and that F-actin and its binding proteins can impact the final steps of insulin secretion.