Anoctamin 1 (Ano1) is required for glucose-induced membrane potential oscillations and insulin secretion by murine β-cells.

Anions such as Cl(-) and HCO3 (-) are well known to play an important role in glucose-stimulated insulin secretion (GSIS). In this study, we demonstrate that glucose-induced Cl(-) efflux from β-cells is mediated by the Ca(2+)-activated Cl(-) channel anoctamin 1 (Ano1). Ano1 expression in rat β-cells is demonstrated by reverse transcriptase-polymerase chain reaction, western blotting, and immunohistochemistry. Typical Ano1 currents are observed in whole-cell and inside-out patches in the presence of intracellular Ca(++): at 1 μM, the Cl(-) current is outwardly rectifying, and at 2 μM, it becomes almost linear. The relative permeabilities of monovalent anions are NO3 (-) (1.83 ± 0.10) > Br(-) (1.42 ± 0.07) > Cl(-) (1.0). A linear single-channel current-voltage relationship shows a conductance of 8.37 pS. These currents are nearly abolished by blocking Ano1 antibodies or by the inhibitors 2-(5-ethyl-4-hydroxy-6-methylpyrimidin-2-ylthio)-N-(4-(4-methoxyphenyl)thiazol-2-yl)acetamide (T-AO1) and tannic acid (TA). These inhibitors induce a strong decrease of 16.7-mM glucose-stimulated action potential rate (at least 87 % on dispersed cells) and a partial membrane repolarization with T-AO1. They abolish or strongly inhibit the GSIS increment at 8.3 mM and at 16.7 mM glucose. Blocking Ano1 antibodies also abolish the 16.7-mM GSIS increment. Combined treatment with bumetanide and acetazolamide in low Cl(-) and HCO3 (-) media provokes a 65 % reduction in action potential (AP) amplitude and a 15-mV AP peak repolarization. Although the mechanism triggering Ano1 opening remains to be established, the present data demonstrate that Ano1 is required to sustain glucose-stimulated membrane potential oscillations and insulin secretion.

Transcription coupled repair of 8-oxoguanine in murine cells: The Ogg1 protein is required for repair in nontranscribed sequences but not in transcribed sequences

To assess the role of the Ogg1 DNA glycosylase in the transcription-coupled repair (TCR) of the mutagenic lesion, 7,8-dihydro-8oxoguanine (8-OxoG), we have investigated the removal of this lesion in wild-type and ogg1−/− null mouse embryo fibroblast (MEF) cell lines. We used nonreplicating plasmids containing a single 8-OxoG·C base pair in a different assay that allowed us to study the removal of 8-OxoG located in a transcribed sequence (TS) or in a nontranscribed sequence (NTS). The results show that the removal of 8-OxoG in a wild-type MEF cell line is faster in the TS than in the NTS, indicating TCR of 8-OxoG in murine cells. In the homozygous ogg1−/− MEF cell line, 8-OxoG was not removed from the NTS whereas there was still efficient 8-OxoG repair in the TS. Expression of the mouse Ogg1 protein in the homozygous ogg1−/− cell line restored the ability to remove 8-OxoG in the NTS. Therefore, we have demonstrated that Ogg1 is essential for the repair of 8-OxoG in the NTS but is not required in the TS. These results indicate the existence of an Ogg1-independent pathway for the TCR of 8-OxoG in vivo.

PHENOTYPIC ASSOCIATION OF GENE AMPLIFICATION WITH MULTIPLE DRUG RESISTANCE IN VINCRISTINE RESISTANT CHINESE HAMSTER OVARY CELLS

Resistance of tumors to pharmacologic agents poses a significant problem in the treatment of human malignancies. This study overviews the scope of clinical resistance and focuses upon current research attempts toward investigation of the phenomenon of multidrug resistance (MDR).^ The objective of this investigation was to determine whether gene amplification had a role in the development of the MDR phenotype in Chinese hamster ovary cells (CHO) primarily selected for resistance to vincristine (VCR). A DNA fragment, previously shown to be amplified in two independently derived Chinese hamster cell lines exhibiting the MDR phenotype, was also amplified in VCR hamster lines. Sequences flanking this fragment were shown to contain coding information for a 4.3 kb transcript overproduced in VCR cells. These sequences were not enriched in double minute DNA preparations isolated from VCR cells. There was an approximately forty-fold increase in both the level of gene amplification and transcript overproduction in the VCR cell lines, independent of the level of primary resistance. This DNA amplification and overproduction of the 4.3 kb transcript was also demonstrated in CHO cells independently selected for resistance to Adriamycin and vinblastine.^ All the DNA sequences of two hamster cDNA clones containing 785 and 932 base pair inserts showed direct homology to the published mouse mdr sequences (about 90%). This sequence conservation held for only portions of the gene when the human mdr1 sequences were compared with those from either the mouse or hamster.^ Somatic cell hybrids, constructed between VCR CHO cells and sensitive murine cells, were used to determine whether there was a functional relationship between the chromosome bearing the amplified sequences and the MDR phenotype. Concordant segregation between vincristine resistance, the MDR phenotype, the presence of MDR-associated amplified sequences, overexpression of the mRNA encoded by these sequences, overexpression of the mRNA encoded by these sequences, and CHO chromosome Z1 was consistent with the hypothesis that there is an amplified gene on chromosome Z1 of the VCR CHO cells which is responsible for MDR in these cells. ^

Regulation of bovine IL-12R beta 2 subunit mRNA expression in bovine lymph node cells

The beta 2 subunit of the interleukin (IL)-12 receptor (IL-12R beta 2) has been shown to play an essential role in differentiation of T helper 1 (Th1) cells in the murine and human system, and antibodies raised against IL-12R beta 2 recognized this molecule on human Th1 but not Th2 cells. However, while the cytokines secreted by clones of murine cells allowed the definition of distinct T helper cell subsets, bovine clones with polarized Th1 and Th2 cytokine profiles were rarely found. This raised important questions about the regulation of immune responses in cattle. We therefore cloned bovine IL-12R beta2 (boIL-12R beta 2) DNA complementary to RNA (cDNA) from the start codon to the 3' end of the mRNA. Comparison of boIL-12R beta 2 cDNA with human and murine IL-12R beta 2 cDNA sequences revealed homologies of 85 and 78%, respectively. The deduced protein sequence showed the hallmark motifs of the cytokine receptor superfamily including the four conserved cysteine residues, the WSXWS motif and fibronectin domains in the extracellular part as well as a STAT4 binding site in the intracellular part of the molecule. Using real-time reverse transcription-polymerase chain reaction, upregulation of mRNA expression of this molecule could be demonstrated in cultured bovine lymph node cells stimulated with phytohemagglutinin. Furthermore, cells with upregulated boIL-12R beta 2 mRNA responded with enhanced expression of interferon gamma to treatment with interleukin 12.

Host range restrictions of oncogenes: myc genes transform avian but not mammalian cells and mht/raf genes transform mammalian but not avian cells.

The host range of retroviral oncogenes is naturally limited by the host range of the retroviral vector. The question of whether the transforming host range of retroviral oncogenes is also restricted by the host species has not been directly addressed. Here we have tested in avian and murine host species the transforming host range of two retroviral onc genes, myc of avian carcinoma viruses MH2 and MC29 and mht/raf of avian carcinoma virus MH2 and murine sarcoma virus MSV 3611. Virus vector-mediated host restriction was bypassed by recombining viral oncogenes with retroviral vectors that can readily infect the host to be tested. It was found that, despite high expression, transforming function of retroviral myc genes is restricted to avian cells, and that of retroviral mht/raf genes is restricted to murine cells. Since retroviral oncogenes encode the same proteins as certain cellular genes, termed protooncogenes, our data must also be relevant to the oncogene hypothesis of cancer. According to this hypothesis, cancer is caused by mutation of protooncogenes. Because protooncogenes are conserved in evolution and are presumed to have conserved functions, the oncogene hypothesis assumes no host range restriction of transforming function. For example, mutated human proto-myc is postulated to cause Burkitt lymphoma, because avian retroviruses with myc genes cause cancer in birds. But there is no evidence that known mutated protooncogenes can transform human cells. The findings reported here indicate that host range restriction appears to be one of the reasons (in addition to insufficient transcriptional activation) why known, mutated protooncogenes lack transforming function in human cells.

Direct continuities between cisternae at different levels of the Golgi complex in glucose-stimulated mouse islet beta cells

Direct continuity between the membranes of cisternae in the Golgi complex in mammalian cells rarely has been observed; when seen, its documentation has been equivocal. Here we have used dual-axis electron microscope tomography to examine the architecture of the Golgi in three dimensions at approximate to6-nm resolution in rapidly frozen, freeze-substituted murine cells that make and secrete insulin in response to glucose challenge. Our data show three types of direct connections between Golgi cisternae that are normally distinct from one another. These connections all bypass interceding cisternae. We propose that when pancreatic beta cells are stimulated to synthesize and secrete insulin rapidly in vivo, such connections provide a continuous lumen that facilitates the rapid transit of large amounts of newly made protein for secretion. The heterotypic fusion of cisternae, even transiently, raises important questions about the molecular mechanisms that (i) facilitate the fusion/fission of cisternal membranes and control the directionality and specificity of such events, and (it) retain Golgi processing enzymes at specific places within individual cisternae when two cisternae at different levels in the Golgi have fused, maintaining the sequential processing hierarchy that is a hallmark of Golgi organization.

BSTA Promotes mTORC2-Mediated Phosphorylation of Akt1 to Suppress Expression of FoxC2 and Stimulate Adipocyte Differentiation

Phosphorylation and activation of Akt1 is a crucial signaling event that promotes adipogenesis. However, neither the complex multistep process that leads to activation of Akt1 through phosphorylation at Thr308 and Ser473 nor the mechanism by which Akt1 stimulates adipogenesis is fully understood. We found that the BSD domain–containing signal transducer and Akt interactor (BSTA) promoted phosphorylation of Akt1 at Ser473 in various human and murine cells, and we uncovered a function for the BSD domain in BSTA-Akt1 complex formation. The mammalian target of rapamycin complex 2 (mTORC2) facilitated the phosphorylation of BSTA and its association with Akt1, and the BSTA-Akt1 interaction promoted the association of mTORC2 with Akt1 and phosphorylation of Akt1 at Ser473 in response to growth factor stimulation. Furthermore, analyses of bsta gene-trap murine embryonic stem cells revealed an essential function for BSTA and phosphorylation of Akt1 at Ser473 in promoting adipocyte differentiation, which required suppression of the expression of the gene encoding the transcription factor FoxC2. These findings indicate that BSTA is a molecular switch that promotes phosphorylation of Akt1 at Ser473 and reveal an mTORC2-BSTA-Akt1-FoxC2–mediated signaling mechanism that is critical for adipocyte differentiation.

Evidence for the direct binding of phosphorylated p53 to sites of DNA breaks in vivo

Despite a clear link between ataxia-telangiectasia mutated (ATM)-dependent phosphorylation of p53 and cell cycle checkpoint control, the intracellular biology and subcellular localization of p53 phosphoforms during the initial sensing of DNA damage is poorly understood. Using GO-G, confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser(15) (p53(Ser15)), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following induction of DNA breaks or DNA base damage. This biologicafly distinct subpool of p53(Ser15) is ATM dependent and resistant to 26S-proteasomal degradation. p53(Ser15) colocalizes and coimmunoprecipitates with gamma-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear micro-beam irradiation studies confirm p53 S,,15 is recruited to sites of DNA damage containing gamma-H2AX, ATM(Ser1981), and DNA-PKcs(Thr2609) in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser(15) or Ser(18) phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21(WAF) decreased gamma-H2AX association, and altered DNA-dsb kinetics following DNA damage. Our results suggest a unique biology for this p53 phosphoform in the initial steps of DNA damage signaling and implicates ATM-p53 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair to prevent carcinogenesis. (Cancer Res 2005; 65(23): 10810-21).

Obesidade e cancro da próstata: Avaliação do efeito dos adipócitos nas células RM1 de carcinoma da próstata

O cancro da próstata é o segundo cancro mais frequente e a sexta causa de morte mundial por cancro no sexo masculino. A obesidade tem sido associada ao aumento da incidência e mortalidade por cancro, com alguma controvérsia. As alterações nas expressões de adipocinas associadas à obesidade têm sido um dos diversos mecanismos propostos para explicar a associação entre a obesidade e o cancro da próstata, nomeadamente na promoção do desenvolvimento e progressão celular do tumor. O objetivo deste trabalho é avaliar o efeito dos fatores produzidos pelos pré-adipócitos e os adipócitos na proliferação, migração e invasão das células de carcinoma da próstata independentes dos androgénios. As células RM1 foram cultivadas na presença de diferentes concentrações de insulina e leptina, bem como em meio condicionado (MC) de pré-adipócitos e adipócitos e co-cultivadas em sistema de transwells, com as mesmas células. A proliferação celular das RM1 foi avaliada recorrendo a contagem celular em camara de Neubauer e em citometro de fluxo, e aos ensaios metabólicos alamar blue e XTT. Efetuou-se um ensaio de migração por dano nas células RM1 na presença dos meios condicionados. A invasão das células foi avaliada recorrendo a um sistema de transwells, com membrana de matrigel, quando cultivadas com pré-adipócitos e adipócitos. A insulina aumentou significativamente a proliferação celular, ao contrário da leptina que não teve efeito. O meio condicionado dos pré-adipócitos aumentou ligeiramente a proliferação, enquanto meio condicionado dos adipócitos de 1 e 2 dias aumentou significativamente a proliferação das células RM1 (p<0.01), quando avaliada por XTT. Na câmara de Neubauer não se verificaram diferenças significativas na proliferação celular. Relativamente à migração celular, observou-se um aumento significativo da migração das células RM1 cultivadas com meio condicionado de adipócitos (MCA) e pré-adipócitos (MCPA) em comparação com o controlo (p<0.01). Observou-se um aumento significativo da invasão de células RM1 cultivadas com adipócitos e pré-adipócitos (p <0.05). Os adipócitos aumentaram significativamente a proliferação das células RM1 em co-cultura (p<0.01). Em conclusão, as células RM1 parecem ser influenciadas por fatores secretados pelos adipócitos, capazes de aumentar a sua capacidade de proliferar, invadir e migrar.

Crucial cytokine interactions in nitric oxide production induced by Mycoplasma arthritidis superantigen

Mycoplasma arthritidis causes autoimmune arthritis in rodents. It produces a superantigen (MAM) that simultaneously activates antigen presenting cells and T cells inducing nitric oxide and cytokine release. Nitric oxide is a key inducer and regulator of the immune system activation. Here, we investigated nitric oxide and cytokine production and interactions of these molecules in MAM-stimulated co-cultures of macrophages (J774A.1 cell line) with spleen lymphocytes. We found that: a) MAM-induced nitric oxide, interferon-gamma, membrane-associated tumor necrosis factor and interleukin-2 production in co-cultures of macrophages with lymphocytes from BALB/c and C3H/HePas but not from C57B1/6 mice; b) production of nitric oxide was dependent on interferon-gamma whereas that of interferon-gamma was dependent on interleukin-2 and membrane-associated tumor necrosis factor; c) these cytokines up regulated MAM-induced nitric oxide production. Unraveling the mechanisms of cell activation induced by MAM might be helpful to design strategies to prevent immune system activation by superantigens and therefore in seeking amelioration of associated immunopathologies. (C) 2008 Elsevier Masson SAS. All rights reserved.

Immunostimulatory and cytotoxic activities of Indigofera suffruticosa (Fabaceae)

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

Obtenção e caracterização de anticorpo monoclonal murino anti-fator VIII da coagulação sangüínea

Entre os avanços da engenharia celular e biotecnologia nas últimas décadas, destaca-se a produção de anticorpos monoclonais murinos (AcMm) utilizados no aprimoramento diagnóstico nas rotinas laboratoriais. A produção de fator VIII de alta pureza sempre foi o desejo e a preocupação das indústrias de hemoderivados para tratamento de pacientes portadores de hemofilia A, porém este produto inexiste no Brasil, sendo necessária sua obtenção no mercado internacional a custos elevados. O trabalho tem por objetivo a produção de AcMm anti-fator VIII humano (FVIII H) através da expansão dos clones e caracterização imunoquímica do anticorpo. Camundongos Balb/c foram imunizados com FVIII H purificado como também proveniente de crioprecipitado e as células esplênicas dos animais foram fusionadas com células mielomatosas murinas segundo o método descrito por Kohler e Milstein para produção de híbridos em cultura. Foram testados 1.983 híbridos dos quais 105 foram submetidos à clonagem. Destes, 39 obtiveram monoclonalidade e 7 destes clones foram caracterizados através de técnicas de immunoblotting. Foram submetidas à purificação por cromatografia três imunoglobulinas de diferentes classes pertencentes aos clones LAMB1-10A1A4, LAMB1-17A1A1 e LAMB1-24A2A1. A imunoglobulina purificada pertencente ao clone LAMB1-10A1A4 foi adsorvida em coluna de imunoafinidade para purificação de concentrado de FVIII proveniente de crioprecipitado plasmático.

Avaliação da citotoxicidade de materiais obturadores de canais radiculares: influência na liberação de fator de necrose tumoral alfa, interferon-y e óxido nítrico em cultura de células murinas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo dos mecanismos envolvidos na expressão de fator de crescimento de células progenitoras e fator de crescimento para fibroblastos-2 por odontoblastos estimulados por lipopolissacarídeos via p42/44, p38 e PI3K

Pós-graduação em Ciência Odontólogica - FOA