Leishmania caracteriza-se por apresentar duas formas morfologicamente distintas em seu ciclo de vida: promastigotas e amastigotas. As formas promastigotas vivem tubo digestório do vetor flebotomíneo, sob as condições de pH 7,0 e temperatura ambiente, ao redor de 25ºC. As formas amastigotas são encontradas no interior dos fagolisossomos de macrófagos infectados onde encontram um ambiente de pH ácido e temperatura ao redor de 34ºC. Leishmania utiliza arginina para a síntese de poliaminas, que desempenham papel fundamental no crescimento, diferenciação celular e sucesso da infecção. A tomada de arginina em L. (L.) amazonensis é feita pela proteína transportadora de aminoácidos - amino acid transporter-like 3 (AAP3), codificada por duas cópias do gene (5.1 aap3 e 4.7 aap3) dispostas em tandem no genoma. Os transcritos de 5.1 aap3 e de 4.7 aap3 apresentam 98% de identidade entre as ORFs, mas diferem nas 5\' e 3\' UTR. O objetivo do presente trabalho foi avaliar se os sinais de temperatura, pH e privação de arginina disparam a regulação da expressão de aap3 em formas promastigotas e amastigotas. Para isso avaliamos o nível dos transcritos e realizamos ensaios de tomada de arginina em células submetidas à privação ou suplementadas com arginina, nas temperaturas de 25°C ou 34°C em pH 7,0 ou 5,0. Constatou-se em formas promastigotas que o transcrito 5.1 aap3 apresentou maior abundância em relação a 4.7 aap3, e que a privação promoveu o aumento da tomada do aminoácido quando os parasitos eram mantidos em pH 7,0 a 25°C, corroborando dados anteriores do nosso grupo. Demonstramos que a mudança de temperatura foi um fator importante para o aumento do número de cópias de 5.1 aap3 em promastigotas privadas, principalmente quando associadas com o pH 5,0. Além disso, o aumento da temperatura favoreceu a tomada de arginina, corroborando com a elevação do número de cópias observada para o transcrito 5.1 aap3. Em amastigotas-like, mantidas a 25°C e pH 7,0 a privação reverteu a expressão de 5.1 aap3 para o mesmo perfil observado para promastigotas. Contudo, não observamos um favorecimento na tomada de arginina. Ainda em amastigotas, o tratamento a 34°C e pH 7,0 favoreceu a tomada de arginina, porém não observamos um aumento correspondente na quantificação do transcrito. O transcrito 4.7 aap3 não apresentou alteração significativa em qualquer tratamento em promastigotas e amastigotas. Os nossos resultados indicam que a variação de temperatura e do pH, além da privação de arginina, podem ser sinais importantes para regulação da expressão diferencial de aap3, principalmente a cópia 5.1 aap3, de forma a assegurar a oferta de arginina em formas promastigotas previamente à entrada no hospedeiro mamífero e em formas amastigotas, na passagem para o vetor, assegurando o sucesso da infecção / Leishmania presents two morphologically distinct forms in its life cycle: promastigote and amastigote. The promastigotes live in the midgut of the sand fly vector under the conditions of pH 7.0 and room temperature, around 25°C. The amastigote forms are found inside the phagolysosomes of infected macrophages where they encounter an environment of acidic pH and temperature around 34°C. Leishmania uses the arginine to synthesize polyamines which play an important role in cell growth, differentiation and in the successful of infection. The arginine uptake in Leishmania (L.) amazonensis is made by an amino acid porter 3-like protein (AAP3), coded by a two copies gene (5.1 aap3 e 4.7 aap3) arranged in tandem in the genome. The transcripts, 5.1 aap3 and 4.7 aap3, present 98% of ORFs identity, but differ in the 5\' and 3\' UTR. The aim of this work was to evaluate if canges in temperature, pH and arginine deprivation represent signals to trigger the regulation expression of aap3 in promastigotes and amastigotes. For this, we evaluated the transcripts level and performed assays of arginine uptake in parasites subjected to arginine starvation or supplemented with arginine, at temperatures of 25°C or 34°C and pH 7.0 or 5.0. In promastigotes we verified that the transcript 5.1 aap3 showed higher abundance in relation to 4.7 aap3, and that the arginine starvation promoted an increase in the amino acid uptake when the parasites were maintained at pH 7.0 and 25°C, confirming previous data from our group. The change of temperature was an important factor to the increase of 5.1 aap3 transcripts - in starved promastigotes, particularly when associated with pH 5.0. In addition, the increase of the temperature led to an increase of the arginine uptake, correlated to the increase of 5.1 aap3 transcript. The amastigotes-like maintained at 25°C and pH 7.0 and submitted to the amino acid starvation reverted 5.1 aap3 expression profile to the same observed for promastigotes. However, those condictions did not favor an increase in arginine uptake. The treatment of amastigotes at 34°C and pH 7.0 facilitated the increased of arginine uptake, but did not correlated with the transcripts level. The 4.7 aap3 transcript did not change significantly in any promastigotes and amastigotes treatments. Our results indicate that variation in temperature and pH, in addition to arginine starvation may be important signals for regulating the aap3 expression, mainly the copy 5.1 aap3, in order to ensure the correct supply of arginine in the previous period in relation to the entry of the promastigotes into the mammalian host or to the amastigotes transition in the vector, ensuring the success of the infection.

Chitin is an abundant polysaccharide found in fungal cell walls, crustacean shells, and insect exoskeletons. The immunological properties of both chitin and its deacetylated derivative chitosan are of relevance due to frequent natural exposure and their increasing use in translational applications. Depending on the preparation studied and the endpoint measured, these compounds have been reported to induce allergic responses, inflammatory responses, or no response at all. Highly purified chitosan and chitin were prepared and the capacity of these glycans to stimulate the release of the inflammasomeassociated cytokine IL-1β was examined. Chitosan was shown to be a potent inflammasome activator in mouse bone marrow macrophages, macrophages polarized towards a M1 or M2 phenotype, dendritic cells, peritoneal cells, and human PBMCs. Acetylation of the chitosan to chitin resulted in a near total loss of IL-1β activity in all cell types tested. The size of the chitosan particles played an important role, with small particles eliciting the greatest activity. An inverse relationship between size and stimulatory activity was demonstrated using chitosan passed through size exclusion filters as well as with chitosan-coated beads of defined size. Partial digestion of chitosan with pepsin resulted in a larger fraction of small phagocytosable particles and more potent inflammasome activity. Inhibition of phagocytosis with cytochalasin D abolished the IL- 1β stimulatory activity of chitosan, offering an explanation for why the largest particles were nearly devoid of activity. Thus, the deacetylated polysaccharide chitosan potently activates the NLRP3 inflammasome in a phagocytosis-dependent manner. The reason for chitin’s inability to elicit IL-1β is unknown, but it does not appear to be due to active inhibition of the inflammasome and while chitin appears to be more readily digested by macrophage cell lysates, it does not occur at a rate which would likely impact inflammasome activation. There are three proposed mechanisms for NLRP3 inflammasome activation: K+ efflux, ROS, and lysosomal destabilization. The contributions of these mechanisms were tested and it was revealed that each of these pathways participated in optimal NLRP3 inflammasome activation by chitosan. Finally, the laminin receptor was evaluated as a potential chitin receptor. These studies provide insight into the activating properties of chitin and chitosan and highlight the importance of matching particle size and degree of acetylation to the level of activity desired for translational applications.

Splicing of mRNA precursors (pre-mRNA) comprises a series of ATP-dependent steps, the first of which is the stable binding of U2 snRNP at the pre-mRNA branchpoint. The basis of ATP use in splicing is not well understood. Several yeast splicing factors belong to DEAD/H box family of RNA-dependent ATPase, and are implicated in dynamic RNA structure rearrangement during spliceosome assembly. In mammals, however, such information is conspicuously lacking. In fact, none of the known mammalian splicing factors has characteristics for ATP hydrolysis. In an attempt to identify mammalian splicing factors involved in ATP usage, we have developed a novel approach to identify and purify spliceosomal ATP binding proteins. Six spliceosomal ATP binding proteins were found, one of them, SAFp56, was purified and microsequenced, and found to be a DEAD box protein containing unique DECD motif instead of the canonical DEAD motif. During the course of this work, a new functional region in U2AF65, an essential splicing factor required for U2 snRNP entry into the spliceosome, was defined. This information was used to clone a human U2AF65 associated protein (UAP). UAP and SAFp56 are identical. We refer to this protein as hUAP56 (human 56 kDa U2AF65 associated protein). We present evidence that hUAP56 is an essential splicing factor required for the U2 snRNP binding to pre-mRNA. Interestingly, UAP56 is recruited to pre-mRNA in a polypyrimidine tract bound U2AF65-dependent fashion. This result underscores a new function of U2AF65, and provides the first description of how a specific DEAD box protein is directed to a pre-mRNA splicing signal, and/or, to the proximity of its substrate at a particular stage. Like an authentic DEAD box protein. hUAP56 has ATP binding, RNA-stimulated ATPase, as well as RNA binding activity. A particularly novel result is that the ATPase activity of UAP56 is stimulated by U2AF65. This observation strongly suggests the role of UAP56 in ATP dependent mechanism during U2 snRNP binding to the pre-mRNA branchpoint, and implies that UAP56 may function through a distinct mechanism. We identify yeast UAP (yUAP), a highly conserved S. cerevisiae homologue of hUAP56. yUAP is essential for viability, can be functionally substituted for by hUAP56, and like its human counterpart, is an essential pre-mRNA splicing factor required for spliceosome assembly. Furthermore, we show that yUAP is required for formation of the branchpoint dependent commitment complex, the precursor for U2 snRNP addition. Site-directed mutagenesis revealed that all DEAD box protein consensus motifs are required for yUAP function. Interestingly, a strain harboring a yUAP mutant in which the DECD sequence, characteristic of UAP members, was changed to canonical sequence, is inviable. Our results demonstrate that UAP is structurally and functionally conserved from yeast to man. In conjunction with previous studies, we conclude that at least two DEAD box proteins, Prp5p and yUAP, are required for the U2 snRNP-branchpoint interaction.

Disruption of cilia proteins results in a range of disorders called ciliopathies. However, the mechanism by which cilia dysfunction contributes to disease is not well understood. Intraflagellar transport (IFT) proteins are required for ciliogenesis. They carry ciliary cargo along the microtubule axoneme while riding microtubule motors. Interestingly, IFT proteins localize to spindle poles in non-ciliated, mitotic cells, suggesting a mitotic function for IFT proteins. Based on their role in cilia, we hypothesized that IFT proteins regulate microtubule-based transport during mitotic spindle assembly. Biochemical investigation revealed that in mitotic cells IFT88, IFT57, IFT52, and IFT20 interact with dynein1, a microtubule motor required for spindle pole maturation. Furthermore, IFT88 co-localizes with dynein1 and its mitotic cargo during spindle assembly, suggesting a role for IFT88 in regulating dynein-mediated transport to spindle poles. Based on these results we analyzed spindle poles after IFT protein depletion and found that IFT88 depletion disrupted EB1, γ-tubulin, and astral microtubule arrays at spindle poles. Unlike IFT88, depletion of IFT57, IFT52, or IFT20 did not disrupt spindle poles. Strikingly, the simultaneous depletion of IFT88 and IFT20 rescued the spindle pole disruption caused by IFT88 depletion alone, suggesting a model in which IFT88 negatively regulates IFT20, and IFT20 negatively regulates microtubulebased transport during mitosis. Our work demonstrates for the first time that IFT proteins function with dynein1 in mitosis, and it also raises the important possibility that mitotic defects caused by IFT protein disruption could contribute to the phenotypes associated with ciliopathies.

No description available.

No description available.

No description available.

Em vacas de corte, folículos pré-ovulatórios (FPO) maiores, maiores concentrações de estradiol (E2) no proestro/estro e progesterona (P4) no diestro favorecem o crescimento do concepto e a fertilidade. Porém, os mecanismos mediados pelos esteroides femininos que influenciam a receptividade uterina ao embrião precisam ser esclarecidos. Os aminoácidos (AA) são componentes das secreções uterinas que são cruciais para a sobrevivência do embrião antes da implantação. A hipótese deste trabalho é que o tamanho do FPO e as concentrações de E2 e P4 modulam a abundância de transcritos relacionados ao transporte e metabolismo de AA no endométrio e afetam a concentração luminal de AA. Para isso, o crescimento folicular de vacas Nelore foi manipulado com o objetivo de formar dois grupos: FPO grande e CL grande (FG-CLG) e FPO pequeno e CL pequeno (FP-CLP). No Dia 4 (D4; Exp 1) e Dia 7 (D7; Exp 2) após a injeção de GnRH para indução da ovulação, foram coletados tecido endometrial e lavado uterino post-mortem. A abundância de transcritos foi avaliada por qRT-PCR e as concentrações de AA nos lavados foram quantificadas por HPLC. No Exp 1, o tamanho do FPO, concentrações plasmáticas de E2 no D-1 e de P4 no D4 foram 15,70mm±0,43 vs. 11,31mm±0,23 (p<0,01), 2,44pg/ml±0,19 vs. 0,65pg/ml (p<0,01) e 1,40ng/ml±0,23 vs. 0,80ng/ml±0,10 (p<0,01) para os grupos FG-CLG vs. FP-CLP, respectivamente. No Exp 2, o tamanho do FPO, concentrações plasmáticas de E2 no D-1 e de P4 no D7 foram 13,18mm±0,44 vs. 10,63mm±0,30 (p<0,01), 2,30pg/ml±0,57 vs. 0,50pg/ml±0,13 (p<0,01) e 3,68ng/ml±0,38 vs. 2,49ng/ml±0,43 (p=0,04) para os grupos FG-CLG vs. FP-CLP, respectivamente. No D4 a abundância de SLC1A4, SLC38A1, SLC6A6, SLC7A4 e SLCY e no D7, SLC1A4, SLC6A1, SLC6A14, SLC7A4, SLC7A8, SLC38A1, SLC38A7, SLC43A2 e DDO foi maior no endométrio dos animais do grupo FG-CLG (p<0,05). No D4, maiores concentrações de taurina, alanina e ácido α-aminobutírico foram observadas no grupo FP-CLP (p<0,05). Em contraste, menores concentrações de valina e cistationina foram encontradas nos lavados uterinos do D7 dos animais do grupo FP-CLP (p<0,05). No D4, os animais do grupo FG-CLG, associado a maior fertilidade, apresentaram menor quantidade de AA nas secreções uterinas, porém, a abundância dos transportadores de AA foi compatível com maior transporte em comparação aos animais do grupo FP-CLP. Esses resultados sugerem que antes do embrião se mover do oviduto ao útero, o transporte e metabolismo dos AA prioriza a preparação das células endometriais para receber o embrião e não o acúmulo nas secreções uterinas. Porém, no D7, quando o embrião está em contato direto com as secreções uterinas, os genes relacionados ao transporte de AA no endométrio e a concentração de AA no histotrofo são estimulados nas vacas do grupo FG-CLG. Portanto o metabolismo e transporte de AA no sentido das células endometriais ou das secreções uterinas pode ser um mecanismo importante para a receptividade materna. / In beef cattle, a large size of the pre-ovulatory follicle (POF) and resulting elevated proestrus/estrus estradiol (E2) and diestrus progesterone (P4) concentrations positively affect conceptus growth and fertility. However, sex-steroid-mediated mechanisms that influence uterine receptivity to the embryo need to be elucidated. Amino acids are important components of maternally-derived secretions that are crucial for embryo survival before implantation. The hypothesis is that the size of the POF, E2 and P4 concentrations modulate endometrial abundance of solute carrier proteins (SLC) transcripts related to AA transport and metabolism and subsequently affect lumenal amino acids concentrations. Therefore, follicle growth of Nelore cows was manipulated to produce two experimental groups: large POF and CL (LF-LCL group) and small POF and CL (SF-SCL group). On Day 4 (D4; Experiment 1) and Day 7 (D7; Experiment 2) post GnRH injection to induce ovulation, endometrial tissue and uterine washings were collected post-mortem. Transcript abundance was evaluated by qRT-PCR and amino acid concentrations were quantified in washings by HPLC. On Experiment 1, POF size, plasma E2 concentration on D-1, and plasma concentration of P4 on D4 were 15.70mm±0.43 vs. 11.31mm±0.23 (p<0.01), 2.44pg/ml±0.19 vs. 0.65pg/ml (p<0.01) and 1.40ng/ml±0.23 vs. 0.80ng/ml±0.10 (p<0.01) for the LF-LCL vs. SF-SCL groups, respectively. For Experiment 2, POF size, plasma E2 concentration on D-1 and plasma P4 concentration on D7 were 13.18mm±0.44 vs. 10.63mm±0.30 (p<0.01), 2.30pg/ml±0.57 vs. 0.50pg/ml±0.13 (p<0.01) and 3.68ng/ml±0.38 vs. 2.49ng/ml±0.43 (p=0.04) for the LF-LCL vs. SF-SCL groups, respectively. On D4, abundance of SLC6A6, SLC7A4, SLC17A5, SLC38A1, SLC38A7 and SLCY and on D7, SLC1A4, SLC6A1, SLC6A14, SLC7A4, SLC7A7, SLC7A8, SLC17A5, SLC38A1, SLC38A7, SLC43A2 and DDO was greater in the endometrium of cows from the LF-LCL group (p<0.05). On D4, higher concentrations of taurine, alanine and α-aminobutiric acid were observed in SF-SCL (p<0.05). In contrast, lower concentrations of valine and cystathionine were quantified in D7 uterine washings from SF-SCL cows (p<0.05). On D4, animals from LF-LCL group, associated with greater fertility, presented less amino acid content in uterine secretion but abundance of transporters was compatible to greater transport in comparison to animals from SF-SCL group. This suggests that before embryo moves from oviduct to uterus, amino acids transport and metabolism pathways prioritizes endometrium cells preparation for receiving the embryo but not accumulation in uterine secretions. However, on D7, when the embryo is in direct contact with uterine secretions, genes related to amino acids transport in endometrium and amino acids concentration in histotroph are up-regulated in LF-LCL cows. The latter insights indicate that amino acids metabolism and transport, towards endometrial cells or uterine secretions, might be mechanisms contributing to maternal receptivity.

A análise das proteínas transportadoras de compostos biliares, antes restrita à fisiologia e à fisiopatologia de colestases, recentemente passou a incluir neoplasias hepato-biliares. O presente estudo teve como objetivo caracterizar a expressão das proteínas ABC de transporte biliar BSEP, MDR3, MRP2 e MRP3 em amostras retrospectivamente colecionadas de 80 casos de autópsias de carcinoma hepatocelular (CHC) e 56 casos de ressecção cirúrgica de colangiocarcinoma (CC). Áreas representativas das neoplasias foram organizadas em tissue microarrays e submetidas à pesquisa imuno-histoquímica (IHQ) com o anticorpo policlonal anti-BSEP (HPA019035) e os anticorpos monoclonais anti-BSEP (F6), anti-MDR3 (P3 II-26), anti-MRP2 (M2 III-6) e anti-MRP3 (DTX-1) com amplificação de sinal mediante uso de sistema de polímeros curtos conjugados à peroxidase. A comparação entre a positividade das reações imuno-histoquímicas para cada anticorpo e as variáveis anatomopatológicas foi realizada através dos testes de qui-quadrado de Pearson ou Exato de Fisher. A positividade das reações IHQ cujos anticorpos propiciaram melhor distinção do sinal positivo vs coloração inespecífica de fundo e detecção de casos positivos e/ou melhor capacidade de discriminar as duas neoplasias hepáticas foi comparada com a positividade observada para as reações IHQ com os anticorpos anti-CEA policlonal, anti-Hep-par-1 e anti-Arginase-1. A expressão canalicular de BSEP nos CHC foi observada em 77,3% (58/75) com o anticorpo monoclonal e 75,9% (60/79) com o anticorpo policlonal. Não foi detectada associação significativa da expressão de BSEP em relação ao tamanho, número dos nódulos e grau de diferenciação de CHC, tendo apenas sido significativamente reduzida (P < 0,05) tal reação nos casos de padrão arquitetural mais complexo. A reatividade dos CHC para o anticorpo monoclonal anti-BSEP foi aparentemente menor que a obtida com a expressão canalicular de CEA, Hep-par-1 e Arginase-1 no CHC, mas esses valores não atingiram significância estatística. Todos os casos de colangiocarcinoma foram negativos para reações IHQ para pesquisa de BSEP, resultado significativamente diferente (P=0,0001) do obtido com uso do Ac policlonal anti-CEA (padrão circunferencial) e Hep-par-1, não tendo sido demonstrada diferença significativa (P=0,222) da expressão de BSEP e de Arginase-1. A expressão canalicular de MDR3 foi observada em 56,4% (44/78) dos casos de CHC, não tendo sido detectada associação significativa quanto ao tamanho e número de nódulos. Foi observada expressão significativamente menor de MDR3 nos casos de CHC de padrão mais complexo (P=0,009), e nos casos de maior grau histológico (P=0,005). A expressão de MDR3 em CHC foi significativamente menor que a de CEA, Hep-par-1 e Arginase-1 (P < 0,05). Todos os casos de colangiocarcinoma foram negativos para a avaliação da expressão de MDR3, diferindo significativamente em relação a expressão de CEA (P=0,001), mas não em comparação a Hep-par-1 e Arginase-1 (P > 0,05). As reações IHQ para detecção de MRP2 exibiram positividade canalicular em 92,3% dos casos de CHC e em 96,3% nos casos de CC. A detecção da alta expressão de MRP2 no CHC foi constante (P > 0,05) em comparação ao tamanho, número dos nódulos, padrão arquitetural e grau histológico de diferenciação de CHC assim como, também não apresentou associação (P > 0,05) com a localização, padrão de crescimento e grau de diferenciação do CC. A reação IHQ para MRP3 resultou positiva em 15/80 casos de CH (18,8%). A reatividade IHQ para MRP foi detectada em 24/54 (44,5%) de CC. Diferente dos transportadores descritos acima, a expressão de MRP3 foi preferencialmente basolateral. A positividade para MRP3 não variou (P > 0,05) em relação ao número, tamanho dos nódulos, padrão arquitetural (inclusive os sólidos), e grau de diferenciação (inclusive os menos diferenciados). A proteína MRP3 esteve expressa regularmente (P > 0,05) em todos os casos de CC, apresentando-se reduzida apenas no subtipo histológico ductular (P=0,023). Em conclusão, o excelente contraste de reação, a frequência razoavelmente alta de positividade de CHC e a plena negatividade de CC para BSEP levam-nos a recomendar a introdução do anticorpo monoclonal anti-BSEP no painel adotado para o diagnóstico diferencial dessas duas neoplasias. A alta expressão de MRP2 no CHC e no CC é conservada independentemente dos parâmetros anatomopatológicos avaliados. A expressão do transportador MRP3 mostrou variação dentre os subtipos histológicos de CC, aspecto que torna promissoras pesquisas futuras para avaliação mais detalhada da expressão deste marcador nos colangiocarcinomas / The assessment of biliary transporters, previously restricted to the physiology and pathophysiology of cholestasis, has recently included hepato-biliary neoplasms. The present study aimed to characterize the expression of BSEP, MDR3, MRP2 and MRP3 biliary transport proteins in retrospectively collected samples from 80 cases of autopsy of hepatocellular carcinoma (HCC) and 56 cases of surgical resection of cholangiocarcinoma (CC). Representative areas of the neoplasms were organized into tissue microarrays and submitted to immunohistochemical (IHC) reaction with polyclonal antibody anti-BSEP (HPA019035) and monoclonal antibodies anti-BSEP (F6), MDR3 (P3 II-26), MRP2 (M2 III-6) and MRP3 (DTX-1). Signal amplification was achieved with a short polymer system conjugated to peroxidase. The comparison between the positivity of the immunohistochemical reactions for each antibody and the pathological variables was performed using the Pearson chi-square test or the Fisher\'s exact test. The performance of antibodies which provided a better distinction of the positive signal vs nonspecific background staining and yield better discrimination between the two hepatic neoplasms was compared with that achieved with the already accepted HCC markers polyclonal anti-CEA, Hep-par-1 and Arginase-1. The canalicular expression of BSEP in HCC was observed in 77.3% (58/75) with the monoclonal antibody and 75.9% (60/79) with the polyclonal antibody. BSEP expression levels were not significantly different according to tumor size, number of nodules and degree of differentiation. The frequency of positive reaction of HCC cases with the monoclonal anti-BSEP was apparently lower than that achieved with the canalicular expression of CEA, Hep-par-1 and Arginase-1, but these values did not reach statistical significance. All cases of cholangiocarcinoma were negative for IHC reactions to BSEP, which was significantly different (P=0.0001) from the results obtained with polyclonal anti-CEA (circumferential pattern) and Hep-par-1, but not from the resultas achieved with Arginase-1 (P=0.222). The canalicular expression of MDR3 was observed in 56.4% (44/78) of HCC cases. Among histological variables, only the finding of more complex architecture (P=0.009) and higher histological grade (P=0.005) of HCC yielded, significantly lower expression of MDR3. The expression of MDR3 in HCC was significantly lower than that of CEA, Hep-par-1 and Arginase-1 (P < 0.05). All cases of cholangiocarcinoma were negative for the evaluation of MDR3 expression, differing significantly with that achieved with polyclonal anti-CEA (P=0.001) but not with that achieved with Hep-par-1 or with Arginase-1 (P > 0.05). The IHC reactions with the MRP2 antibody exhibited canalicular positivity in 92.3% of HCC cases and 96.3% in CC cases. High expression of MRP2 in HCC was constant (P > 0.05) despite changes in size, number of nodules, architectural pattern and histological degree of HCC differentiation, as well as no association (P > 0.05) with the location, pattern of growth and degree of differentiation of CC. The IHC reaction for MRP3 was positive in 15/80 cases of HCC (18.8%) and in 24/54 (44.5%) of CC. Unlike the carriers described above, the hepatocellular expression of MRP3 was preferentially basolateral. Positivity for MRP3 did not vary (P > 0.05) in relation to number, nodule size, architectural standard (including solids), and degree of differentiation. The MRP3 protein was expressed regularly (P > 0.05) in different presentations of CC, but significant lower frequency of positivity was found in the ductular histological subtype (P=0.023). In conclusion, the excellent signal-to-noise ratio, reasonably high frequency of HCC positivity and full negativity of CC to BSEP lead us to recommend the introduction of the anti-BSEP monoclonal antibody in the panel adopted for the differential diagnosis of these two neoplasms. The high expression of MRP2 in HCC and in CC is conserved independently of the pathological parameters evaluated herein. The frequency of expression of the MRP3 transporter varied among the histological subtypes of CC, which makes promising future research for a more detailed assessment of the expression of this marker in the cholangiocarcinomas

CopA, a thermophilic membrane ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu+ across cellular membranes. CopA contains at least two metal binding domains, a regulatory N-terminal Metal Binding Domain (N-MBD) and an occlusion/coordinating metal binding site in the 6th, 7th and 8th transmembrane segments. Previous studies showed that the presence of millimolar concentration of Cys is essential for CopA activity. The high affinity of CopA for metal in the presence of millimolar concentration of Cys suggests a multifaceted interaction of the enzyme with Cys. To elucidate the role of Cys, we studied its effect on the partial reactions of the catalytic cycle of CopA. We observed that 2-50 mM Cys accelerates enzyme turnover with little effect on the Cu+ affinity of CopA. Cys accelerates enzyme phosphorylation, but has no effect on the dephosphorylation rates. Thus, Cys increases steady state phosphoenzyme levels. Besides, Cys has no significant effect on E1¡ÃƒÂªE2 equilibrium. Similar results were observed in truncated CopA lacking the N-MBD suggesting that enzyme activation by Cys is independent of the regulatory metal binding sites. These results and the kinetic analysis of activation curves suggest that while Cu+ is delivered to the transport site as a Cu-Cys complex, Cys in the mM range stimulates the ATPase acting as a non-essential activator.