The liver injury following ischemia and reperfusion is worse in experimental knockout heterozygote mouse model for expression of connexin 43

Purpose:To evaluate that Connexin (Cx43) plays a role in lesions after hepatic ischemia/reperfusion (IR) injury.Methods:We use Cx43 deficient model (heterozygotes mice) and compared to a wild group. The groups underwent 1 hour ischemia and 24 hours reperfusion. The heterozygote genotype was confirmed by PCR. We analyzed the hepatic enzymes (AST, ALT, GGT) and histology.Results:The mice with Cx43 deficiency showed an ALT mean value of 4166 vs. 307 in the control group (p<0.001); AST mean value of 7231 vs. 471 in the control group (p<0.001); GGT mean value of 9.4 vs. 1.7 in the control group (p=0.001); histology showed necrosis and inflammation in the knockout group.Conclusions:This research demonstrated that the deficiency of Cx43 worses the prognosis for liver injury. The topic is a promising target for therapeutics advancements in liver diseases and procedures.(AU)

The variable success of in vitro maturation: can we do better?

The efficiency of in vitro assisted reproductive technologies, consisting of the transfer of embryos obtained in vitro through in vitro maturation, in vitro fertilization and early embryo culture is still limited. The quality of the oocytes is pivotal for assisted reproductive efficiency and the maturation of the oocyte represents the first key limiting step of the in vitro embryo production system. At the time of removal from the antral follicles, the oocyte is still completing the final growth and differentiation steps, needed to provide the so-called developmental competence, i.e. the machinery required to sustain fertilization and embryo development. In mono-ovular species only one oocyte per cycle is available for procreation, therefore the current assisted reproduction techniques strive to overcome this natural boundary. However, the success is still limited and overall the effectiveness does not exceed the efficiency achieved in millions of years of mammalian evolution. One of the problems lies in the intrinsic heterogeneity of the oocytes that are subjected to in vitro maturation and in the lack of dedicated in vitro approaches to finalize the differentiation process. In this review we will try to overview some of the salient aspects of current practices by emphasizing the most critical and fundamental features in oocyte differentiation that should be carefully considered for improving current techniques

The variable success of in vitro maturation: can we do better?

The efficiency of in vitro assisted reproductive technologies, consisting of the transfer of embryos obtained in vitro through in vitro maturation, in vitro fertilization and early embryo culture is still limited. The quality of the oocytes is pivotal for assisted reproductive efficiency and the maturation of the oocyte represents the first key limiting step of the in vitro embryo production system. At the time of removal from the antral follicles, the oocyte is still completing the final growth and differentiation steps, needed to provide the so-called developmental competence, i.e. the machinery required to sustain fertilization and embryo development. In mono-ovular species only one oocyte per cycle is available for procreation, therefore the current assisted reproduction techniques strive to overcome this natural boundary. However, the success is still limited and overall the effectiveness does not exceed the efficiency achieved in millions of years of mammalian evolution. One of the problems lies in the intrinsic heterogeneity of the oocytes that are subjected to in vitro maturation and in the lack of dedicated in vitro approaches to finalize the differentiation process. In this review we will try to overview some of the salient aspects of current practices by emphasizing the most critical and fundamental features in oocyte differentiation that should be carefully considered for improving current techniques(AU)

INFLUÊNCIA DE GRÂNULOS CITOPLASMÁTICOS EM OÓCITOS BOVINOS SOBRE O DESENVOLVIMENTO EMBRIONÁRIO E FETAL

Análises morfológicas para seleção de oócitos podem subestimar os gametas femininos em relação ao seu potencial de desenvolvimento, pela subjetividade da técnica por identificar apenas alterações aparentes em microscopia de luz. Este método pode não ser capaz de avaliar por exemplo, a competência a nível molecular, nuclear e citoplasmática do oócito, como perfil de transcritos acumulados, organização de organelas e vias de apoptose ativas. Portanto, sob a hipótese que grânulos citoplasmáticos em oócitos não alteram a capacidade de desenvolvimento, a qualidade dos embriões gerados e o desenvolvimento embrionário e fetal, o objetivo do nosso estudo foi avaliar os efeitos de grânulos citoplasmáticos na produção in vitro de embriões. Para testar esta hipótese, utilizamos o modelo bovino em dois experimentos para avaliar grupos de oócitos com citoplasma homogêneo (controle) e oócitos que apresentam granulações no citoplasma (granular). No experimento 1, através de amostras de ovários de abatedouro, observamos que: i. Nas análises de perfil funcional do oócito, estruturas que possuem grânulos citoplasmáticos apresentam maiores níveis de caspase 3 ativa (indicador da cascata de apoptose), no entanto não demonstram nenhuma diferença em relação a atividade de junções GAP (comunicação oócito-células do cumulus), ii: Nas análises de expressão de genes marcadores de qualidade realizadas em oócitos, blastocistos e células do cumulus, apenas o transcrito ZAR1 foi diferencialmente expresso, sendo pouco detectado em oócitos do grupo granular e iii: Em análises de taxas e cinética de desenvolvimento embrionário (clivagem, blastocisto), não detectamos diferenças entre os grupos. No experimento 2, através de amostras obtidas por aspirações foliculares, observamos que, i: As taxas de desenvolvimento embrionário (clivagem, blastocisto e prenhez) não foram diferentes em relação a presença de grânulos citoplasmáticos e ii: O desenvolvimento fetal (diâmetro de vesícula embrionária, diâmetro biparietal e comprimento crânio-caudal) também foi similar. Portanto, concluímos que apesar de oócitos que apresentam grânulos citoplasmático, possuírem maiores níveis de caspase 3 ativa e menor abundância de transcritos de ZAR1, não foi detectada diferença no potencial de desenvolvimento pré e pós-implantação, bem como alterações no desenvolvimento fetal. Nossos resultados demonstram que o citoplasma granular no modelo bovino não afeta o potencial de desenvolvimento embrionário, e estas estruturas podem ser incluídas para produção in vitro de embriões sem prejuízos aparentes.

Morphological analysis for oocyte selection may underestimate female gametes in regarding their developmental potential, due to the technique subjectivity for only identifies apparent changes in light microscopy. This method may not be able to evaluate for example the molecular, nuclear and cellular oocyte competence, such as transcript profile accumulations, organelles organization and active apoptosis pathways. Therefore, under the hypothesis that oocytes cytoplasmic granules do not alter the development capacity, embryos quality and embryonic and fetal development, the study objective was to evaluate cytoplasmic granules effects on in vitro embryo production. To test this hypothesis we used bovine model in two experiments to evaluate groups of homogeneous cytoplasm oocytes (control) and granular cytoplasm oocytes (granular). In experiment 1, through slaughterhouse ovarian samples we observed: i. In the analysis of oocyte functional profile structures that have cytoplasmic granules have higher active caspase 3 levels (apoptosis cascade indicator), but do not show any difference in GAP junction activity (oocyte-cumulus cell communication). ii. In the analysis of quality marker gene expression performed in oocytes, blastocysts and cumulus cells, only the ZAR1 transcript was differentially expressed and poorly detected in granular group oocytes and iii. In embryonic development rates (cleavage, blastocyst) and kinetics analysis we did not detect differences between the groups. In experiment 2, through samples obtained by follicular aspirations, we observed that: i. Embryonic development rates (cleavage, blastocyst and pregnancy) were not different in relation to the presence of cytoplasmic granules and ii: Fetal development (embryonic vesicle diameter, biparietal diameter and craniocaudal length) was also similar. Therefore, we conclude that although oocytes that have cytoplasmic granules have higher levels of active caspase 3 and lower abundance of ZAR1 transcripts, no difference was detected in pre- and post-implantation developmental potential, as well as changes in fetal development. Our results demonstrate that granular cytoplasm in the bovine model does not affect embryonic development potential and these structures can be included for in vitro embryo production without apparent damage.

Large-scale chromatin structure and function changes during oogenesis: theinterplay between oocyte and companioncumulus cell

The process of chromatin configuration remodeling within the mammalian oocyte nucleus or germinal vesicle (GV), which occurs towards the end of its differentiation phase before meiotic resumption, has received much attention and has been studied in several mammals. This review is aimed to highlight the relationship between changes in chromatin configurations and to both functional and structural modifications occurring in the oocyte nuclear compartment. During the extensive phase of meiotic arrest at the diplotene stage, the chromatin enclosed within the GV is subjected to several levels of regulation. Morphologically, the chromosomes lose their individuality and form a loose chromatin mass. Then the decondensed chromatin undergoes profound rearrangements during the final stages of oocyte growth in tight association with the acquisition of meiotic and developmental competence. Functionally, the discrete stages of chromatin condensation are characterized by different level of transcriptional activity, DNA methylation and covalent histone modifications. Interestingly, the program of chromatin rearrangement is not completely intrinsic to the oocyte, but follicular cells exert their regulatory actions through gap junction mediated communications and intracellular messenger dependent mechanism(s). With this in mind and since oocyte growth mostly relies on the bidirectional crosstalk with the follicular cells, experimental manipulation of large-scale chromatin configuration is discussed. Besides providing tools to determine the key cellular pathways involved in genome-wide chromatin modifications , the present findings will aid to the refinement of physiological culture systems that can have important implications in treating human infertility as well as managing breeding schemes in animal husband .

Large-scale chromatin structure and function changes during oogenesis: theinterplay between oocyte and companioncumulus cell

The process of chromatin configuration remodeling within the mammalian oocyte nucleus or germinal vesicle (GV), which occurs towards the end of its differentiation phase before meiotic resumption, has received much attention and has been studied in several mammals. This review is aimed to highlight the relationship between changes in chromatin configurations and to both functional and structural modifications occurring in the oocyte nuclear compartment. During the extensive phase of meiotic arrest at the diplotene stage, the chromatin enclosed within the GV is subjected to several levels of regulation. Morphologically, the chromosomes lose their individuality and form a loose chromatin mass. Then the decondensed chromatin undergoes profound rearrangements during the final stages of oocyte growth in tight association with the acquisition of meiotic and developmental competence. Functionally, the discrete stages of chromatin condensation are characterized by different level of transcriptional activity, DNA methylation and covalent histone modifications. Interestingly, the program of chromatin rearrangement is not completely intrinsic to the oocyte, but follicular cells exert their regulatory actions through gap junction mediated communications and intracellular messenger dependent mechanism(s). With this in mind and since oocyte growth mostly relies on the bidirectional crosstalk with the follicular cells, experimental manipulation of large-scale chromatin configuration is discussed. Besides providing tools to determine the key cellular pathways involved in genome-wide chromatin modifications , the present findings will aid to the refinement of physiological culture systems that can have important implications in treating human infertility as well as managing breeding schemes in animal husband .(AU)

Influence of dietary vegetable crops on rainbow trout (Oncorhynchus mykiss) immune system and growth performance / Influence of dietary vegetable crops on rainbow trout (Oncorhynchus mykiss) immune system and growth performance

Background: Carotenoids such as -carotene, -carotene, lycopene, lutein and -cryptoxanthin are a family of pigmented compounds which are synthesized by many vegetable crops and microorganisms but not animals. In human and murine models, carotenoids are shown to mediate their effects via different mechanisms such as gap junction communication, cell growth regulation, modulating gene expression and immune response. In some fish species, the immunomodulating action of synthetic carotenoids has been the subject of some research. However, studies on the effects of carotenoids from natural sources on fish growth performance and immune parameters are lacking. In the current study, a preliminary study with 60 days feeding was conducted to study the influence of different natural sources of carotenoids from some vegetable crops on growth and some immune indices in rainbow trout (Oncorhynchus mykiss). Materials, Methods & Results: Purified isonitrigenous (crude protein: 40.16%) and isocaloric (3660 kcal kg-1) diet with 4.5 g of powdered crops namely tomato (Solanum lycopersicum) and sweet pepper (Capsicum annuum) per kg feed or control diet without any treatment was prepared. Rainbow trout weighing 150 ± 9 g were distributed equally into 2 groups with 33 fish in each group. Each group contained 11 fish in triplicate reared in individual ponds. In treatment group, fish were fed diet

Background: Carotenoids such as -carotene, -carotene, lycopene, lutein and -cryptoxanthin are a family of pigmented compounds which are synthesized by many vegetable crops and microorganisms but not animals. In human and murine models, carotenoids are shown to mediate their effects via different mechanisms such as gap junction communication, cell growth regulation, modulating gene expression and immune response. In some fish species, the immunomodulating action of synthetic carotenoids has been the subject of some research. However, studies on the effects of carotenoids from natural sources on fish growth performance and immune parameters are lacking. In the current study, a preliminary study with 60 days feeding was conducted to study the influence of different natural sources of carotenoids from some vegetable crops on growth and some immune indices in rainbow trout (Oncorhynchus mykiss). Materials, Methods & Results: Purified isonitrigenous (crude protein: 40.16%) and isocaloric (3660 kcal kg-1) diet with 4.5 g of powdered crops namely tomato (Solanum lycopersicum) and sweet pepper (Capsicum annuum) per kg feed or control diet without any treatment was prepared. Rainbow trout weighing 150 ± 9 g were distributed equally into 2 groups with 33 fish in each group. Each group contained 11 fish in triplicate reared in individual ponds. In treatment group, fish were fed diet

IDENTIFICAÇÃO E CARACTERIZAÇÃO DA EXPRESSÃO DAS CONEXINAS 26, 32, 43 E PANEXINA-1 EM LINHAGENS DE CÉLULAS MAMÁRIAS CANINA E HUMANA

As conexinas, proteínas formadoras de junções gap, são fortemente implicadas na manutenção da homeostase tecidual devido à importante função de comunicação intercelular. As panexinas, proteínas formadoras de hemicanais, foram descobertas a pouco mais de uma década e não têm seu papel bem compreendido. São descritas diversas alterações nas conexinas durante o processo da carcinogênese e relaciona-se a panexina1 ao processo oncogênico. Considera-se a diminuição da expressão das conexinas ou a deficiência na sua capacidade de comunicação intercelular como o evento mais comumente associado às neoplasias. Além disso, pode ocorrer a localização citoplasmática incomum destas proteínas em células cancerosas. As conexinas 26, 32 e 43 podem ser observadas em glândulas mamárias de roedores e humanos, mas durante processos neoplásicos a expressão destas proteínas pode variar. Neoplasias mamárias são o segundo tipo de câncer mais freqüente em cadelas e são responsáveis por 50% dos casos malignos. Neste contexto, objetivou-se identificar a presença de panexina 1 em tecido mamário hígido e neoplásico de cadelas e identificar a presença de Cx26, 32 e 43 e panexina 1 em linhagens de células mamárias canina e humana. Para tanto, foram realizadas as técnicas de imunofluorescência indireta em tecido mamário hígido e neoplásico de cadelas e em linhagens de células mamárias canina e humana, além de avaliação através de microscopia confocal nestes materiais. Obteve-se diferentes padrões de marcação das Cxs 26, 32 e 43 e da Px1 em linhagens de células mamárias hígida e tumoral canina e humana. A expressão das Cxs 26, 32 e 43 e da Px1 nestas linhagens mostrou distribuição nuclear, citoplasmática e de membrana celular. Novos estudos são necessários para confirmar e ampliar os achados deste trabalho.

Connexins, gap junction forming proteins, are strongly implicated in the maintenance of tissue homeostasis due to their important intercellular communication function. The panexins, hemicysmal-forming proteins, were discovered within a little over a decade and have their role not well understood. It has been described changes in connexins during the carcinogenesis process and related panexin 1 to the oncogenic process. The decrease in the expression of the connexins or the deficiency in their capacity of intercellular communication is considered as the event most commonly associated with the neoplasias. In addition, inusual cytoplasmic localization of these proteins can occur in cancer cells. Connexins 26, 32 and 43 may be observed in mammary glands of rodents and humans, but during neoplastic processes the expression of these proteins may vary. Breast neoplasms are the second most frequent type of cancer in bitches and account for 50% of malignant cases. In this context, the objective was to identify the presence of panexin 1 in healthy and neoplastic breast tissue of bitches and to identify the presence of connexins 26, 32 and 43 and panexin 1 in canine and human mammary cell lines. Indirect immunofluorescence in healthy and neoplastic mammary tissue of bitches and in canine and human mammary cell lines were performed, as well as evaluation by confocal microscopy in these materials was done. Different marking patterns of Cxs 26, 32 and 43 and of Px1 were obtained in healthy and canine mammary and human breast cell lines. The expression of Cxs 26, 32 and 43 and Px1 in these lines showed nuclear, cytoplasmic and cell membrane distribution. Further studies are needed to confirm and amplify the findings of this work.

Influence of dietary vegetable crops on rainbow trout (Oncorhynchus mykiss) immune system and growth performance

Background: Carotenoids such as ß-carotene, α-carotene, lycopene, lutein and ß-cryptoxanthin are a family of pigmented compounds which are synthesized by many vegetable crops and microorganisms but not animals. In human and murine models, carotenoids are shown to mediate their effects via different mechanisms such as gap junction communication, cell growth regulation, modulating gene expression and immune response. In some fish species, the immunomodulating action of synthetic carotenoids has been the subject of some research. How ever, studies on the effects of carotenoids from natural sources on fish growth performance and immune parameters are lacking. In the current study, a preliminary study with 60 days feeding was conducted to study the influence of different natural sources of carotenoids from some vegetable crops on growth and some immune indices in rainbow trout (Oncorhynchus mykiss). Materials, Methods & Results: Purified isonitrigenous (crude protein: 40.16%) and isocaloric (3660 kcal kg-1) diet with 4.5 g of powdered crops namely tomato (Solanum lycopersicum) and sweet pepper (Capsicum annuum) per kg feed or control diet without any treatment was prepared. Rainbow trout weighing 150 ± 9 g were distributed equally into 2 groups with 33 fish in each group. Each group contained 11 fish in triplicate reared in individual ponds. In treatment group, fish were fed diet containing 4.5 g of powdered crops for 60 days while in control group, basal diet without any treatments was fed. At the end of the feeding trial, 4 fish per tank were sampled to analyze the growth parameters. Seven fish were also bled from the caudal vein to collect serum sample for immune parameters. During this study no mortality of fish was observed in different groups. Results of this study showed that condition factor and feed intake were similar among the groups while specific growth rate and weight gain showed a significant increase in treatment group compared to control group. Immunological parameters namely peroxidase content, antibacterial activity, α1-antiprotease, total antiprotease activities and total protein did not vary among the groups, even though slight decrease in serum peroxidase content was shown in treatment group. On the other hand, serum lysozyme activity of fish fed treatment diet was significantly higher than control group. Discussion: Enhanced growth performance in the current study might be attributed to some intermediary metabolism which could enhance nutrient utilization and may ultimately result in improved fi sh growth. Lysozyme is secreted by leukocytes and is a marker of leukocyte activity, increasing concomitantly with phagocytic activity. Administration of natural carotenoids in fish diet exerts a stabilizing or protective effect against oxidative damage, and enhances the proliferation of these cells, which could result in increased serum lysozyme level. Feeding natural carotenoids might act as a direct scavenger of reactive oxygen species and decrease the body's need for certain antioxidant enzymes. Therefore, slight decrease in serum peroxidase content can be attributed to this point. In conclusion, this study showed that rainbow trout appear to benefit from inclusion of crops in diet in terms of improved growth performance and immune system.

Effects of Deletion of the Connexin 32 gene on pro and Anti-inflammatories Aspects in the Ethidium Bromide Toxic Demyelination Model

Gap junctions are cellular structures that allow transit of molecules between cells, allowing intercellular signaling and transportation. They are formed by proteins denominated connexins and represent key structures in highly complex and integrated tissues, such as the central nervous system (CNS). The present study evaluates the effects of connexin 32 (Cx32) deletion upon CNS inflammation and regeneration/repair after 1, 3, 7, 10 and 20 days after intracerebral injection of ethidium bromide in Cx32 Knock Out and normal mice. To accomplish so, Real Time PCR gene expression quantification was performed upon Tumour Necrosis Factor alpha (TNFα), Transforming Growth Factor beta 1 (TGFß1), Metalloproteinase 3 (MMP3), Metalloproteinase 9 (MMP9) and Tissue Inhibitor of Metalloproteinases 1 (TIMP1) genes. Results indicate varying differences in the expression pattern, including difference in expression of all evaluated genes in the 3 days post injection period, apex of the acute inflammation mechanisms. These results suggest that Cx32 may perform important functions on molecular, inflammatory and regenerative/repair signalling in the CNS.(AU)

THE ROLE OF CONNEXINS, PANNEXINS AND THEIR CHANNELS IN ACUTE LIVER FAILURE

A homeostase tecidual é controlada por uma série de mecanismos de comunicação. Ao nível intercelular, as junções gap permitem o tráfego de mensageiros pequenos e hidrofílicos, menores do que 1.5 kilodalton, entre duas células adjacentes. As junções gap são formadas por dois hemicanais, que por sua vez são compostos por seis proteínas denominadas conexinas (Cx). No fígado, os hepatócitos produzem principalmente Cx32, enquanto as células hepáticas não parenquimatosas contem predominantemente Cx43. As junções gap hepáticas são conhecidas por serem essenciais em diversas funções específicas do fígado, tais como secreção de albumina e metabolismo de xenobióticos. Na última década, tem-se tornado claro que os hemicanais de conexinas não são apenas precursores das junções gap, mas eles também podem participar da via de comunicação, contudo entre o citoplasma de células individuais e seus ambientes extracelulares. Além disso, foi descrita uma nova classe de proteínas semelhantes às conexinas, as panexinas (Panx), das quais a Panx1 é expressa no fígado. As pannexins se reúnem em uma configuração que lembra os hemicanais de conexinas e também participam da sinalização extracelular. Acredita-se que tanto os hemicanais de conexinas como os canais de pannexinas se tornam ativados preferencialmente em condições patológicas e, deste modo, promovam a morte celular e inflamação. O presente projeto de doutorado foi estabelecido para verificar se esta hipótese também é verdadeira nas doenças hepáticas, em particular na insuficiência hepática aguda. Inicialmente, o padrão de expressão das conexinas hepáticas foi totalmente caracterizado em um modelo murino de insuficiência hepática aguda induzida pelo paracetamol (APAP). Isto revelou uma mudança da Cx32 para Cx43 após a hepatotoxicidade induzida por APAP, com expressão de novo de Cx43 nos hepatócitos. Subsequentemente, a relevância funcional da expressão aumentada de Cx43 na insuficiência hepática aguda foi testada utilizando camundongos deficientes em Cx43. Verificou-se que a Cx43 tem um efeito protetor, uma vez que a lesão hepática induzida por APAP se agravou após a ablação genética da Cx43. Em paralelo, foi realizado um estudo semelhante utilizando camundongos knockout para Cx32, não mostrando diferenças significativas na extensão da hepatotoxicidade induzida pelo APAP em comparação aos animais selvagens. Uma vez que os animais deficientes em conexina não permitem diferenciar entre a comunicação via junções gap e a sinalização pelos hemicanais de conexina, uma série de experimentos seguintes baseou-se na utilização de inibidores dos hemicanais de Cx32 e Cx43, chamados de TAT-Gap24 e TAT-Gap19, respectivamente. Após os testes in vitro quanto a sua especificidade e eficiência in vitro, ambos os peptídeos foram administrados em camundongos com overdose pelo APAP. Enquanto TAT-Gap24 reduziu claramente a morte celular e inflamação, TAT-Gap19 apresentou apenas efeitos discretos sobre a lesão hepática. Além disso, o co-tratamento 12 de camundongos intoxicados pelo APAP com ambos os peptídeos revelou um efeito aditivo na redução da lesão hepática. Um estudo final foi focado na elucidação da expressão hepática e função da Panx1 na hepatotoxicidade induzida pelo APAP, utilizando o peptídeo inibidor da Panx1, 10Panx1. Verificou-se que a inibição dos canais de Panx1 minimiza as características clínicas da hepatotoxicidade desencadeada pelo APAP, em particular a relacionada com morte celular e inflamação. Em geral, este projeto de doutorado demonstra, pela primeira vez, o envolvimento dos hemicanais de conexina e canais de pannexina na insuficiência hepática aguda induzida pelo APAP, o que sugere um papel como alvos terapêuticos para fármacos.

Tissue homeostasis is controlled by a plethora of communication mechanisms. At the intercellular level, gap junctions allow the trafficking of small and hydrophilic messengers of less than 1.5 kilodalton between two adjacent cells. Gap junctions are built up by two hemichannels, which in turn are composed of six connexin (Cx) proteins. In liver, hepatocytes mainly produce Cx32, while non-parenchymal hepatic cells predominantly harbor Cx43. Hepatic gap junctions are known to underlie several liver-specific functions, such as albumin secretion and xenobiotic metabolism. In the last decade, it has become clear that the connexin hemichannels are not only precursors of gap junctions, but that they can also provide a communication pathway, albeit between the cytosol of individual cells and their extracellular environment. In addition, a novel class of connexin-like proteins has been identified, the pannexins (Panx), of which Panx1 is expressed in the liver. Pannexins gather in a configuration reminiscent of connexin hemichannels and also support extracellular signaling. Both connexin hemichannels and pannexin channels are believed to become preferably activated in pathological conditions and thereby to promote cell death and inflammation. The present doctoral project was set up to verify the hypothesis that this also holds true for liver disease, in particular acute liver failure. In a first instance, the hepatic connexin expression pattern was fully characterized in a mouse model of acute liver failure induced by acetaminophen (APAP). This revealed a switch from Cx32-to-Cx43 upon APAP-induced hepatotoxicity with de novo expression of Cx43 in hepatocytes. Subsequently, the functional relevance of enhanced Cx43 expression in acute liver failure was tested using Cx43-deficient mice. It was found that Cx43 has a protective effect, since liver APAP-induced injury became aggravated upon genetic ablation of Cx43. In parallel, a similar study was set up using Cx32 knock-out mice, showing no major differences in the extent of APAP-induced hepatotoxicity compared to wild type animals. Since connexin-deficient animals do not allow to discriminate between gap junction communication and connexin hemichannel signaling, a next set of experiments relied on the use of claimed peptide-based inhibitors of Cx32 and Cx43 hemichannels, namely TAT-Gap24 and TAT-Gap19, respectively. Following testing of their specificity and efficiency in vitro, both peptides were administered to APAPoverdosed mice. While TAT-Gap24 clearly reduced liver cell death and inflammation, TAT-Gap19 only had marginal effects on liver injury. Furthermore, co-treatment of APAPintoxicated mice with both peptides revealed an additive effect in lowering liver injury. A final study was focused on elucidating the hepatic expression and role of Panx1 in APAPinduced hepatotoxicity using the Panx1 inhibiting peptide 10Panx1. It was found that inhibition of Panx1 channels counteracts the clinical features of APAP-triggered hepatotoxicity, in particular related to cell death and inflammation. Overall, this doctoral 10 project shows for the first time the involvement of connexin hemichannels and pannexin channels in APAP-induced acute liver failure, which suggests a role as therapeutic drug targets.

Expression of Connexins 43, 26 and 32 in normal, hyperplastic and neoplastic perianal dog glands

Connexin (Cx) expression is reportedly altered in neoplasms. This study aimed to investigate the expression of Cx43, 26 and 32 in normal and pathological canine perianal glands. Thirty perianal glands bearing pathological processes and ten normal canine perianal glands were submitted to immunohistochemistry to search for presence of Cx43, Cx26 and Cx32. Both Cx43 and Cx26 expressions were observed in normal, hyperplastic glands, and in well and moderately differentiated adenomas. However, in poorly differentiated adenomas, expressions were reduced, and they were absent in carcinomas. Cx26 was located in the cytoplasm of normal, hyperplastic perianal gland cells, and in well and moderately differentiated adenomas. Cx32 was not observed in any neoplasm neither in normal or hyperplastic glands. Our results show that Cx43 and Cx26 expressions are altered in more aggressive canine perianal gland neoplasms, and we conclude that they may be related to the perianal gland carcinogenesis process

Expression of Connexins 43, 26 and 32 in normal, hyperplastic and neoplastic perianal dog glands

Connexin (Cx) expression is reportedly altered in neoplasms. This study aimed to investigate the expression of Cx43, 26 and 32 in normal and pathological canine perianal glands. Thirty perianal glands bearing pathological processes and ten normal canine perianal glands were submitted to immunohistochemistry to search for presence of Cx43, Cx26 and Cx32. Both Cx43 and Cx26 expressions were observed in normal, hyperplastic glands, and in well and moderately differentiated adenomas. However, in poorly differentiated adenomas, expressions were reduced, and they were absent in carcinomas. Cx26 was located in the cytoplasm of normal, hyperplastic perianal gland cells, and in well and moderately differentiated adenomas. Cx32 was not observed in any neoplasm neither in normal or hyperplastic glands. Our results show that Cx43 and Cx26 expressions are altered in more aggressive canine perianal gland neoplasms, and we conclude that they may be related to the perianal gland carcinogenesis process (AU)

Diminished angiogenesis in the cornea of mice with heterologous deletion of Connexin 43 gene (Gja1)

Angiogenesis is involved in several physiological and pathological processes, and the proliferation of endothelial cells is a central event in the generation of new blood vessels. Gap junctions (GJ) are membrane structures that communicate adjacent cells, contribute to tissue homeostasis, and are important to the control of cell proliferation. Connexins (Cxs) are the proteins that form gap junctions. Endothelial cells may express Cx43, Cx37 and Cx40. In this study, we analyzed the effect of the heterologous deletion of the Gja1 (Cx43 gene) on the development of newly formed blood vessels (NFBV) in the mouse cornea. Heterozygous (Cx43+/-) and wild-type (Cx43+/+) mice were submitted to the silver crystal corneal cauterization model. Two parameters were quantified by image analysis 2 or 6 days after cauterization: NFBV density and length. At days 2 and 6 after corneal cauterization, Cx43+/- mice showed smaller density of NFBV as compared to Cx43+/+ mice. Therefore, deletion of one Gja1 allele (connexin-43 gene) may lead to impaired cell-cell communication in endothelial cells, diminishing angiogenesis after cauterization of the mouse cornea

Diminished angiogenesis in the cornea of mice with heterologous deletion of Connexin 43 gene (Gja1)

Angiogenesis is involved in several physiological and pathological processes, and the proliferation of endothelial cells is a central event in the generation of new blood vessels. Gap junctions (GJ) are membrane structures that communicate adjacent cells, contribute to tissue homeostasis, and are important to the control of cell proliferation. Connexins (Cxs) are the proteins that form gap junctions. Endothelial cells may express Cx43, Cx37 and Cx40. In this study, we analyzed the effect of the heterologous deletion of the Gja1 (Cx43 gene) on the development of newly formed blood vessels (NFBV) in the mouse cornea. Heterozygous (Cx43+/-) and wild-type (Cx43+/+) mice were submitted to the silver crystal corneal cauterization model. Two parameters were quantified by image analysis 2 or 6 days after cauterization: NFBV density and length. At days 2 and 6 after corneal cauterization, Cx43+/- mice showed smaller density of NFBV as compared to Cx43+/+ mice. Therefore, deletion of one Gja1 allele (connexin-43 gene) may lead to impaired cell-cell communication in endothelial cells, diminishing angiogenesis after cauterization of the mouse cornea(AU)

Expression and distribution of connexin 32 in rat liver with experimentally induced fibrosis / Expressão e distribuição da conexina 32 em fígados de ratos com fibrose induzida experimentalmente

The connexin 32 (Cx32) is a protein that forms the channels that promote the gap junction intercellular communication (GJIC) in the liver, allowing the diffusion of small molecules through cytosol from cell-to-cell. Hepatic fibrosis is characterized by a disruption of normal tissue architeture by cellular lesions, and may alter the GJIC. This work aimed to study the expression and distribution of Cx32 in liver fibrosis induced by the oral administration of dimethylnitrosamine in female Wistar rats. The necropsy of the rats was carried out after five weeks of drug administration. They presented a hepatic fibrosis state. Sections from livers with fibrosis and from control livers were submitted to immunohistochemical, Real Time-PCR and Western-Blot analysis to Cx32. In fibrotic livers the Cxs were diffusely scattered in the cytoplasm, contrasting with the control livers, where the Cx32 formed junction plaques at the cell membrane. Also it was found a decrease in the gene expression of Cx32 without reduction in the protein quantity when compared with controls. These results suggest that there the mechanism of intercellular communication between hepatocytes was reduced by the fibrotic process, which may predispose to the occurrence of a neoplastic process, taken in account that connexins are considered tumor suppressing genes.(AU)

A conexina 32 (Cx32) é uma proteína que constitui os canais que promovem as comunicações intercelulares via junções comunicantes (CIJC) no fígado, permitindo difusão de pequenas moléculas citoplasmáticas de uma célula à outra. A fibrose hepática caracteriza-se pela alteração da arquitetura normal do fígado e podem alterar as CIJCs. O objetivo deste trabalho foi estudar a expressão e distribuição de Cx32 na fibrose hepática. O objetivo do presente trabalho foi estudar a expressão e distribuição da Cx32 em fígados com fibrose induzida pela administração oral de dimetilnitrosamina em fêmeas de ratos Wistar. A necropsia foi realizada após cinco semanas da última administração da droga e observou-se um quadro de fibrose hepática. Amostras dos fígados com fibrose e de animais controle foram submetidas à análise imunoistoquímica, por Real Time-PCR e por Western-Blot verificando-se a presença de Cx32 difusa e dispersa no citoplasma dos fígados com fibrose. No grupo controle a Cx32 localizou-se na membrana citoplasmática com a formação de placas juncionais. O fígado com fibrose também revelou diminuição da expressão gênica de Cx32, embora sem a redução da quantidade do produto protéico, quando comparado ao grupo controle. Estes resultados sugerem que o mecanismo de comunicação intercelular entre os hepatócitos reduziu-se durante o processo fibrótico, o que pode predispor a ocorrência de processos neoplásicos, uma vez que as conexinas são consideradas genes supressores de tumores.(AU)

Gap junctions and communication within the nervous system

Gap junctions are sites on the cellular membrane with intercellular channels build up by twelve protein subunits called connexins. Each connected cell contributes with a hemichannel made up by six connexins subunits. This kind of connection represents and efficient way of intercellular communication in most tissues, including the nervous system. It works as a passage for ions, secondary messenger and metabolites exchange between the cells. In a complex tissue like the nervous tissue they are particularly important because they connect the various cellular types composing a panglial syncytium that performs neuronal protection and tissue homeostasis. The expression of connexins and the intercellular communication through gap junctions are crucial to regulate vital functions as cellular motility, proliferations and survival; changes in the conformational expression of connexins may be involved in diseases as Alzheimer's disease, neoplasms, bacterial and parasitic infections, or even affect cellular groups when they occur as genetic mutations leading to functional defects of the nervous system as demyelination in the PNS (Charcot-Marie-Tooth disease), hereditary epilepsy, non-syndromic deafness and senile cataract.

Compensatory kidney hypertrophy/hyperplasia after nephrectomy in mice: alterations of connexin 43 (Cx43) phosphorylated isoforms

Compensatory kidney hypertrophy/hyperplasia leads to several changes in kidney structure and function, as increased glomeruli filtration. The aim of this study was to evaluate connexin 43 in remnant mouse kidneys after unilateral nephrectomy. The right kidney was surgically removed from BALB/c mice. Groups were euthanized at 24, 48 and 72 hours, and at 7 and 30 days. Kidney sections of the reminiscent kidneys were stained with Periodic Acid/Schiff and additional slides were submitted to BrdU and Cx43 immunohistochemistry. The results demonstrated an increase in kidney weight as early as 24 hours through 30 post-nephrectomy. In addition, BrdU positive epithelial cells increased at 24 and 48 hours post-nephrectomy. Cx43 was detected in the cytoplasm and membrane of epithelial cells and vasculature. Taking into consideration the quantity, intensity and localization of Cx43 immunostaining pattern, we observed that nephrectomized mice presented lower Cx43 expression and a cytoplasmic localization after 24 hours, peaking in 48 hours. Furthermore, western blot revealed that during the first 24 and 48 hours after nephrectomy, PO (unphosphorylated) and P1 (phosphorylated) Cx43 disappeared, and the products of Cx43 degradation were reduced. On the other hand, after 72 hours the PO and P1 state reappeared and the amount of degraded peptides also increased. Seven and thirty days after nephrectomy, a higher intensity of PO and P1 state and a lower P2 (hyperphosphorylated) band were observed. In conclusion, our results suggest that Cx43 phosphorylation results in the retention of Cx43 in cytoplasm and its increased degradation during compensatory renal hyperplasia/hypertrophy.

Análise proteômica do intestino primitivo de embriões bovinos / Proteomic analysis of primitive gut from bovine embryo

O desenvolvimento de biotecnologia de embriões em animais de produção é prejudicado por perdas no primeiro trimestre da gestação, idade em que o intestino primitivo está sendo estabelecido. O estudo das proteínas contidas no intestino primitivo nesta fase inicial da gestação pode aumentar o conhecimento sobre as vias moleculares envolvidas no desenvolvimento embrionário normal e em perdas de embriões, assim como a sua participação na organogênese e diferenciação celular. Intestino primitivo de embriões de bovinos a partir dos 39 SD ± 4 dias de desenvolvimento (variando de 33 a 45 dias) foram coletados em um matadouro local. As amostras foram processadas e agrupadas para análise proteômica shotgun label-free usando MudPIT (Multidimensional Protein Identification Technology). Análise funcional e de via foram feitas usando FatiGO (www.babelomics.org); Pathway Express (http://vortex.cs.wayne.edu/ ontoexpress) para identificar as ontologias relevantes e vias canônicas ou não-canônicas representada pelas proteínas expressas no Intestino primitivo. Um total de 74 proteínas ou sequências randômicas foram identificadas, correspondentes a 30 proteínas específicas expressas pelo Intestino primitivo bovino. Das 30 proteínas únicas, 21 proteínas foram utilizadas na ontologia e análise de vias. As análises mostraram um enriquecimento de ontologias relacionadas com a ligação (N = 5); atividade catalítica (N = 6); organela intracelular (N = 6). Houve um enriquecimento de ontologias associado às modificações do citoesqueleto; processo de diferenciação celular (N = 3), a migração celular (N = 4) e no metabolismo celular (N = 6). Além disso, a via e a análise de rede mostraram um enriquecimento de vias de comunicação entre células, tais como junções comunicantes e tight e as vias de adesão focal. Além disso, as vias envolvidas no movimento celular (por exemplo, vias de regulação do citoesqueleto de actina e a migração transendotelial de leucócitos) foram extremamente enriquecimento no grupo de proteínas expressas pelo Intestino primitivo bovino. Nossos resultados sugerem que as células do intestino primitivo tem alto perfil migratório e são compostas de células não totalmente diferenciadas, com alto metabolismo celular. A migração e a diferenciação destas células poderiam determinar o destino do embrião em desenvolvimento. Além disso, a compreensão da função e interação de proteínas expressas pelo embrião normal fornecerá informações sobre o impacto das biotecnologias reprodutivas no desenvolvimento do embrião durante a implantação e placentação.

The development of embryo biotechnology in farm animals is hampered by embryo losses in first trimester of gestation, period in which the primitive gut is being established. The study of proteins contained in the primitive gut at this early stage of pregnancy may increase the knowledge about the molecular pathways involved in normal embryonic development and loss of embryos, as well as their involvement in organogenesis and cell differentiation.primitive gut from bovine embryos on day 39 SD± 4 of development (ranging from 33 to 45 days) were collected at a local slaugtherhouse. The samples were processed and pooled for label-free shotgun proteomics analysis using MudPIT (Multidimensional Protein Identification Technology) tandem MSE acquisition. Functional and pathway analysis using FatiGo (www.babelomics.org); Pathway Express (http://vortex.cs.wayne.edu/ ontoexpress) and Ingenuity Pathway Analysis (www.ingenuity.com) were used to identify relevant ontologies and canonical or noncanonical pathways represented by the expressed proteins in the primitive gut. A total of 74 protein sequences were identified corresponding to 30 unique proteins expressed by the bovine primitive gut. out of 30 unique proteins, 21 proteins were used on the ontology and pathway analysis. The ontology analysis showed an enrichment of ontologies related to binding (N=5); catalytic activity (N=6); intracellular organelle (N=6). There was an enrichment of ontologies associated to cytoskeleton modifications; cell differentiation process (N=3); cellular migration (N=4) and cell metabolism (N=6). Furthermore, the pathway and the network analysis showed an enrichment of cell-to-cell communication pathways such as gap and tight junction, and focal adhesion pathways. In addition, pathways involved in cellular movement (regulation of actin cytoskeleton and leukocyte transendothelial migration) were extremely enrichment in the group of proteins expressed by the bovine primitive gut. Our results suggested that the cells from primitive gut have high migratory profile and are composed of not fully differentiated cells with high cellular metabolism. The proper migration and differentiation of these cells would dictate the fate of the developing embryo. Moreover, understanding the function and interaction of proteins expressed by normal embryo will give clues of the impact of the reproductive biotechnologies in embryo development during the window between implantation and placentation.

Análise proteômica do intestino primitivo de embriões bovinos / Proteomic analysis of primitive gut from bovine embryo

O desenvolvimento de biotecnologia de embriões em animais de produção é prejudicado por perdas no primeiro trimestre da gestação, idade em que o intestino primitivo está sendo estabelecido. O estudo das proteínas contidas no intestino primitivo nesta fase inicial da gestação pode aumentar o conhecimento sobre as vias moleculares envolvidas no desenvolvimento embrionário normal e em perdas de embriões, assim como a sua participação na organogênese e diferenciação celular. Intestino primitivo de embriões de bovinos a partir dos 39 SD ± 4 dias de desenvolvimento (variando de 33 a 45 dias) foram coletados em um matadouro local. As amostras foram processadas e agrupadas para análise proteômica shotgun label-free usando MudPIT (Multidimensional Protein Identification Technology). Análise funcional e de via foram feitas usando FatiGO (www.babelomics.org); Pathway Express (http://vortex.cs.wayne.edu/ ontoexpress) para identificar as ontologias relevantes e vias canônicas ou não-canônicas representada pelas proteínas expressas no Intestino primitivo. Um total de 74 proteínas ou sequências randômicas foram identificadas, correspondentes a 30 proteínas específicas expressas pelo Intestino primitivo bovino. Das 30 proteínas únicas, 21 proteínas foram utilizadas na ontologia e análise de vias. As análises mostraram um enriquecimento de ontologias relacionadas com a ligação (N = 5); atividade catalítica (N = 6); organela intracelular (N = 6). Houve um enriquecimento de ontologias associado às modificações do citoesqueleto; processo de diferenciação celular (N = 3), a migração celular (N = 4) e no metabolismo celular (N = 6). Além disso, a via e a análise de rede mostraram um enriquecimento de vias de comunicação entre células, tais como junções comunicantes e tight e as vias de adesão focal. Além disso, as vias envolvidas no movimento celular (por exemplo, vias de regulação do citoesqueleto de actina e a migração transendotelial de leucócitos) foram extremamente enriquecimento no grupo de proteínas expressas pelo Intestino primitivo bovino. Nossos resultados sugerem que as células do intestino primitivo tem alto perfil migratório e são compostas de células não totalmente diferenciadas, com alto metabolismo celular. A migração e a diferenciação destas células poderiam determinar o destino do embrião em desenvolvimento. Além disso, a compreensão da função e interação de proteínas expressas pelo embrião normal fornecerá informações sobre o impacto das biotecnologias reprodutivas no desenvolvimento do embrião durante a implantação e placentação. (AU)

The development of embryo biotechnology in farm animals is hampered by embryo losses in first trimester of gestation, period in which the primitive gut is being established. The study of proteins contained in the primitive gut at this early stage of pregnancy may increase the knowledge about the molecular pathways involved in normal embryonic development and loss of embryos, as well as their involvement in organogenesis and cell differentiation.primitive gut from bovine embryos on day 39 SD± 4 of development (ranging from 33 to 45 days) were collected at a local slaugtherhouse. The samples were processed and pooled for label-free shotgun proteomics analysis using MudPIT (Multidimensional Protein Identification Technology) tandem MSE acquisition. Functional and pathway analysis using FatiGo (www.babelomics.org); Pathway Express (http://vortex.cs.wayne.edu/ ontoexpress) and Ingenuity Pathway Analysis (www.ingenuity.com) were used to identify relevant ontologies and canonical or noncanonical pathways represented by the expressed proteins in the primitive gut. A total of 74 protein sequences were identified corresponding to 30 unique proteins expressed by the bovine primitive gut. out of 30 unique proteins, 21 proteins were used on the ontology and pathway analysis. The ontology analysis showed an enrichment of ontologies related to binding (N=5); catalytic activity (N=6); intracellular organelle (N=6). There was an enrichment of ontologies associated to cytoskeleton modifications; cell differentiation process (N=3); cellular migration (N=4) and cell metabolism (N=6). Furthermore, the pathway and the network analysis showed an enrichment of cell-to-cell communication pathways such as gap and tight junction, and focal adhesion pathways. In addition, pathways involved in cellular movement (regulation of actin cytoskeleton and leukocyte transendothelial migration) were extremely enrichment in the group of proteins expressed by the bovine primitive gut. Our results suggested that the cells from primitive gut have high migratory profile and are composed of not fully differentiated cells with high cellular metabolism. The proper migration and differentiation of these cells would dictate the fate of the developing embryo. Moreover, understanding the function and interaction of proteins expressed by normal embryo will give clues of the impact of the reproductive biotechnologies in embryo development during the window between implantation and placentation. (AU)