RESUMO
A progesterona é um hormônio fundamental na manutenção da gestação nos mamíferos. Em bovinos clonados, perdas gestacionais significativas ocorrem devido à deficiência placentária. Tendo em vista esse problema, o objetivo do trabalho foi analisar em diferentes idades gestacionais a imunolocalização para receptores de progesterona em placentas de animais não clonados e clonados. Tecidos placentomais de bovinos não clonados foram obtidos da linha de abate no Matadouro Mantiqueira, enquanto os tecidos placentomais dos animais clonados com idades gestacionais de 68, 135, 225 e 270 dias de gestação foram obtidos em empresas especializadas em clonagem animal. Os tecidos foram processados e embebidos em paraplast para localização de receptores de progesterona, utilizando-se a técnica de imuno-histoquímica pelo anticorpo monoclonal (Progesterone receptor SER 294). Pela imuno-histoquímica, comparando-se a concentração de receptores de progesterona na placenta de animais clonados com as de animais não clonados, em diferentes idades gestacionais, observou-se que as células dos placentomas dos animais clonados apresentaram uma marcação mais intensa de receptor de progesterona nas idades de 135 e 270 dias de gestação em relação aos não clonados. Tal fato sugere que a presença de progesterona no final da gestação pode ser mais uma das possíveis causas de gestações prolongadas.
To progesterone is a fundamental hormone in the maintenance of the gestation in the mammals. In bovine they cloned, losses gestational significant, occur due to the placental deficiency. Having in mind that problem, the objective of the work was analyze in different gestational ages to immune stained for receivers of progesterone in placentas of normal animals and cloned. Placentomal tissues of bovine normal were obtained of the line of counts on in the Slaughterhouse Mantiqueira, while the placentomal tissues of the animals cloned with gestational ages of 68, 135, 225 and 270 days of gestation were obtained in companies specialized in animal cloning. The fabrics were prosecuted and absorbed in paraplast for progesterone receivers location, utilizing itself to technical of immunohistochemistry by the antibody monoclonal (Progesterone receiver BE 294). By the immunohistochemistry comparing the progesterone receivers concentration in the placenta of animals cloned, with the of animals done not manipulate, in different gestational ages we observe that the cells of the placentomal tissues of the animals manipulated (cloned) presented a more intense marking of receiver of progesterone in the ages of 135 and 270 days of gestation regarding the done not manipulate. Suggesting that the presence of progesterone in the end of the gestation can be more one of the possible cause of gestations prolonged.
Assuntos
Feminino , Animais , Bovinos , Células Clonais/enzimologia , Células Clonais/imunologia , Hormônios Placentários/deficiência , Imuno-Histoquímica/veterinária , Receptores de Progesterona/imunologia , Esteroides/imunologiaRESUMO
A progesterona é um hormônio fundamental na manutenção da gestação nos mamíferos. Em bovinos clonados, perdas gestacionais significativas ocorrem devido à deficiência placentária. Tendo em vista esse problema, o objetivo do trabalho foi analisar em diferentes idades gestacionais a imunolocalização para receptores de progesterona em placentas de animais não clonados e clonados. Tecidos placentomais de bovinos não clonados foram obtidos da linha de abate no Matadouro Mantiqueira, enquanto os tecidos placentomais dos animais clonados com idades gestacionais de 68, 135, 225 e 270 dias de gestação foram obtidos em empresas especializadas em clonagem animal. Os tecidos foram processados e embebidos em paraplast para localização de receptores de progesterona, utilizando-se a técnica de imuno-histoquímica pelo anticorpo monoclonal (Progesterone receptor SER 294). Pela imuno-histoquímica, comparando-se a concentração de receptores de progesterona na placenta de animais clonados com as de animais não clonados, em diferentes idades gestacionais, observou-se que as células dos placentomas dos animais clonados apresentaram uma marcação mais intensa de receptor de progesterona nas idades de 135 e 270 dias de gestação em relação aos não clonados. Tal fato sugere que a presença de progesterona no final da gestação pode ser mais uma das possíveis causas de gestações prolongadas.(AU)
To progesterone is a fundamental hormone in the maintenance of the gestation in the mammals. In bovine they cloned, losses gestational significant, occur due to the placental deficiency. Having in mind that problem, the objective of the work was analyze in different gestational ages to immune stained for receivers of progesterone in placentas of normal animals and cloned. Placentomal tissues of bovine normal were obtained of the line of counts on in the Slaughterhouse Mantiqueira, while the placentomal tissues of the animals cloned with gestational ages of 68, 135, 225 and 270 days of gestation were obtained in companies specialized in animal cloning. The fabrics were prosecuted and absorbed in paraplast for progesterone receivers location, utilizing itself to technical of immunohistochemistry by the antibody monoclonal (Progesterone receiver BE 294). By the immunohistochemistry comparing the progesterone receivers concentration in the placenta of animals cloned, with the of animals done not manipulate, in different gestational ages we observe that the cells of the placentomal tissues of the animals manipulated (cloned) presented a more intense marking of receiver of progesterone in the ages of 135 and 270 days of gestation regarding the done not manipulate. Suggesting that the presence of progesterone in the end of the gestation can be more one of the possible cause of gestations prolonged.(AU)
Assuntos
Animais , Feminino , Bovinos , Hormônios Placentários/deficiência , /química , Receptores de Progesterona/imunologia , Células Clonais/enzimologia , Células Clonais/imunologia , Imuno-Histoquímica/veterinária , Esteroides/imunologiaRESUMO
Ganglioside glycosyltransferases organize as multienzyme complexes that localize in different sub-Golgi compartments. Here we studied whether in CHO-K1 cells lacking CMP-NeuAc: GM3 sialyltransferase (SialT2), the sub-Golgi localization of UDP-Gal:glucosylceramide beta-1,4-galactosyltransferase (GalT1) and CMP-NeuAc:lactosylceramide sialyltransferase (SialT1) complex is affected when SialT2, another member of this complex, is coexpressed. GalT1 and SialT1 sub-Golgi localization was determined by studying the effect of brefeldin A (BFA) and monensin on the synthesis of glycolipids and on the sub-Golgi localization of GalT1(1-52)-CFP (cyan fluorescent protein) and SialT1(1-54)-YFP (yellow fluorescent protein) chimeras by single cell fluorescence microscopy and by isopycnic subfractionation. We found that BFA, and also monensin, impair the synthesis of glycolipids beyond GM3 ganglioside in wild type (WT) cells but beyond GlcCer in SialT2(+) cells. Although BFA redistributed GalT1-CFP and SialT1-YFP to the endoplasmic reticulum in WT cells, a fraction of these chimeras remained associated with a distal Golgi compartment, enriched in trans Golgi network, and recycling endosome markers in SialT2(+) cells. In BFA-treated cells, the percentage of GalT1-CFP and SialT1-YFP associated with Golgi-like membrane fractions separated by isopycnic subfractionation was higher in SialT2(+) cells than in WT cells. These effects were reverted by knocking down the expression of SialT2 with specific siRNA. Results indicate that sub-Golgi localization of glycosyltransferase complexes may change according to the relative levels of the expression of participating enzymes and reveal a capacity of the organelle to adapt the topology of the glycolipid synthesis machinery to functional states of the cell.
Assuntos
Galactosiltransferases/metabolismo , Glicolipídeos/biossíntese , Complexo de Golgi/enzimologia , N-Acetilgalactosaminiltransferases/metabolismo , Sialiltransferases/metabolismo , Animais , Antibacterianos/farmacologia , Antiprotozoários/farmacologia , Biomarcadores/metabolismo , Brefeldina A/farmacologia , Células CHO , Centrifugação Isopícnica , Células Clonais/enzimologia , Cricetinae , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Monensin/farmacologia , N-Acetilgalactosaminiltransferases/química , N-Acetilgalactosaminiltransferases/genética , RNA Interferente Pequeno/farmacologia , Sialiltransferases/química , Sialiltransferases/genética , Frações Subcelulares/metabolismo , TransfecçãoRESUMO
The aim of this study was to investigate the effect of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on NADPH oxidase activity and gp91-phox gene expression in HL-60 clone 15 cells as they differentiate along the eosinophilic lineage. The results were compared to the eosoniphilic inducers interleukin-5 (IL-5) and butyric acid. IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml) or IL-5 (200 pM) caused a significant increase in the expression of the eosinophil peroxidase (EPO) and the major basic protein (MBP) genes. Similar results were observed when the cells were cultured with 0.5 mM butyric acid for 5 days. IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml) also caused a significant increase in superoxide release by HL-60 clone 15 cells after 2 days compared with control or with butyric acid-induced cells. After 5 days, these cytokines and butyric acid induced an even stronger release of superoxide. HL-60 clone 15 cells cultured with IFN-gamma and TNF-alpha for 2 days showed a significant increase in gp91-phox gene expression. We conclude that IFN-gamma and TNF-alpha are sufficient to induce the differentiation of HL-60 clone 15 cells to the eosinophilic lineage and to upregulate gp91-phox gene expression and activity of the NADPH oxidase system.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Eosinófilos/efeitos dos fármacos , Interferon gama/farmacologia , NADPH Oxidases/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Biomarcadores/análise , Ácido Butírico/farmacologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem da Célula , Células Clonais/efeitos dos fármacos , Células Clonais/enzimologia , Células Clonais/metabolismo , Ativação Enzimática/efeitos dos fármacos , Eosinófilos/citologia , Eosinófilos/enzimologia , Eosinófilos/metabolismo , Eritropoetina/genética , Eritropoetina/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Interleucina-5/farmacologia , Proteínas Ligantes de Maltose , Glicoproteínas de Membrana/genética , NADPH Oxidase 2 , NADPH Oxidases/genética , Peroxidase/genética , Peroxidase/metabolismoRESUMO
UDP-GalNAc:lactosylceramide/GM3/GD3 beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T) transforms its acceptors into the gangliosides GA2, GM2 and GD2. It is well established that it is a Golgi-located glycosyltransferase, but its sub-Golgi localization is still unclear. We addressed this question in Chinese hamster ovary K1 cell clones stably transfected with a c-myc-tagged version of GalNAc-T which express the enzyme at different levels of activity. In these cell clones we examined the effect of brefeldin A (BFA) on the synthesis of glycolipids (in metabolic-labelling experiments) and on the sub-Golgi localization of the GalNAc-T (by immunocytochemistry). We found that in cell clones expressing moderate levels of activity, GalNAc-T immunoreactivity behaved as the trans-Golgi network (TGN) marker mannose-6-P receptor (M6PR) both in BFA-treated and untreated cells, and that BFA completely blocked the synthesis of GM2, GM1 and GD1a. On the other hand, in cell clones expressing high levels of activity and treated with BFA, most GalNAc-T immunoreactivity redistributed to the endoplasmic reticulum, as did the medial-Golgi marker mannosidase II, and the synthesis of GM2, GM1 and GD1a was not completely blocked. These results indicate that GalNAc-T is a TGN-located enzyme and that the mechanism that localizes it to this compartment involves steps that, when saturated, lead to its mislocalization to the cis-, medial- or trans-Golgi. Changes of Golgi membrane properties by modification of local glycolipid composition due to the activity of the expressed enzyme were not the main cause of mislocalization, since it persists when glycolipid synthesis is inhibited with d, l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol-HCl.
Assuntos
Complexo de Golgi/enzimologia , N-Acetilgalactosaminiltransferases/metabolismo , Animais , Western Blotting , Células CHO , Células Clonais/enzimologia , Cricetinae , Inibidores Enzimáticos/farmacologia , Glicolipídeos/biossíntese , Complexo de Golgi/efeitos dos fármacos , Imuno-Histoquímica , N-Acetilgalactosaminiltransferases/antagonistas & inibidores , N-Acetilgalactosaminiltransferases/genética , Propanolaminas/farmacologia , Pirrolidinas/farmacologia , Transfecção , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
A informaçäo disponível sobre a epidemiologia molecular e as interrelaçöes genéticas dos Haemophilus influenzae serotipo b (Hib) recuperados de pacientes que vivem na América do Sul é escassa. Quarenta e três cepas isoladas de pacientes com doença invasiva no Uruguai entre 1987 e 1992 foram caracterizadas por genótipo enzimático multilocus e por subtipo de proteína de membrana externa (outer membrana protein: OMP). As análises de OMP identificaram 3 dos subtipos conhecidos, incluindo 3L (70 por cento), 2L (18 por cento) e 18L(2 por cento). Também se encontrou dois padröes, näo descritos anteriormente, relacionados à famílias dos subtipos L e U, cada um representando 5 por cento dos organismos. Quatro tipos electroforéticos de enzimas multilocus foram identificados, incluindo ET 12,5 (67,3 por cento), 12,8 (23,3 por cento), 12,7 (4,7 por cento), e um ET 12.5/3L foi responsável por 63 por cento de todos os isolamentos, e causou 83 por cento dos casos de meningite mas somente 55 por cento dos episódios de pneumonia e septicemia. Em geral, a frequência da ocorrência de subclones Hib no Uruguai foi igual à registrada em vários países da Europa
Assuntos
Humanos , Haemophilus influenzae/isolamento & purificação , Epidemiologia Molecular , Genótipo , Células Clonais/enzimologia , Pneumonia/etiologia , Pneumonia/epidemiologia , Sepse/etiologia , Sepse/epidemiologia , Meningite por Haemophilus/epidemiologiaRESUMO
Clones have been derived by limiting dilution from recent isolates of Plasmodium falciparum from Africa and Brazil. These clones have been characterized for pyrimethamine and chloroquine sensitivity and/or isoenzyme type. Clones obtained from an isolate containing a mixture of GPI isoenzyme types were found to have (with one exception) only GPI-1 or GPI-2. Clones differing 1,000-fold in pyrimethamine resistance were derived from a single isolate from Brazil which was resistant to both pyrimethamine and chloroquine. Several levels of chloroquine sensitivity were observed in the isolates studied. All clones showed levels of chloroquine sensitivity similar to that of their parent isolates. The KZ1 isolate from East Africa had an intermediate level of chloroquine resistance and a flat dose-response curve and each of its clones had a similar dose-response curve, indicating that the intermediate level of chloroquine resistance did not result from the KZ1 isolate's being a mixture of clones with high and low sensitivity.