Localization of Diacylglycerol Kinase ζ in the Kidney of Adult Rats: Its Dominant Expression in Collecting Tubules and its Selective Lack in Proximal Tubules / Localización de Diacilglicerol Quinasa ζ en el Riñón de Ratas Adultas: su Expresión Dominante en Túbulos Colectores y su Carencia Selectiva en Túbulos Proximales

SUMMARY: Diacylglycerol kinase (DGK) exerts balancing the intracellular level between two-second messengers, diacylglycerol and phosphatidic acid, by its phosphorylation activity. DGK ζ is often localized in cell nuclei, suggesting its involvement in the regulation of intranuclear activities, including mitosis and apoptosis. The present immunohistochemical study of rat kidneys first revealed no detection levels of DGK ζ -immunoreactivity in nuclei of most proximal tubule epithelia in contrast to its distinct occurrence in cell nuclei of collecting and distal tubules with the former more dominant. This finding suggests that DGK ζ is a key factor regulating vulnerability to acute kidney injury in various renal tubules: its low expression represents the high vulnerability of proximal tubule cells, and its distinct expression does the resistance of collecting and distal tubule cells. In addition, this isozyme was more or less localized in nuclei of cells forming glomeruli as well as in endothelial nuclei of peritubular capillaries and other intrarenal blood vessels, and epithelial nuclei of glomerular capsules (Bowman's capsules) and renal calyces, including intrarenal interstitial cells.

La diacilglicerol quinasa (DGK) ejerce el equilibrio del nivel intracelular entre dos segundos mensajeros, diacilglicerol y ácido fosfatídico, por su actividad de fosforilación. La DGK ζ a menudo se localiza en los núcleos celulares, lo que sugiere su participación en la regulación de las actividades intranucleares, incluidas la mitosis y la apoptosis. El presente estudio inmunohistoquímico en riñones de rata no reveló niveles de detección de inmunorreactividad de DGK ζ en los núcleos de la mayoría de los epitelios de los túbulos proximales, en contraste a la detección en los núcleos celulares de los túbulos colectores y distales, siendo el primero más dominante. Este hallazgo sugiere que DGK ζ es un factor clave que regula la vulnerabilidad a la lesión renal aguda en varios túbulos renales: su baja expresión representa la alta vulnerabilidad de las células del túbulo proximal, y su expresión distinta hace a la resistencia de las células del túbulo colector y distal. Además, esta isoenzima estaba más o menos localizada en los núcleos de las células que forman los glomérulos, así como en los núcleos endoteliales de los capilares peritubulares y otros vasos sanguíneos intrarrenales, y en los núcleos epiteliales de las cápsulas glomerulares (cápsulas de Bowman) y los cálices renales, incluidas las células intersticiales intrarrenales.

Spatiotemporally differential expression of HER (human epithelial growth factor receptor)-2 with sexual dimorphism in submandibular gland of mice

SUMMARY: Considering that the submandibular gland (SMG) of postnatal mice performs active cell proliferation, apoptosis and differentiation which are regulated by proto-oncogene products in cancerous cells, the expression and localization of a proto-oncogene product HER (human epidermal growth factor receptor)-2 was examined in SMG of postnatal mice. In Western blot analysis, the expression for HER-2 was high until pre-puberty, and it decreased from puberty to young adult stages with male SMG more dominant. In immunohistochemistry, the immunoreactivity was positive in acinar and ductal cells of newborn SMG with distinct localization at the intercellular apposition sites. The immunoreactivity in acinar cells progressively decreased to negligible levels by pre-pubertal stage, while it remained positive in most ductal cells throughout the postnatal time-course. The immunoreactivity in cells of terminal tubules and intercalated ducts, both of which have a high potential to produce cells, were seen at levels similar to those of more proximal ducts, while the immunoreactivity in ductal basal cells was significantly high, but the granular convoluted tubule cells were seen at negligible levels in male and at faint levels in female. In immuno-electron microscopy of excretory ducts, the immunoreactivity was dominantly localized on the basal infolding membranes as well as vesicles and vacuoles of various sizes, but rarely in Golgi apparatus and mitochondria. The immunoreactivity without association to any membranous structures were also seen, though not numerous. The relation of expression levels of HER-2 in various portions of normal SMG to those in their cancerous ones is briefly discussed.

RESUMEN: Considerando que la glándula submandibular (GSM) de ratones postnatales realiza la proliferación celular activa, apoptosis y diferenciación que están reguladas por productos protooncogénicos en células cancerosas, la expresión y localización de un producto protooncogénico HER (receptor del factor de crecimiento epidérmico humano) - 2 se examinó en GSM de estos ratones. En el análisis de Western blot, la expresión de HER-2 fue alta hasta la prepubertad, y disminuyó desde la pubertad hasta las etapas de adultos jóvenes con GSM macho más dominante. En inmunohistoquímica, la inmunorreactividad fue positiva en las células acinares y ductales de GSM de recién nacido con una localización distinta en los sitios de aposición intercelular. La inmunorreactividad en las células acinares disminuyó progresivamente a niveles insignificantes en la etapa prepuberal, mientras que permaneció positiva en la mayoría de las células ductales durante el transcurso del tiempo posnatal. La inmunorreactividad en las células de los túbulos terminales y los conductos intercalados, los cuales tienen un alto potencial para producir células, se obser- vó a niveles similares a los de los conductos más proximales, mientras que la inmunorreactividad en las células basales ductales fue significativamente alta, pero en el túbulo contorneado granular las células se observaron en niveles insignificantes en los machos y en niveles débiles en las hembras. En la microscopía inmunoelectrónica de los conductos excretores, la inmunorreactividad se localizó de manera predominante en las membranas de pliegues basales, así como en vesículas y vacuolas de varios tamaños, pero raramente en el aparato de Golgi y en las mitocondrias. También se observó la inmunorreactividad sin asociación a ninguna estructura membranosa, aunque no numerosa. Se discute brevemente la relación de los niveles de expresión de HER-2 en varias porciones de GSM normal con aquellos en sus cancerosos.

Identification in immuno-electron microscopy of solitary multi-polar peripheral neurons of adult Opisthorchis viverrini by antibody against rat diacylglycerol kinase / Identificación por microscopía imuno-electrónica de las neuronas periféricas multipolares solitarias del Opisthorchis viverrini adulto mediante anticuerpo contra la diacilglicerol kinasa de ratas

By utilizing the antibody for rat DGKz a substantial number of immunopositive cells were found in the OV (Opisthorchis viverrini). The immunopositive cells appeared solitarily and they were distributed rather symmetrically to the longitudinal axis of the OV. Some of them were located in close proximity to internal organs such as uterus, ovary, testes, vitelline glands and guts. The immunostained cells extended tapering processes horizontally or obliquely to the OV longitudinal axis. In immuno-electron microscopy, the immunopositive cells were characterized by intensely immunostained mitochondria and weakly immunostained cytoplasm and immunonegative chromatin-poor nucleus. Vacuoles of various sizes without the immunoreactivity were also contained in the cells. Thin cellular processes without the immunoreactivity were found to enclose thinly the entire surfaces of the immunostained cells and processes, and they were in continuity with the interstitial partition-like processes which contained nuclei and aggregation of microfibrils at some distance from the cytoplasmic envelopes. The present finding suggests the possibility that the immunostained cells were peripheral neurons enveloped by peripheral glia and that the glia are of mesenchymal origin because of their cytoplasmic continuity to the interstitial partition-like processes. The motor or sensory nature of the neurons remains to be elucidated.

Mediante el uso del anticuerpos DGK para rata se determinó un número considerable de células inmunopositivas en el Opisthorchis viverrini (OV). Las células inmunopositivas aparecían solitarias y se distribuían simétricamente al eje longitudinal de la OV. Algunas estaban ubicadas en las proximidades de los órganos internos como el útero, ovarios, testículos, glándulas vitelinas e intestino. Las células inmunoteñidas extendían sus procesos horizontalmente u oblicuamente al eje longitudinal de la OV. Por microscopía inmunoelectrónica, las células inmunopositivas se caracterizaron por presentar mitocondrias intensamente teñidas, citoplasma con tinción débil e inmunonegatividad en núcleos pobres en cromatina. También se observó en las células, vacuolas de diversos tamaños sin inmunorreactividad. Se encontraron procesos celulares sin inmunorreactividad para cerrar finamente todas las superficies de las células y procesos, y se continuaron con los procesos de partición intersticiales que contenían núcleos y agregación de microfibrillas a cierta distancia de las envolturas citoplásmicas. El presente hallazgo sugiere la posibilidad de que las células inmunoteñidas son neuronas periféricas envueltas por glia periférica y que la glía presenta origen mesenquimal debido a su continuidad citoplasmática con los procesos de partición intersticiales. La naturaleza motora o sensorial de las neuronas aún no se ha dilucidado.

Propuesta de reformas a los prestadores públicos de servicios médicos en Chile: “fortaleciendo la opción pública” / A reform proposal to strengthen public health care

Currently, there is no discussion on the need to improve and strengthen the institutional health care modality of FONASA (MAI), the health care system used by the public services net and by most of the population, despite the widely known and long lasting problems such as waiting lists, hospital debt with suppliers, lack of specialists and increasing services purchase transference to the private sector, etc. In a dichotomous sectorial context, such as the one of health’s social security in Chile (the state on one side and the market on the other), points of view are polarized and stances tend to seek refuge within themselves. As a consequence, to protect the public solution is commonly associated with protecting the “status quo”, creating an environment that is reluctant to change. The author proposes a solution based on three basic core ideas, which, if proven effective, can strengthen each other if combined properly. These are: network financing management, governance of health care services in MAI and investments and human resources in networked self-managed institutions. The proposal of these core ideas was done introducing a reality testing that minimizes the politic complexity of their implementation.

Diagnosis, classification and treatment of gestational trophoblastic neoplasia / Diagnóstico, classificação e tratamento da neoplasia trofoblástica gestacional

Gestational trophoblastic neoplasia (GTN) is the term to describe a set of malignant placental diseases, including invasive mole, choriocarcinoma, placental site trophoblastic tumor and epithelioid trophoblastic tumor. Both invasive mole and choriocarcinoma respond well to chemotherapy, and cure rates are greater than 90%. Since the advent of chemotherapy, low-risk GTN has been treated with a single agent, usually methotrexate or actinomycin D. Cases of high-risk GTN, however, should be treated with multiagent chemotherapy, and the regimen usually selected is EMA-CO, which combines etoposide, methotrexate, actinomycin D, cyclophosphamide and vincristine. This study reviews the literature about GTN to discuss current knowledge about its diagnosis and treatment.

Neoplasia trofoblástica gestacional (NTG) é o termo que descreve o conjunto de anomalias malignas da placenta, incluindo a mola invasora, coriocarcinoma, tumor trofoblástico do sítio placentário e tumor trofoblástico epitelióide. Ambos a mola invasora e o coriocarcinoma respondem bem à quimioterapia, com taxas de cura superiores a 90%. Desde o advento da quimioterapia, NTG de baixo risco tem sido tratada com monoquimioterapia, pelo geral methotrexate ou actinomicina-D. Casos de NTG de alto risco, contudo, devem ser tratados com poliquimioterapia, e o regime usualmente escolhido é o EMA-CO que combina etoposide, methotrexate, actinomicina-D, ciclofosfamida e vincristina. Esse estudo revê a literatura sobre NTG a fim de discutir os conhecimentos atuais sobre seu diagnóstico e tratamento.

Immunoelectron microscopy study of superficial skin nerves in drug-induced acute urticaria / Estudo de microscopia imunoeletrônica dos nervos superficiais da pele na urticária aguda induzida por medicamentos

BACKGROUND: Few studies have evaluated the ultrastructure of the superficial skin nerves in urticaria. OBJECTIVE: The objective of this study was to describe findings in superficial skin nerves in cases of drug-induced acute urticaria. METHODS: Seven patients with drug-induced acute urticaria were included in the study. Skin biopsies were obtained from the urticarial lesion and from the apparently normal skin. The 14 fragments collected were processed for immunogold electron microscopy using single stains for antitryptase and anti-FXIIIa antibodies, as well as double immunogold labeling for both. RESULTS: Some sections showed mast cells in the process of degranulation. Following double immunogold staining, 10 nm (FXIIIa) and 15 nm (Tryptase) gold particles were found together throughout the granules in mast cells, indicating that tryptase and FXIIIa are located inside each one of the granules of these cells. Interestingly, we found strong evidence of the presence of tryptase and factor XIIIa in the superficial skin nerves of these patients, both in cases of urticarial lesions (wheals) and in the apparently normal skin. CONCLUSIONS: Tryptase and FXIIIa are present in the superficial nerves of the skin in drug-induced acute urticaria. This is the first report of tryptase and FXIIIa expression in the superficial skin nerves of patients with urticaria. Tryptase may be participating in neural activation in these patients, while FXIIIa may be present in the nerves to guarantee the functional integrity of structures.

FUNDAMENTOS: Poucos autores têm estudado a ultraestrutura dos nervos superficiais na urticária. OBJETIVO: Descrever os achados nos nervos cutâneos superficiais em casos de urticária aguda induzida por medicamentos. MÉTODOS: Sete pacientes com urticária aguda induzida por medicamentos foram incluídos no estudo. Foram obtidas biopsias da pele da lesão urticariforme e da pele aparentemente normal. Os 14 fragmentos coletados foram processados usando imunomarcação com ouro para anticorpos anti-triptase e anti-FXIIIa separadamente, além da dupla imunomarcação com ambos anticorpos. A seguir as amostras foram submetidas à análise por microscopia imunoeletrônica. RESULTADOS: Alguns cortes demonstraram mastócitos em processo de degranulação. Após a imunomarcação dupla, partículas de ouro de 10 nm (FXIIIa) e partículas de ouro de 15 nm (Triptase) apresentavam-se juntas em grânulos de mastócitos indicando que a triptase e o FXIIIa se localizam dentro de cada um dos grânulos dessas células. Curiosamente, foi encontrada uma forte evidência da presença da triptase e do fator XIIIa nos nervos superficiais dos pacientes avaliados, tanto em lesões urticadas, como na pele aparentemente normal. CONCLUSÕES: A triptase e o FXIIIa estão presentes nos nervos superficiais da pele na urticária aguda medicamentosa. Este é o primeiro relato da expressão de triptase e de FXIIIa nos nervos superficiais na urticária. A triptase poderia estar participando da ativação neural nos pacientes estudados. O FXIIIa poderia estar presente nos nervos, com a finalidade de manter a integridade funcional dessas estruturas.

Correlative microscopy of Purkinje cells

The Purkinje cell and their synaptic contacts have been described using (1) light microsocopy, (2) transmission and scanning electron microscopy, and freeze etching technique, (3) conventional and field emission scanning electron microscopy and cryofracture methods, (4) confocal laser scanning microscopy using intravital stain FM64, and (5) immunocytochemical techniques for Synapsin-I, PSD9-5, GluR1 subunit of AMPA receptors, N-cadherin, and CamKII alpha. The outer surface and inner content of plasma membrane, cell organelles, cytoskeleton, nucleus, dendritic and axonal processes have been exposed and analyzed in a three-dimensional view. The intramembrane morphology, in bi- and three-dimensional views, and immunocytochemical labeling of synaptic contacts with parallel and climbing fibers, basket and stellate cell axons have been characterized. Freeze etching technique, field emission scanning microscopy and cryofracture methods, and GluR1 immunohistochemistry showed the morphology and localization of postsynaptic receptors. Purkinje cell shows N-cadherin and CamKII alpha immunoreactivity. The correlative microscopy approach provides a deeper understanding of structure and function of the Purkinje cell, a new three-dimensional outer and inner vision, a more detailed study of afferent and intrinsic synaptic junctions, and of intracortical circuits.

Toxoplasma gondii: further studies on the subpellicular network

The association of the pellicle with cytoskeletal elements in Toxoplasma gondii allows this parasite to maintain its mechanical integrity and makes possible its gliding motility and cell invasion. The inner membrane complex (IMC) resembles the flattened membrane sacs observed in free-living protozoa and these sacs have been found to associate with cytoskeletal proteins such as articulins. We used immunofluorescence microscopy to characterise the presence and distribution of plateins, a sub-family of articulins, in T. gondii tachyzoites. A dispersed labelling of the whole protozoan body was observed. Electron microscopy of detergent-extracted cells revealed the presence of a network of 10 nm filaments distributed throughout the parasite. These filaments were labelled with anti-platein antibodies. Screening the sequenced T. gondii genome, we obtained the sequence of an IMC predicted protein with 25 percent identity and 42 percent similarity to the platein isoform alpha 1 present in Euplotes aediculatus, but with 42 percent identity and 55 percent similarity to that found in Euglena gracilis, suggesting strong resemblance to articulins.

Efeito do Peróxido de Carbamida a 16 % na mucosa oral de ratos diabéticos e não diabéticos / Effects of 16 % carbamide peroxide in the oral mucosa of diabetic and non diabetic rats

Objetivo: avaliar os efeitos do peróxido de carbamida a 16% aplicado na mucosa oral de ratos diabéticos e não diabéticos. Métodos: foram utilizados trinta ratos albinos sendo 15 diabéticos induzidos por estreptozotocina e 15 normais, machos, adultos jovens, variedade Wistar. Os animais foram dividos em 6 grupos: NDC (não diabético controle), NDIP (não diabético imediato peróxido), ND7DP (não diabético 7 dias peróxido), DC (diabético controle), DIP (diabético imediato peróxido), D7DP (diabético 7 dias peróxido). Os grupos NDC e DC não receberam qualquer tratamento. Nos grupos NDIP e DIP foi aplicado o gel de peróxido de carbamida a 16% por 7dias (2 horas/dia) e os animais foram sacrificados no sétimo dia do experimento. Nos grupos ND7DP e D7DP foi aplicado o gel de peróxido de carbamida a 16% por 7 dias (2 horas dia) e os animais foram sacrificados 7 dias após (reparo). Após o sacrifício dos animais, a região mentual foi retirada cirurgicamente e preparada para estudo ao microscópio de luz com estereologia e imunohistoquímica para α-actina de músculo liso (ACML, detecção de vasos). Resultados: todos os animais tratados com estreptozotocina tornaram-se diabéticos. A determinação da densidade de volume de tecido conjuntivo (Vv[tc]), da densidade de volume de vasos (Vv[vasos]) e da densidade de mastócitos na área-teste (QA[mast]) foram realizadas através do programa Image Pro Plus Version 5.0.5 – 2004 Media Cybernetics Inc. O Vv[tc] se apresentou 23% maior no grupo DC, em relação ao grupo NDC. A Vv[vasos] foi 125% maior no grupo DC em relação ao grupo NDC, e 108% maior no grupo DP7D em relação ao grupo NDP7D, todos com diferença estatística significante. A (QA[mast]) se apresentou maior nos grupos diabéticos, porém sem diferença estatística significante...

Purpose: to evaluate the effects of the application of 16% carbamide peroxide in the oral mucosa of diabetic and non diabetic rats. Methods: we studied 30 young, male and adult Wistar albino rats, 15 diabetics induced by streptozotocin, and 15 non diabetics. The animals were divided in 6 groups: NDC (non diabetic control), NDIP (non diabetic immediately peroxide), ND7DP (non diabetic 7 days peroxide), DC (diabetic control), DIP (diabetic immediately peroxide), D7DP (diabetic 7 days peroxide). Groups NDC and DC had no treatment. In groups NDIP e DIP were applied the 16% carbamide peroxide gel (for 2 hours a day) and the animals were sacrificed in the seventh day of the experimentation. In groups ND7DP e D7DP were applied the 16% carbamide peroxide gel (for 2 hours a day) and the animals were sacrificed 7 days after (repair). After the sacrifice, the material from the jugal mucosa of the animals was removed for study with light microscope, stereology and immunohistochemistry for smooth muscle alfa-actin (SMAA, vessel detection). Results: all the specimens treated with streptozotocin became diabetics. For determination of the density of connective tissue (Vv[tc]), of the density of vessel volume (Vv[vasos]) and the density of mast cells (QA[mast]) was used the program Image Pro Plus Version 5.0.5 – 2004 Media Cybernetics Inc. The Vv[tc] were 23% higher in group DC than the group NDC. The Vv[vasos] was 125% higher in group DC than the group NDC, and 108% higher in group DP7D than the group NDP7D, all data with statistically significant difference. The (QA[mast]) was higher in diabetic groups but without statistically significant difference...

Estudos ultra-estruturais e imunocitoquímicos da infecção pelo vírus da hepatite C em macacos rhesus / Ultrastructural and immunocitochemistry studies of the infection for the virus of hepatites C in rhesus monkey

Alterações morfológicas do tecido hepático de macaco rhesus (Macaca mulatta) infectado experimentalmente com o vírus da hepatite C (HCV) foram analisadas utilizando técnicas em microscopia eletrônica. A localização do RNA e de proteínas específicas do HCV nos hepatócitos foi demonstrada por meio das técnicas de hibridização in situ e imuno-microscopia eletrônica. As modificações no tecido hepático mais marcantes foram observadas entre a quinta e sétima semana pós-inoculação. Numerosos hepatócitos apresentaram, progressivamente, citoplasma rarefeito e vacuolizado, culminando com a citólise na sétima semana. Na oitava semana alguns hepatócitos já exibiam sinais regenerativos, indicados pelo aumento de células binucleadas e recuperação da individualização celular. A proliferação reticular de membranas e vésículas derivadas do retículo endoplasmático rugoso (REr) observada no citoplasma dos hepatócitos do macaco rhesus infectado evidenciou semelhança com a do HCV encontrado em chimpanzé, em culturas celulares que abrigam os réplicons do HCV. Para a localização das proteínas virais, cortes de amostras de fígado incluídas em LR-gold, foram incubados com anticorpos monoclonais contra a proteína do nucleocapsídeo e a proteína NS5A e com anticorpo policlonal contra as proteínas E1, E2, NS3 e NS4, seguido de incubação com o conjugado proteína A-ouro coloidal. A proteína do nucleocapsídeo foi localizada sobre membranas do REr que envolvem as mitocôndrias e diretamente associada à membrana externa da mitocôndria. As proteínas não-estruturais foram localizadas em vesículas e cisternas dilatadas do REr. Na localização do RNA viral pela técnica de hibridização in situ foram utilizadas sondas de fita negativa complementar e fita positiva, utilizando para ambas, oligonucleotídeos específicos. As observações ultra-estruturais evidenciaram marcações específicas sobre membranas do REr e sobre vesículas dentro das cisternas do REr que se formam durante a infecção por HCV...

A resposta inflamatória na urticária aguda associada a medicamentos: avaliação imunoistoquímica e imunoeletrônica da unidade microvascular da derme / The inflammatory response in acute drug-induced urticaria: immunohistochemistry and ultrastructure of dermal microvascular unit

Os objetivos deste estudo foram orientados no sentido de descreverem-se os tipos de degranulação na urticária, e se analisarem as interações entre dendrócitos da derme e mastócitos. Sete doentes com urticária aguda associada a medicamentos foram incluídos. Foram biopsiadas lesões urticadas e a pele normal, sendo uma das partes processada pela coloração de hematoxilina-eosina, pela coloração de Azul de Toluidina e imunoistoquímica com anti-CD34, antifator XIIIa e antitriptase e a outra para a microscopia imunoeletrônica. Este estudo é inédito pela demonstração da expressão do FXIIIa nos grânulos intracitoplasmáticos e nos grânulos extruídos dos mastócitos, dispersos no extracelular, nos doentes com urticária. Outro fato inédito foi a fagocitose dos grânulos extruídos dos mastócitos pelos dendrócitos.

Background: The knowledge about the cell types involved in urticaria is an essential element for understanding the pathophysiology of this disease. Few authors have been attempting on interactions among mast cells and dermal dendrocytes in urticaria...

Expression of the rice hoja blanca virus (RHBV) non-structural protein 3 (NS3) in Escherichia coli and its in situ localization in RHBV-infected rice tissues

The non-structural NS3 protein gene from the rice hoja blanca virus (RHBV) was fused to the glutathione-S-transferase carboxilic end and expressed in Escherichia coli strain JM83. Large quantities of fusion protein were produced in insoluble form. The fusion protein was fractionated in SDS-PAGE and purified by electroelution, polyclonal antibodies were raised in rabbit and the antiserum was absorbed with bacterial crude extract. A band of similar size as that of NS3 protein was observed in Western blots using extracts from RHBV-infected rice plants. Immunoelectron microscopy with colloidal gold-labeled antibodies against NS3 protein and the viral nucleocapsid protein revealed in situ accumulation of NS3 protein in the cytoplasm but not in the viral inclusion bodies, vacuoles or chloroplasts of RHBV-infected plants, following the same pattern of distribution as the RHBV nucleocapsid protein.

Análise do padrão de expressão do produto de PKHD1, o gene mutado na doença renal policística autossômica recessiva / Analysis of the expression pattern of the PKHD1 gene product, mutated in autosomal recessive polycistic kidney disease

O gene PKHD1, mutado na doença renal policística autossômica recessiva, apresenta um padrão de splicing complexo associado a múltiplos transcritos alternativos. Neste trabalho estudamos o perfil de expressão de seu produto, poliductina. Análises por western blot revelaram produtos putativos de membrana de >440 kDa e aproximadamente 230 kDa, e de aproximadamente 140 kDa em frações solúveis de rim, fígado e pâncreas. Estudos imunoistoquímicos mostraram marcação em ductos coletores renais e porção ascendente espessa da alça de Henle, em epitélios ductais biliar e pancreático e, no período embrionário, em broto ureteral, ductos biliar e pancreático e glândula salivar. /PKHD1, the gene mutated in autosomal recessive polycystic kidney disease, presents a complex splicing pattern, associated with multiple alternative transcripts. In this work we have studied the expression profile of its product, polyductin. Western blot analysis revealed putative membrane products of >440 kDa and 230 kDa, and of about 140 kDa in soluble fractions in kidney, liver and pancreas. Immunohistochemistry studies showed staining in renal collecting duct and thick ascending limb of Henle, in biliary and pancreatic ductal epithelia and, in the embryonic period, in ureteric bud, biliary and pancreatic ducts and salivary gland...

Antigen incorporation on Cryptosporidium parvum oocyst walls

Cryptosporidium parvum oocysts are the infective stages responsible for transmission and survival of the organism in the environment. In the present work we show that the oocyst wall, far from being a static structure, is able to incorporate antigens by a mechanism involving vesicle fusion with the wall, and the incorporation of the antigen to the outer oocyst wall. Using immunoelectron microscopy we show that the antigen recognized by a monoclonal antibody used for diagnosis of cryptosporidiosis (Merifluor®, Meridian Diagnostic Inc.) could be found associated with vesicles in the space between the sporozoites and the oocysts wall, and incorporated to the outer oocyst wall by an unknown mechanism

Serotonergic ependymal fibres in rat and monkey: light and electron microscopic immunocytochemical study

The distribution of serotonin (5HT) immunoreactive fibres in the ependymal epithelium of aqueductus cerebri in adult rat and monkey was studied by means of immunocytochemical methods using specific antibodies against 5HT. Light microscopic examination of immunostained coronal sections of mesencephalon showed, in tryptophan and pargylin pretreated animals, abundant serotonergic fibres distributed along the ependymal cells of the aqueduct, forming supraependymal and subependymal plexi. Serotonin fibres lying either supraependymally or ending freely in the aqueduct lumen contributed to the formation of a rich 5HT containing network on the ependymal surface. Electron microscope images showed dense 5HT-immunoreactive (5HT-IR) profiles with ultrastructural characteristics of axon terminals ending on the ependymal cells. Dense diaminobenzidine (DAB) deposits were found in the axoplasm, on outer mitochondrial surface and in vesicles. No synaptic contacts were observed between 5HT-IR terminals and ependymal cells. Unstained microvilli and cilia were also observed in the aqueduct lumen. Serotonin immunoreactivity disappeared from ependymal fibres in animals treated with parachlorophenylalanine, an inhibitor of 5HT synthesis. 5HT containing fibres described in this paper may be the source of 5HT and its metabolite 5-hydroxyindoleacetic acid (5HIAA) found in cerebrospinal fluid (CSF) and of clinical relevance in some psychiatric conditions such as depression, suicidal attempts, etc

Distribution of GABA in the golden hamster retina: An immunocytochemical study

Distribution of the inhibitory neurotransmitter GABA was studied in the golden hamster retina using immunocytochemistry at cellular and subcellular levels. GABA-immunoreactivity was observed in somata of amacrine, displaced amacrine and horizontal cells. GABA immunoreactive fibers were abundant in the inner plexiform layer. Ultrastructural analysis exhibited dense GABA-immunoreactive deposits in amacrine cell somata, processes and terminals. Immunolabelling was also observed in the cytoplasm of horizontal or interplexiform cells and displaced amacrine cells. In every case DAB deposits were observed in the cytosolic compartment, attached to the inner surface of cell membranes and to outer mitochondrial membranes. Immunolabeled terminals predominated in the inner plexiform layer and immunoprecipitates were also observed attached to the outer face of vesicle membranes as well as completely filling synaptic vesicles. Both clear and dense core vesicles were observed. The present results are similar to those obtained in other mammalian species showing GABA immunoreactivity in amacrine, displaced amacrine and horizontal cells

Ultrastructural aspects of the human T-lymphotropic virus type I (HTLV-I) in Brazil

Nowadays, Brazil may be considered an endemic area for HTLV-I infection. Several studies showed a high incidence of HTLV-I infection among the general population and different groups such as blood donors, hemophiliacs, hematological and neurological patients. Cases of adult T-cell leukemia/lymphoma as well as tropical spastic paraparesis/HTLV-I associated myelopathy have been already described. Therefore the use of different technical approaches, to characterize Brazilian HTLV strains has become important. HTLV particles were characterized and recognized by the use of transmission electron microscopy in lymphocyte cocultures. The ultra-structural analysis revealed typical virus particles close to the lymphocyte membrane. The immunoelectron microscopy allowed the identification of the virus as HTLV, a type C oncovirus, group HTLV-BLV. The ethanol phosphotungstic acid technique showed structures similar to the virus budding process and the routine preparations have contributed to the analysis of the first virus-cell interaction events. Structures related to the endocytic route HTLV entrance are also pointed out.

Nephrotoxicity of human Bence Jones protein in rats: proteinuria and enzymuria profile

The effect of intravenous administration of 80 mg purified human Bence Jones protein twice weekly for 5 weeks was investigated in male Wistar rats (N = 7; 2 months old). A state of immunological tolerance was demonstrated by the absence of a B-cell response (plaque-forming cells and hemagglutination titers) and by the absence of detectable antigen or antibody deposition in glomeruli, as indicated by light and electron microscopy. No rise in blood urea level was detected (33.9 +/- 4.3 vs 32.8 +/- 1.3 mg per cent ). There was an increase in proteinuria (5.3 +/- 0.9 vs 32.8 +/- 4.0 mg/day), mainly due to Bence Jones protein excretion (0 vs 29.2 +/- 5.2 mg/day), with a slight but significant increase in albuminuria (0.2 +/- 0.1 vs 1.0 +/- 0.2 mg/day). There was a significant increase of lysosomal N-acetyl-beta-D-glucosaminidase in the urine (6.1 +/- 1.3 vs 72.7 +/- 18.8 mU/mg in creatinine). Lysosomal accumulation of Bence Jones protein in proximal tubular cells was evidenced by immunoelectronmicroscopy with protein A-gold. These results clearly showed proximal tubular dysfunction induced by chronic Bence Jones protein administration, without interference of autologous immune response as demonstrated by immunological state of tolerance

Prevalence of HTLV-I antibody among two distinct ethnic groups inhabiting the Amazon region of Brazil

HTLV-I seroprevalences of 3.63% (02/55), 12.19% (10/82) and 13.88% (10/72) were demonstrated among Tiryio, Mekranoiti and Xicrin Amazonian Indians, respectively, by the Western blotting enzyme assay (WBEI). By indirect immunoelectron microscopy (IIEM), 2 Tiriyo, 9 Mekranoiti and 6 Xicrin Amerindians were reactive. Of 44 serum samples from Japanese immigrants, none reacted by any of the techniques before mentioned. One, 8 and 6 serum samples from Tiryio, Mekranoiti and Xicrin Indians, respectively, were both WBEI and IIEM positive. Our results strongly suggest that HTLV-I and/or an HTLV-I antigenic variant circulate (s) among populations living in the Amazon region of Brazil