Antioxidant effect of endothelin-1 receptor antagonist protects the rat kidney against chronic injury induced by hypertension and hyperglycemia / Efeito antioxidante de antagonista dos receptores de endotelina-1 protege ratos contra lesão renal crônica induzida por hipertensão e hiperglicemia

ABSTRACT Hypertension and Diabetes mellitus are the two main causes of chronic kidney disease that culminate in the final stage of kidney disease. Since these two risk factors are common and can overlap, new approaches to prevent or treat them are needed. Macitentan (MAC) is a new non-selective antagonist of the endothelin-1 (ET-1) receptor. This study aimed to evaluate the effect of chronic blockade of ET-1 receptor with MAC on the alteration of renal function observed in hypertensive and hyperglycemic animals. Genetically hypertensive rats were divided into control hypertensive (HT-CTL) group, hypertensive and hyperglycemic (HT+DIAB) group, and hypertensive and hyperglycemic group that received 25 mg/kg macitentan (HT-DIAB+MAC25) via gavage for 60 days. Kidney function and parameters associated with oxidative and nitrosative stress were evaluated. Immunohistochemistry for neutrophil gelatinase-associated lipocalin (NGAL), ET-1, and catalase in the renal cortex was performed. The HT+DIAB group showed a decrease in kidney function and an increase in NGAL expression in the renal cortex, as well as an increase in oxidative stress. MAC treatment was associated with attenuated ET-1 and NGAL production and increases in antioxidant defense (catalase expression) and nitric oxide production. In addition, MAC prevented an increase in oxidant injury (as measured by urinary hydroperoxide and lipid peroxidation), thus improving renal function. Our results suggest that the antioxidant effect of the ET-1 receptor antagonist MAC is involved in the improvement of kidney function observed in hypertensive and hyperglycemic rats.

RESUMO Hipertensão e Diabetes Mellitus figuram como as duas principais causas de doença renal crônica que culmina em doença renal terminal. Uma vez que os dois fatores de risco são comuns e podem se sobrepor, novas abordagens preventivas e terapêuticas se fazem necessárias. O macitentan (MAC) é um novo antagonista não-seletivo dos receptores da endotelina-1 (ET-1). O presente estudo teve como objetivo avaliar os efeitos do bloqueio crônico dos receptores da ET-1 com MAC sobre a alteração da função renal em animais hipertensos e hiperglicêmicos. Ratos geneticamente hipertensos foram divididos em grupos com animais hipertensos de controle (HT-CTL), hipertensos e hiperglicêmicos (HT+DIAB) e hipertensos e hiperglicêmicos tratados com 25 mg/kg de macitentan (HT-DIAB+MAC25) via gavagem por 60 dias. Foram avaliados função renal e parâmetros associados ao estresse oxidativo e nitrosativo. Exames de imunoistoquímica foram realizados para lipocalina associada à gelatinase neutrofílica (NGAL), ET-1 e catalase no córtex renal. O grupo HT+DIAB exibiu diminuição da função renal e aumento na expressão de NGAL no córtex renal, bem como estresse oxidativo aumentado. O tratamento com MAC foi associado a atenuação da produção de ET-1 e NGAL e maior ativação das defesas antioxidantes (expressão de catalase) e elevação da produção de óxido nítrico. Além disso, o MAC evitou exacerbação da lesão oxidante (medida por hidroperóxidos urinários e peroxidação lipídica), melhorando assim a função renal. Nossos resultados sugerem que o efeito antioxidante do antagonista dos receptores da ET-1 MAC esteja imbricado no aprimoramento da função renal observada em ratos hipertensos e hiperglicêmicos.

Antioxidant effect of endothelin-1 receptor antagonist protects the rat kidney against chronic injury induced by hypertension and hyperglycemia.

Hypertension and Diabetes mellitus are the two main causes of chronic kidney disease that culminate in the final stage of kidney disease. Since these two risk factors are common and can overlap, new approaches to prevent or treat them are needed. Macitentan (MAC) is a new non-selective antagonist of the endothelin-1 (ET-1) receptor. This study aimed to evaluate the effect of chronic blockade of ET-1 receptor with MAC on the alteration of renal function observed in hypertensive and hyperglycemic animals. Genetically hypertensive rats were divided into control hypertensive (HT-CTL) group, hypertensive and hyperglycemic (HT+DIAB) group, and hypertensive and hyperglycemic group that received 25 mg/kg macitentan (HT-DIAB+MAC25) via gavage for 60 days. Kidney function and parameters associated with oxidative and nitrosative stress were evaluated. Immunohistochemistry for neutrophil gelatinase-associated lipocalin (NGAL), ET-1, and catalase in the renal cortex was performed. The HT+DIAB group showed a decrease in kidney function and an increase in NGAL expression in the renal cortex, as well as an increase in oxidative stress. MAC treatment was associated with attenuated ET-1 and NGAL production and increases in antioxidant defense (catalase expression) and nitric oxide production. In addition, MAC prevented an increase in oxidant injury (as measured by urinary hydroperoxide and lipid peroxidation), thus improving renal function. Our results suggest that the antioxidant effect of the ET-1 receptor antagonist MAC is involved in the improvement of kidney function observed in hypertensive and hyperglycemic rats.

The influence of chromosome 4 on metabolism and spatial memory in SHR and SLA16 rat strains.

The Spontaneously Hypertensive Rat (SHR) has been proposed as a good model to study the pathways related to neurodegenerative diseases and glucose intolerance. Our research group developed the SLA16 (SHR.LEW-Anxrr16) congenic strain, which is genetically identical to the SHR strain, except for a locus on chromosome 4 (DGR). We applied in silico analysis on DGR to evaluate the association of their genes with neurobiological and metabolic pathways. After, we characterized cholesterol, triglycerides, metabolism of glucose and the behavioral performance of young (2 months old) and adult (8 months old) SHR and SLA16 rats in the open field, object location and water maze tasks. Finally, naïve young rats were repeatedly treated with metformin (200 mg/kg; v.o.) and evaluated in the same tests. Bioinformatics analysis showed that DGR presents genes related to glucose metabolism, oxidative damage and neurodegenerative diseases. Young SLA16 presented higher cholesterol, triglycerides, glucose and locomotion in the open field than SHR rats. In adulthood, SLA16 rats presented high triglycerides and locomotion in the open field and impairment on spatial learning and memory. Finally, the treatment with metformin decreased the glucose tolerance curve and also improved long-term memory in SLA16 rats. These results indicate that DGR presents genes associated with metabolic pathways and neurobiological processes that may produce alterations in glucose metabolism and spatial learning/memory. Therefore, we suggest that SHR and SLA16 strains could be important for the study of genes and subsequent mechanisms that produce metabolic glucose alterations and age-related cognitive deficits.

Experimental exposure to 3-monochloropropane-1,2-diol from the pre-puberty causes damage in sperm production and motility in adulthood / Exposição experimental ao 3-monocloropropano-1,2-diol, a partir da pré-puberdade, causaalteração na produção e motilidade espermática na idade adulta

3-Monochloropropane-1,2-diol (3-MCPD) is a food contaminant that can be formed during the thermic processing of various foodstuffs. Studies of reproductive toxicology of 3-MCPD are mainly concentrated in the evaluation of possible insults caused by exposure of adult animals. However, the prepuberty might be a period of different susceptibility to chemicals. The aim of this study was to evaluate the effects on reproductive endpoints of the 3-MCPD-exposure prepubertal male rats. Wistar male rats were assigned to 4 groups: control and exposed to 2.5; 5 or 10 mg kg-1 day-1 of 3-MCPD for 30 days by gavage. Testis and epididymis were used for sperm counts and histology analysis. Sertoli cell number and dynamic of the spermatogenesis were evaluated. Sperm were collected from the vas deferens for evaluation of the sperm motility and morphology. Number of sperm with progressive movement, number of Sertoli cells and germ cells and relative daily sperm production were decreased in the groups exposed to 5 and 10 mg kg-1 day-1 of 3-MCPD. Sperm morphology, testicular and epididymal histology were comparable among groups. Results show that 3-MCPD-exposure of rats from prepuberty might cause alterations in spermatogenesis and sperm maturation, similarly to exposure in adulthood.(AU)

3-monocloropropano-1,2-diol (3-MCPD) é um contaminante alimentar formado durante o processamento térmico de vários produtos. Estudos de toxicologia reprodutiva do 3-MCPD estão concentrados principalmente na avaliação de danos causados pela exposição de animais adultos. No entanto, a peri-puberdade pode ser um período de susceptibilidade diferente a produtos químicos. O objetivo deste estudo foi avaliar os efeitos reprodutivos da exposição de ratos machos pré-púberes ao 3- MCPD. Ratos Wistar machos foram divididos em quatro grupos: controle e expostos a 2,5; 5 ou 10 mg kg-1 dia-1 de 3-MCPD durante 30 dias, por gavagem. Os testículos e epidídimos foram usados para contagem espermática e análise histológica. Foram avaliados o número de células de Sertoli e a dinâmica da espermatogênese. Espermatozoides foram coletados a partir dos ductos deferentes para avaliação da motilidade e morfologia. O número de espermatozoides com movimento progressivo, o número de células de Sertoli e de células germinativas e a produção espermática foram reduzidos nos grupos expostos a 5 e 10 mg kg-1 dia-1 de 3-MCPD. A morfologia espermática e histologia testicular e epididimária foram semelhantes entre os grupos. Os resultados mostram que a exposição de ratos ao 3-MCPD, a partir da prépuberdade, pode causar alterações na espermatogênese e na maturação espermática, de forma semelhante à exposição na idade adulta.(AU)

Roles of the ERK1/2 and PI3K/PKB signaling pathways in regulating the expression of extracellular matrix genes in rat pulmonary artery smooth muscle cells

Purpose: To investigate the mechanisms by which PD98059 and LY294002 interfere with the abnormal deposition of extracellular matrix regulated by connective tissue growth factor (CTGF) of rat pulmonary artery smooth muscle cells (PASMCs). Methods: Rat PASMCs were cultured and separated into a control group. Real-time fluorescence quantitative PCR was performed to detect the expression of collagen III and fibronectin mRNA. Immunohistochemistry and western blot analyses were performed to detect the expression of collagen III protein. Results: The expression of collagen III and fibronectin mRNA was greater in PASMCs stimulated with CTGF for 48 h, than in the control group. After 72h of stimulation, the expression of collagen III protein in the PASMCs was greater than in the control. The equivalent gene and protein expression of the CPL group were much more significant. Conclusions: CTGF can stimulate the gene expression of collagen III and fibronectin in PASMCs, which may be one of the factors that promote pulmonary vascular remodeling (PVR) under the conditions of pulmonary arterial hypertension (PAH). PD98059 and LY294002 can inhibit the ERK1/2 and PI3K/PKB signaling pathways, respectively, thus interfering with the biological effects of CTGF. This may be a new way to reduce PAH-PVR.(AU)

Maternal plasma levels of vascular endothelial growth factor and interleukin-6 in the term-pregnant and non-pregnant rats

Female reproductive functions are associated with changes in the level of systemic cytokines which play pivotal roles during ovulation, development of corpus luteum, embryo implantation, and labor. Although many studies reported elevated level of VEGF during early pregnancy and IL-6 at the onset of labors, there is still incomplete elucidation of the level of these systemic cytokines in the very-late pregnancy or in the non-pregnant states. The purpose of this study was to determine the maternal plasma level of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) in the term-pregnant and non-pregnant rats to see if their levels are changed at the very end of pregnancy. Blood samples were collected from twelve term-pregnant rats and compared by aged-matched seven non-pregnant rats. Plasma was separated carefully from all samples and the concentrations of VEGF and IL-6 were determined by ELISA techniques. The plasma concentration of VEGF was significantly higher in the non-pregnant rats (0.237 pg/ ml) than in the term-pregnant (0.2 pg/ ml; P < 0.001). The plasma concentration of IL-6 was significantly higher in the term-pregnant rats (50.12 pg/ ml) than in the non-pregnant ones (40.19 pg/ ml; P < 0.05). These findings suggest that the level of VEGF is decreased few days before labor possibly to decrease the ovarian blood flow and to cause luteolysis while the level of IL-6 is increased at the end of pregnancy possibly to cause a local inflammation. Together, these changes are necessary to stimulate the onset of labor.(AU)

Maternal plasma levels of vascular endothelial growth factor and interleukin-6 in the term-pregnant and non-pregnant rats

Female reproductive functions are associated with changes in the level of systemic cytokines which play pivotal roles during ovulation, development of corpus luteum, embryo implantation, and labor. Although many studies reported elevated level of VEGF during early pregnancy and IL-6 at the onset of labors, there is still incomplete elucidation of the level of these systemic cytokines in the very-late pregnancy or in the non-pregnant states. The purpose of this study was to determine the maternal plasma level of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) in the term-pregnant and non-pregnant rats to see if their levels are changed at the very end of pregnancy. Blood samples were collected from twelve term-pregnant rats and compared by aged-matched seven non-pregnant rats. Plasma was separated carefully from all samples and the concentrations of VEGF and IL-6 were determined by ELISA techniques. The plasma concentration of VEGF was significantly higher in the non-pregnant rats (0.237 pg/ ml) than in the term-pregnant (0.2 pg/ ml; P < 0.001). The plasma concentration of IL-6 was significantly higher in the term-pregnant rats (50.12 pg/ ml) than in the non-pregnant ones (40.19 pg/ ml; P < 0.05). These findings suggest that the level of VEGF is decreased few days before labor possibly to decrease the ovarian blood flow and to cause luteolysis while the level of IL-6 is increased at the end of pregnancy possibly to cause a local inflammation. Together, these changes are necessary to stimulate the onset of labor.

Temporal and spatial expression of Muc2 and Muc5ac mucins during rat respiratory and digestive tracts development.

Secreted mucins constitute a crucial part of the gel that protects respiratory and digestive epithelia, being MUC2/Muc2 the predominant gel-forming mucin of the intestine while MUC5AC/Muc5ac is one of the gel-forming mucins most expressed at the airways. In this study, we have analyzed Muc2 and Muc5ac during rat development by using immunohistochemistry, Western blotting and RT-PCR. We demonstrated that rat Muc2 was expressed in fetal intestinal goblet cells of surface epithelium of villi and developing Lieberkühn crypts. In neonates and adults, Muc2 was expressed at luminal goblet cells of small and large intestine and at gastric mucous and glandular cells. Muc5ac protein was observed in embryonic gastric and lung samples; expression increased during development and postnatal and adult life. After birth, a low reaction was detected at the tracheal surface epithelium and glands, which increased in adults.

Multiple Paternity in the Norway Rat, Rattus norvegicus, from Urban Slums in Salvador, Brazil.

The Norway rat, Rattus norvegicus, is one of the most important pest species globally and the main reservoir of leptospires causing human leptospirosis in the urban slums of tropical regions. Rodent control is a frequent strategy in those settings to prevent the disease but rapid growth from residual populations and immigration limit the long-term effectiveness of interventions. To characterize the breeding ecology of R. norvegicus and provide needed information for the level of genetic mixing, which can help identify inter-connected eradication units, we estimated the occurrence of multiple paternity, distances between mothers and sires, and inbreeding in rats from urban slum habitat in Salvador, Brazil. We genotyped 9 pregnant females, their 66 offspring, and 371 males at 16 microsatellite loci. Multiple paternity was observed in 22% (2/9) of the study litters. Of the 12 sires that contributed to the 9 litters, we identified 5 (42%) of those sires among our genotyped males. Related males were captured in close proximity to pregnant females (the mean inter-parent trapping distance per litter was 70 m, ±58 m SD). Levels of relatedness between mother-sire pairs were higher than expected and significantly higher than relatedness between all females and non-sire males. Our findings indicate multiple paternity is common, inbreeding is apparent, and that mother-sire dyads occur in close proximity within the study area. This information is relevant to improve the spatial definition of the eradication units that may enhance the effectiveness of rodent management programs aimed at preventing human leptospirosis. High levels of inbreeding may also be a sign that eradication efforts are successful.

DHEA and non-alcoholic fat liver disease: increased gene expression of peroxisome proliferation-activated receptor (PPAR) and fatty acid synthase (FAS) / DHEA e doença gordurosa hepática não-alcoólica: aumento na expressão gênica do peroxisome proliferation-activated receptor (PPAR) e ácido graxo sintase (FAS)

Dehydroespiandrosterone (DHEA) is associated with improvements in chronic degenerative diseases, including obesity, insulin resistance, and cardiovascular diseases. Nevertheless, it is observed an increase in its concentration in individuals with liver lipid infiltration, but it is not precise if this condition emerges as a cause or a consequence. In this way, we aimed to identify gene expression alterations in lipid and glucose liver metabolism markers, as well as oxidative stress markers. For this purpose, male Wistar rats, 12-14 months old were treated with subcutaneous injections of DHEA (only dose of 10 mg kg-1); and after 7 days, hepatic gene expression by PCR real time were performed for the following genes:  G6Pase, PEPCK, FAS, PPAR, malic enzyme, ChREBP, LXR, catalase, GPx, iNOS, NADPH oxidase subunits and PCNA. We observed a tendency of reduction in G6Pase gene expression in treated group (p = 0.08). In addition, it was identified an increase in liver PPAR and FAS gene expressions, two markers of increased activity of lipogenic pathway. We also observed an increase in iNOS gene expression, a known inductor of systemic and hepatic insulin resistance. In conclusion, our data indicates that the treatment with DHEA can be associated with the development of liver lipid infiltration and hepatic insulin resistance.(AU)

A deidroepiandrosterona (DHEA) encontra-se associada a melhorias em quadros de obesidade, resistência à insulina e doenças cardiovasculares. Porém, observa-se um aumento na sua concentração em indivíduos portadores de infiltração lipídica hepática, mas sem saber precisar se o mesmo surge como causa ou consequência. Assim, objetivamos identificar alterações na expressão gênica hepática de marcadores relacionados ao metabolismo lipídico e glicídico e de estresse oxidativo. Para tanto, ratos machos com 12-14 meses de idade foram tratados com injeção subcutânea de DHEA (dose única 10 mg kg-1), e após 7 dias foram feitas análises da expressão gênica hepática por PCR em tempo real das seguintes proteínas: G6Pase, PEPCK, FAS, PPAR, enzima málica, ChREBP, LXR, catalase, GPx, iNOS, subunidades da NADPHoxidase e PCNA. Observamos uma tendência à redução da expressão gênica da G6Pase no grupo tratado (p = 0,08). Também identificamos um aumento na expressão gênica hepática do PPAR e FAS, dois indicadores de aumento da atividade das vias de lipogênese. Observamos um aumento na expressão gênica da iNOS, um conhecido agente indutor de resistência à insulina sistêmica e hepática. Em conclusão, nossos dados indicam que o tratamento com DHEA pode estar associado com o desenvolvimento de um quadro de infiltração lipídica hepática e resistência à insulina hepática.(AU)

Urban population genetics of slum-dwelling rats (Rattus norvegicus) in Salvador, Brazil.

Throughout the developing world, urban centres with sprawling slum settlements are rapidly expanding and invading previously forested ecosystems. Slum communities are characterized by untended refuse, open sewers and overgrown vegetation, which promote rodent infestation. Norway rats (Rattus norvegicus) are reservoirs for epidemic transmission of many zoonotic pathogens of public health importance. Understanding the population ecology of R. norvegicus is essential to formulate effective rodent control strategies, as this knowledge aids estimation of the temporal stability and spatial connectivity of populations. We screened for genetic variation, characterized the population genetic structure and evaluated the extent and patterns of gene flow in the urban landscape using 17 microsatellite loci in 146 rats from nine sites in the city of Salvador, Brazil. These sites were divided between three neighbourhoods within the city spaced an average of 2.7 km apart. Surprisingly, we detected very little relatedness among animals trapped at the same site and found high levels of genetic diversity, as well as structuring across small geographical distances. Most F(ST) comparisons among sites were statistically significant, including sites <400 m apart. Bayesian analyses grouped the samples in three genetic clusters, each associated with distinct sampling sites from different neighbourhoods or valleys within neighbourhoods. These data indicate the existence of complex genetic structure in R. norvegicus in Salvador, linked to the heterogeneous urban landscape. Future rodent control measures need to take into account the spatial and temporal linkage of rat populations in Salvador, as revealed by genetic data, to develop informed eradication strategies.

Genotype and expression of the enhanced gree fluorescent protein in LEW-Tg (EGFP) F455.5/Rrrc rats / Genótipo e expressão da proteína fluorescente verde melhorada em ratos da linhagem LEW-Tg (EGFP) F455.5/Rrrc

The Green fluorescent protein (GFP) was first described after being extracted from Aequorea victoria in 1987; Since then, GFP and its derivatives have been widely used in several experiments as cell and protein marker. In the present study it was verified the genotype of the offspring from crosses between heterozygote Lewis LEW-Tg (EGFP) F455.5/Rrrc rats and analyzed the expression of the enhanced green fluorescent protein (EGFP) in different cell types and genotypes. The genotype of the offspring was assessed by PCR and analysis of EGFP expression in different cells and genotypes, including mesenchymal stem cells (MSC) derived from adipose tissue and calvarial osteoblast cells. Expression of EGFP was verified by flow cytometry, fluorescence microscopy, and immunostaining. Through these methods, it was identified the genotypes of the offspring and determined the levels of expression of EGFP in two cell types. A difference in expression between the (EGFP +/+) and (EGFP +/-) genotypes was also observed in addition to the presence of autofluorescence. Further studies on the natural fluorescence of cells with the (EGFP +/-) genotype and that induced by presence of the EGFP are necessary.(AU)

A proteína fluorescente verde (GFP) foi descrita pela primeira vez após ter sido extraída de Aequorea victoria em 1987. Desde então, a GFP e seus derivados têm sido amplamente utilizados em várias experiências como marcador celular e de proteínas. O objetivo do presente estudo foi o de verificar o genótipo dos descendentes de cruzamentos entre ratos Lewis LEW-Tg (EGFP) F455.5/Rrrc heterozigotos e de analisar a expressão da proteína fluorescente verde melhorada (EGFP) em diferentes tipos celulares e genótipos. O genótipo da descendência foi avaliado por PCR e pela análise da expressão da EGFP em diferentes células e genótipos, incluindo-se as células-tronco mesenquimais (MSC) derivadas de tecido adiposo e de osteoblastos de calvária. A expressão da EGFP foi verificada por citometria de fluxo, microscopia de fluorescência e imunocoloração. Foram, identificados os genótipos da descendência e determinados os níveis de expressão de EGFP em dois tipos de células. Foi também constatada uma diferença de expressão entre os genótipos (EGFP +/+) e (EGFP +/-) além da presença de autofluorescência. Mais estudos são necessários para esclarecer a fluorescência natural de células com o genótipo (EGFP +/-) e aquela induzida pela presença da EGFP.(AU)

Genotype and expression of the enhanced gree fluorescent protein in LEW-Tg (EGFP) F455.5/Rrrc rats / Genótipo e expressão da proteína fluorescente verde melhorada em ratos da linhagem LEW-Tg (EGFP) F455.5/Rrrc

The Green fluorescent protein (GFP) was first described after being extracted from Aequorea victoria in 1987; Since then, GFP and its derivatives have been widely used in several experiments as cell and protein marker. In the present study it was verified the genotype of the offspring from crosses between heterozygote Lewis LEW-Tg (EGFP) F455.5/Rrrc rats and analyzed the expression of the enhanced green fluorescent protein (EGFP) in different cell types and genotypes. The genotype of the offspring was assessed by PCR and analysis of EGFP expression in different cells and genotypes, including mesenchymal stem cells (MSC) derived from adipose tissue and calvarial osteoblast cells. Expression of EGFP was verified by flow cytometry, fluorescence microscopy, and immunostaining. Through these methods, it was identified the genotypes of the offspring and determined the levels of expression of EGFP in two cell types. A difference in expression between the (EGFP +/+) and (EGFP +/-) genotypes was also observed in addition to the presence of autofluorescence. Further studies on the natural fluorescence of cells with the (EGFP +/-) genotype and that induced by presence of the EGFP are necessary.

A proteína fluorescente verde (GFP) foi descrita pela primeira vez após ter sido extraída de Aequorea victoria em 1987. Desde então, a GFP e seus derivados têm sido amplamente utilizados em várias experiências como marcador celular e de proteínas. O objetivo do presente estudo foi o de verificar o genótipo dos descendentes de cruzamentos entre ratos Lewis LEW-Tg (EGFP) F455.5/Rrrc heterozigotos e de analisar a expressão da proteína fluorescente verde melhorada (EGFP) em diferentes tipos celulares e genótipos. O genótipo da descendência foi avaliado por PCR e pela análise da expressão da EGFP em diferentes células e genótipos, incluindo-se as células-tronco mesenquimais (MSC) derivadas de tecido adiposo e de osteoblastos de calvária. A expressão da EGFP foi verificada por citometria de fluxo, microscopia de fluorescência e imunocoloração. Foram, identificados os genótipos da descendência e determinados os níveis de expressão de EGFP em dois tipos de células. Foi também constatada uma diferença de expressão entre os genótipos (EGFP +/+) e (EGFP +/-) além da presença de autofluorescência. Mais estudos são necessários para esclarecer a fluorescência natural de células com o genótipo (EGFP +/-) e aquela induzida pela presença da EGFP.

Cultivo de células estromales de rata. Influencia del suero fetal bovino / Stromal rat cell culture. Influence of fetal calf serum

Las células madre estromales humanas y de roedores cultivadas pueden ser inducidas a diferenciarse en neuronas, enfatizando su utilidad potencial en la terapia celular neurorrestaurativa. Los sistemas de cultivo para la expansión de estas células describen el uso de diferentes proporciones de suero fetal, lo que motivó a estudiar qué concentración de suero fetal bovino era capaz de garantizar un adecuado rendimiento celular. Las células de la médula ósea de rata se cultivaron en medio a-MEM suplementado con 10 y 20 por cientode suero fetal bovino y se subcultivaron hasta 3 veces. La viabilidad celular de los cultivos primarios y los subcultivos estuvo por encima del 98 por ciento en ambos experimentos. Los cultivos primarios demoraron 17,4 días en confluir y los subcultivos 7,7 días. La concentración de suero fetal al 20 por ciento no aumentó significativamente la velocidad de multiplicación celular; no obstante, se obtuvo un mayor número de células estromales. El sistema de expansión in vitro podría utilizarse en estudios futuros para la expansión de las células estromales humanas, lo que sienta mejores bases para su aplicación clínica

Cultured human and rodents stromal stem cells can be induced to differentiate into neurons, emphasizing its potential use in neurorestorative cell therapy. Cropping systems for the expansion of these cells describe the use of different ratios of fetal serum, which led to study what concentration of fetal calf serum was able to ensure an adequate cell yield. Cells from rat bone marrow were cultured in medium supplemented with a-MEM 10 and 20 percent fetal bovine serum and subcultured up to 3 times. Cell viability of primary cultures and subcultures was above 98 percent in both experiments. Primary cultures converge delayed in 17.4 days and 7.7 days subcultures. The concentration of 20 percent fetal calf serum did not significantly increase the speed of cell division, however, we obtained a greater number of stromal cells. The expansion in vitro system could be used in future studies for the expansion of human stromal cells, which feels better basis for clinical application

Population structure and genetic variability of mainland and insular populations of the Neotropical water rat, Nectomys squamipes (Rodentia, Sigmodontinae)

Seven microsatellite loci were used to investigate the genetic variability and structure of six mainland and two island populations of the Neotropical water rat Nectomys squamipes, a South American semi-aquatic rodent species with a wide distribution. High levels of variability were found within mainland populations while island populations were less variable but the more differentiated in respect to allele number and frequency. The time of biological divergence between mainland and island populations coincided with geological data. A significant geographic structure was found in mainland populations (q = 0.099; r = 0.086) although the degree of differentiation was relatively low in respect to the distance between surveyed localities (24 to 740 km). Genetic and geographic distances were not positively correlated as previously found with random amplified polymorphic DNA (RAPD) markers. Significant but low genetic differentiation in the mainland and lack of isolation by distance can be explained by large population size and/or recent population expansion. Additionally, the agreement between the age of geologic events (sea level fluctuations) and divergence times for insular populations points to a good reference for molecular clock calibration to associate recent environmental changes and the distribution pattern of small mammals in the Brazilian Atlantic Forest

Nine karyomorphs for spiny rats of the genus Proechimys (Echimyidae, Rodentia) from North and Central Brazil

Spiny rats of the genus Proechimys are morphologically diverse, widely distributed and have diploid numbers ranging from 2n = 14-16 to 2n = 62. In this paper we present cytogenetical data and brief comments on morphological and biogeographical issues related to spiny rats. In our sample of 42 spiny rats collected from 12 Brazilian Amazonian tropical rainforest and the Cerrado (Brazilian savanna) sites we detected nine karyological entities: four different karyomorphs with 2n = 30, three with 2n = 28, one with 2n = 15 and one with 2n = 44. Based on qualitative morphological characters these karyomorphs can be allocated to five species within the goeldii, guyannensis and longicaudatus species groups

Dispersal in a parasitic worm and its two hosts: consequence for local adaptation.

Characterizing host and parasite population genetic structure and estimating gene flow among populations is essential for understanding coevolutionary interactions between hosts and parasites. We examined the population genetic structure of the trematode Schistosoma mansoni and its two host species (the definitive host Rattus rattus and the intermediate host Biomphalaria glabrata) using microsatellite markers. Parasites were sampled from rats. The study was conducted in five sites of the Guadeloupe Island, Lesser Antilles. Mollusks display a pattern of isolation by distance whereas such a pattern is not found neither in schistosomes nor in rats. The comparison of the distribution of genetic variability in S. mansoni and its two host species strongly suggests that migration of parasites is principally determined by that of the vertebrate host in the marshy focus of Guadeloupe. However, the comparison between genetic differentiation values in schistosomes and rats suggests that the efficacy of the schistosome rat-mediated dispersal between transmission sites is lower than expected given the prevalence, parasitic load and migration rate of rats among sites. This could notably suggest that rat migration rate could be negatively correlated to the age or the infection status of individuals. Models made about the evolution of local adaptation in function of the dispersal rates of hosts and parasites suggest that rats and mollusks should be locally adapted to their parasites.

The mammalian gene pecanex 1 is differentially expressed during spermatogenesis.

Using mRNA differential display and cDNA library screening approaches we have identified differential gene expression of pecanex 1--a mammalian homologue of pecanex gene from Drosophila--in the testes of the rat. Northern blot analyses showed that the transcript is only present in the germ line and not in the somatic cells of the testis, reaching its peak at the pachytene stage of the meiotic prophase. Moreover, nonradioactive in situ hybridization did not detect the expression of the gene in any cell type of the testis other than pachytene spermatocytes. Northern blot assays did not allow the detection of the transcript in nine other tissues. Remarkably, although pecanex exerts a neurogenic role in Drosophila, the transcript was not detectable by Northern blotting in the nervous tissue of adult rats, nor in the brain of neonate and embryonal stages. The protein product of the pecanex 1 gene was detected by immunoblotting in pachytene spermatocytes and round spermatids as well, but not in liver nor brain. From genomic analysis we conclude that, although only one pecanex gene exists in Drosophila, mammalian pecanex 1 belongs to a gene family with three related genes in different chromosomes. We speculate that pecanex 1 could play an important role in the testis, related to spermatogenesis.

BCG modulation of anaphylactic antibody response, airwayinflammation and lung hyperreactivity in geneticallyselected mouse strains (Selection IV-A)

The effect of Bacillus Calmette–Gue´rin (BCG) treatment in allergic pulmonary reaction was studied in micegenetically selected accordingly to a High (H-IVA) or Low (L-IVA) antibody responsiveness. Mice wereimmunized with ovalbumin (OVA) or OVA plus BCG. Two days after nasal antigenic challenge, seric IgE andIgG1 anti-OVA, eosinophils in pulmonary tissue, inflammatory cells in bronchoalveolar lavage and the complianceand conductance of respiratory system were evaluated. H-IVA mice were found more susceptible than L-IVA, andBCG was able to inhibit simultaneously the production of IgE, the bronchopulmonary inflammation and bronchialhyperresponsiveness in these genetically selected mice.