P2X7 siRNA targeted to the kidneys increases klotho and delays the progression of experimental diabetic nephropathy.

Previous studies in our laboratory have suggested that P2X7 could contribute to the progression of diabetic nephropathy and modulated klotho expression. The aim of this study was to investigate if P2X7 receptor is related to the expression of klotho in the onset of diabetic nephropathy in rats. Seven-week-old male Wistar rats weighing 210 g were all uninephrectomized; two-third of the animals were induced to diabetes with 60 mg/kg streptozotocin i.v., and one-third received its vehicle (control rats). At 4th day of the fifth week of the protocol, half of the diabetic rats received a small interfering RNA targeting for P2X7 mRNA, and the other half received its vehicle. Euthanasia was made at the eighth week. Diabetic animals reproduced all classic symptoms of the disease; besides, they showed reduced renal function and low NO bioavailability; also, SOD1, SOD2, and catalase were increased, probably due to the oxidative stress which was elevated in this situation. Metabolic data of diabetic rats did not change by silencing P2X7 receptor. For the other hand, silencing P2X7 was able to contribute to balance oxidative and nitrosative profile, ultimately improving the renal function and increasing plasma and membrane forms of klotho. These findings suggest that the management of P2X7 receptor can benefit the kidneys with diabetic nephropathy. Further studies are needed to show the therapeutic potential of this receptor inhibition to provide a better quality of life for the diabetic patient.

Respiratory gases, air pollution and epilepsy.

A growing number of studies have shown that exposure to air pollutants such as particulate matter and gases can cause cardiovascular, neurodegenerative and psychiatric diseases. The severity of the changes depends on several factors such as exposure time, age and gender. Inflammation has been considered as one of the main factors associated with the generation of these diseases. Here we present some cellular mechanisms activated by air pollution that may represent risk factors for epilepsy and drug resistance associated to epilepsy.

P2X7 receptor and klotho expressions in diabetic nephropathy progression.

Diabetes mellitus is characterized by increased levels of reactive oxygen species (ROS), leading to high levels of adenosine triphosphate (ATP) and the activation of purinergic receptors (P2X7), which results in cell death. Klotho was recently described as a modulator of oxidative stress and as having anti-apoptotic properties, among others. However, the roles of P2X7 and klotho in the progression of diabetic nephropathy are still unclear. In this context, the aim of the present study was to characterize P2X7 and klotho in several stages of diabetes in rats. Diabetes was induced in Wistar rats by streptozotocin, while the control group rats received the drug vehicle. From the 1st to 8th weeks after the diabetes induction, the animals were placed in metabolic cages on the 1st day of each week for 24 h to analyze metabolic parameters and for the urine collection. Then, blood samples and the kidneys were collected for biochemical analysis, including Western blotting and qPCR for P2X7 and klotho. Diabetic rats presented a progressive loss of renal function, with reduced nitric oxide and increased lipid peroxidation. The P2X7 and klotho expressions were similar up to the 4th week; then, P2X7 expression increased in diabetes mellitus (DM), but klotho expression presented an opposite behavior, until the 8th week. Our data show an inverse correlation between P2X7 and klotho expressions through the development of DM, which suggests that the management of these molecules could be useful for controlling the progression of this disease and diabetic nephropathy.

Richness of endophytic fungi isolated from Opuntia ficus-indica Mill. (Cactaceae) and preliminary screening for enzyme production.

Opuntia ficus-indica Mill. (forage cactus) is farmed with relative success in the semi-arid region of the Brazilian northeast for commercial purposes, particularly as forage and food. Endophytic microorganisms are those that can be isolated inside plant tissues and can be a new source to production of enzymes with different potentialities. The objective of this study was to describe the richness of endophytic fungi from O. ficus-indica and to detect the capacity of these species to produce extracellular hydrolytic enzymes. Forty-four endophytic fungi species were isolated. Among them, the most commonly found were Cladosporium cladosporioides (20.43%) and C. sphaerospermum (15.99%). Acremonium terricola, Monodictys castaneae, Penicillium glandicola, Phoma tropica and Tetraploa aristata are being reported for the first time as endophytic fungi for Brazil. The majority of isolated fungi exhibited enzymatic potential. Aspergillus japonicus and P. glandicola presented pectinolytic activity. Xylaria sp. was the most important among the other 14 species with positive cellulase activity. All 24 isolates analysed were xylanase-positive. Protease was best produced by isolate PF103. The results indicate that there is a significant richness of endophytic fungi in O. ficus-indica, and that these isolates indicate promising potential for deployment in biotechnological processes involving production of pectinases, cellulases, xylanases and proteases.

Isolation of cellulolytic fungi from waste of castor (Ricinus communis L.).

This is the first report of isolation of fungi present in fatty and defatted castor bean meal as well as the first of crop's selection to test the cellulolytic potential, in order to verify the diversity and potential of cellulolytic fungi in castor bean waste (Ricinus communis L.). For the screening on solid medium, it was used carboxymethylcellulose (CMC) as the sole carbon source. The microcrystalline cellulose (Avicel) was used as a substrate for submerged fermentation for production of cellobiohydrolase (FPase) and the CMC to produce endoglucanases (CMCase) and ß-glycosidases (BG). 189 cultures of fungi were isolated, including 40 species of filamentous fungi and three yeasts. The Aspergillus was the most frequent found genus. Regarding the distribution of isolated species from defatted castor bean meal, the A. niger was the most frequent one; and within the fatty castor bean meal, the Emericela variecolor prevailed among other species. Among the 67 fungal cultures tested in the initial screening on solid media to assess the cellulolytic potential, 54 disclosed Cellulolytic Index (CI) ranging from 1.04 to 6.00 mm. The isolates were selected for enzyme production in liquid medium with values above 2.0 CI. They were obtained with A. japonicus URM5620 FPase activity (4.99 U/ml) and BG (0.05 U/ml), and Rhodotorula glutinis URM5724 activity of CMCase 3.58 U/ml. These cases occurred after 168 h of submersion for both species of fungi. In our study, we could conclude that the castor bean is a promising source of fungi capable of producing cellulolytic enzymes.

Glutamate-induced alterations in Ca2+ signaling are modulated by mitochondrial Ca2+ handling capacity in brain slices of R6/1 transgenic mice.

Huntington's disease is a neurodegenerative disorder caused by an expansion of CAGs repeats and characterized by alterations in mitochondrial functions. Although changes in Ca(2+) handling have been suggested, the mechanisms involved are not completely understood. The aim of this study was to investigate the possible alterations in Ca(2+) handling capacity and the relationship with mitochondrial dysfunction evaluated by NAD(P)H fluorescence, reactive oxygen species levels, mitochondrial membrane potential (DeltaPsi(m)) measurements and respiration in whole brain slices from R6/1 mice of different ages, evaluated in situ by real-time real-space microscopy. We show that the cortex and striatum of the 9-month-old R6/1 transgenic mice present a significant sustained increase in cytosolic Ca(2+) induced by glutamate (Glu). This difference in Glu response was partially reduced in R6/1 when in the absence of extracellular Ca(2+), indicating that N-methyl-D-aspartate receptors participation in this response is more important in transgenic mice. In addition, Glu also lead to a decrease in NAD(P)H fluorescence, a loss in DeltaPsi(m) and a further increase in respiration, which may have evoked a decrease in mitochondrial Ca(2+) Ca(2+)(m) uptake capacity. Taken together, these results show that alterations in Ca(2+) homeostasis in transgenic mice are associated with a decrease in Ca(2+)(m) uptake mechanism with a diminished Ca(2+) handling ability that ultimately causes dysfunctions and worsening of the neurodegenerative and the disease processes.

Alterations in calcium signaling and a decrease in Bcl-2 expression: possible correlation with apoptosis in aged striatum.

Aging is a multifaceted process associated with various functional and structural deficits that might be evolved in degenerative diseases. It has been shown that neurodegenerative disorders are associated with alterations in Ca(2+) homeostasis. Thus, in the present work, we have investigated Ca(2+) signaling and apoptosis in aged striatum. Our results show that glutamate and NMDA evoke a greater Ca(2+) rise in striatum slices from aged animals. However, this difference is not present when glutamate is tested in the absence of external Ca(2+). Immunostaining of glutamate receptors shows that only NMDA receptors (NR1) are increased in the striatum of aged rats. Increases in mitochondrial Ca(2+) content and in the reactive oxygen species levels were also observed in aged animals, which could be associated with tissue vulnerability. In addition, a decrease in the Bcl-2 protein expression and an enhancement in apoptosis were also present in aged striatum. Together the results indicate that, in aged animals, alterations in Ca(2+) handling coupled to an increase in ROS accumulation and a decrease in the prosurvival protein Bcl-2 may contribute to apoptosis induction and cell death in rat striatum.

Alterations in Calcium Signalingand a Decrease in Bcl-2 Expression: Possible Correlation With Apoptosisin Aged Striatum

Aging is a multifaceted process associated with various functional and structural deficits that might be evolved in degenerative diseases. It has been shown that neurodegenerative disorders are associated with alterations in Ca2+ homeostasis. Thus, in the present work, we have investigated Ca2+ signaling and apoptosis in aged striatum. Our results show that glutamate and NMDA evoke a greater Ca2+ rise in striatum slices from aged animals. However, this difference is not present when glutamate is tested in the absence of external Ca2+. Immunostaining of glutamate receptors shows that only NMDA receptors (NR1) are increased in the striatum of aged rats. Increases in mitochondrial Ca2+ content and in the reactive oxygen species levels were also observed in aged animals, which could be associated with tissue vulnerability. In addition, a decrease in the Bcl-2 protein expression and an enhancement in apoptosis were also present in aged striatum. Together the results indicate that, in aged animals, alterations in Ca2+ handling coupled to an increase in ROS accumulation and a decrease in the prosurvival protein Bcl-2 may contribute to apoptosis induction and cell death in rat striatum.

Filamentous fungi isolated from sand and water of "Bairro Novo" and "Casa Caiada" beaches, Olinda, Pernambuco, Brazil.

Fungi comprehend a heterogeneous group of heterotrophic microorganisms that act as saprobes or parasites or, less frequently, as symbionts living in association with other organisms. They are cosmopolitan and important components of ecosystems. Considering the small number of Brazilian papers on the filamentous mycota in marine environments, and the need to improve knowledge of the diversity of these microfungi in "Casa Caiada" and "Bairro Novo" beaches, Olinda, PE, the aim of this work was to isolate and identify the fungi from sand and water samples of these ecosystems. Thirty two samplings of sand (surface and 20 cm deep) and water (surface and 1 m deep) were carried out during the dry and rainy seasons, in low and high tide. From each sand sample, a suspension was made with 50 g of sand diluted in 90 mL of sterilized distilled water. From each sand suspension and water sample, 0.5 mL was spread, in triplicate, onto Petri dishes containing Sabouraud Agar added of chloramphenicol. The plates were incubated at 28 degrees C (+/-2 degrees C). Fifty seven species were isolated, identified, and classified in 20 genera. Aspergillus and Penicillium were the most frequent genera in both sand and water, with a total of 11 and 19 species, respectively.

Filamentous fungi isolated from sand and water of "Bairro Novo" and "Casa Caiada" beaches, Olinda, Pernambuco, Brazil / Fungos filamentosos isolados do solo e da água nas praias de Bairro Novo e Casa Caiada, Olinda, Pernambuco, Brasil

Fungi comprehend a heterogeneous group of heterotrophic microorganisms that act as saprobes or parasites or, less frequently, as symbionts living in association with other organisms. They are cosmopolitan and important components of ecosystems. Considering the small number of Brazilian papers on the filamentous mycota in marine environments, and the need to improve knowledge of the diversity of these microfungi in "Casa Caiada" and "Bairro Novo" beaches, Olinda, PE, the aim of this work was to isolate and identify the fungi from sand and water samples of these ecosystems. Thirty two samplings of sand (surface and 20 cm deep) and water (surface and 1 m deep) were carried out during the dry and rainy seasons, in low and high tide. From each sand sample, a suspension was made with 50 g of sand diluted in 90 mL of sterilized distilled water. From each sand suspension and water sample, 0.5 mL was spread, in triplicate, onto Petri dishes containing Sabouraud Agar added of chloramphenicol. The plates were incubated at 28 °C (±2 °C). Fifty seven species were isolated, identified, and classified in 20 genera. Aspergillus and Penicillium were the most frequent genera in both sand and water, with a total of 11 and 19 species, respectively.

Os fungos compreendem um grupo heterogêneo de microorganismos heterotróficos, atuando como sapróbios ou parasitas, ou menos freqüentemente como simbiontes, vivendo em associação com outros organismos. São cosmopolitas e componentes importantes dos ecossistemas. Considerando-se a escassez de trabalhos no Brasil que tratam da micota filamentosa em ambientes marinhos, e ainda a necessidade do conhecimento da diversidade desses microfungos nas praias de Casa Caiada e Bairro Novo, Olinda, PE, este trabalho teve como objetivo o isolamento e identificação de fungos, em amostras de solo e de água, desses ecossistemas. Foram realizadas 32 coletas do solo (em superfície e a 20 cm de profundidade) e da água (em superfície e a 1 m de profundidade), nos períodos de estiagem e chuvoso, na baixa-mar e preamar. De cada amostra de solo, foi feita uma suspensão de 50 g de solo em 90 mL de água destilada esterilizada, e de cada amostra de solo e água foi retirado 0,5 mL para o semeio em triplicata em placas de Petri contendo ágar Sabouraud acrescido de cloranfenicol e incubadas sob temperatura ambiente (28 ± 2 °C). Foram isoladas e identificadas 57 espécies correspondentes a 20 gêneros. Aspergillus e Penicillium dominaram tanto no solo quanto na água, com um total de 11 e 19 espécies, respectivamente.

Apoptosis in alpha interferon (IFN-alpha) intratumoral chemotherapy for cystic craniopharyngiomas.

OBJECTIVES: The aim of this study was to verify whether intracystic injections of alpha-Interferon (IFN-alpha) in cystic craniopharyngiomas were able to reduce the tumor by activating the Fas apoptotic pathway. MATERIALS AND METHODS: Twenty-one patients with cystic craniopharyngiomas treated at the Pediatric Oncology Institute (IOP/GRAACC) of Federal University of São Paulo were submitted to intracystic chemotherapy with IFN-alpha. The tumor sizes of all patients were monitored and the apoptotic factor soluble FasL (sFasL) concentration was determined by ELISA (enzyme-linked immunosorbent assay) in tumor fluid samples from eight patients. RESULTS: There was a complete reduction in 11 patients, a partial response in seven, and a minor response in three patients. The concentration of sFasL was increased in all the eight patients examined concomitantly with the tumor size reduction. CONCLUSIONS: Our data demonstrated that the IFN-alpha was able to induce Fas-mediated apoptosis together with a reduction in the tumor size; such an observation may suggest the importance to investigate still unexplored mechanisms to be exploited in craniopharyngioma therapy.

Deficit in hippocampal long-term potentiation in monosodium glutamate-treated rats.

Rats subjected to monosodium glutamate (MSG) administration during the neonatal period present chronic neuroendocrine dysfunction associated with marked cognitive deficits. Long-term potentiation (LTP) in the hippocampus provides a model suited for the study of mammalian brain plasticity and memory formation. In the present work, we used the LTP protocol to investigate the synaptic plasticity in the hippocampal CA1 area of adult rats subjected to MSG treatment during the first 10 days of life. Synaptic transmission in CA1 area was analyzed using extracellular field recordings in response to Schaffer's collateral fiber stimulation in hippocampal slices. Animals injected with MSG exhibited a dramatic decrement of LTP field excitatory postsynaptic potentials (fEPSPs) compared to control group. Analysis of percent enhancement of fEPSP slope at 2 min after high frequency stimulation (HFS) increased by 189.3 +/- 33.2% in slices from control rats and 129.45 +/- 18.5% (p < 0.01) in slices from MSG-treated rats. Additionally, MSG-treated animals failed to maintain or consolidate LTP as revealed by a significant reduction in fEPSP slope enhancement over time after HFS. The mean fEPSP slope, 60 min after HFS, was 154.28 +/- 21% of the average baseline slope in control slices versus only 124.4 +/- 15% in MSG-treated rats (p < 0.01). At 90 min after HFS, slices from controls reached a potentiation of 44.5 +/- 2.9%, whereas the MSG group displayed an overall response enhancement of 17.65 +/- 2.7% of basal levels (p < 0.01). These findings indicate that MSG-treated rats display a chronic impairment of CA1 synaptic plasticity.

Evidence that ATP participates in the pathophysiology of pilocarpine-induced temporal lobe epilepsy: fluorimetric, immunohistochemical, and Western blot studies.

PURPOSE: This study was performed to study the role of adenosine triphosphate (ATP) in the brain of pilocarpine-induced chronic epileptic rats. METHODS: ATP-mediated changes in intracellular calcium were studied by the fura-2 method. Immunohistochemistry and Western blotting methods were used to localize and quantify P2X7 receptors in these animals. RESULTS: The fluorimetric study in chronic rats revealed a biphasic response indicating the presence of P2X7 receptors. The Western blotting study showed an increase of 80% of P2X7 expression in chronic rats compared with the control group. P2X7 immunoreactivity resembled mossy fiber sprouting at the dentate gyrus of epileptic animals. CONCLUSIONS: These results suggest that purinergic receptors may participate in the pathophysiology of temporal lobe epilepsy.

Serum serotonin levels of normal and autistic children

1. Serum serotonin (5HT) was determined in normal and autistic children by high performance liquid chromatography with electrochemical detection (HPLC-ED), after serum deproteinization. 2. The sample deproteinization was carried out by the addition of 3.4 M HCLO4 to a small volume of the serum, followed by freezing, centrifugation, dilution and injection of sample into the HPLC. 3. Serum 5HT concentration was significantly increased in autistic children (303 +/- 92 ng/ml) (N = 19) when compared to that of normal children (215 +/- 67 ng/ml) (N = 46). The data of normal children analyzed by ANOVA for sex or age showed no difference. 4. The method employed in this study showed high resolution, good sensitivity and can be used for routine determination of serum 5HT in the clinical investigation of hyperserotonemia in autism

Serum catecholamine levels determined by high performance liquid chromatography coupled with electrochemical detection

Este trabalho apresenta um método rápido para a quantificaçäo de catecolaminas utilizando a técnica de cromatografia líquida de fase reversa acoplada à detecçäo eletroquímica. Separaçäo isocrática rápida foi obtida empregando como fase móvel a soluçäo: 0,02M de fosfato de sódio dibásico, 0,02M de ácido nítrico, pH 2,64, metanol a 10 por cento, 0,12mM de EDTA sódico e 556 mg/L de ácido heptanosulfônico. Delineou-se o procedimento de preparaçäo das amostras com extraçäo das monoaminas em alumina, para melhorar a recuperaçäo e diminuir fatores de diluiçäo. O tempo total de análise é de 15 minutos, com boa separaçäo dos picos de monoaminas. O limite de detecçäo obtido para as monoaminas séricas é de 40 a 50 pg/mL, com uma taxa de recuperaçäo de 70-75 por cento.

Implante de células embrionárias em um modelo experimental de epilepsia / Embryons cells implanted in a experimental model of epilepsy

Estudos experimentais mostram que celulas nervosas provenientes de embriöes säo capazes de sobreviver, em diversas regiöes do SNC de animais adultos. Uma vez integrados ao hospedeiro, esses neurônios podem liberar neurotransmissores específicos em regiöes previamente desnervadas. Essa abordagem oferece a possibilidade de se restabelecer a neurotransmissäo, em patologias nervosas diversas, particularmente as degenerativas. Resultados bastantes favoráveis têm sido obtidos utilizando modelos experimentais de patologias extrapiramidais e quadros demenciais. Em epileptologia, no entanto, resultados favoráveis e desfavoráveis têm sido encontrados. Uma das razöes para a ocorrência de dados conflitantes pode estar relacionada a escolha de modelos experimentais, que nem sempre reproduzem os mesmos fenômenos fisiopatológicos. O objetivo deste trabalho é o de avaliar o efeito do implante de células embrionárias noradrenérgicas sobre um modelo de crises espontâneas no rato. Neste modelo, as crises espontâneas säo observadas após a induçäo de status epilepticus através da administraçäo sistêmica de pilocarpina. Estudos realizados têm indicado ser este modelo muito semelhante às crises parciais complexas humanas associadas a escleose mesial temporal. Células embrionárias provenientes do locus coeruleus foram implantadas no hipocampo de animais epilépticos, sendo os animais acompanhados por 3 meses. A avaliaçäo histológica (imunohistoquímica para tirosina hidroxilase), 90 dias após o implante, demonstrou a presença de neurônios noradrenérgicos em várias regiöes do hipocampo. No entanto, o procedimento näo interferiu na freqüência de crises. Estes resultados diferem de estudos utilizando outros modelos animais, onde o mesmo tipo de transplante apresentou efeito positivo e indicam que, no modelo da pilocarpina, é provável que outros sistemas neurotransmissores estejam envolvidos, além da simples participaçäo isolada do sistema noradrenérgico

Fungos isolados do ar e do piso de ambientes fechados do Hospital Escola da Universidade Federal de Pernambuco, Recife, Brasil-I / Fungi isolated from air and floor in closed enviroment in the Hospital Escola da Universidade Federal de Pernambuco, Recife, Brasil-I

Del aire y del piso de ambientes confinados del "Hospital das Clínicas da Universidade Federal de Pernambuco, Recife, Brasil", fueron aisladas 3.330 colonias de hongos correspondientes a 123 entidades taxonómicas pertenecientes en su mayoría a los Asco-Deuteromycotina (95,3%), estando los demás grupos escasamente representados Zigomycotina (5,6%); Basidiomycotina (0,8%) y Micelia sterilla (0,2%). No hubo diferencias significativas entre la cantidad de colonias aisladas durante los períodos de pluviosidad y verano. Observándose el mayor número de colonias en el piso más que en aquellas aisladas del aire atmosférico. Los géneros encontrados con mayor frecuencia fueron Aspergillus, Penicillium y Fusarium. Entre los Hyphomycetes aislados, 14 especies son referidas como agentes etiológicos de micosis

Cromomicose por Fonsecaea pedrosoi apresentando as tres fases conidiais, acrógena, acropleurógena e semi-endógena / Chromomycosis due to Fonsecaea pedrosoi presenting 3 conidial stages: acrogenous, acropleurogenous and semi-endogenous

Una muestra de Fonsecaea pedrosoi (Brumpt) Negroni 1936, fue aislada de un caso de cromomicosis forma plana y presentó tres fases de esporulación: Cladosporium, Rhinocladiella y Phialophora