P2X7 receptor large pore signaling in avian Müller glial cells.

ATP is a pleiotropic molecule that promotes extra- and intracellular signaling to regulate numerous functions. This nucleotide activates purine and pyrimidine receptors at the plasma membrane, categorized as ionotropic P2X or G-protein-coupled receptor (GPCR) P2Y receptors. P2X are ligand-gated ion channel receptors, expressed in both retinal neurons and Müller cells leading to neuron-glia communication, calcium waves and neurovascular coupling. However, how P2X pore is formed upon ATP activation and how signaling pathways regulates the complex is still a matter of controversy. Here we studied the properties of the P2X7 receptor (P2X7R) using electrophysiology, single cell Ca2+ imaging, and dye uptake assay in purified avian Müller glia in culture. Our data show that ATP (or benzoyl-benzoyl ATP, BzATP) evoked large inward currents in patch-clamp studies while addition of P2X7R antagonist such as brilliant Blue G (BBG), abolished these currents. Ruthenium red (RU-2), a general transient receptor potential (TRP) inhibitor, reduced currents induced by ATP. Our data also point to the involvement of mitogen activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), Ca2+-calmodulin kinase II (CAMKII), microtubules or protein kinase C (PKC) modulating ATP-induced ionic current in Müller cells. We show that ATP induced Ca2+ influx, partially inhibited by P2X7R antagonists (oxidized ATP or BBG), and totally inhibited by blockers of other pores such as transient receptor potential (TRPs) or connexin hemichannel. Additionally, MAPK, PKC, PI3K or CAMKII inhibitors also are involved in the modulation of intracellular calcium signaling. Finally, ATP induced 80-90% of dye uptake in Muller glia cells, while oxidized ATP (oATP), BBG or A740003 inhibited this effect. We conclude that large conductance channel and other P2XRs are not involved in the ATP-induced dye uptake, but signaling pathways such as MAPK, PI3-K, microtubules or PKC are involved in pore formation.

Role of melatonin in the oxidative damage prevention at different times of hepatic regeneration.

The process of regenerating liver is the result of a balance between stimulating factors and inhibitors of hepatocyte proliferation. Melatonin and its metabolites have been found to protect tissues against oxidative damage generated by a variety of toxic agents and metabolic processes. Furthermore, studies in liver of rats showed a decrease in the liver mitochondrial hydroxylation of drugs returning to the normal state after the administration of antioxidants. This study was designed to determine, in experimental animals, whether the administration of an antioxidant agent such as melatonin could prevent cells events leading to tissue injury and hepatic dysfunction after partial hepatectomy (PH). Biliary flow (BF), oxidative stress in hepatic tissue and Na⁺/K⁺ ATPase activities in whole plasma membrane were determined. PH decreased the Na⁺/K⁺ ATPase activity. PH significantly reduced the BF (36%) and promoted oxidative stress with an increase of lipoperoxidation and decrease of glutathione peroxidase and catalase activities. Treatment with melatonin prevented the decrease of BF in rats with hepatectomy and normalized the Na⁺/K⁺ ATPase activity. Moreover, melatonin markedly attenuated oxidative stress produced by PH. This may be the results of the higher efficacy of melatonin in scavenging various free radicals and also because of its ability in stimulating the antioxidant enzymes. We suggest that oxidative stress before and during liver regeneration has a crucial role in cholestasis, apoptotic/necrotic hepatocellular damage and the impairment in liver transport function induced by PH and that melatonin could modulate the degree of oxidative stress and through it prevent the alterations in liver function carrier.

Vasoinhibin gene transfer by adenoassociated virus type 2 protects against VEGF- and diabetes-induced retinal vasopermeability.

PURPOSE: Specific proteolytic cleavages of the hormone prolactin (PRL) generate vasoinhibins, a family of peptides (including 16-kDa PRL) that are able to inhibit the pathologic increase in retinal vasopermeability (RVP) associated with diabetes. Here the authors tested the ability of an adenoassociated virus type 2 (AAV2) vasoinhibin vector to inhibit vascular endothelial growth factor (VEGF)- and diabetes-induced RVP. METHODS: AAV2 vectors encoding vasoinhibin, PRL, or soluble VEGF receptor 1 (soluble FMS-like tyrosine kinase-1 [sFlt-1]) were injected intravitreally into the eyes of rats. Four weeks later, either VEGF was injected intravitreally or diabetes was induced with streptozotocin. Tracer accumulation was evaluated as an index of RVP using fluorescein angiography or the Evans blue dye method. RT-PCR verified transgene expression in the retina, and the intravitreal injection of an AAV2 vector encoding green fluorescent protein revealed transduced cells in the retinal ganglion cell layer. In addition, Western blot analysis of AAV2-transduced HEK293 cells confirmed the expression and secretion of the vector-encoded proteins. RESULTS: The AAV2-vasoinhibin vector prevented the increase in tracer accumulation that occurs 24 hours after the intravitreal injection of VEGF. Diabetes induced a significant increase in tracer accumulation compared with nondiabetic controls. This increase was blocked by the AAV2-vasoinhibin vector and reduced by the AAV2-sFlt-1 vector. The AAV2-PRL vector had no effect. CONCLUSIONS: These results show that an AAV2-vasoinhibin vector prevents pathologic RVP and suggest it could have therapeutic value in patients with diabetic retinopathy.

Evaluation of RbCl and CrCl3 as markers of Triatoma brasiliensis (Hemiptera: Reduviidae) nymphs: persistence and influence of Rb and Cr on triatomine biology.

In order to mark Triatoma brasiliensis, the vector of Chagas disease in Brazil, two chemical compounds, rubidium chloride (RbCl) and chromium chloride (CrCl3), were tested. First, 199 N2-N5 nymphs were fed on blood with 0.025M RbCl. Rb marker positivity ranged from 2.5% (N3)-70% (N2), with a maximum persistence of 98 days. Second, 265 N2-N5 nymphs were fed on blood containing 0.0015M CrCl3. Cr marker positivity ranged up to 93% (N5), with a maximum persistence of 119 days. Finally, we blood fed 213 T. brasiliensis to investigate whether CrCl3 altered the biology of this insect. The developmental time of T. brasiliensis was unaltered, but the survival of the Cr-marked group was lower than that of the control group. Differences in the mean fecundity of the control (mean of 156.1) and experimental (mean of 135.6) groups were not statistically significant and 100% of the egg batches of females Cr-marked as nymphs were positive. In conclusion, CrCl3 is a useful tool for marking T. brasiliensis nymphs due to its high positivity and persistence.

Evaluation of RbCl and CrCl3 as markers of Triatoma brasiliensis (Hemiptera: Reduviidae) nymphs: persistence and influence of Rb and Cr on triatomine biology

In order to mark Triatoma brasiliensis, the vector of Chagas disease in Brazil, two chemical compounds, rubidium chloride (RbCl) and chromium chloride (CrCl3), were tested. First, 199 N2-N5 nymphs were fed on blood with 0.025M RbCl. Rb marker positivity ranged from 2.5 percent (N3)-70 percent (N2), with a maximum persistence of 98 days. Second, 265 N2-N5 nymphs were fed on blood containing 0.0015M CrCl3. Cr marker positivity ranged up to 93 percent (N5), with a maximum persistence of 119 days. Finally, we blood fed 213 T. brasiliensis to investigate whether CrCl3 altered the biology of this insect. The developmental time of T. brasiliensis was unaltered, but the survival of the Cr-marked group was lower than that of the control group. Differences in the mean fecundity of the control (mean of 156.1) and experimental (mean of 135.6) groups were not statistically significant and 100 percent of the egg batches of females Cr-marked as nymphs were positive. In conclusion, CrCl3 is a useful tool for marking T. brasiliensis nymphs due to its high positivity and persistence.

Dentin permeability of the apical third in different groups of teeth.

This ex vivo study evaluated dentin permeability of the root canal in the apical third of different human groups of teeth. Eighty teeth were used, 8 from each dental group: maxillary and mandibular central incisors, lateral incisors and canines, maxillary first premolars (buccal and palatal roots), mandibular first premolars, and maxillary and mandibular second premolars, totalizing 88 roots that were distributed in 11 groups. The root canals were instrumented, irrigated with 1% NaOCl and 15% EDTA. Roots were immersed in 10% copper sulfate for 30 min and then in 1% rubeanic acid alcohol solution for the same period; this chemical reaction reveals dentin permeability by the formation of copper rubeanate, which is a dark-colored compound. Semi-serial 100-µm-thick cross-sections were obtained from the apical third of the roots. Five sections of each apical third were washed, dehydrated, cleared and mounted on glass slides for examination under optical microscopy. The percentage of copper ion infiltration and the amount of tubular dentin were quantified by morphometric analysis. The penetration of copper ions in the apical third ranged from 4.60 to 16.66%. The mandibular central and lateral incisors presented the highest dentin permeability (16.66%), while the maxillary canines and mandibular second and first premolars presented the lowest dentin permeability (4.60%, 4.80% and 5.71%, respectively; p<0.001). The other teeth presented intermediate permeability. In conclusion, dye penetration into dentin tubules at the apical region is strongly dependent on the group of teeth evaluated.

Dentin permeability of the apical third in different groups of teeth

This ex vivo study evaluated dentin permeability of the root canal in the apical third of different human groups of teeth. Eighty teeth were used, 8 from each dental group: maxillary and mandibular central incisors, lateral incisors and canines, maxillary first premolars (buccal and palatal roots), mandibular first premolars, and maxillary and mandibular second premolars, totalizing 88 roots that were distributed in 11 groups. The root canals were instrumented, irrigated with 1 percent NaOCl and 15 percent EDTA. Roots were immersed in 10 percent copper sulfate for 30 min and then in 1 percent rubeanic acid alcohol solution for the same period; this chemical reaction reveals dentin permeability by the formation of copper rubeanate, which is a dark-colored compound. Semi-serial 100-µm-thick cross-sections were obtained from the apical third of the roots. Five sections of each apical third were washed, dehydrated, cleared and mounted on glass slides for examination under optical microscopy. The percentage of copper ion infiltration and the amount of tubular dentin were quantified by morphometric analysis. The penetration of copper ions in the apical third ranged from 4.60 to 16.66 percent. The mandibular central and lateral incisors presented the highest dentin permeability (16.66 percent), while the maxillary canines and mandibular second and first premolars presented the lowest dentin permeability (4.60 percent, 4.80 percent and 5.71 percent, respectively; p<0.001). The other teeth presented intermediate permeability. In conclusion, dye penetration into dentin tubules at the apical region is strongly dependent on the group of teeth evaluated.

Este estudo ex vivo avaliou a permeabilidade da dentina do canal radicular do terço apical de diferentes grupos de dentes humanos. Foram utilizados 80 dentes, sendo 8 de cada grupo dental superior e inferior: incisivos centrais, incisivos laterais, caninos, primeiros pré-molares superiores (raízes vestibulares e palatinas), primeiros pré-molares inferiores, segundos pré-molares superiores e inferiores, totalizando 88 raízes, as quais foram distribuídas em 11 grupos. Os canais foram instrumentados, irrigados com NaOCl a 1 por cento e EDTA a 15 por cento. As raízes foram imersas em sulfato de cobre a 10 por cento por 30 min e acido rubeânico a 1 por cento pelo mesmo período. Esta reação química revela a permeabilidade da dentina por meio da formação de um complexo escurecido denominado rubeanato de cobre. Hemi-secções de 100 µm de espessura foram obtidas do terço apical da raiz. Cinco secções do terço apical foram lavadas, desidratadas, diafanizadas e montadas em lâminas para análise em microscopia óptica. A porcentagem de infiltração de íons cobre e a quantidade de dentina tubular foram quantificadas por meio de análise morfométrica. A penetração de íons cobre no terço apical da raiz variou de 4,60 por cento a 16,66 por cento. Os incisivos centrais e laterais apresentaram a maior permeabilidade dentinária (16,66 por cento), e os caninos superiores e segundos e primeiros pré-molares inferiores as menores (4,60 por cento, 4,80 por cento e 5,71 por cento, respectivamente; p<0,001). Os outros dentes apresentaram permeabilidade intermediaria. Conclui-se que a penetração de corante nos túbulos dentinários da região apical é extremamente dependente do grupo de dentes avaliado.

Bioremediation of dyes in textile effluents by Aspergillus oryzae.

In this study Aspergillus oryzae was utilized to remove azo dyes from aqueous solution. Physically induced in its paramorphogenic form to produce standardized mycelial pellets, the non-autoclaved and autoclaved hyphae biomass was applied to biosorb the reactive dyes Procion Red HE7B (PR-HE7B) and Procion Violet H3R (PV-H3R) at different pH values (2.50, 4.50, and 6.50). The best pH for biosorption was 2.50, though the autoclaved demonstrated a higher biosorption capacity than the non-autoclaved pellets. The toxicity level was determined using the Trimmed Spearman-Karber method with Daphnia similis in all bioassays. The calculated toxicity of PV-H3R (LC100 62.50 microg mL(-1)) was higher than to PR-HE7B (LC100 300.00 microg mL(-1)), and its results brought out that the decrease of toxicity levels to zero might be accomplished by adding small quantities of pelletized A. oryzae to the solutions.

Indocyanine green nanoparticles useful for photomedicine.

OBJECTIVE: The aim of this study was to evaluate the potential application of biodegradable nanoparticles (NPs) containing indocyanine green (ICG) in photodynamic therapy (PDT). METHODS: Important parameters, such as particle size and external morphology, were established by dynamic light scattering (DLS) and scanning electron microscopy (SEM). Also, drug encapsulation efficiency and in vitro release behavior were evaluated by spectroscopic methods. RESULTS: The particles are spherical in shape, they exhibit an 817-nm diameter, and they have a low tendency to aggregate. The loading efficiency was 65%. ICG photophysical parameters showed a bathocromic shift in ICG-loaded nanoparticles (ICG-NP). Analysis of the cell P388-D1 in the presence of the ICG-NP by SEM showed that the majority of the nanoparticles were uptaken by phagocytic cells after 2 h of incubation. After laser irradiation photodamage was observed in P388-D1 cells where ICG-NPs had been uptaken by phagocytic cells. CONCLUSION: Polymeric NPs work as an efficient drug delivery system for PDT drugs, and this approach can be used in the administration of amphiphilic photosensitizers in the treatment of neoplasic cells.

Natural medicaments in endodontics -- a comparative study of the anti-inflammatory action.

The objective of this study was to evaluate the irritant potential of propolis, Casearia sylvestris, Otosporin and saline solution (control). Twenty-eight male Wistar rats were selected, anesthetized and four experimental sites were designed on their backs. Injections of 2% Evans blue were intravenously administered in the lateral caudal vein and 0.1 ml of the tested solutions was injected intradermally into the experimental sites. The animals were killed 1/2, 1, 3 and 6 hours after the injection of the solutions. Each piece of skin containing the lesion was immersed in formamide and incubated at 45 masculine C for 72 h. After filtration, optical density was measured in a spectrophotometer. Data were statistically analyzed by a 2-way non-parametric test. The highest values of extracted dye were observed at 3 hours characterizing a peak in the inflammatory process. Propolis was the least irritant solution. The natural medicaments tested in this study may be a valuable alternative for endodontic treatment.

Natural medicaments in endodontics: a comparative study of the anti-inflammatory action

O objetivo desta pesquisa foi avaliar o potencial irritativo de própolis, Casearia sylvestris, Otosporin e soro fisiológico (controle). Foram utilizados 28 ratos machos da linhagem Wistar. Os animais foram anestesiados e, em seguida, receberam a injeção do corante azul de Evans (2%) por via intravenosa na veia caudal. Em quatro pontos predeterminados e depilados da região dorsal de cada animal, foram injetados 0,1 ml das substâncias teste. Os animais foram sacrificados meia, uma, três e seis horas após a injeção das substâncias, e cada pedaço de pele contendo a lesão foi colocado em frascos contendo formamida, que foram incubados a 45oC por 72 h. Após esse período, as amostras foram filtradas e submetidas à análise em espectrofotômetro. Os dados foram submetidos ao teste estatístico não-paramétrico de medidas repetidas. No período de 3 h, foram observados os maiores valores de corante extraído, caracterizando, assim, o pico do processo inflamatório. A própolis foi a substância que apresentou menor potencial irritativo. Os produtos naturais testados neste trabalho podem ser uma alternativa para o tratamento endodôntico.

Comparative study of dentine permeability after apicectomy and surface treatment with 9.6 microm TEA CO2 and Er:YAG laser irradiation.

Failure of apicectomies is generally attributed to dentine surface permeability as well as to the lack of an adequate marginal sealing of the retrofilling material, which allows the percolation of microorganisms and their products from the root canal system to the periodontal region, thus compromising periapical healing. The purpose of this study was to evaluate dentine and the marginal permeability after apicectomy and surface treatment with 9.6 micro m TEA CO(2) or Er:YAG 2.94 micro m laser irradiation. Sixty-five single rooted human endodontically treated teeth were divided into five experimental groups: group I (control), apicectomy with high speed bur; group II, similar procedure to that of group I, followed by dentinal surface treatment with 9.6 micro m CO(2) laser; group III, similar procedure to group I followed by dentinal surface treatment with Er:YAG laser 2.94 micro m; group IV, apicectomy and surface treatment with CO(2) 9.6 micro m laser; and group V, apicectomy and surface treatment with Er:YAG laser 2.94 micro m. The analysis of methylene blue dye infiltration through the dentinal surface and the retrofilling material demonstrated that the samples from the groups that were irradiated with the lasers showed significantly lower infiltration indexes than the ones from the control group. These results were compatible with the structural morphological changes evidenced through SEM analysis. Samples from groups II and IV (9.6 micro m CO(2)) showed clean smooth surfaces, fusion, and recrystallized dentine distributed homogeneously throughout the irradiated area sealing the dentinal tubules. Samples from groups III and V (Er:YAG 2.94 micro m) also presented clean surfaces, without smear layer, but roughly compatible to the ablationed dentine and without evidence of dentinal tubules. Through the conditions of this study, the Er:YAG 2.94 micro m and the 9.6 micro m CO(2) laser used for root canal resection and dentine surface treatment showed a reduction of permeability to methylene blue dye.

Sildenafil, a phosphodiesterase-5 inhibitor, delays gastric emptying and gastrointestinal transit of liquid in awake rats.

We studied the effect of sildenafil on gastric emptying (GE) and gastrointestinal (GI) transit in awake rats. After cervical vessel cannulation and 24 hr of fasting, the animals received an intravenous (IV) injection of sildenafil (4 mg/kg) or vehicle. Next they were gavage fed (1.5 ml) with a test meal (phenol red in 5% glucose solution, 0.5 mg/ml) and sacrificed 10, 20, or 30 min later. Experimental and control subsets consisted of 5-10 rats. Gastric and proximal, medial, and distal small intestine dye retentions (GDR and IDR, respectively) were obtained by spectrophotometry. Data were compared by ANOVA and Student-Newman-Keuls test. In sildenafil-treated animals, GDR increased (P < 0.05) by 20.3%, 46.9%, and 55,5% while medial IDR decreased (P < 0.05) by 35.1%, 43.4%, and 41.6%, respectively, at 10, 20, and 30-min intervals. Proximal and distal IDR values did not change in sildenafil-treated animals. Mean arterial pressure (MAP) decreased 25% (P < 0.05) right after sildenafil administration but normalized afterwards while in controls MAP remained unchanged. In conclusion, sildenafil delays GE and GI transit of a liquid meal while transiently decreases MAP in awake rats.

Applicability of the equations of Freundlich and Langmuir to the adsorption of the azo dye procion scarlet on paramorphic colonies of Neurospora crassa.

Experiments on the adsorption of Procion Scarlet MX-G by normal hyphae and by paramorphic colonies of Neurospora crassa were performed at pH 2.5, 4.5 and 6.5 at 30 degrees C. The measured adsorption isotherms were evaluated by the Freundlich and Langmuir equations. The removal of dye was most effective at pH 2.5 and more dye was adsorbed per unit mass of cells in the paramorphic cultures than in the normal hyphae. The statistical tests showed Langmuir's equation to give a better fit to the adsorption data.