In vitro effect of deoxynivalenol (DON) mycotoxin on porcine reproductive and respiratory syndrome virus replication.

Deoxynivalenol (DON) is a mycotoxin produced by Fusarium spp. Among monogastric farm animals, swine are the most susceptible to DON as it markedly reduces feed intake and decreases weight gain. DON has also been shown to increase susceptibility to viral infections; therefore the objective of this study was to investigate in vitro impact of DON on porcine reproductive and respiratory syndrome virus (PRRSV). Permissive cells were infected or not with PRRSV and were treated with increasing concentrations of DON. Cell survival and mortality were evaluated by determining the number of viable cells with a tetrazolium compound and by measuring lactate dehydrogenase (LDH) release, respectively. Virus titration and antiviral cytokines mRNA expression were evaluated by quantitative PCR. DON significantly affected the survival of noninfected cells in a dose dependent manner. However, DON concentrations between 140 and 280 significantly increased the survival of cells infected with PRRSV. These concentrations significantly decreased PRRSV replication by inducing a pro-inflammatory cytokines environment and an early activation of apoptosis, which in turn seem to interrupt viral replication. For the first time, this study showed that DON had significant effects on the survival of PRRSV infected cells and on virus replication, in a dose dependent manner.

Convergencia de vías de señalización en un modelo de apoptosis

La sepsis es un evento inflamatorio generalizado del organismo inducido por un daño causado generalmente por un agente infeccioso. El patógeno más frecuentemente asociado con esta entidad es el Staphylococcus aureus, responsable de la inducción de apoptosis en células endoteliales debida a la producción de ceramida. Se ha descrito el efecto protector de la proteína C activada (PCA) en sepsis y su relación con la disminución de la apoptosis de las células endoteliales. En este trabajo se analizó la activación de las quinasas AKT, ASK1, SAPK/JNK y p38 en un modelo de apoptosis endotelial usando las técnicas de Western Blotting y ELISA. Las células endoteliales (EA.hy926), se trataron con C2-ceramida (130μM) en presencia de inhibidores químicos de cada una de estas quinasas y PCA. La supervivencia de las células en presencia de inhibidores químicos y PCA fue evaluada por medio de ensayos de activación de las caspasas 3, 7 y 9, que verificaban la muerte celular por apoptosis. Los resultados evidencian que la ceramida reduce la activación de AKT y aumenta la activación de las quinasas ASK, SAPK/JNK y p38, en tanto que PCA ejerce el efecto contrario. Adicionalmente se encontró que la tiorredoxina incrementa la activación/fosforilación de AKT, mientras que la quinasa p38 induce la defosforilación de AKT.

Inhibition of human mesangial cell proliferation by targeting T-Type calcium channels

Background: Aberrant glomerular mesangial cell (MC) proliferation is a common finding in renal diseases. T-type calcium channels (T-CaCN) play an important role in the proliferation of a number of cell types, including vascular smooth muscle cells. The hypothesis that T-CaCN may play a role in the proliferation of human MC was investigated. Methods: The presence of T-CaCN in primary cultures of human MC was examined using voltage clamping and by RT-PCR. The effect of calcium channel inhibitors, and of siRNA directed against the Cav3.2 T-CaCN isoform, on MC proliferation was assessed using the microculture tetrazolium assay and nuclear BrdU incorporation. Results: Human MC express only the Cav3.2 T-CaCN isoform. Co-incubation of MC with a T-CaCN inhibitor (mibefradil, TH1177 or Ni2+) results in a concentration-dependent attenuation of proliferation. This effect cannot be attributed to direct drug-induced cytotoxicity or apoptosis and is not seen with verapamil, an L-type channel blocker. Transfection of MC with siRNA results in knockdown of T-CaCN Cav3.2 mRNA and a clear attenuation of MC proliferation. Conclusions: These results demonstrate for the first time an important role for T-CaCN in human MC proliferation. This could potentially lead to a novel therapy in the treatment of proliferative renal diseases.

In Vitro Evaluation of Acellular Dermal Matrix as a Three-Dimensional Scaffold for Gingival Fibroblasts Seeding

Background: Tissue engineering principles could improve the incorporation of acellular dermal matrix (ADM). The aim of this study is to verify if ADM is a suitable three-dimensional matrix for gingival fibroblasts and cancerous cells ingrowth, and also if cultured medium conditioned in ADM affect cellular behavior. Methods: Canine gingival fibroblasts (CGF), human gingival fibroblasts (HGF), and murine melanoma cell line (B16F10) were seeded on ADM for up to 14 days. The following parameters were assessed: morphology and distribution of CGF, HGF, and B16F10; CGF and HGF viability; and the effect of ADM conditioned medium (CM) on CGF viability. Results: Epifluorescence revealed that CGF were unevenly distributed on the ADM surface, showing no increase in cell number over the periods of study; HGF formed a monolayer on the ADM surface in a higher number at 14 days (P<0.05); B16F10 exhibited an increase in cell number within 7 days (P<0.05), and were mainly arranged in cell aggregates on the ADM, forming a continuous layer at 14 days. A higher percentage of cells on the ADM surface (P<0.05) compared to inside was observed for all cell types. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MU) values indicated higher cell viability in samples cultured with HGF compared to CGF (P=0.024). A significantly lower cell viability for CGF grown in CM compared to cells grown in non-CM was observed at 48 and 72 hours (P<0.05). Conclusions: ADM is not suitable as a three-dimensional matrix for gingival fibroblasts ingrowth. Gingival fibroblasts and highly proliferative cells as B16F10 can only be superficially located on ADM, and CGF are negatively affected by culture medium conditioned in ADM, reducing its viability. J Periodontol 2011;82:293-301.

Bcl-2 expression and apoptosis induction in human HL60 leukaemic cells treated with a novel organotellurium(IV) compound RT-04

Organotellurium(]V) compounds have been reported to have multiple biological activities including cysteine protease-inhibitory activity, mainly cathepsin B. As cathepsin B is a highly predictive indicator for prognosis and diagnosis of cancer, a possible antitumor potential for these new compounds is expected. In this work, it was investigated the effectiveness of organotellurium(IV) RT-04 to produce lethal effects in the human promyelocytic leukaemia cell line HL60. Using the MTT tetrazolium reduction test, and trypan blue exclusion assay, the IC50 for the compound after 24 h incubation was 6.8 and 0.35 mu M, respectively. Moreover, the compound was found to trigger apoptosis in HL60 cells, inducing DNA fragmentation and caspase-3, -6, and -9 activations. The apoptsosis-induced by RT-04 is probably related to the diminished Bcl-2 expression, observed by RT-PCR, in HL60-treated cells. In vivo studies demonstrated that the RT-04 treatment (2.76 mg/kg given for three consecutive days) produces no significant toxic effects for bone marrow and spleen CFU-GM. However, higher doses (5.0 and 10 mg/kg) produced a dose-dependent reduction in the number of CFU-GM of RT-04-treated mice. These results suggest that RT-04 is able to induce apoptosis in HL60 cells by Bcl-2 expression down-modulation. Further studies are necessary to better clarify the effects of this compound on bone marrow normal cells. (C) 2008 Elsevier Ltd. All rights reserved.

Pharmacological and local toxicity studies of a liposomal formulation for the novel local anaesthetic ropivacaine

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Adesão, proliferação e genotoxicidade celular de celulose bacteriana modificada por plasma

Bacterial cellulose (BC) has a wide range of potential applications, namely as temporary substitute skin in the treatment of skin wounds, such as burns, ulcers and grafts. Surface properties determine the functional response of cells, an important factor for the successful development of biomaterials. This work evaluates the influence of bacterial cellulose surface treatment by plasma (BCP) on the cellular behavior and its genotoxicity potential. The modified surface was produced by plasma discharge in N2 and O2 atmosphere, and the roughness produced by ion bombardment characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Cell adhesion, viability and proliferation on BCP were analysed using crystal violet staining and the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium (MTT) method. Genotoxicity was evaluated using the comet and cytokinesis block micronucleus assay. The results show that the plasma treatment changed surface roughness, producing an ideal cell attachment, evidenced by more elongated cell morphology and improved proliferation. The excellent biocompatibility of BCP was confirmed by genotoxicity tests, which showed no significant DNA damage. The BCP has therefore great potential as a new artificial implant

Viabilidade de sementes de acerola (Malpighia emarginata DC): avaliação da vitalidade dos tecidos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Controle em pós-emergência e características germinativas de agriãozinho

O agriãozinho é uma planta daninha de grande importância em pastagens do Brasil e apresenta destacada agressividade, sendo seu controle, portanto, desejável para o sucesso da produção forrageira. O objetivo deste trabalho foi avaliar o controle químico de Synedrellopsis grisebachii na fase reprodutiva e as suas consequências sobre as características germinativas dos aquênios da planta daninha. Os tratamentos constaram da aplicação dos herbicidas glyphosate (100, 200, 900 e 1.800 g ha-1), paraquat (34, 68, 300 e 600 g ha-1) e triclopyr (75, 150, 667 e 1.334 g ha-1), além da testemunha sem aplicação. Foram coletados aquênios aos 15 dias após a aplicação, sendo estes submetidos ao teste de germinação, determinando-se a porcentagem e o índice de velocidade de germinação. Após 29 dias em germinação, verificou-se a viabilidade dos aquênios não germinados, através do teste de tetrazólio. A eficácia dos herbicidas foi avaliada por meio de notas visuais de controle aos 7, 14, 21 e 28 DAA. Conclui-se que para o controle total de S. grisebachii, em estádio reprodutivo, é necessária a aplicação de 1.334 g ha-1 de triclopyr. Nesse estádio, a planta apresentou grande tolerância ao glyphosate e também ao paraquat. Quanto às características germinativas da progênie, o herbicida triclopyr nas doses de 150 e 667 g ha-1 promoveu redução na velocidade de germinação e na viabilidade, enquanto o glyphosate e paraquat não proporcionaram efeito.

Preliminary study on age- and sex-dependent alterations in the composition of skeletal muscle fibers of Brasileiro de Hipismo horses

The aim of this work was to characterize the distribution of myofibers in the gluteus medius muscle of inactive horses of the Brasileiro de Hipismo (BH) breed at different ages by means of histochemical analyses, according to sex and depth of the biopsy. A total of 78 inactive horses (9 castrated males, 35 stallions, and 34 females) of the BH breed, aged 1 to 4 years, were used. A percutaneous muscle biopsy was obtained with a 6.0-mm Bergstrom-type needle, which allowed the removal of muscle fragments at depths of 20 and 60 mm. Myofiber types were determined based on myofibrillar adenosine triphosphatase (mATPase) and nicotinamide dinucleotide tetrazolium reductase (NADH-TR) techniques. Morphometry of the fibers was determined based on cross-sectional area (CSA), mean frequency (F), and relative cross-sectional area (RCSA). The current study demonstrated that BH horses 3 and 4 years of age show a greater percentage of, and area occupied by, type IIA fibers and lower percentage of type IIX fibers in the gluteus medius muscle compared with horses 1 and 2 years of age. No difference was found between sexes in the frequency of and area occupied by the different fiber types at any of the depths and ages studied. In this study, females showed a greater CSA for all fibers in comparison with males, at 1 year of age. The results of the current study indicate that the gluteus medius muscle of inactive BH horses shows modifications in its structural and biochemical composition during the growth of the animals, leading to a better oxidative capacity.

Light exercise causes adaptive changes in muscles of young Brasileiro de Hipismo horses

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Metodologia alternativa para condução do teste de envelhecimento acelerado em sementes de milho

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Alternative procedure for the cold test for soybean seeds

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Método alternativo para submeter sementes de amendoim à solução de tetrazólio

A técnica para a determinação da viabilidade de sementes de amendoim por meio do tetrazólio (TZ) é, atualmente, aquela pela qual as sementes, após embebição em água, têm o tegumento removido. A seguir as sementes são, em grupo, imersas na solução de TZ. Essa técnica dificulta o contato entre os tecidos das faces internas dos cotilédones e a solução de TZ em virtude de que o período prévio de embebição em água não é suficiente para que os cotilédones das sementes de amendoim se separem. Assim, o acesso da solução de TZ aos tecidos das faces internas dos cotilédones e ao próprio eixo embrionário, que fica entre as duas faces cotiledonares internas, é consideravelmente dificultado. Na suposição de que este procedimento levaria a resultados de testes de TZ que se correlacionam mal com o desempenho germinativo das sementes no campo, tentou-se uma nova técnica pela qual as sementes, após a embebição em água, além de terem o tegumento removido, são manualmente separadas em seus cotilédones, com o eixo embrionário ligado a um deles, e os cotilédones de uma determinada semente, agora separados, imersos na solução de TZ contida em uma célula individual de uma bandeja de PVC. A hipótese aqui formulada mostrou-se correta : a técnica alternativa de submissão das sementes de amendoim à solução de tetrazólio permitiu a obtenção de resultados que, comparados com os da técnica convencional, refletiram de modo mais preciso o desempenho germinativo das sementes no campo.

Pré-condicionamento das sementes de mamoneira para o teste de tetrazólio

Preconditioning of castor bean seeds for the tetrazolium test. This research had the objective of standardizing the methodology for preconditioning of castor bean (Ricinus communis L.) seeds for the evaluation of their physiological potential by the tetrazolium test. The evaluated seed preconditioning methods were: seeds with coat between moist paper towel at 30, 35 and 40 degrees C for 6, 8, 10, 12, 14, 16 and 18 hours; and seeds without coat between moist paper towel; and seeds with coat immersed in water, at 25, 30, 35 and 40 degrees C for 1, 2, 3, 4, 5 and 6 hours. After preconditioning, the seed coat was removed, the seeds were cut lengthwise, and immersed in tetrazolium solution at a concentration of 0.2% for 120 minutes at 35 degrees C. The seeds' germination percentage, moisture content before and after preconditioning, and staining uniformity after the tetrazolium were evaluated. The statistical design was completely randomized, and the means comparisons were accomplished by the Tukey test at 0.05 level of probability. It was concluded that castor bean seeds should be preconditioned with coat between moist paper towel at 35 degrees C for 12 hours, so that the results of the tetrazolium test will be similar to those obtained in the germination test.