In vitro study of the Nd : YAG laser effect on human dental enamel: Optical and scanning electron microscope analysis

Objective: the Nd:YAG laser irradiation of dental enamel was evaluated in enamel demineralization experiments in a Streptococcus mutans culture media. Summary Background Data: Previous studies had shown that a continuous wave Nd:YAG laser at an energy of approximately 67 mJ may induce an increased acid resistance in human dental enamel when exposed to severe demineralization conditions. Methods: Enamel windows of 3 x 4 cm in the buccal surface were irradiated with a continuous wave Nd:YAG laser at a wavelength of 1,064 Ecm using energy densities of from 83.75 to 187.50 J/cm(2), Enamel windows of 3 x 4 cm on the lingual surface served as control (without the laser irradiation). The enamel windows were then exposed to a Streptococcus mutans culture media at a temperature of 37 degrees C for 15 and 21 days. The laser effects and demineralization were examined both by optical microscopy and scanning electron microscopy (SEM), Results: A comparison between the lased and the unlased windows of enamel showed fusion and recrystalization of the enamel and increased acid-resistance in all groups irradiated with the Nd:YAG laser, on the other hand, the 3 x 4 delimited enamel surfaces from the control group (not irradiated with the Nd:YAG laser) showed 100% deminerization, Conclusions: These findings are consistent with the finding that laser irradiation of dental results in significant reduction of the effective solubility of enamel mineral.

Penicillium expansum versus antagonist yeasts and patulin degradation in vitro

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Ultrastructural analysis of in vitro direct and indirect organogenesis

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

Influência do diâmetro e da fase folicular sobre a competência in vitro de oócitos obtidos de novilhas da raça Nelore

Avaliou-se o efeito do diâmetro e da fase do desenvolvimento folicular sobre a competência de oócitos para a produção in vitro de embriões bovinos. A primeira onda folicular foi sincronizada com progestógeno por nove dias e 24 horas após a sua retirada aplicou-se LH. Os ovários foram recuperados 60h (G-60), 96h (G-96) e 108h (G-108) após a ovulação induzida pelo LH. Os folículos foram dissecados ou aspirados e medidos e os oócitos recuperados e submetidos à maturação, fecundação e cultivo in vitro. Os ovários do G-60 apresentaram mais oócitos viáveis (graus I, II e III) (96,6%). A taxa de clivagem teve efeito significativo sobre o diâmetro folicular, sendo maior nos oócitos oriundos de folículos classe 3 (>7mm). Na taxa de produção de blastocisto observou-se interação diâmetro versus fase de desenvolvimento folicular. A taxa de produção de blastocisto foi maior em oócitos obtidos de folículos com diâmetros <5mm (classe 1) no G-60 (64,5%), de 5-7mm (classe 2) no G-96 (33,3%) e >7mm (classe 3) no G-108 (50%). Conclui-se que o diâmetro e a fase de desenvolvimento folicular influenciam a competência oocitária para o desenvolvimento in vitro. Nos estádios iniciais da onda folicular a produção de blastocisto foi maior em oócitos de folículos pequenos; com o avanço da onda, a produção de blastocistos foi maior em oócitos obtidos de folículos maiores.

Efeitos da escarificação química e da concentração de nitrogênio sobre a germinação e o desenvolvimento in vitro de Vanilla planifolia Jack ex Andr. (Orchidaceae: Vanilloideae)

Vanilla planifolia é uma espécie com grande valor comercial, porém sua propagação é dificultada devido à baixa germinação de suas sementes. No presente estudo procurou-se avaliar a influência da escarificação destas sementes por meio da imersão em H2SO4 concentrado durante 60, 120 e 180 segundos, bem como de diferentes concentrações de nitrogênio sobre a germinação e o desenvolvimento das plântulas. Sessenta segundos de escarificação e 25% de nitrogênio no meio de cultura proporcionaram os melhores resultados sobre a germinação e desenvolvimento de V. planifolia, constituindo-se em um procedimento viável para a produção comercial dessa espécie.

Estudo das poliaminas na morfogênese in vitro de Hemerocallis sp.

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Efeito do boro sobre os teores de proteína e atividade da peroxidade em calos de cana-de-açúcar (Saccharum spp. var NA56-79) in vitro

Calos de cana-de-açúcar variedade NA56-79, foram desenvolvidos em meio de cultura contendo sais minerais acrescidos de hormônios e vitaminas. Após a obtenção de quantidade suficiente de material, foram submetidos a três níveis diferentes de boro (omisso, 6,2 mg/L, 12,4mg/L). Foram analisados o teor protéico e a atividade da peroxidase. Os resultados mostraram que, em relação ao teor protéico, este foi menor na deficiência e maior em níveis mais elevados de boro. A variação da atividade da peroxidase mostrou um resultado inverso ao da proteína.

Multiplicação in vitro DE Ficus carica L.: efeito da cinetina e do ácido giberélico

A cultura da figueira é afetada pelo vírus-do-mosaico e a cultura de tecidos é uma alternativa para se proceder à limpeza clonal. Neste trabalho, objetivou-se estudar o efeito da cinetina e GA3 na multiplicação in vitro da figueira. Segmentos nodais foram inoculados em meio de cultura WPM contendo as seguintes combinações de cinetina (0; 0,5; 1; 2 e 4 mg.L-1) e GA3 (0, 2, 4, 6 e 8 mg.L-1). Avaliaram-se número e comprimento dos brotos, peso da matéria fresca e seca da parte aérea, número de raízes, peso da matéria fresca e seca do sistema radicular e de calos. A utilização de 0,5 mg.L-1 de cinetina promoveu melhor taxa de multiplicação in vitro de Ficus carica. O GA3 reduziu a formação e multiplicação dos brotos e induziu ao estiolamento, à hiperidricidade, clorose e necrose apical das plântulas.

Multiplicação in vitro de amoreira-preta cultivar Brazos

A micropropagação da amoreira-preta pode gerar plantas livres de vírus e em curto espaço de tempo. Com o objetivo de aprimorar técnicas de micropropagação de amoreira-preta cultivar Brazos (Rubus idaeus L.), segmentos nodais, oriundos de plântulas preestabelecidas in vitro foram excisados e inoculados em meio WPM (0, 50, 100, 150 e 200%), suplementado com diferentes concentrações de BAP (0; 0,5; 1,0; 2,0 e 4,0 mg L-1). Após a inoculação, os explantes foram transferidos para sala de crescimento a 27±1ºC, irradiância de 35 mmol m² s¹ e fotoperíodo de 16 horas, onde permaneceram por 60 dias. O delineamento experimental utilizado foi inteiramente casualizado, utilizando-se de quatro repetições com quatro explantes cada. Maior número de brotos foi proporcionado com 1,0 mg L-1 de BAP associado a 100% de meio WPM e maior comprimento médio dos brotos após 60 dias foi verificado em 1,0 mg L-1 de BAP associado a 200% de meio WPM. Maior peso de matéria seca da parte aérea foi obtido em meio WPM 200% acrescido de 0,5 mg L-1 de BAP.

Expression of pathogenicity-related genes of Xylella fastidiosa in vitro and in planta

Xylella fastidiosa is responsible for several economically important plant diseases. It is currently assumed that the symptoms are caused by vascular occlusion due to biofilm formation. Microarray technology was previously used to examine the global gene expression profile of X. filstidiosa freshly isolated from symptomatic plants or after several passages by axenic culture medium, and different pathogenicity profiles have been obtained. In the present study the expression of some pathogenicity-related genes was evaluated in vitro and in planta by RT-PCR. The results suggest that adhesion is important at the beginning of biofilm formation, while the genes related to adaptation are essential for the organism's maintenance in planta. Similar results were observed in vitro mainly for the adhesion genes. The pattern of expression observed suggests that adhesion modulates biofilm formation whereas the expression of some adaptation genes may be related to the environment in which the organism is living.

New insights into the in vitro organogenesis process: the case of Passiflora

We present evidences that ultrastructural electron microscope findings are valuable ways to understand the in vitro regeneration process, in particular in the yellow passion fruit. Shoot-regeneration was induced in hypocotyl and leaf-derived explants using 4.44 mu M BAP, and the entire organogenic process was analyzed using conventional histology, scanning and transmission electronic microscopy. Both direct and indirect regeneration modes were observed in hypocotyl explants, but only direct regeneration occurred in leaf-derived cultures. In the direct pathway from both explant types, meristemoids developed into globular structures, here called protuberances. The peripheral meristematic layers of the protuberances displayed ultrastructural characteristics indicative of a high metabolic activity, and only these cells originated shoots and leaf primordia, the latter being frequent when leaf explants were used. Moreover, the peripheral cells of the protuberances derived from leaf explants lost adhesion during the culture, diminishing the regeneration rates. We recommend the use of hypocotyls as a source of explant to obtain shoots as well as a genetic transformation system for the yellow passion fruit. However, the direct pathway is preferred because a type of amitosis occurred in the peripheral cells of hypocotyl-derived calli, which has the potential to result in genetic instability of the regenerating plants/tissue.

Synthesis and in vitro evaluation of potential antichagasic dipeptide prodrugs of primaquine

American trypanosomiasis (Chagas' disease) is an endemic parasitic disease afflicting more than 20 million people in Latin America. Currently, therapy is unsatisfactory and only two drugs are available. Primaquine, an antimalarial drug, has trypanocidal activity. Dipeptide derivatives of primaquine, Phe-Arg-PQ, Lys-Arg-PQ, and Phe-Ala-PQ, were synthesized. The choice of the peptides was based on the primary specificity of cruzipain, the major cysteine proteinase from T. cruzi. The prodrugs obtained were tested on the LLC-MK2 cell culture infected with trypomastigotes forms of T. cruzi Phe-Arg-PQ, Lys-Arg-PQ, and Phe-Ala-PQ were active in all stages.

Biocompatibility of gutta-percha solvents using in vitro mammalian test-system

Objectives. Taking into consideration that DNA damage and cellular death play important roles during carcinogenesis, the purpose of the present study was to evaluate in vitro genotoxic or cytotoxic effects of chloroform and eucalyptol by single cell gel (comet) assay and trypan blue exclusion test, respectively.Study design. Chloroform and eucalyptol were exposed to Chinese hamster ovary cells in culture directly for 3 hours at 37 degrees C at final concentrations ranging from 1.25 to 10 mu L/mL. The negative control group was treated with vehicle control (phosphate-buffered solution), and the positive control group was treated with methyl metasulfonate (MMS, at 1 mu g/mL concentration). All data were analyzed by the Kruskal-Wallis nonparametric test followed by the Dunn test.Results. The results showed that both gutta-percha solvents were cytotoxic at concentrations of 2.5, 5, and 10 mu L/mL (P < .05). on the other hand, both solvents did not induce DNA breakage at 1.25 mu L/mL concentration.Conclusions. These results suggest that both chloroform or eucalyptol are strong cytotoxicants, but they may not be a factor that increases the level of DNA lesions in mammalian cells.

In vitro effects of endodontic irrigants on endotoxins in root canals

Objective. The objective of this study was to evaluate the effects of endodontic irrigants on endotoxins in root canals.Study design. Ninety-eight single-root human teeth were used. Escherichia coli endotoxin was inoculated into 84 root canals. All root canals were enlarged and assigned to 7 groups (n = 14), according to solution used. Group 1 (G1): 2.5% NaOCl; G2: 5.25% NaOCl; G3: 2% chlorhexidine; G4: 0.14% calcium hydroxide; G5: polymyxin B; G6: positive control, saline solution; G7: negative control (no endotoxin). Two samplings of root canal were accomplished: immediate and after 7 days. Detoxification of endotoxin was evaluated by Limulus assay and antibody production in B-lymphocyte culture. Results were analyzed by Kruskal-Wallis/Dunn and ANOVA/Tukey.Results. At the immediate and second samplings, groups G4, G5, and G7 presented the best results, significantly different from groups G1, G2, G3, and G6 (P = .05).Conclusions. Calcium hydroxide and polymyxin B detoxified endotoxin in root canals and altered properties of LPS to stimulate the antibody production by B-lymphocytes. Sodium hypochlorite and chlorhexidine did not detoxify endotoxin.

Interleukin-10 secreted by B-1 cells modulates the phagocytic activity of murine macrophages in vitro

As demonstrated previously in our laboratory, B-1 cells migrate from the peritoneal cavity of mice and home to a distant site of inflammation to become macrophage-like cells. However, the influence that these cells might have on the kinetics and fate of the inflammatory process is not known. Considering that macrophages are pivotal in the inflammatory reaction, we decided to investigate the possible influence B-1 cells could have on macrophage activities in vitro. Our results show that peritoneal macrophages from Xid mice, a mouse strain deprived of B-1 cells, have higher phagocytic indexes for zymozan particles when compared with macrophages from wild-type mice. Moreover, macrophages from wild-type mice have a lower ability to release nitric oxide and hydrogen peroxide when compared with macrophages from Xid mice. Experiments using cocultures of B-1 cells and macrophages from Xid mice in transwell plates demonstrated that B-1 cells down-regulate macrophage activities. These observations also indicate that this phenomenon is not due to a physical interaction between these two cell populations. As B-1 cells are one of the main sources of interleukin (IL)-10, we demonstrate in this study that adherent peritoneal cells from Xid mice produce significantly less amounts of this cytokine in culture when compared with IL-10 production by cells from wild-type mice. When B-1 cells from IL-10 knock-out mice and macrophages from wild-type mice were cocultured in transwell plates, the phagocytic index of macrophages was not altered demonstrating that B-1 cells can influence the effector functions of macrophages in vitro via IL-10 secretion.