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.

In vitro biocompatibility of a novel membrane of the composite poly(vinylidene-trifluoroethylene)/barium titanate

This study was aimed at investigating the in vitro biocompatibility of a novel membrane of the composite poly(vinylidene-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT). Osteoblastic cells were obtained from human alveolar bone fragments and cultured under standard osteogenic condition until subconfluence. First passaged cells were cultured on P(VDF-TrFE)/BT and expanded polytetrafluoroethylene (e-PTFE - control) membranes in 24-well plates. Cell adhesion and spreading were evaluated at 30 min, and 4 and 24 h. For proliferation assay, cells were cultured for 1, 7, and 10 days. Cell viability was detected by trypan blue at 7 and 10 days. Total protein content and alkaline phosphatase (ALP) activity were measured at 7, 14, and 21 days. Cultures were stained with Alizarin red at 21 days, for detection of mineralized matrix. Data were compared by ANOVA and Student t test. Cell attachment (p = 0.001), cell number (p = 0.001), and ALP activity (p = 0.0001) were greater on P(VDF-TrFE)/BT. Additionally, doubling time was greater on P(VDF-TrFE)/BT (p = 0.03), indicating a decreased proliferation rate. Bone-like nodule formation took place only on P(VDF-TrFE)/BT. The present results showed that both membranes are biocompatible. However, P(VDF-TrFE)/BT presented a better in vitro biocompatibility and allowed bone-like nodule formation. Therefore, P(VDF-TrFE)/BT could be an alternative membrane to be used in guided tissue regeneration. (c) 2006 Wiley Periodicals, Inc.

Role of roscovitine and IBMX on kinetics of nuclear and cytoplasmic maturation of bovine oocytes in vitro

The 3-isobutyl-1-methylxanthine (IBMX) is able to prevent resumption of meiosis by maintaining elevated cyclic AMP (cAMP) concentrations in the oocyte, and roscovitine, a purine known to specifically inhibit MPF kinase activity, maintains bovine oocytes at the germinal vesicle (GV) stage. The present study was conducted to analyze whether cytoplasmic maturation (examined by the pattern of cortical granule (CG) distribution) of bovine oocytes is improved during meiotic arrest with IBMX and roscovitine. Oocytes were matured in vitro in a 10% Knockout(SR) supplemented TCM-199 medium (Control) with either 0.5 mM IBMX or 25 mu M roscovitine (ROSC). Oocytes were stained with fluorescein isothiocyanate conjugated Lens culinaris agglutinin (FITC-LCA) for CG evaluation and with Hoechst 33342 for nuclear stage assessment. At 16 h of culture, the percentage of oocytes remaining in the GV stage was higher (P < 0.05) in the ROSC group (32.41%) compared with the Control and IBMX groups (8.61% and 9.73%, respectively). At 24h of culture, progression of meiosis to M II stage was retarded (P < 0.05) in the ROSC group (24.05%) compared to the Control (60.20%), whereas the IBMX group (33.88%) showed no significant difference to the other two groups. At 16h of maturation, the proportion of oocytes with CG in clusters (immature cytoplasm) was similar between the groups, as was the percentage of peripheral CG (mature) at 24h of maturation. The results of the present study demonstrated that the meiotic inhibitors IBMX and roscovitine delay the progression of nuclear maturation without affecting cytoplasmic maturation, assessed by the analysis of CG repositioning. (c) 2006 Elsevier B.V. All rights reserved.

THE EFFECT OF SERUM ON IN-VITRO MATURATION, IN-VITRO FERTILIZATION AND STEROIDOGENESIS OF BOVINE OOCYTES COCULTURED WITH GRANULOSA-CELLS

The influence of fetal calf serum alone (FCS) or associated with proestrous (FCS+PCS), estrous (FCS+ECS) or metaestrous (FCS+MCS) cow serum added to the culture medium and of the steroids produced by co-cultured granulosa cells were evaluated in terms of the in vitro maturation (TVM) and fertilization (IVF) of bovine oocytes. Supplementation of the medium with FCS+ECS and FCS+MCS resulted in higher proportions of oocytes that reached metaphase II (96.0% and 93.3%, respectively) and in higher proportions of embryos that reached the four- and eight-cell/morula stages (51.9% and 65.6%, respectively), whereas the supplementation with FCS and FCS+PCS resulted in only 79.2% and 67.5%, respectively, of matured oocytes and 26.7% and 34.3%, respectively, of cleaved embryos. These findings show that the best IVM and IVF were obtained at lower concentrations of estradiol produced by co-cultured granulosa cells (supplementation with FCS+ECS: 10.3 ng/ml and FCS+MCS: 2.1 ng/ml), whereas the worst-results in IVM and IVF occurred at higher concentrations of estradiol that were obtained with FCS (33.1 ng/ml) and FCS+PCS (19.9 ng/ml) supplementation. These data suggest an inhibitory effect of estradiol on resumption of oocyte meiosis in vitro.

In vitro study of antiviral activity of mycophenolic acid on Brazilian orthobunyaviruses

Objective: Oropouche, Caraparu, Guama, Guaroa and Tacaiuma are ssRNA viruses that belong to the genus Orthobunyavirus and have been associated with human febrile illnesses and/or encephalitis. In this study, we evaluated the antiviral action of mycophenolic acid (MPA) on these orthobunyaviruses to achieve a therapeutic agent to treat the diseases caused by these viruses. Methods: the in vitro antiviral evaluation to MPA was done by using plaque assay at different periods of treatment. Results: Results showed that MPA at a concentration of 10 mu g/ml has significant antiviral activity on Tacaiuma virus when treatment was initiated either 24 h before or 2 h after viral infection. Moreover, MPA has an inhibitory effect on Guama virus replication, but only when treatment was initiated before cell infection. Addition of guanosine in the culture reverted the inhibitory effect of MPA on Tacaiuma and Guama viruses, suggesting that the antiviral activity of this substance was via depletion of the intracellular guanosine pool. Conclusion: Our results suggest that MPA would not be a good therapeutic agent to treat the diseases caused by Oropouche, Caraparu, Guama, Guaroa, and Tacaiuma viruses.

In vitro biocompatibility of poly(vinylidene fluoride-trifluoroethylene)/barium titanate composite using cultures of human periodontal ligament fibroblasts and keratinocytes

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

Cytotoxicity, genotoxicity and antimutagenicity of hexane extracts of Agaricus blazei determined in vitro by the comet assay and CHO/HGPRT gene mutation assay

Agaricus blazei Murrill ss. Heinem, known as the sun mushroom or himematsutake, is a basidiomycete native to Brazil, which is popular for its medicinal properties. The aim of this study was to test hexane extracts (one fraction and its four sub-fractions) of A. blazei for bioactivity in cultured mammalian cells (CHO-K1). The comet assay, the colony forming assay (CFA) and CHO/HGPRT gene mutation assay were used respectively to determine genotoxicity, cytotoxicity and antimutagenicity of these extracts at different concentrations. The cells were incubated in culture medium and treated for 3 h according to the standard protocol for each assay. The DNA damage-inducing agent ethylmethane sulfonate (EMS) was utilized as the positive control and also in combination with extracts to test for a protective effect. Statistical analysis of the data was performed using analysis of variance (ANOVA) and Tukey's test. A relationship between cytotoxicity and genotoxicity could be established and two extracts EH6B and EH6D showed a protective tendency, while the others did not, with the primary extract EH6 causing the most substantial damage to genetic material. These findings warrant more in-depth studies of the active principles of this mushroom. (c) 2005 Elsevier Ltd. All rights reserved.