ABSTRACT
This manuscript aims to investigate the effects of resibufogenin on the proliferation, migration and invasion of human hepatocellular carcinoma cells and its related mechanisms. MTT assay was used to determine the inhibitory effect of resibufogenin on the growth of four hepatocellular carcinoma cells in vitro. Wound-healing assay and Transwell assay were used to evaluate the migration and invasion ability of resibufogenin on MHCC-97H cells. Western blot assay was used to detect the expression of migration and invasion related proteins in MHCC-97H cells treated with different concentrations of resibufogenin. The results showed that resibufogenin significantly inhibited the proliferation of hepatocellular carcinoma cells in vitro. The half maximal inhibitory concentration (IC50) values on MHCC-97H, HepG2, SK-Hep-1 and Huh-7 cells were 0.55 ± 0.06, 2.83 ± 0.24, 5.25 ± 0.49, 14.89 ± 2.28 μmol·L-1, respectively. Resibufogenin also suppressed the migration and invasion of MHCC-97H cells in a concentration-dependent manner. The protein expression of integrin α2, integrin α6, integrin β1, N-cadherin, matrix metalloproteinase 2 (MMP2) and transcription factor Twist in MHCC-97H cells were decreased significantly with the increase of the concentration of resibufogenin, while the protein expression of E-cadherin increased. In addition, we found that p-PI3K/PI3K and p-AKT/AKT ratios were significantly reduced after treatment with resibufogenin. In conclusion, resibufogenin can inhibit the proliferation, migration and invasion of hepatocellular carcinoma MHCC-97H cells in vitro, which is related to the regulation of intracellular migration and invasion protein expression and PI3K/AKT signaling pathway.
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ABSTRACT: The goal of the present study was to evaluate the germination, initial growth, and in vitro co-cultivation of Comanthera curralensis Moldenke, a "sempre viva" native of the Chapada Diamantina state of Bahia. Full strength (MS) and half-strength MS (MS1/2) growth media supplemented with two different sucrose concentrations (15 and 30g L-1) were tested for germination and initial plant growth. Three different plant densities were tested by in vitro culture (8, 10 and 12 plants per container). MS1/2 medium with 15g L-1 sucrose resulted in a higher percentage of germination and plant growth for the in vitro establishment of C. curralensis. The use of 12 plants per container is indicated for cost reduction in C. curralensis in vitro production.
RESUMO: Este trabalho teve como objetivo avaliar a germinação, o crescimento inicial e o co-cultivo in vitro de Comanthera curralensis Moldenke, uma "sempre viva" nativa da Chapada Diamantina-BA. Para germinação e crescimento inicial, foram testados os meios de cultura MS completo e MS1/2 suplementados com duas concentrações de sacarose (15 e 30gL-1); no cultivo in vitro, foram testadas três quantidades de plantas por recipiente (8,10 e 12). A utilização do meio MS1/2 com 15gL-1 de sacarose proporcionou maiores porcentagem de germinação crescimento das plantas no estabelecimento in vitro de C. curralensis , e o uso de 12 plantas por recipiente é indicado para a redução de custos na produção in vitro da espécie.
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Hyptis leucocephala e Hyptis platanifolia (Lamiaceae) são espécies aromáticas endêmicas do semiárido nordestino e possuem grande importância econômica devido ao seu potencial medicinal. O presente trabalho teve por objetivo obter o protocolo para o estabelecimento inicial in vitro das espécies H. leucocephala e H. platanifolia. O meio de cultura WPM promoveu a maior porcentagem de germinação (96,81%) para H. leucocephala, porém não diferiu estatisticamente dos resultados obtidos no MS½ na última semana de cultivo. Para a germinação de sementes de H. platanifolia, o MS½ foi o meio que proporcionou a maior porcentagem (40,83%). O tipo de esterilização não interferiu na germinação in vitro para as duas espécies. Para a espécie H. platanifolia, quando se utilizou o fechamento dos tubos de ensaio com tampão de algodão, a hiperidricidade foi eliminada; porém, a porcentagem de germinação das sementes foi menor. Avaliando-se o tipo de meio de cultura no crescimento in vitro de H. leucocephala observou-se que este fator não influenciou no número de brotações, nem o comprimento de parte aérea e da matéria seca de raiz. O meio MS proporcionou incremento para o número de folhas, matéria seca da parte aérea, e comprimento da maior raiz, enquanto o meio MS½ foi o que promoveu maior número de raízes. O meio de cultura ideal para a germinação de H. leucocephala e H. platanifolia foi o MS½, podendo ser esterilizado com hipoclorito de sódio, enquanto que o MS foi o melhor meio de cultura para o crescimento in vitro de H. leucocephala.
The Hyptis leucocephala and Hyptis platanifolia (Lamiaceae) are aromatic species, endemic to the Brazilian Semiarid northeast, and they have great economic importance becaude of their pharmacological potential. This study aimed to obtain the protocol for the in vitro establishment of H. leucocephala and H. platanifolia plants. The WPM promoted higher germination percentage (96.81%) for H. leucocephala, but there was no difference between the results obtained in ½MS, in the last week of cultivation. For the germination of H. platanifolia, ½MS was the one that provided the highest percentage (40.83%). The type of sterilization did not affect the in vitro germination of both species. For the H. platanifolia species, when we closed the tubes with a cotton plug, hyperhydricity was eliminated, but the percentage of seed germination was the lowest one. Evaluating the type of culture medium on the in vitro growth of H. leucocephala, it did not influence the number of shoots, length of shoot or root dry matter. The MS medium promoted an increase of the number of leaves, dry shoot and longest root length, and the ½MS medium was more suitable for root induction. The optimal culture medium for the germination of H. leucocephala and H. platanifolia is ½MS, as it can be sterilized with sodium hypochlorite, while the MS was the best culture medium for the in vitro growth of H. leucocephala.
Subject(s)
In Vitro Techniques/methods , Germination , Hyptis/growth & developmentABSTRACT
Aims: Basal stem rot (BSR) disease caused by Ganoderma pathogenic fungi, especially Ganoderma boninense is thriving rapidly in both areas with coastal and inland soils. The objectives of this study were to isolate and characterize Ganoderma isolates collected from various locations in Peninsular Malaysia through the comparison of their growth rate in vitroly on conventional and novel palm extract media, and to determine the degree of virulence caused by the isolates in oil palm seedlings. Methodology and results: In this study, 12 Ganoderma isolates were collected from infected oil palm trees, from various locations – Johor, Negeri Sembilan, Kedah, Perak, Pahang, and Kelantan, in Malaysia in year 2011. Twelve Ganoderma isolates were identified using molecular method with primer set that targeted at small-subunit 18S rDNA fragment, and characterized by determining the in vitro growth rate, and degree of virulence in 2-month-old oil palm seedlings in the nursery using both disease incidence (DI) and disease severity index (DSI) as the measurements to quantify the infection. All the Ganoderma isolates were identified as G. boninense and sequences of the respective isolates were deposited in GenBank. In general, all the isolates proliferated faster on oil palm extract medium (OPEM) compared to malt extract agar (MEA). Twelve G. boninense isolates were observed to illustrate different degree of virulence ranging from highly pathogenic to least pathogenic. Conclusion, significance and impact of study: Cultures of 12 G. boninense isolates were observed to show faster growth rate (P < 0.014) on OPEM under in vitro conditions compared to conventional MEA medium, except Bt Lintang G10 and GBA G12 isolates. OPEM medium could provide a better alternative for maintaining and culturing Ganoderma strains. In the current study, both DI and DSI were highly correlated. However, there were low linear relationships (R2 < 0.423) between mycelia growth rate (on MEA and OPEM) and degree of virulence (DI and DSI) at 12-, 14- and 16- weeks after treatments among the G. boninense isolates tested. Furthermore, different degrees of virulence in twelve separate Ganoderma isolates were reported. Therefore, it is crucial to incorporate more than one isolate into any researches on screening for Ganoderma resistance or tolerance planting materials, searching for potential biological control agents, and studying bitrophic or tri-trophic interactions. In addition, this study was aimed to isolate G. boninense strains with various virulence levels for future studies.
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Foi efetuada a comparação em hamsters da proteção conferida e dos níveis de anticorpos induzidos por duas bacterinas comerciais antileptospirose. Os ensaios empregados foram o teste oficial de potência com desafio (TP), o ensaio proposto, teste de inibição de crescimento de leptospiras in vitro (ICLIV) e a soroaglutinação microscópica (SAM). O protocolo de imunização foi representado por duas aplicações individuais de 0,25 mL das bacterinas, puras ou de suas diluições geométricas de razão dois variando de 200 a 51.200 para a bacterina A e de 200 a 3.200 para a bacterina B, por via subcutânea com o intervalo de 15 dias. Decorridos 15 dias da segunda aplicação de vacina, um grupo de animais foi desafiado com 0,2 mL de cultivos de leptospiras, por indivíduo, respectivamente dos sorovares Canicola (bacterinas A e B) ou Kennewicki (bacterina A). Os números de doses infectantes empregados nos desafios foram de 100 e 631 respectivamente, para os sorovares Canicola e Kennewicki. Decorridos 21 dias do desafio, os grupos de animais utilizados nos testes de ICLIV e SAM foram sangrados e os seus soros foram reunidos em pools (n = 5). No TP, adotando-se os critérios internacionais, as bacterinas foram aprovadas. A comparação do desempenho das bacterinas para os sorovares adotados, segundo sua concentração, por meio das proporções de animais sobreviventes ao TP e a média dos títulos de anticorpos identificados no teste de ICLIV, indicou que um título igual ou superior a 0,77 log corresponde ao nível de aprovação da bacterina no TP.
It was performed a comparison between the protection afforded in hamsters and the antibody levels induced by two commercial vaccines against leptospirosis. The assays used were the official challenge test (TP), the in vitro leptospires growth inhibition test (ICLIV) and microscopic agglutination test (MAT). The immunization protocol consisted of two single applications, 15 days from each other, of 0.25 mL of the bacterins, pure or its two-fold serial dilutions: 200 to 51,200 for bacterin A and 200 to 3.200 bacterin B, both of them administered subcutaneously. A group of animals was challenged, after 15 days from the second vaccine application, with 0.2 mL/animal of live leptospire cultures, with Canicola (bacterin A and B) or Kennewicki (bacterin A) serovars. The numbers of infective doses employed in the challenges were 100 and 631 for Canicola and Kennewicki serovars, respectively. After 15 days from the second vaccine dose the groups of animals used in ICLIV and SAM tests were bled and their sera were collected in pools (n = 5). In TP, adopting the criteria established by the Code Federal Regulation, both bacterins were approved. The comparison of the performance of the tested bacterins with the adopted serovars, according to its concentration, by the proportions of surviving animals to the challenge assay and the average of the neutralizing antibodies titers, established a neutralizing antibodies titer equal or higher than 0.77 log corresponding with the bacterin level of approval in the potency assay.
Subject(s)
Animals , Antibodies/administration & dosage , Antibodies/analysis , Leptospirosis/immunology , Leptospirosis/veterinary , Mesocricetus/immunology , Vaccine Potency , Vaccines/administration & dosageABSTRACT
Aiming at evaluating genotype influence on the concentration of protein and oil, immature seeds of cultivars CD 202 and CD 206 were removed from the mother-plant, in the stage R5, and were grown in vitro, in a liquid culture medium which contained 20, 40 and 60 mM of glutamine, during eight days. Afterwards, the concentrations of oil and protein were compared to the contents of the seeds cultivated in vivo. With a higher availability of glutamine for the seed, there was an increase of protein content. The genotypes were statistically different as far as the protein concentration was concerned,which confirmed that the genotype had influence on the concentration of protein in the seed. Oil and protein concentrations were inversely related when a variation of glutamine concentration occurred.
Com o objetivo de avaliar a influência do genótipo sobre as concentrações de proteína e óleo, sementes imaturas das cultivares de soja CD 202 e CD 206 foram retiradas da planta mãe no estádio R5, cultivadas in vitro em meio de cultura líquido por oito dias, contendo 20, 40 e 60 mM de glutamina. Depois disso, as concentrações de óleo e proteína foram comparadas com as das sementes que continuaram seu desenvolvimento in vivo. Com a maior disponibilidade de glutamina para a semente, houve um aumento do conteúdo protéico nas sementes. Quanto às concentrações de proteína, os genótipos foram estatisticamente diferentes, levando a afirmar a sua influência na concentração de proteína na semente. As concentrações de proteína e óleo foram inversamente relacionadas quando variou a concentração de glutamina.
ABSTRACT
OBJECTIVE: This present study was conducted to examine the effects on in vitro growth of pre-antral mouse follicles by thecal-stromal cells attached to around the follicular membrane in culture medium without hormones. METHODS: Pre-antral follicles (100-130 micro meter) used in our studies were isolated mechanically by fine 30 G needles attached to 1 ml insulin syringe from mice ovaries of 20-25 days old female ICR strain. Isolated pre-antral follicles were divided into three groups by attached status of thecal-stromal cell layers to follicular membrane. Follicular Initial diameters of group I was 112.5 (mean) +/- 6.2 (SD) micro meter (n=31), group II was 112.8 +/- 7.8 micro meter (n=23), and group III was 115.1 +/- 6.5 micro meter (n=27). Divided pre-antral follicles were washed in fresh Ham's F-10 medium and cultured in 20 micro liter droplets of DMEM with glutamine, glucose and pyruvate under mineral oil on the 60 mm culture dish. All experimental media were supplemented with 10% FBS without hormones. Media were exchanged wholly by fresh media every 2 days in culture. Diameters of intrafollicular oocytes and cultured pre-antral follicles were measured using an precalibrated cross ocular micrometer at X200 every day during 6 days in vitro culture. Results were considered statistically significant when p value was less than 0.05 using Student's t-test. RESULTS: The mean and standard deviation (SD) of initial diameters of pre-antral follicles was 113.5 +/- 2.2 micro meter in three groups. Follicular growth rate of group III was significantly (p<0.05) higher in whole culture periods than group I and group II. Rate of follicular growth between group I and group II were not significant difference in culture for 1 to 3 days. However, group II was significantly higher than group I in culture for 4 days. Diameters of grown follicles for 6 days was 155.2 +/- 18.7 micro meter, 196.9 +/- 24.1 micro meter and 284.2 +/- 47.6 micro meter in group I, group II and group III, respectively. Growth rate of intrafollicular oocytes between three groups were not difference and revealed to continuous growth patterns in whole culture periods. Follicular diameter after culture for 7 days were not measured because of disruption of follicular structure or outgrowth of granulosa cells. CONCLUSION: Pre-antral follicles were grown very well in DMEM medium without hormones. Thecal- stromal cells attached to the surface of follicular membrane has acted effectively in vitro growth of follicles.
Subject(s)
Animals , Female , Humans , Mice , Glucose , Glutamine , Granulosa Cells , Insulin , Membranes , Mineral Oil , Needles , Oocytes , Ovary , Pyruvic Acid , Stromal Cells , SyringesABSTRACT
No abstract available.