RESUMO
The nonclinical safety evaluation of therapeutic drug candidates is commonly conducted in two species (rodent and non-rodent) in keeping with international health authority guidance. Biologic drugs typically have restricted species cross-reactivity, necessitating the evaluation of safety in non-human primates and thus limiting the utility of lower order species. Safety studies of cross-reactive ocular biologic drug candidates have been conducted in rabbits as a second toxicology species, despite the fact that rabbits are not a rodent species. Such studies are often confounded by the development of anti-drug antibodies and severe ocular inflammation, the latter requiring studies to be terminated prematurely for animal welfare reasons. Notably, these confounding factors preclude the interpretation of safety. Nonclinical toxicology programs should be designed with consideration of ethical animal use and 3Rs principles (Replacement, Reduction and Refinement). The experience of several pharmaceutical sponsors, demonstrating that toxicology studies of ocular (intravitreal and topical ocular) biologic drug candidates in the rabbit are of limited interpretive value, calls into question the utility of such studies in this species and indicates that such studies should not be conducted.
Assuntos
Produtos Biológicos/efeitos adversos , Avaliação Pré-Clínica de Medicamentos/métodos , Oftalmopatias/imunologia , Coelhos , Animais , Olho/imunologia , Inflamação/imunologia , Especificidade da EspécieRESUMO
PURPOSE: To assess the clinical safety, tolerability, and efficacy of topically administered MGV354, a soluble guanylate cyclase (sGC) activator, in patients with ocular hypertension (OH) or glaucoma. DESIGN: Double-masked, randomized, and vehicle-controlled study. METHODS: Parts 1 and 2 evaluated safety and tolerability to identify the maximum tolerated dose (MTD) of once-daily MGV354 in 32 healthy volunteers (Part 1) and 16 patients with OH or glaucoma (Part 2) at a single clinical site. Part 3 was a multisite trial that evaluated intraocular pressure (IOP)-lowering efficacy of the MTD administered nightly for 1 week in 50 patients with minimum IOP of 24 mm Hg at 8 AM, with a main outcome measure of mean diurnal IOP at day 8 compared to baseline (ClinicalTrials.govNCT02743780). RESULTS: There was no difference in favor of MGV354 for IOP lowering; change from baseline to day 8 in mean diurnal IOP was -0.6 mm Hg for MGV354-treated patients and -1.1 mm Hg for vehicle-treated patients in Part 3, with a confidence interval of -0.7 to 1.7. The most common adverse events reported after MGV354 administration were conjunctival and ocular hyperemia. CONCLUSIONS: Overall, MGV354 0.1% demonstrated no statistically significant effect compared to vehicle in lowering IOP based on the study's main outcome measure. MGV354 produced ocular hyperemia consistent with its pharmacology.
Assuntos
Ativadores de Enzimas/uso terapêutico , Glaucoma de Ângulo Aberto/tratamento farmacológico , Guanilato Ciclase/metabolismo , Pressão Intraocular/efeitos dos fármacos , Piperidinas/uso terapêutico , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Administração Oftálmica , Adolescente , Adulto , Idoso , Método Duplo-Cego , Ativadores de Enzimas/efeitos adversos , Feminino , Glaucoma de Ângulo Aberto/fisiopatologia , Humanos , Pressão Intraocular/fisiologia , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Nível de Efeito Adverso não Observado , Hipertensão Ocular/tratamento farmacológico , Hipertensão Ocular/fisiopatologia , Soluções Oftálmicas , Piperidinas/efeitos adversos , Pirazóis/efeitos adversos , Piridinas/efeitos adversos , Tonometria Ocular , Resultado do Tratamento , Acuidade Visual/fisiologia , Adulto JovemRESUMO
Purpose: The nitric oxide/soluble guanylate cyclase/protein kinase G (NO/sGC/PKG) is known to be involved in the regulation of intraocular pressure (IOP) and may be dysregulated in glaucoma. The purpose is to demonstrate that the sGC activator MGV354 lowers IOP in a monkey model of glaucoma and could be considered as a possible new clinical drug candidate. Methods: Changes to cGMP were assessed in primary human trabecular meshwork (hNTM) cells and binding studies were conducted using human sGC full-length protein. Ocular safety tolerability, exposure, and efficacy studies were conducted in rabbit and monkey models following topical ocular dosing of MGV354. Results: sGC was highly expressed in the human and cynomolgus monkey outflow pathways. MGV354 had a 7-fold greater Bmax to oxidized sGC compared to that of reduced sGC and generated an 8- to 10-fold greater cGMP compared to that of a reduced condition in hTM cells. A single topical ocular dose with MGV354 caused a significant dose-dependent reduction of 20% to 40% (versus vehicle), lasting up to 6 hours in pigmented rabbits and 24 hours postdose in a cynomolgus monkey model of glaucoma. The MGV354-induced IOP lowering was sustained up to 7 days following once-daily dosing in a monkey model of glaucoma and was greater in magnitude compared to Travatan (travoprost)-induced IOP reduction. Mild to moderate ocular hyperemia was the main adverse effect noted. Conclusions: MGV354 represents a novel class of sGC activators that can lower IOP in preclinical models of glaucoma. The potential for sGC activators to be used as effective IOP-lowering drugs in glaucoma patients could be further determined in clinical studies.
Assuntos
Anti-Hipertensivos/farmacologia , Ativadores de Enzimas/farmacologia , Glaucoma/tratamento farmacológico , Pressão Intraocular/efeitos dos fármacos , Piperidinas/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Guanilil Ciclase Solúvel/metabolismo , Administração Oftálmica , Animais , Anti-Hipertensivos/administração & dosagem , Células Cultivadas , GMP Cíclico/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ativadores de Enzimas/administração & dosagem , Glaucoma/fisiopatologia , Humanos , Imuno-Histoquímica , Macaca fascicularis , Hipotensão Ocular/tratamento farmacológico , Soluções Oftálmicas , Piperidinas/administração & dosagem , Pirazóis/administração & dosagem , Piridinas/administração & dosagem , Coelhos , Malha Trabecular/metabolismoRESUMO
The highly specific S1 serine protease factor D (FD) plays a central role in the amplification of the complement alternative pathway (AP) of the innate immune system. Genetic associations in humans have implicated AP activation in age-related macular degeneration (AMD), and AP dysfunction predisposes individuals to disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). The combination of structure-based hit identification and subsequent optimization of the center (S)-proline-based lead 7 has led to the discovery of noncovalent reversible and selective human factor D (FD) inhibitors with drug-like properties. The orally bioavailable compound 2 exerted excellent potency in 50% human whole blood in vitro and blocked AP activity ex vivo after oral administration to monkeys as demonstrated by inhibition of membrane attack complex (MAC) formation. Inhibitor 2 demonstrated sustained oral and ocular efficacy in a model of lipopolysaccharide (LPS)-induced systemic AP activation in mice expressing human FD.
Assuntos
Fator D do Complemento/antagonistas & inibidores , Via Alternativa do Complemento/efeitos dos fármacos , Prolina/análogos & derivados , Prolina/farmacologia , Administração Oral , Animais , Síndrome Hemolítico-Urêmica Atípica/tratamento farmacológico , Síndrome Hemolítico-Urêmica Atípica/imunologia , Fator D do Complemento/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/antagonistas & inibidores , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Feminino , Haplorrinos , Humanos , Macaca fascicularis , Degeneração Macular/tratamento farmacológico , Degeneração Macular/imunologia , Masculino , Camundongos , Prolina/administração & dosagem , Prolina/farmacocinéticaRESUMO
Small interfering RNA (siRNA) induced posttranscriptional gene silencing (PTGS) has been an efficient method for genetic and molecular analysis of certain developmental and physiological processes and represented a potential strategy for both controlling virus replication and developing therapeutic products. However, there are limitations for the methods currently used to deliver siRNA into cells. We report here, to our knowledge, the first efficient delivery of siRNA to plant cells by a nanosecond pulsed laser-induced stress wave (LISW) for posttranscriptional gene silencing. Using LISW, we are able to silence gene expression in cell cultures of three different plant species rice (Oryza sativa L.), cotton (Gossypium hirsutum L.), and slash pine (Pinus elliottii Engelm.). Gene silencing induced by siRNA has been confirmed by northern blot, laser scanning microscopy, and siRNA analysis. These data suggested that LISW-mediated siRNA delivery can be a reliable and effective method for inducing PTGS in cultured cells.
RESUMO
Mevalonate kinase (MK) catalyzes a step in the isoprenoid biosynthetic pathway, which leads to a huge number of compounds that play important roles in plant growth and development. Here, we report on changes in MK activity in white pine (Pinus strobus L.) during plant regeneration by adventitious shoot organogenesis from cotyledons of mature embryos, including nodular callus induction, shoot formation and rooting. Nodular calli were induced from Pinus strobus (PS) embryos by culture in nodular callus induction medium in a 0-, 8- or 16-h photoperiod. Mevalonate kinase activity peaked in nodular calli after three weeks of culture on nodular callus induction medium in a 16-h photoperiod, whereas frequency of nodular callus formation peaked after 4 weeks of culture on nodular callus induction medium in darkness. During adventitious shoot formation, MK activity peaked in shoots derived from dark-grown nodular calli after 3 weeks on bud formation medium, and frequency of shoot formation was highest in dark-grown nodular calli cultured on bud formation medium for 4 weeks. During rooting, MK activity peaked 2 weeks after transfer of adventitious shoots to rooting medium and rooting frequency was highest in adventitious shoots after 3 weeks on rooting medium. Although during nodular callus induction in darkness MK activity was inversely related to frequency of nodular callus formation, MK activity was highly correlated with frequency of shoot formation and with rooting frequency. The observed increase in MK activity preceding rooting suggests that MK could serve as a marker for rooting of white pine shoots in vitro.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Pinus/fisiologia , Brotos de Planta/fisiologia , Compostos de Benzil , Cotilédone/efeitos dos fármacos , Cotilédone/fisiologia , Ácidos Indolacéticos/farmacologia , Indóis/farmacologia , Cinetina/farmacologia , Ácidos Naftalenoacéticos/farmacologia , Pinus/enzimologia , Reguladores de Crescimento de Plantas/farmacologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/fisiologia , Brotos de Planta/enzimologia , Purinas , Regeneração/efeitos dos fármacos , Plântula/efeitos dos fármacos , Plântula/fisiologia , Técnicas de Cultura de TecidosRESUMO
A plant regeneration system through multiple adventitious shoot differentiation from callus cultures has been established in slash pine (Pinus elliottii). Influences of seven different basal media on callus induction, adventitious shoot formation, and rooting were investigated. Among the different basal media, B5, SH, and TE proved to be suitable for callus induction and plantlet regeneration. Multiple adventitious shoot formation was obtained from callus cultures of slash pine on B5, SH, and TE media containing indole-3-butyric acid, N6-benzyladenine, and thidiazuron. Scanning electron microscopy demonstrated the early development of adventitious shoots derived from callus cultures. These results indicate that an efficient plant regeneration protocol for micropropagation of slash pine had been established. This protocol could be most useful for future studies on genetic transformation of slash pine.
Assuntos
Pinus/fisiologia , Brotos de Planta/fisiologia , Regeneração/fisiologia , Técnicas de Cultura de Tecidos , Pinus/ultraestrutura , Brotos de Planta/ultraestruturaRESUMO
We reported establishment of an efficient plant regeneration procedure through direct adventitious shoot (DAS) formation from cotyledons and hypocotyls of eastern white pine (Pinus strobus L.) mature embryos in this investigation. Multiple DASs were initiated from cotyledons of embryos on PS medium containing N6-benzyladenine (BA), thidiazuron (TDZ), or kinetin (KIN). Among different concentrations of casein enzymatic hydrosylate (CH) and glutamine used in this study, 500 mg l(-1) CH or 600 mg l(-1) glutamine induced the highest frequency of DAS formation. Rooting of regenerated shoots was obtained on PS medium supplemented with 0.01-0.1 microM indole-3-acetic acid (IAA) with the highest frequency on medium containing 0.01 muM IAA. No DASs were obtained on medium without TDZ. Measurement of peroxidase (POD) and catalase (CAT) activity during direct shoot induction and differentiation demonstrated that the lowest POD activity appeared in the 5-6th week of culture and lowest CAT activity occurred in the 7-8th week of culture on medium with TDZ. No such a change in POD and CAT activities was observed on medium without TDZ. These results demonstrated that POD and CAT activities were involved in DAS formation induced by TDZ in eastern white pine.
Assuntos
Catalase/metabolismo , Peroxidase/metabolismo , Compostos de Fenilureia/farmacologia , Pinus/efeitos dos fármacos , Pinus/enzimologia , Brotos de Planta/crescimento & desenvolvimento , Sementes/enzimologia , Tiadiazóis/farmacologia , Compostos de Benzil , Cinetina/farmacologia , Pinus/crescimento & desenvolvimento , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/enzimologia , Purinas , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimentoRESUMO
A reproducible approach to improve salt tolerance of conifers has been established by using the technology of plant genetic transformation and using loblolly pine (Pinus taeda L.) as a model plant. Mature zygotic embryos of three genotypes of loblolly pine were infected with Agrobacterium tumefaciens strain LBA 4404 harboring the plasmid pBIGM which carrying two bacterial genes encoding the mannitol-1-phosphate dehydrogenase (Mt1D, EC 1.1.1.17) and glucitol-6-phosphate dehydrogenase (GutD) (EC 1.1.1.140), respectively. Transgenic plantlets were produced on selection medium containing 15 mg l(-1) kanamycin and confirmed by polymerase chain reaction (PCR) and Southern blot analysis of genomic DNA. The Mt1D and GutD genes were expressed and translated into functional enzymes that resulted in the synthesis and accumulation of mannitol and glucitol in transgenic plants. Salt tolerance assays demonstrated that transgenic plantlets producing mannitol and glucitol had an increased ability to tolerate high salinity. These results suggested that an efficient A. tumefaciens-mediated transformation protocol for stable integration of bacterial Mt1D and GutD genes into loblolly pine has been developed and this could be useful for the future studies on engineering breeding of conifers.
Assuntos
Pinus taeda/enzimologia , Cloreto de Sódio/farmacologia , Desidrogenase do Álcool de Açúcar/biossíntese , Agrobacterium tumefaciens/genética , Manitol/metabolismo , Pressão Osmótica , Pinus taeda/genética , Plantas Geneticamente Modificadas , Biossíntese de Proteínas , Sorbitol/metabolismo , Desidrogenase do Álcool de Açúcar/genéticaRESUMO
Genotoxic stress causes a variety of cellular and molecular responses in mammalian cells, including cell cycle arrest, DNA repair, and apoptosis. These responses result from the interplay between the genotoxic events themselves, and the biological context in which they occur. To better understand this interplay, we investigated cytotoxicty, mutagenesis, cell cycle profile, and global gene expression in the human TK6 lymphoblastoid cell line exposed to six genotoxicants. The six compounds have broad structural diversity and cause genotoxic stress by many different mechanisms, including covalent modification (methyl methanesulfonate, mitomycin C), reactive oxygen species (hydrogen peroxide, bleomycin), and topoisomerase II inhibition (etoposide and doxorubicin). Cell cycle analysis was performed 4 and 20 h following a 4 h chemical exposure. Cells exposed to all compounds experienced S-phase arrest at the 8h time point, but by 24 h had markedly different cell cycle responses. Cells exposed to compounds that cause covalent modification had a strong G2/M arrest at 24 h. These cells also had a robust (>25-fold) increase in mutant frequency, and had a moderate but sustained p53 response at 4, 8, and 24h, detectable as approximately 2-5-fold increases in transcript levels for p21WAF1/CIP1, GADD45alpha, BTG2, and cyclin G1. In contrast, cells exposed to the reactive oxygen compounds had little or no G2/M arrest at 24 h and no increase in mutant frequency. In addition, these compounds caused a strong but transient induction of the p53 pathway, detectable as 15-25-fold increases in p21WAF1/CIP1 transcription at 4 h that decreased dramatically by 8h and was near control levels at 24 h. Thus, the mutagenic effect of compounds was consistent with G2/M arrest and sustained kinetics of p53 pathway activation. Global gene expression data were also consistent with the mutagenesis data. Activation of genes associated with cell cycle arrest, the p53 and TNF-related pathways, and chemokines and chemokine receptors, were particularly evident for the reactive oxygen compounds. In contrast, the most mutagenic compounds caused fewer and less robust changes in global gene expression. There was therefore an inverse relationship between global gene expression and mutagenic potency.
Assuntos
Ciclo Celular/efeitos dos fármacos , Dano ao DNA , Genoma Humano , Linfócitos/efeitos dos fármacos , Mutagênicos/toxicidade , Linhagem Celular Tumoral , Fase G2 , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes p53/efeitos dos fármacos , Humanos , Cinética , Linfócitos/citologia , Linfócitos/fisiologia , Análise em Microsséries , Modelos Biológicos , Fase S , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
Transgenic cell lines of loblolly pine (Pinus taeda L.) were analyzed by a compact laser-tweezers-Raman-spectroscopy (LTRS) system in this investigation. A low power diode laser at 785 nm was used for both laser optical trapping of single transgenic cells and excitation for near-infrared Raman spectroscopy of the nuclei of synchronized cells, which were treated as single organic particles, at the S-phase of the cell cycle. Transgenic living cells with gfp and uidA genes were used as biological samples to test this LTRS technique. As expected, different Raman spectra were observed from the tested biological samples. This technique provides a high sensitivity and enables real-time spectroscopic measurements of transgenic cell lines. It could be a valuable tool for the study of the fundamental cell and molecular biological process by trapping single nucleus and by providing a wealth of molecular information about the nuclei of cells.
Assuntos
Núcleo Celular , Lasers , Micromanipulação/métodos , Pinus taeda/citologia , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Análise Espectral Raman/métodos , Ciclo Celular/fisiologia , Micromanipulação/instrumentação , Plantas Geneticamente Modificadas , Espectroscopia de Luz Próxima ao Infravermelho/instrumentação , Análise Espectral Raman/instrumentaçãoRESUMO
Microarrays provide an unprecedented opportunity for comprehensive concurrent analysis of thousands of genes. The global analysis of the response of genes to a toxic insult (toxicogenomics), as opposed to the historical method of examining a few select genes, provides a more complete picture of toxicologically significant events. Here we examine the utility of microarrays for providing mechanistic insights into the response of cells to DNA damage. Our data indicate that the value of the technology is in its potential to provide mechanistic insight into the mode of action of a genotoxic compound. Array-based expression profiling may be useful for differentiating compounds that interact directly with DNA from those compounds that are genotoxic via a secondary mechanism. As such, genomic microarrays may serve as a valuable alternative methodology that helps discriminate between these two classes of compounds. Key words: biomarkers, gene expression profile, genetic toxicology, mechanism of action, toxicogenomics.
Assuntos
Dano ao DNA , Poluentes Ambientais/intoxicação , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Toxicogenética/tendências , Animais , Biomarcadores/análise , Adutos de DNA , Humanos , Linfoma/patologia , Camundongos , Células Tumorais CultivadasRESUMO
Exposure to DNA-damaging agents can elicit a variety of stress-related responses that may alter the gene expression of numerous biological pathways. We used Affymetrix microarrays to detect gene expression changes in mouse lymphoma (L5178Y) and human lymphoblastoid (TK6) cells in response to methyl methanesulfonate (MMS; a prototypical alkylating agent) and bleomycin (a prototypical oxidative mutagen). Cells were treated for 4 hr, and RNA was isolated either at the end of the treatment or after a 20-hr recovery period. Two concentrations of each agent were used based on cytotoxicity levels and Tk mutant frequencies. Our microarray data analysis indicated that MMS and bleomycin gene expression responses were considerably different in mouse cells versus human cells. The results also suggested that more comprehensive cellular responses to MMS and bleomycin occurred in TK6 cells than in L5178Y cells. In contrast to L5178Y cells, the response of TK6 cells to MMS and bleomycin was characterized by the induction of p53-dependent genes that are involved in DNA repair, cell cycle regulation, and apoptosis. It appears that the induction of DNA damage by MMS in human TK6 cells mediated cytotoxicity and led to decreased cell survival. This may explain the greater sensitivity of TK6 cells to cytotoxic effects of MMS compared to L5178Y cells. Bleomycin exerted comparable cytotoxic effects in the two cell lines. Overall, these studies were unable to identify distinctive gene expression changes that differentiated bleomycin from MMS in either TK6 cells or mouse lymphoma cells.
Assuntos
Alquilantes/toxicidade , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Linfoma/genética , Metanossulfonato de Metila/toxicidade , Animais , DNA de Neoplasias/análise , Humanos , Camundongos , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Short interfering RNA (siRNA) is widely used for studying post-transcriptional gene silencing and holds great promise as a tool for both identifying function of novel genes and validating drug targets. Two siRNA fragments (siRNA-a and -b), which were designed against different specific areas of coding region of the same target green fluorescent protein (GFP) gene, were used to silence GFP expression in cultured gfp transgenic cells of rice (Oryza sativa L.; OS), cotton (Gossypium hirsutum L.; GH), Fraser fir [Abies fraseri (Pursh) Poir; AF], and Virginia pine (Pinus virginiana Mill.; PV). Differential gene silencing was observed in the bombarded transgenic cells between two siRNAs, and these results were consistent with the inactivation of GFP confirmed by laser scanning microscopy, Northern blot, and siRNA analysis in tested transgenic cell cultures. These data suggest that siRNA-mediated gene inactivation can be the siRNA specific in different plant species. These results indicate that siRNA is a highly specific tool for targeted gene knockdown and for establishing siRNA-mediated gene silencing, which could be a reliable approach for large-scale screening of gene function and drug target validation.
Assuntos
Plantas/genética , Interferência de RNA/fisiologia , RNA Interferente Pequeno/metabolismo , Células Cultivadas , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Plantas/metabolismo , Fatores de TempoRESUMO
Mature zygotic embryos of recalcitrant Christmas tree species eastern white pine (Pinus strobus L.) were used as explants for Agrobacterium tumefaciens strain GV3101-mediated transformation using the uidA (beta-Glucuronidase) gene as a reporter. Influence of the time of sonication and the concentrations of protein phosphatase inhibitor (okadaic acid) and kinase inhibitor (trifluoperazine) on Agrobacterium-mediated transformation have been evaluated. A high transformation frequency was obtained after embryos were sonicated for 45-50 s, or treated with 1.5-2.0 microM okadaic acid or treated with 100-200 microM trifluoperazine, respectively. Protein phosphatase and kinase inhibitors enhance Agrobacterium-mediated transformation in eastern white pine. A 2-3.5-fold higher rate of hygromycin-resistant callus was obtained with an addition of 2 microM okadaic acid or 150 microM trifluoperazine or sonicated embryos for 45 s. Stable integration of the uidA gene in the plant genome of eastern white pine was confirmed by polymerase chain reaction (PCR), Southern and northern blot analyses. These results demonstrated that a stable and enhanced transformation system has been established in eastern white pine and this system would provide an opportunity to transfer economically important genes into this Christmas tree species.
Assuntos
Agrobacterium tumefaciens/efeitos dos fármacos , Agrobacterium tumefaciens/fisiologia , Ácido Okadáico/farmacologia , Pinus/efeitos dos fármacos , Pinus/genética , Transformação Genética/efeitos dos fármacos , Trifluoperazina/farmacologia , Cinamatos/metabolismo , DNA Bacteriano/metabolismo , Resistência a Medicamentos , Glucuronidase/metabolismo , Higromicina B/análogos & derivados , Higromicina B/metabolismo , Plantas Geneticamente Modificadas , Sonicação , Técnicas de Cultura de TecidosRESUMO
An efficient transgenic eastern white pine (Pinus strobus L.) plant regeneration system has been established using Agrobacterium tumefaciens strain GV3850-mediated transformation and the green fluorescent protein (gfp) gene as a reporter in this investigation. Stable integration of transgenes in the plant genome of pine was confirmed by polymerase chain reaction (PCR), Southern blot, and northern blot analyses. Transgene expression was analysed in pine T-DNA transformants carrying different numbers of copies of T-DNA insertions. Post-transcriptional gene silencing (PTGS) was mostly obtained in transgenic lines with more than three copies of T-DNA, but not in transgenic lines with one copy of T-DNA. In situ hybridization chromosome analysis of transgenic lines demonstrated that silenced transgenic lines had two or more T-DNA insertions in the same chromosome. These results suggest that two or more T-DNA insertions in the same chromosome facilitate efficient gene silencing in transgenic pine cells expressing green fluorescent protein. There were no differences in shoot differentiation and development between transgenic lines with multiple T-DNA copies and transgenic lines with one or two T-DNA copies.
Assuntos
Inativação Gênica , Pinus/genética , Transformação Genética , Transgenes , Agrobacterium tumefaciens/genética , Cromossomos de Plantas , DNA Bacteriano/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/análise , Hibridização In Situ , Microscopia Confocal , Pinus/crescimento & desenvolvimento , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , SoloRESUMO
Transcription factors regulating the stress-responsive gene expression play an important role in plant stress adaptation. In this study, we examined the salt stress tolerance of transgenic Virginia pine (Pinus virginiana Mill.) overexpressing a Capsicum annuum ERF/AP2-type transcription factor (CaPF1), which may enhance the ability of transgenic plants to tolerate various kinds of stresses during vegetative growth. CaPF1 transgene increased the salt and oxidative stress tolerances of pine tissues and counteracted the inhibitory effects of salt stress on the growth of transgenic Virginia pine calli, shoots, and plants. To our surprise, the ability of shoot formation was enhanced in three CaPF1 transgenic Virginia pine cell lines under stress of different NaCl concentrations. NaCl at 200 mM significantly increased the frequency of adventitious shoot formation and the number of shoots per gram calli. Measurement of plant hormone demonstrated that the levels of cytokinin was altered in CaPF1-overexpressed Virginia pine calli, compared to the control. Based on our results, we speculate that the altered level of cytokinin may result in enhancing adventitious shoot formation of transgenic calli exposed to salt for 1 week via an unknown mechanism.
Assuntos
Capsicum , Pinus/efeitos dos fármacos , Pinus/metabolismo , Brotos de Planta/efeitos dos fármacos , Cloreto de Sódio/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Capsicum/genética , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica , Estresse Oxidativo/efeitos dos fármacos , Pinus/genética , Pinus/crescimento & desenvolvimento , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/ultraestrutura , Plantas Geneticamente ModificadasRESUMO
The double-stranded short interfering RNA (siRNA) molecules can silence targeted genes through sequence-specific cleavage of the cognate RNA transcript. The rapid adoption of technologies based on this siRNA interference mechanism has been a widely used method to analyze gene function in plants, invertebrates, and mammalian systems. In order to understand the dynamics of siRNA-mediated gene inactivation during cell division, we have investigated the relationship between the cell cycle phase and the post-transcriptional gene silencing mediated by siRNA in gfp transgenic Virginia pine (Pinus virginiana Mill.) cells. Among the different phases of the cell cycle, transgenic cells at the M phase gave 2-3 times lower gfp silencing than those at the G1, S, and G2 phases. The similar results of the siRNA-mediated gfp silencing were obtained in three transgenic cell lines. Differential gfp silencing induced by siRNA has been confirmed by northern blot, laser scanning microscopy, and siRNA analysis. These data suggested that siRNA-mediated gene inactivation is associated with the cell cycle phase in Virginia pine.
Assuntos
Ciclo Celular/genética , Pinus/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Células Cultivadas , Dosagem de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Confocal , Pinus/citologia , Pinus/metabolismo , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/análiseRESUMO
Plant regeneration via adventitious shoot organogenesis from callus cultures initiated from mature embryos in white pine (Pinus strobus L.) was achieved in this study. Callus cultures were induced from mature embryos cultured on PS medium supplemented with 2,4-dichlorophenoxyacetic acid, alpha-naphthaleneacetic acid, or indole-3-acetic acid. Adventitious shoot regeneration from callus cultures was induced on medium containing 2 microM indole-3-butyric acid (IBA) and 3-12 microM N(6)-benzylaminopurine, thidiazuron (TDZ), or 6-(gamma,gamma-dimethylallylamino) purine. Sucrose was the most suitable sugar for adventitious shoot organogenesis in white pine. Shoot organogenesis was improved by treatment at 4 degrees C for 6 weeks. The frequency of adventitious shoot formation increased when 0.1 mM putrescine was added to basal medium supplemented with 6 microM TDZ and 2 microM IBA. Putrescine improved adventitious shoot organogenesis by decreasing lipid peroxidation. These findings provide useful information on adventitious shoot organogenesis and may be valuable to genetic transformation in white pine.