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Pseudemys alabamensis is one of the most endangered freshwater turtle species in the United States due to its restricted geographic distribution in coastal Alabama and Mississippi. Populations of P. alabamensis are geographically isolated from one another by land and saltwater, which could act as barriers to gene flow. It is currently unknown how differentiated these populations are from one another and whether they have experienced reductions in population size. Previous work found morphological differences between Alabama and Mississippi populations, suggesting that they may be evolutionarily distinct. Other Pseudemys turtles such as P. concinna and P. floridana occur naturally within the same geographic area as P. alabamensis and are known to hybridize with each other. These more abundant species could threaten the unique genetic identity of P. alabamensis through introgression. In order to evaluate the endangered status of P. alabamensis and the level of hybridization with other species, we used mitochondrial and nuclear microsatellite markers to assess genetic variation within and among populations of P. alabamensis throughout its range and estimate admixture with co-occurring Pseudemys species. In P. alabamensis, we found no variation in mitochondrial DNA and an excess of homozygosity in microsatellite data. Our results show genetic differentiation between Alabama and Mississippi populations of P. alabamensis, and low estimated breeding sizes and signs of inbreeding for two populations (Fowl River, Alabama and Biloxi, Mississippi). We also found evidence of admixture between P. alabamensis and P. concinna/P. floridana. Based on our results, P. alabamensis is highly endangered throughout its range and threatened by both low population sizes and hybridization. In order to improve the species' chances of survival, focus should be placed on habitat preservation, maintenance of genetic diversity within both the Mississippi and Alabama populations, and routine population-monitoring activities such as nest surveillance and estimates of recruitment.
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Ecological risk assessments of chemicals are frequently based on laboratory toxicity data from a small number of model species that may be reared in labs for years or decades. These populations can undergo many processes in the lab including artificial selection, founder effect, and genetic drift, and may not adequately represent their wild counterparts, potentially undermining the goal of protecting natural populations. Here we measure variation in lethality to copper chloride among strains of an emerging model species in toxicology, Caenorhabditis elegans. We tested four wild strains from Chile, Germany, Kenya, and Madeira (Portugal) against several versions of the standard laboratory N2 strain from Bristol, UK used in molecular biology. The four wild strains were more sensitive than any of the N2 strains tested with copper. We also found that the standard N2 strain cultured in the laboratory for >1 year was less sensitive than a recently cultured N2 strain as well as a cataloged ancestral version of the N2 strain. These results suggest that toxicologists should be cognizant of performing toxicity testing with long-held animal cultures, and should perhaps use multiple strains as well as renew cultures periodically in the laboratory. This study also shows that multi-strain toxicity testing with nematodes is highly achievable and useful for understanding variation in intra- and interspecific chemical sensitivity.
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Caenorhabditis elegans , Nematoides , Animais , Caenorhabditis elegans/genética , Cobre/toxicidade , Laboratórios , Testes de ToxicidadeRESUMO
Twenty-four hour median lethal concentration (LC50) toxicity tests were performed with five species of nematodes (Caenorhabditis elegans, Caenorhabditis briggsae, Pristionchus pacificus, Oscheius tipulae, and Oscheius myriophila) in response to copper chloride and zinc chloride. In addition, lethality tests were also performed with seven strains of C. elegans (N2 > 1 year in culture, N2 newly acquired, N2 ancestral, ED3053, JU258, JU1171, and MY1) exposed to copper chloride. Nominal chemical concentrations were validated and analyzed according to U.S. Environmental Protection Agency method 6010 using inductively coupled plasma-atomic emission spectroscopy (ICP-AES). This paper combines the datasets previously published separately by Heaton et al. (2020, 2022). The goal is to catalog all raw and analyzed toxicity data collected from both studies in a single consistent information source for use by the scientific community.
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Performing toxicity testing on multiple species with differing degrees of evolutionary relatedness can provide important information on how chemical sensitivity varies among species and can help pinpoint the biological drivers of species sensitivity. Such knowledge could ultimately be used to design better multispecies predictive ecological risk assessment models and identify particularly sensitive species. However, laboratory toxicity tests involving multiple species can also be resource intensive, especially when each species has unique husbandry conditions. We performed lethality tests with 2 metals, copper chloride and zinc chloride, on 5 different nematode species, which are nested in their degree of evolutionary relatedness: Caenorhabditis briggsae, Caenorhabditis elegans, Oscheius myriophila, Oscheius tipulae, and Pristionchus pacificus. All species were successfully cultured and tested concurrently with limited resources, demonstrating that inexpensive, multispecies nematode toxicity testing systems are achievable. The results indicate that P. pacificus is the most sensitive to both metals. Conversely, C. elegans is the least sensitive species to copper, but the second most sensitive to zinc, indicating that species relationships do not necessarily predict species sensitivity. Toxicity testing with additional nematode species and types of chemicals is feasible and will help form more generalizable conclusions about relative species sensitivity. Environ Toxicol Chem 2020;39:1006-1016. © 2020 SETAC.
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Metais/toxicidade , Nematoides/efeitos dos fármacos , Animais , Caenorhabditis elegans/efeitos dos fármacos , Cobre/toxicidade , Filogenia , Especificidade da Espécie , Zinco/toxicidadeRESUMO
We examine the theoretical implications of incorporating metasurfaces on solar sails, and the effect they can have on the forces applied to the sail. This would enable a significant enhancement over state-of-the- art attitude control by demonstrating a novel, propellant-free and low-mass approach to induce a roll torque on the sail, which is a current limitation in present state-of-the-art technology. We do so by utilizing anomalous optical reflections from the metasurfaces to generate a net in-plane lateral force, which can lead to a net torque along the roll axis of the sail, in addition to the other spatial movements exhibited by the sail from solar radiation pressure. We characterize this net lateral force as a function of incidence angle. In addition, the influence of the phase gradients and anomalous conversion efficiencies characteristics of the metasurfaces are independently considered. The optimum incidence angle that corresponded with the maximum net lateral-to-normal force ratio was found to be -30° for a metasurface exhibiting 75% anomalous conversion efficiency with a phase gradient of 0:71k0.
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The co-occurrence of the Deepwater Horizon oil spill and the northern Gulf of Mexico cetacean Unusual Mortality Event have raised questions about the stability of inshore bottlenose dolphin (Tursiops truncatus) populations throughout the region. Several factors could have contributed to the ongoing event, but little attention has been paid to the potential effects of increased search effort and reporting of strandings associated with oil spill response activities, which were widespread for an extended period. This study quantified the influence of increased search effort by estimating the number of bottlenose dolphin strandings reported by oil spill responders and comparing monthly stranding rates with and without response-related records. Results showed that response teams reported an estimated 58% of strandings during the Active Response period within the study area. Comparison of Poisson rates tests showed that when responder-influenced stranding records were removed, the monthly stranding rates from the Active Response period (May 2010 -April 2014) were similar to the Post-Removal Actions Deemed Complete period (May 2013 -March 2015) (e.g., p = 0.83 for remote areas in Louisiana). Further, analyses using the Getis-Ord Gi* spatial statistic showed that when response-related stranding reports were removed from the Active Response period, significant spatial clustering of strandings (p < 0.05) was reduced by 48% in coastal Louisiana. Collectively, these results suggest that increased search effort resulting from the Deepwater Horizon oil spill response throughout remote portions of the Unusual Mortality Event geographic region had the capacity to increase reporting and recovery of marine mammal strandings to unusually high levels. To better understand how stranding data relates to actual mortality, more work is needed to quantify dolphin population size, population trends, and carcass detection rates including the role of search effort. This is vital for understanding the status of a protected species within the northern Gulf of Mexico.
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Comportamento Animal/fisiologia , Golfinho Nariz-de-Garrafa/fisiologia , Monitoramento Ambiental , Poluição por Petróleo/efeitos adversos , Relatório de Pesquisa , Alabama , Animais , Intervalos de Confiança , Geografia , Golfo do MéxicoRESUMO
DNA damage has been linked to genomic instability and the progressive breakdown of cellular and organismal homeostasis, leading to the onset of disease and reduced longevity. Insults to DNA from endogenous sources include base deamination, base hydrolysis, base alkylation, and metabolism-induced oxidative damage that can lead to single-strand and double-strand DNA breaks. Alternatively, exposure to environmental pollutants, radiation or ultra-violet light, can also contribute to exogenously derived DNA damage. We previously validated a novel, high through-put approach to measure levels of DNA damage in cultured mammalian cells. This new CometChip Platform builds on the classical single cell gel electrophoresis or comet methodology used extensively in environmental toxicology and molecular biology. We asked whether the CometChip Platform could be used to measure DNA damage in samples derived from environmental field studies. To this end, we determined that nucleated erythrocytes from multiple species of turtle could be successfully evaluated in the CometChip Platform to quantify levels of DNA damage. In total, we compared levels of DNA damage in 40 animals from two species: the box turtle (Terrapene carolina) and the red-eared slider (Trachemys scripta elegans). Endogenous levels of DNA damage were identical between the two species, yet we did discover some sex-linked differences and changes in DNA damage accumulation. Based on these results, we confirm that the CometChip Platform allows for the measurement of DNA damage in a large number of samples quickly and accurately, and is particularly adaptable to environmental studies using field-collected samples. Environ. Mol. Mutagen. 59:322-333, 2018. © 2018 Wiley Periodicals, Inc.
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Bioensaio/instrumentação , Dano ao DNA/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Eritrócitos/efeitos dos fármacos , Animais , Coleta de Amostras Sanguíneas , Feminino , Masculino , Fatores Sexuais , TartarugasRESUMO
Successive rounds of chemical modification in three generations of benzopyran molecules have shown to select for different mechanisms of actions and progressive increases in anti-cancer activity. In this study, we investigated the mechanism of action of the third-generation benzopyran compounds, TRX-E-002-1 and TRX-E-009-1. High-content screening of a panel of 240 cancer cell lines treated with TRX-E-009-1 demonstrated it has broad anti-cancer potential. Within this screen, melanoma cell lines showed a range of sensitivities and subsequently a second independent panel of 21 melanoma 3D spheroid lines were assessed for their responses to both TRX-E-002-1 and TRX-E-009-1 compounds. Time-lapse microscopy illustrated both of these compounds caused mitotic delays in treated cells, resulting in either mitotic slippage or apoptosis. This finding along with immunostaining, in vitro polymerization assays, and animal experiments in both athymic and immunocompetent mice, demonstrates that these third-generation benzopyran compounds are potent tubulin polymerization inhibitors in vitro and in vivo, and this is the molecular basis of their anti-cancer activity in melanoma. These findings indicate these BP compounds may offer a novel anti-microtubule strategy for cancer intervention and provides the basis for further investigation into biomarkers of clinical sensitivity.
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Benzopiranos , Flavonoides , Melanoma Experimental/tratamento farmacológico , Moduladores de Tubulina , Animais , Benzopiranos/química , Benzopiranos/farmacologia , Linhagem Celular Tumoral , Flavonoides/química , Flavonoides/farmacologia , Humanos , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Nus , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Actin filaments, with their associated tropomyosin polymers, and microtubules are dynamic cytoskeletal systems regulating numerous cell functions. While antimicrotubule drugs are well-established, antiactin drugs have been more elusive. We previously targeted actin in cancer cells by inhibiting the function of a tropomyosin isoform enriched in cancer cells, Tpm3.1, using a first-in-class compound, TR100. Here, we screened over 200 other antitropomyosin analogues for anticancer and on-target activity using a series of in vitro cell-based and biochemical assays. ATM-3507 was selected as the new lead based on its ability to disable Tpm3.1-containing filaments, its cytotoxicity potency, and more favorable drug-like characteristics. We tested ATM-3507 and TR100 alone and in combination with antimicrotubule agents against neuroblastoma models in vitro and in vivo Both ATM-3507 and TR100 showed a high degree of synergy in vitro with vinca alkaloid and taxane antimicrotubule agents. In vivo, combination-treated animals bearing human neuroblastoma xenografts treated with antitropomyosin combined with vincristine showed minimal weight loss, a significant and profound regression of tumor growth and improved survival compared with control and either drug alone. Antitropomyosin combined with vincristine resulted in G2-M phase arrest, disruption of mitotic spindle formation, and cellular apoptosis. Our data suggest that small molecules targeting the actin cytoskeleton via tropomyosin sensitize cancer cells to antimicrotubule agents and are tolerated together in vivo This combination warrants further study. Mol Cancer Ther; 16(8); 1555-65. ©2017 AACR.
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Antineoplásicos/uso terapêutico , Microtúbulos/metabolismo , Neoplasias/tratamento farmacológico , Tropomiosina/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Fase G2/efeitos dos fármacos , Humanos , Camundongos Nus , Microtúbulos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Neoplasias/patologia , Tropomiosina/metabolismo , Vincristina/farmacologiaRESUMO
PURPOSE: Recurrent, chemo-resistant ovarian cancer is thought to be due to a subgroup of slow-growing, drug-resistant cancer cells with stem-like properties and a high capacity for tumour repair. Cantrixil targets this sub-population of cells and is being developed as an intraperitoneal therapy to be used as first-line therapy in combination with carboplatin for epithelial ovarian cancer. The studies presented here justify further development. METHODS: A GLP dog CV study using a 4 × 4 Latin Square Crossover study was conducted using telemetric ECG recordings from dogs post IP administration to assess for cardiac abnormalities. Mutagenic potential was assessed using the bacterial reverse mutation assay. Clastogenicity was assessed by determining micronuclei formation in the bone marrow of SPF Arc(S) Swiss mice dosed at clinical concentrations. TRX-E-002-1 toxicology was evaluated in GLP-compliant MTD and 28-day repeat-dose studies in rats and dogs. RESULTS: In vitro TRX-E-002-1 has potent cytotoxic activity against human cancer cells including CD44+/MyD88+ ovarian cancer stem cells. TRX-E-002-1 increased phosphorylated c-Jun levels in these cancer cells resulting in caspase-mediated apoptosis. In vivo, Cantrixil was active in a model of disseminated ovarian cancer as a monotherapy and in combination with Cisplatin. Cantrixil was active as maintenance therapy in a model of drug-resistant, recurrent ovarian cancer and in an orthotopic model of pancreatic cancer. CONCLUSIONS: In animals, this clinical formulation and route of administration of Cantrixil demonstrated acceptable activity, safety pharmacology, genotoxicity and toxicology profile and constituted a successful Investigational New Drug application to the US Food and Drug Administration.
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Antineoplásicos/farmacologia , Benzopiranos/farmacologia , Flavonoides/farmacologia , Animais , Antineoplásicos/toxicidade , Benzopiranos/farmacocinética , Benzopiranos/toxicidade , Linhagem Celular Tumoral , Estudos Cross-Over , Cães , Interações Medicamentosas , Flavonoides/farmacocinética , Flavonoides/toxicidade , Humanos , Camundongos , Ratos , Especificidade da EspécieRESUMO
In circulation, shedding of microparticles from a variety of viable cells can be triggered by pathological activation of inflammatory processes, by activation of coagulation or complement systems, or by physical stress. Elevated microparticle content (MPC) in donor blood might therefore indicate a clinical condition of the donor which, upon transfusion, might affect the recipient. In blood products, elevated MPC might also represent product stress. Surprisingly, the MPC in blood collected from normal blood donors is highly variable, which raises the question whether donor microparticles are present in-vivo and transfer into the final blood component, and how production methods and post-production processing might affect the MPC. We measured MPC using ThromboLUX in (a) platelet-rich plasma (PRP) of 54 apheresis donors and the corresponding apheresis products, (b) 651 apheresis and 646 pooled platelet concentrates (PCs) with plasma and 414 apheresis PCs in platelet additive solution (PAS), and (c) apheresis PCs before and after transportation, gamma irradiation, and pathogen inactivation (N = 8, 7, and 12 respectively). ThromboLUX-measured MPC in donor PRP and their corresponding apheresis PC samples were highly correlated (r = 0.82, P = .001). The average MPC in pooled PC was slightly lower than that in apheresis PC and substantially lower in apheresis PC stored with PAS rather than plasma. Mirasol Pathogen Reduction treatment significantly increased MPC with age. Thus, MPC measured in donor samples might be a useful predictor of product stability, especially if post-production processes are necessary.
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Doadores de Sangue , Plaquetas/citologia , Plaquetas/metabolismo , Segurança do Sangue/métodos , Micropartículas Derivadas de Células/metabolismo , Plasma Rico em Plaquetas , Plaquetoferese , Desinfecção/métodos , Feminino , Humanos , MasculinoRESUMO
Chemoresistance is a major hurdle in the management of patients with epithelial ovarian cancer and is responsible for its high mortality. Studies have shown that chemoresistance is due to the presence of a subgroup of cancer cells with stemness properties and a high capacity for tumor repair. We have developed a library of super-benzopyran analogues to generate potent compounds that can induce cell death in chemoresistant cancer stem cells. TRX-E-002-1 is identified as the most potent analogue and can induce cell death in all chemoresistant CD44(+)/MyD88(+) ovarian cancer stem cells tested (IC50 = 50 nmol/L). TRX-E-002-1 is also potent against spheroid cultures formed from cancer stem cells, chemosensitive CD44(-)/MyD88(-) ovarian cancer cells, and heterogeneous cultures of ovarian cancer cells. Cell death was associated with the phosphorylation and increased levels of c-Jun and induction of caspases. In vivo, TRX-E-002-1 given as daily intraperitoneal monotherapy at 100 mg/kg significantly decreased intraperitoneal tumor burden compared with vehicle control. When given in combination with cisplatin, animals receiving the combination of cisplatin and TRX-E-002-1 showed decreased tumor burden compared with each monotherapy. Finally, TRX-E-002-1 given as maintenance treatment after paclitaxel significantly delayed disease recurrence. Our results suggest that TRX-E-002-1 may fill the current need for better therapeutic options in the control and management of recurrent ovarian cancer and may help improve patient survival. Mol Cancer Ther; 15(6); 1279-90. ©2016 AACR.
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Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Flavonoides/administração & dosagem , Recidiva Local de Neoplasia/prevenção & controle , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-jun/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/administração & dosagem , Cisplatino/farmacologia , Sinergismo Farmacológico , Feminino , Flavonoides/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A self-deferral form has been used to screen Chiang Mai University Hospital blood donors and was improved in 2005. It has never been evaluated. The study aimed to assess the self-deferral form procedures in detecting infected donors. Sera from 5,083 donors, who passed the self-deferral screening form, were tested with the routine immuno-assays (serology) for HIV 1 and 2 antibodies, P24 antigen, HCV antibodies, HBV surface antigen, and syphilis. Antibody negative sera were also tested individually with the the Procleix Ultrio Assay for HIV-1 DNA, HCV RNA, and HBV DNA. The donors who had discrepant results between serology and NAT were evaluated with additional tests, including a more sensitive Alternative Nucleic Acid Test, AntiBcore IgM, AntiBcore IgG, HBsAg and Anti HBs. Among 5,083 donors, 331 (6.5%) had at least one positive marker. In multiple logistic regression analysis, the statistically significant factors (adjusted odds ratio and 95% CI) for infection were age 30 years or below [1.45 (1.03, 2.03)], male gender [2.73 (1.64, 4.56)], primary school or lower education [1.56 (1.09, 2.23)], first-time donation [1.82 (1.25, 2.67)], and frequent donation [0.80 (0.70, 0.92)]. The safest donors were females, older than 30 years, with an education more than primary school, and frequent donation. Because of missing responses to some sensitive questions, there remains a need for further improvement of the self-deferral form.
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Doadores de Sangue , Programas de Rastreamento/métodos , Inquéritos e Questionários , Adolescente , Adulto , Fatores Etários , Idoso , Feminino , Proteína do Núcleo p24 do HIV/sangue , HIV-1/isolamento & purificação , HIV-2/isolamento & purificação , Antígenos de Superfície da Hepatite B/sangue , Anticorpos Anti-Hepatite C/sangue , Hospitais Universitários , Humanos , Masculino , Pessoa de Meia-Idade , Fatores Sexuais , Fatores Socioeconômicos , Sífilis/sangue , Tailândia , Adulto JovemRESUMO
BACKGROUND: Currently used formulas for estimation of a person's red cell volume (RCV) by weight and height are decades old and were based on the use of 51Cr isotopes and on a sample population, which may not be reflective of today's population. In this study, the accuracy and precision of the use of 99mTc RCV measurements in volunteers more typical of today's population were evaluated. STUDY DESIGN AND METHODS: The subjects were volunteers who met the requirements for a standard blood donation. RESULTS: The mean +/- standard deviation (SD) 99mTc RCV for 127 males (mean weight, 83.2 kg; height, 180 cm) was 2062 +/- 339 mL, and for 101 females (mean weight, 69.5 kg; height, 166 cm) it was 1320 +/- 201 mL. These results were highly correlated with RCV results with the standard extrapolation 51Cr method with stored red blood cells (RBCs) and highly consistent (within +/-10%) by repeated measurements with the same 22 donors over a 3.5-year period. The RCV results correlated with estimates from the current formulas, but were on average 11 to 14 percent lower. CONCLUSION: The studies demonstrated that 99mTc is a reproducible and precise method for determination of a person's RCV and that current formulas may significantly overestimate the RCV of today's population. This is likely the result of a shift in population characteristics over the past four decades as reflected by an increased mean body mass index (from 25 to 28 kg/m2), which has not resulted in a proportionally increased RCV.
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Doadores de Sangue , Determinação do Volume Sanguíneo/normas , Volume Sanguíneo , Hematócrito/normas , Modelos Biológicos , Adolescente , Adulto , Idoso , Armazenamento de Sangue/métodos , Radioisótopos de Cromo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , TecnécioRESUMO
Fractionation of cytotoxic extracts of specimens of a newly described sponge genus, Candidaspongia, has yielded the candidaspongiolides (3), a complex mixture of acyl esters of a macrolide related to tedanolide. The general structure of the candidaspongiolides was determined by analyses of various 2D NMR and MS data sets. The acyl ester components were identified by GC-MS analysis of the derived fatty acid methyl esters. The mixture could be selectively converted to the deacylated macrolide core (4) by enzymolysis with immobilized porcine lipase, with the structure of the candidaspongiolide core then secured by NMR and MS analysis. The candidaspongiolide mixture was potently cytotoxic, exhibiting a mean panel 50% growth inhibition (GI50) of 14 ng/mL in the National Cancer Institute's 60-cell-line in vitro antitumor screen.
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Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Poríferos/química , Animais , Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Lactonas/química , Lactonas/isolamento & purificação , Lactonas/farmacologia , Lipase/metabolismo , Macrolídeos , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Poríferos/classificação , SuínosRESUMO
Eastern cottonwood (Populus deltoides Bartr. ex Marsh.) trees were engineered to express merA (mercuric ion reductase) and merB (organomercury lyase) transgenes in order to be used for the phytoremediation of mercury-contaminated soils. Earlier studies with Arabidopsis thaliana and Nicotiana tabacum showed that this gene combination resulted in more efficient detoxification of organomercurial compounds than did merB alone, but neither species is optimal for long-term field applications. Leaf discs from in vitro-grown merA, nptII (neomycin phosphotransferase) transgenic cottonwood plantlets were inoculated with Agrobacterium tumefaciens strain C58 carrying the merB and hygromycin resistance (hptII) genes. Polymerase chain reaction of shoots regenerated from the leaf discs under selection indicated an overall transformation frequency of 20%. Western blotting of leaves showed that MerA and MerB proteins were produced. In vitro-grown merA/merB plants were highly resistant to phenylmercuric acetate, and detoxified organic mercury compounds two to three times more rapidly than did controls, as shown by mercury volatilization assay. This indicates that these cottonwood trees are reasonable candidates for the remediation of organomercury-contaminated sites.
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Liases/genética , Oxirredutases/genética , Acetato de Fenilmercúrio/metabolismo , Populus/genética , Biodegradação Ambiental , Western Blotting , Cinamatos/farmacologia , Resistência a Medicamentos , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Compostos Organomercúricos/metabolismo , Acetato de Fenilmercúrio/farmacologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Populus/enzimologia , Populus/crescimento & desenvolvimento , Rhizobium/genética , Transformação Genética , TransgenesRESUMO
Plants have many natural properties that make them ideally suited to clean up polluted soil, water, and air, in a process called phytoremediation. We are in the early stages of testing genetic engineering-based phytoremediation strategies for elemental pollutants like mercury and arsenic using the model plant Arabidopsis. The long-term goal is to develop and test vigorous, field-adapted plant species that can prevent elemental pollutants from entering the food-chain by extracting them to aboveground tissues, where they can be managed. To achieve this goal for arsenic and mercury, and pave the way for the remediation of other challenging elemental pollutants like lead or radionucleides, research and development on native hyperaccumulators and engineered model plants needs to proceed in at least eight focus areas: (1) Plant tolerance to toxic elementals is essential if plant roots are to penetrate and extract pollutants efficiently from heterogeneous contaminated soils. Only the roots of mercury- and arsenic-tolerant plants efficiently contact substrates heavily contaminated with these elements. (2) Plants alter their rhizosphere by secreting various enzymes and small molecules, and by adjusting pH in order to enhance extraction of both essential nutrients and toxic elements. Acidification favors greater mobility and uptake of mercury and arsenic. (3) Short distance transport systems for nutrients in roots and root hairs requires numerous endogenous transporters. It is likely that root plasma membrane transporters for iron, copper, zinc, and phosphate take up ionic mercuric ions and arsenate. (4) The electrochemical state and chemical speciation of elemental pollutants can enhance their mobility from roots up to shoots. Initial data suggest that elemental and ionic mercury and the oxyanion arsenate will be the most mobile species of these two toxic elements. (5) The long-distance transport of nutrients requires efficient xylem loading in roots, movement through the xylem up to leaves, and efficient xylem unloading aboveground. These systems can be enhanced for the movement of arsenic and mercury. (6) Aboveground control over the electrochemical state and chemical speciation of elemental pollutants will maximize their storage in leaves, stems, and vascular tissues. Our research suggests ionic Hg(II) and arsenite will be the best chemical species to trap aboveground. (7) Chemical sinks can increase the storage capacity for essential nutrients like iron, zinc, copper, sulfate, and phosphate. Organic acids and thiol-rich chelators are among the important chemical sinks that could trap maximal levels of mercury and arsenic aboveground. (8) Physical sinks such as subcellular vacuoles, epidermal trichome cells, and dead vascular elements have shown the evolutionary capacity to store large quantities of a few toxic pollutants aboveground in various native hyperaccumulators. Specific plant transporters may already recognize gluthione conjugates of Hg(II) or arsenite and pump them into vacuole.
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Arabidopsis/metabolismo , Arsênio/metabolismo , Proteínas de Bactérias/metabolismo , Poluentes Ambientais/metabolismo , Mercúrio/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Arabidopsis/genética , Proteínas de Bactérias/genética , Biodegradação Ambiental , Biotecnologia/métodos , Liases/genética , Liases/metabolismo , Plantas/genética , Plantas/metabolismo , Plantas Geneticamente Modificadas/genéticaRESUMO
Strong, tissue-specific and genetically regulated expression systems are essential tools in plant biotechnology. An expression system tool called a 'repressor-operator gene complex' (ROC) has diverse applications in plant biotechnology fields including phytoremediation, disease resistance, plant nutrition, food safety, and hybrid seed production. To test this concept, we assembled a root-specific ROC using a strategy that could be used to construct almost any gene expression pattern. When a modified E. coli lac repressor with a nuclear localization signal was expressed from a rubisco small subunit expression vector, S1pt::lacIn, LacIn protein was localized to the nuclei of leaf and stem cells, but not to root cells. A LacIn repressible Arabidopsis actin expression vector A2pot was assembled containing upstream bacterial lacO operator sequences, and it was tested for organ and tissue specificity using beta-glucuronidase (GUS) and mercuric ion reductase (merA) gene reporters. Strong GUS enzyme expression was restricted to root tissues of A2pot::GUS/S1pt::lacIn ROC plants, while GUS activity was high in all vegetative tissues of plants lacking the repressor. Repression of shoot GUS expression exceeded 99.9% with no evidence of root repression, among a large percentage of doubly transformed plants. Similarly, MerA was strongly expressed in the roots, but not the shoots of A2pot::merA/S1pt::lacIn plants, while MerA levels remained high in both shoots and roots of plants lacking repressor. Plants with MerA expression restricted to roots were approximately as tolerant to ionic mercury as plants constitutively expressing MerA in roots and shoots. The superiority of this ROC over the previously described root-specific tobacco RB7 promoter is demonstrated.
RESUMO
There are a number of diseases where the 5-alpha-reductase (5AR) enzyme is of therapeutic interest as a drug target. Currently the crystal structure for 5-alpha-reductase is unavailable, thus ligand-based pharmacophore techniques are beneficial in the drug development process. We have developed pharmacophores to aid inhibitor design for both human types I (preliminary) and II 5-alpha-reductase isozymes and also the rat type II isozyme. To our knowledge, these are the first published pharmacophores for inhibitors of the human type I and rat type II enzymes. A comparison between isozymes and the previously published human type II isozyme pharmacophore is also presented.