RESUMO
Current human leukocyte antigen (HLA) DNA typing methods such as the sequence-based typing (SBT) and sequence-specific oligonucleotide (SSO) methods generally yield ambiguous typing results because of oligonucleotide probe design limitations or phase ambiguity for HLA allele assignment. Here we describe the development and application of the super high-resolution single-molecule sequence-based typing (SS-SBT) of HLA loci at the 8-digit level using next generation sequencing (NGS). NGS which can determine an HLA allele sequence derived from a single DNA molecule is expected to solve the phase ambiguity problem. Eight classical HLA loci-specific polymerase chain reaction (PCR) primers were designed to amplify the entire gene sequences from the enhancer-promoter region to the 3' untranslated region. Phase ambiguities of HLA-A, -B, -C, -DRB1 and -DQB1 were completely resolved and unequivocally assigned without ambiguity to single HLA alleles. Therefore, the SS-SBT method described here is a superior and effective HLA DNA typing method to efficiently detect new HLA alleles and null alleles without ambiguity.
Assuntos
Loci Gênicos , Antígenos HLA/análise , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Tipagem de Sequências Multilocus/métodos , Regiões 3' não Traduzidas , Alelos , Primers do DNA , Antígenos HLA/genética , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Humanos , Tipagem de Sequências Multilocus/instrumentação , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Análise de Sequência de DNARESUMO
Fish bioconcentration factors (BCFs) are often used to assess substance-specific bioaccumulation. However, reliable BCF data are limited given the practical challenges of conducting such tests. The objectives of this paper are to describe nine rainbow trout studies performed in our lab using tailored dosing and test designs for obtaining empirical BCFs for 21 test substances; gain insights into the structural features and processes determining the magnitude and uncertainty in observed BCFs; and assess performance of six quantitative structure property relationships (QSPRs) for correctly categorizing bioaccumulation given current regulatory triggers. Resulting mean steady-state BCFs, adjusted to a 5% lipid content, ranged from 12 Lkg-1 for isodecanol to 15,448 Lkg-1 for hexachlorobenzene which served as a positive control. BCFs for hydrocarbons depended on aromatic and saturated ring configurations and position. Uptake clearances appeared to be modulated by gill metabolism and substance bioavailability, while elimination rates were likely influenced by somatic biotransformation. Current approaches for quantifying uncertainty in experimental BCFs, which take into account only variability in measured fish concentrations, were found to underestimate the true uncertainty in this endpoint with important implications for decision-making. The Vega (KNN/Read-Across) QSPR and Arnot-Gobas model yielded the best model performance when compared to measured BCFs generated in this study.
Assuntos
Biotransformação/fisiologia , Hidrocarbonetos/análise , Hidrocarbonetos/metabolismo , Oncorhynchus mykiss/metabolismo , Petróleo/análise , Petróleo/metabolismo , Poluentes Químicos da Água/análise , Animais , Hexaclorobenzeno/metabolismo , Cinética , Modelos Teóricos , Relação Quantitativa Estrutura-Atividade , IncertezaRESUMO
Solid-phase microextraction fibers coated with polydimethylsiloxane (PDMS) provide a convenient passive sampling format to characterize bioavailability of petroleum substances. Hydrocarbons absorb onto PDMS in proportion to both freely dissolved concentrations and partitioning properties of the individual constituents, which parallels the mechanistic basis used to predict aquatic toxicity in the PETROTOX model. When deployed in a non-depletive manner, combining SPME with thermal desorption and quantification using gas chromatography-flame ionization creates a biomimetic extraction (BE) procedure that has the potential to simplify aquatic hazard assessments of petroleum substances since the total moles of all hydrocarbons sorbed to the fiber can be related to toxic thresholds in target lipid of aquatic organisms. The objective of this work is to describe the technical basis for applying BE measurements to predict toxicity of petroleum substances. Critical BE-based PDMS concentrations corresponding to adverse effects were empirically derived from toxicity tests on different petroleum substances with multiple test species. The resulting species sensitivity distribution (SSD) of PDMS effect concentrations was then compared and found consistent with the previously reported target lipid-based SSD. Further, BE data collected on samples of aqueous media dosed with a wide range of petroleum substances were highly correlated to predicted toxic units derived using the PETROTOX model. These findings provide justification for applying BE in environmental hazard and risk evaluations of petroleum substances and related mixtures.
Assuntos
Biomimética/métodos , Petróleo/toxicidade , Microextração em Fase Sólida/métodos , Disponibilidade Biológica , Cromatografia Gasosa , Dimetilpolisiloxanos/química , Hidrocarbonetos/química , Hidrocarbonetos/isolamento & purificação , Petróleo/análise , Poluentes da Água/toxicidade , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/toxicidadeRESUMO
The aim of the present study was to elucidate consistent patterns in chronic polycyclic aromatic compound (PAC) toxicity to soil and sediment inhabiting invertebrates. Therefore we examined our experimental dataset, consisting of twenty-one chronic effect concentrations for two soil invertebrates (Folsomia candida and Enchytraeus cripticus) and two sediment invertebrates (Lumbriculus variegatus and Chironomus riparius) exposed to six PACs (two homocyclic isomers, anthracene and phenanthrene; two azaarene isomers: acridine and phenanthridine; and two azaarene transformation products, acridone and phenanthridone). In order to determine if effect concentrations were accurately predicted by existing toxicity-K(ow) relationships describing narcosis, chronic pore water effect concentrations were plotted jointly against logK(ow). Fifteen of the twenty-one effect concentrations (71%) were above the lower limit for narcosis, showing that narcosis was the main mode of action for the majority of the tested homo- and heterocyclic PACs during chronic exposure. Toxicity of all tested compounds to soil organisms was accurately described by the toxicity-K(ow) relationship. However, for the sediment invertebrates exposed to some of the tested heterocyclic PACs deviations from narcosis were identified, related to specific physicochemical properties of the test compounds and/or species specific sensitivities. It is concluded that existing toxicity-K(ow) relationships describing narcosis in some cases underestimate chronic PAC toxicity to sediment inhabiting invertebrates.
Assuntos
Compostos Heterocíclicos/toxicidade , Invertebrados/efeitos dos fármacos , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Poluentes do Solo/toxicidade , Animais , Sedimentos Geológicos/química , Compostos Heterocíclicos/química , Dose Letal Mediana , Estrutura Molecular , Hidrocarbonetos Policíclicos Aromáticos/química , Valor Preditivo dos Testes , Poluentes do Solo/química , Relação Estrutura-Atividade , Testes de Toxicidade CrônicaRESUMO
To identify cell death-induced genes, we employed a subtractive hybridization approach and isolated a cDNA encoding a mouse homolog of carnitine palmitoyltransferase I (CPT I), an enzyme that resides at the outer mitochondrial membrane and facilitates passage of long-chain fatty acids into mitochondria for beta-oxidation. Induced expression of CPT I mRNA was observed upon programmed cell death in the murine hematopoietic cell lines LyD9 and WEHI-231. To elucidate the role of CPT I in programmed cell death, we examined the effects of long-chain fatty acids and found that the addition of palmitate or stearate to cultured cells led to activation of a death program with a morphology resembling that of apoptosis. Other naturally occurring fatty acids, including myristate and palmitoleate, had no effect. Since both palmitate and stearate are sphingolipid precursors, the effect of these fatty acids on sphingolipid metabolism was tested. Our results indicate that apoptosis induced by palmitate or stearate is correlated with de novo synthesis of ceramide. Inhibition of CPT I by etomoxir enhanced palmitate-induced cell death and led to a further increase in ceramide synthesis.
Assuntos
Apoptose/efeitos dos fármacos , Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Ácido Palmítico/farmacologia , Esfingolipídeos/biossíntese , Animais , Linhagem Celular , Ceramidas/biossíntese , Fígado/enzimologia , Camundongos , Esfingomielinas/biossíntese , Ácidos Esteáricos/farmacologiaRESUMO
To demonstrate interaction between the mitochondrial membrane proteins CPT I and Bcl-2 the yeast two-hybrid system was used. Full-length CPT I was required for binding to Bcl-2. Direct protein interaction was confirmed in a GST binding assay and in coimmunoprecipitations using two different kinds of anti-Bcl-2 antibodies.
Assuntos
Apoptose , Carnitina O-Palmitoiltransferase/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Membranas Intracelulares/metabolismo , Camundongos , Testes de Precipitina , Ligação Proteica , Proteínas Recombinantes , Relação Estrutura-AtividadeRESUMO
The alymphoplasia (aly) mutation of mice causes the systemic absence of lymph nodes, Peyer's patches and well-defined lymphoid follicles in the spleen. We found that antibody responses are elicited, albeit weakly, to either T cell-dependent or T cell-independent antigen by aly/aly mutants. However, isotype switching was defective. The T cell-dependent immune response was not elicited in splenectomized aly/aly mice. Neither hypermutation nor germinal center formation was observed in aly/aly mice. These results suggest that T-B collaboration requires either lymph nodes or spleen, and that hypermutation and affinity maturation depend on germinal center formation.