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2.
Phys Chem Chem Phys ; 21(38): 21355-21369, 2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31531502

RESUMO

Laser-induced fluorescence (LIF) excitation, dispersed fluorescence (DFL), UV-UV-hole burning, and UV-depletion spectra have been collected on methyl anthranilate (MA, methyl 2-aminobenzoate) and its water-containing complex (MA-H2O), under jet-cooled conditions in the gas phase. As a close structural analog of a sunscreen agent, MA has a strong absorption due to the S0-S1 transition that begins in the UV-A region, with the electronic origin at 28 852 cm-1 (346.6 nm). Unlike most sunscreens that have fast non-radiative pathways back to the ground state, MA fluoresces efficiently, with an excited state lifetime of 27 ns. Relative to methyl benzoate, inter-system crossing to the triplet manifold is shut off in MA by the strong intramolecular NHO[double bond, length as m-dash]C H-bond, which shifts the 3nπ* state well above the 1ππ* S1 state. Single vibronic level DFL spectra are used to obtain a near-complete assignment of the vibronic structure in the excited state. Much of the vibrational structure in the excitation spectrum is Franck-Condon activity due to three in-plane vibrations that modulate the distance between the NH2 and CO2Me groups, ν33 (421 cm-1), ν34 (366 cm-1), and ν36 (179 cm-1). Based on the close correspondence between experiment and theory at the TD-DFT B3LYP-D3BJ/def2TZVP level of theory, the major structural changes associated with electronic excitation are evaluated, leading to the conclusion that the major motion is a reorientation and constriction of the 6-membered H-bonded ring closed by the intramolecular NHO[double bond, length as m-dash]C H-bond. This leads to a shortening of the NHO[double bond, length as m-dash]C H-bond distance from 1.926 Å to 1.723 Å, equivalent to about a 25% reduction in the HO distance compared to full H-atom transfer. As a result, the excited state process near the S1 origin is a hydrogen atom dislocation that is brought about primarily by heavy atom motion, since the shortened H-bond distance results from extensive heavy-atom motion, with only a 0.03 Å increase in the NH bond length relative to its ground state value.

3.
J Phys Chem A ; 123(19): 4178-4187, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-30973730

RESUMO

Aminoisobutyric acid (Aib) oligomers are known to form racemic mixtures of enantiomeric left- and right-handed structures. The introduction of a chiral cap converts the enantiomeric structures into diastereomers that, in principle, afford spectroscopic differentiation. Here, we screen different C-terminal caps based on a model Aib dipeptide using double resonance laser spectroscopy in the gas phase to record IR and UV spectra of individual conformations present in the supersonic expansion: NH-benzyl (NHBn) as a reference structure because of its common use as a fluorophore in similar studies, NH- p-fluorobenzyl (NHBn-F), and α-methylbenzylamine (AMBA). For both the NHBn and NHBn-F caps, a single conformer is observed, with infrared spectra assignable to an enantiomeric pair of type II/II' ß-turns in these molecules lacking a chiral center. The higher oscillator strength of the NHBn-F cap enabled UV-UV hole burning, not readily accomplished with the NHBn cap. The AMBA-capped structure, with its chiral center, produced two unique conformers, one of which was a nearly identical left-handed type II ß-turn, while the minor conformer is assigned to a C7-C7 sequential double ring, which is an emergent form of a 27-ribbon. Although not observed, the type II' ß-turn diastereomer, with opposite handedness, is calculated to be 11 kJ/mol higher in energy, a surprisingly large difference. This destabilization is attributed primarily to steric interference between the C-terminal acyl oxygen of the peptide and the chirality-inducing methyl of the AMBA group. Last, computational evidence indicates that the use of an N-terminal aromatic cap hinders the formation of a 310-helix in Ac-Aib2 dipeptides.


Assuntos
Ácidos Aminoisobutíricos/química , Dipeptídeos/química , Cristalografia por Raios X , Teoria da Densidade Funcional , Modelos Moleculares , Conformação Proteica , Espectrofotometria Infravermelho , Espectrofotometria Ultravioleta
4.
J Phys Chem A ; 122(44): 8762-8775, 2018 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-30343572

RESUMO

The infrared and ultraviolet spectra of a series of capped asparagine-containing peptides, Ac-Asn-NHBn, Ac-Ala-Asn-NHBn, and Ac-Asn-Asn-NHBn, have been recorded under jet-cooled conditions in the gas phase in order to probe the influence of the Asn residue, with its -CH2-C(═O)-NH2 side chain, on the local conformational preferences of a peptide backbone. The double-resonance methods of resonant ion-dip infrared (RIDIR) spectroscopy and infrared-ultraviolet hole-burning (IR-UV HB) spectroscopy were used to record single-conformation spectra in the infrared and ultraviolet, respectively, free from interference from other conformations present in the molecular beam. Ac-Asn-NHBn spreads its population over two conformations, both of which are stabilized by a pair of H-bonds that form a bridge between the Asn carboxamide group and the NH and C═O groups on the peptide backbone. In one the peptide backbone engages in a 7-membered H-bonded ring (labeled C7eq), thereby forming an inverse γ-turn, stabilized by a C6/C7 Asn bridge. In the other the Asn carboxamide group forms a C8/C7 H-bonded bridge with the carboxamide group facing in the opposite direction across an extended peptide backbone involving a C5 interaction. Both Ac-Ala-Asn-NHBn and Ac-Asn-Asn-NHBn are found exclusively in a single conformation in which the peptide backbone engages in a type I ß-turn with its C10 H-bond. The Asn residue(s) stabilize this ß-turn via C6 H-bond(s) between the carboxamide C═O group and the same residue's amide NH. These structures are closely analogous to the corresponding structures in Gln-containing peptides studied previously [Walsh, P. S. et al. PCCP 2016, 18, 11306-11322; Walsh, P. S. et al. Angew. Chem. Int. Ed. 2016, 55, 14618-14622], indicating that the Asn and Gln side chains can each configure so as to stabilize the same backbone conformations. Spectroscopic and computational evidence suggest that glutamine is more predisposed than asparagine to ß-turn formation via unusually strong side-chain-backbone hydrogen-bond formation. Further spectral and structural similarities and differences due to the side-chain length difference of these similar amino acids are presented and discussed.

5.
J Phys Chem A ; 122(8): 2096-2107, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29464953

RESUMO

The single-conformation spectroscopy and infrared-induced conformational isomerization of a model protonated pentapeptide [YGPAA + H]+ is studied under cryo-cooled conditions in the gas phase. Building on recent results ( DeBlase , A. F. ; J. Am. Chem. Soc. 2017 , 139 , 5481 - 5493 ), firm assignments are established for the presence of two conformer families with distinct infrared and ultraviolet spectra, using IR-UV depletion spectroscopy. Families (A and B) share a similar structure near the N-terminus but differ in the way that the C-terminal COOH group configures itself (cis versus trans) in forming H-bonds with the peptide backbone. Infrared population transfer (IR-PT) spectroscopy is used to study the IR-induced conformational isomerization following single-conformer infrared excitation. IR-induced isomerization is accomplished in both directions (A → B and B → A) in the hydride stretch region and is used to determine fractional abundances for the two conformer families (FA = 0.65 ± 0.04, FB = 0.35 ± 0.04, 2σ error bars). The time scale for collisional cooling of the room-temperature ions to Tvib = 10 K by cold helium in the octupole trap is established as 1.0 ms. Key stationary points on the isomerization potential energy surface are calculated at the DFT B3LYP/6-31+G(d) G3DBJ level of theory. Using RRKM theory, the energy-dependent isomerization rates and populations are calculated as a function of energy. According to the model, the observed population distribution after collisional cooling is close to that of the 298 K Boltzmann distribution and is in near-quantitative agreement with experiment. On the basis of this success, inferences are drawn for the circumstances that govern the population distribution in the trap, concluding that, in ions the size of [YGPAA + H]+ and larger, the observed distributions will be near those at 298 K.

6.
J Phys Chem Lett ; 8(21): 5296-5300, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-28994601

RESUMO

An ultraviolet-infrared (UV-IR) double-resonance method for recording conformation-specific excited-state infrared spectra is described. The method takes advantage of an increase in fluorescence signal in phenylalkanes produced by infrared excitation of the S1 origin levels of different conformational isomers. The shorter lifetimes of these IR-excited molecules, combined with their red-shifted emission, provides a way to discriminate the fluorescence due to the infrared-excited molecules from the S1 origin fluorescence, resulting in spectra with high signal-to-noise ratios. Spectra for a series of phenylalkanes and a capped phenylalanine derivative (Ac-Phe-NHMe) demonstrate the potential of the method. The excited-state spectrum in the alkyl CH stretch region of ethylbenzene is well-fit by an anharmonic model developed for the ground electronic state, which explicitly takes into account stretch-bend Fermi resonance.

7.
Environ Toxicol Chem ; 36(3): 727-734, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27530554

RESUMO

Determining the rate of biodegradation of double-stranded RNA (dsRNA) in the environment is an essential element of a comprehensive risk assessment of an RNA-based agricultural product. This information is used during problem formulation to define relevant routes and durations of environmental exposure for in planta-expressed dsRNA. Although exposure to biotechnology-derived crops expressing dsRNA traits in the aquatic environment is predicted to be minimal, little is known regarding the fate of dsRNA in these environments. To assess exposure to aquatic environments, a study was conducted to measure the rate of biodegradation of DvSnf7 dsRNA in aerobic water-sediment systems. Aquatic systems containing natural water and sediments that varied in physical and chemical characteristics were treated with dsRNA by applying DvSnf7 dsRNA directly to the water column. In the present study, DvSnf7 dsRNA dissipated rapidly from the water phase and was undetectable within 7 d as measured by QuantiGene (Affymetrix) and a sensitive insect bioassay in these diverse systems. Degradation kinetics estimated a half-life (time to 50% dissipation [DT50]) of less than 3 d and a time to 90% dissipation of approximately 4 d. Further analysis indicated that DvSnf7 dsRNA had DT50 values of less than 6 d in both sediment-free systems containing natural water and systems with only sediment. Taken together, the results of the present study indicate that dsRNA-based agricultural products rapidly degrade and consequently are unlikely to persist in aquatic environments. Environ Toxicol Chem 2017;36:727-734. © 2016 SETAC.


Assuntos
Sedimentos Geológicos/química , RNA de Cadeia Dupla/análise , RNA de Plantas/análise , Poluentes Químicos da Água/análise , Agricultura , Animais , Biodegradação Ambiental , Bioensaio , Produtos Agrícolas , Meia-Vida , Insetos/efeitos dos fármacos
8.
Chemosphere ; 161: 319-324, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27441991

RESUMO

Performing environmental assessments for double-stranded RNA-based agricultural products require the development of sensitive and selective methods to measure biodegradation rates of dsRNAs. We developed and characterized a novel analytical procedure that uses a molecular hybridization assay (QuantiGene(®)) to accurately measure dsRNA extracted from diverse soils. In this report, we utilize this method to demonstrate that two dsRNAs with distinct size, structure, and sequence degrade rapidly in soil with indistinguishable kinetics.


Assuntos
Monitoramento Ambiental/métodos , Hibridização de Ácido Nucleico/métodos , RNA de Cadeia Dupla/análise , Solo/química , Agricultura , Proteínas de Insetos/genética , Limite de Detecção , Controle Biológico de Vetores , Plantas Geneticamente Modificadas/genética , RNA de Plantas/análise , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Microbiologia do Solo
9.
Anal Chem ; 88(22): 10831-10836, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-26938428

RESUMO

Matrix-assisted ionization (MAI)-mass spectrometry (MS) eliminates the need for high voltage, a heat source, lasers, and compressed gases in the ionization process and uses minimal solvents in sample preparation, thus making MAI ideal for field-portable mass spectrometers. The broad applicability of MAI is demonstrated by simple, rapid, and robust positive and negative detection mode analyses of low and high mass compounds including some pesticides, dyes, drugs, lipids, and proteins (186 Da to 8.5 kDa) from various materials including urine, biological tissue sections, paper, and plant material on a low pumping capacity, single-quadrupole mass spectrometer. Different sample introduction methods are applicable, including the use of a pipet tip or glass melting point tube, allowing integration of sample preparation with sample introduction for increased analytical utility and ease of operation, even when sampling directly from surfaces.


Assuntos
Corantes/análise , Lipídeos/análise , Praguicidas/análise , Preparações Farmacêuticas/análise , Proteínas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação
10.
Conf Proc IEEE Eng Med Biol Soc ; 2016: 4361-4364, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-28269244

RESUMO

Concussion is currently a serious health problem and can lead to severe brain dysfunction. There is a definite need for sensitive and reliable tests to detect and evaluate the subtle changes in brain function caused by concussion. This study details the development of a low cost portable eye tracking device that can aid in the detection of concussions. The device evaluates ocular motor function and can be used on the sidelines of sporting events to give a quick indication of the severity of head trauma. It can further be used to evaluate and monitor mild Traumatic Brain Injuries and aid in the return to play decision making process. Preliminary results show good accuracy and a fact test administration time. The device shows promise and will be tested in a large scale clinical trial.


Assuntos
Concussão Encefálica/diagnóstico , Movimentos Oculares/fisiologia , Gravação em Vídeo/métodos , Algoritmos , Traumatismos em Atletas/diagnóstico , Humanos , Gravação em Vídeo/instrumentação
11.
J Am Soc Mass Spectrom ; 26(12): 2086-95, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26453417

RESUMO

The analytical utility of a new and simple to use ionization method, matrix-assisted ionization (MAI), coupled with ion mobility spectrometry (IMS) and mass spectrometry (MS) is used to characterize a 2-armed europium(III)-containing poly(ethylene glycol) (Eu-PEG) complex directly from a crude sample. MAI was used with the matrix 1,2-dicyanobenzene, which affords low chemical background relative to matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). MAI provides high ion abundance of desired products in comparison to ESI and MALDI. Inductively coupled plasma-MS measurements were used to estimate a maximum of 10% of the crude sample by mass was the 2-arm Eu-PEG complex, supporting evidence of selective ionization of Eu-PEG complexes using the new MAI matrix, 1,2-dicyanobenzene. Multiply charged ions formed in MAI enhance the IMS gas-phase separation, especially relative to the singly charged ions observed with MALDI. Individual components are cleanly separated and readily identified, allowing characterization of the 2-arm Eu-PEG conjugate from a mixture of the 1-arm Eu-PEG complex and unreacted starting materials. Size-exclusion chromatography, liquid chromatography at critical conditions, MALDI-MS, ESI-MS, and ESI-IMS-MS had difficulties with this analysis, or failed. Graphical Abstract ᅟ.


Assuntos
Complexos de Coordenação/análise , Európio/análise , Polietilenoglicóis/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Cromatografia Líquida/métodos , Desenho de Equipamento , Nitrilos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação
12.
Bioorg Med Chem ; 23(15): 5035-49, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26048026

RESUMO

The combination of (1α,25)-dihydroxyvitamin D3 (1,25D) and histone deacetylase inhibitor (HDACi) trichostatin A is highly antiproliferative in numerous cancer cell lines. We have previously prepared novel non-secosteroidal hybrid molecules which simultaneously act as both vitamin D receptor (VDR) agonists and HDACi. These molecules function as cytostatic and cytotoxic agents in 1,25D-resistant SCC4 squamous carcinoma cells. Here we have extended the scope of the hybrids by making several modifications to the diarylpentane core and to the aliphatic spacer unit to develop molecules with increased potency towards HDACs while maintaining VDR agonist activity. Notably, hybrid DK-366 (33a), a direct analog of first-generation hybrids but lacking a methyl group on one aryl ring possesses low micromolar potency for HDAC3 and HDAC6 and is a highly effective antiproliferative agent in SCC4 cells. Chain extended hybrids such as DK-367 (33c) possess even greater HDAC potency and are also highly antiproliferative. These results show that we can optimize HDACi potency in hybrid molecules without sacrificing VDR agonism.


Assuntos
Citostáticos/química , Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Ácidos Hidroxâmicos/química , Receptores de Calcitriol/agonistas , Vitamina D/análogos & derivados , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citostáticos/síntese química , Citostáticos/toxicidade , Desenho de Drogas , Histona Desacetilases/metabolismo , Humanos , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Receptores de Calcitriol/metabolismo , Relação Estrutura-Atividade , Vitamina D/química
13.
Cell Microbiol ; 17(6): 860-75, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25486989

RESUMO

Microbial pathogens that colonize multiple tissues commonly produce adhesive surface proteins that mediate attachment to cells and/or extracellular matrix in target organs. Many of these 'adhesins' bind to multiple ligands, complicating efforts to understand the role of each ligand-binding activity. Borrelia burgdorferi, the causative agent of Lyme disease, produces BBK32, first identified as a fibronectin-binding adhesin that promotes skin and joint colonization. BBK32 also binds to glycosaminoglycan (GAG), which, like fibronectin is ubiquitously present on cell surfaces. To determine which binding activity is relevant for BBK32-promoted infectivity, we generated a panel of BBK32 truncation and internal deletion mutants, and identified variants specifically defective for binding to either fibronectin or GAG. These variants promoted bacterial attachment to different mammalian cell types in vitro, suggesting that fibronectin and GAG binding may play distinct roles during infection. Intravenous inoculation of mice with a high-passage non-infectious B. burgdorferi strain that produced wild-type BBK32 or BBK32 mutants defective for GAG or fibronectin binding, revealed that only GAG-binding activity was required for significant localization to joints at 60 min post-infection. An otherwise infectious B. burgdorferi strain producing BBK32 specifically deficient in fibronectin binding was fully capable of both skin and joint colonization in the murine model, whereas a strain producing BBK32 selectively attenuated for GAG binding colonized the inoculation site but not knee or tibiotarsus joints. Thus, the BBK32 fibronectin- and GAG-binding activities are separable in vivo, and BBK32-mediated GAG binding, but not fibronectin binding, contributes to joint colonization.


Assuntos
Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/metabolismo , Glicosaminoglicanos/metabolismo , Adesinas Bacterianas/genética , Animais , Proteínas de Bactérias/genética , Borrelia burgdorferi/genética , Análise Mutacional de DNA , Modelos Animais de Doenças , Fibronectinas/metabolismo , Articulações/microbiologia , Doença de Lyme , Camundongos , Ligação Proteica , Deleção de Sequência
14.
PLoS One ; 9(3): e93155, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24676387

RESUMO

A laboratory soil degradation study was conducted to determine the biodegradation potential of a DvSnf7 dsRNA transcript derived from a Monsanto genetically modified (GM) maize product that confers resistance to corn rootworm (CRW; Diabrotica spp.). This study provides new information to improve the environmental assessment of dsRNAs that become pesticidal through an RNAi process. Three agricultural soils differing in their physicochemical characteristics were obtained from the U.S., Illinois (IL; silt loam), Missouri (MO; loamy sand) and North Dakota (ND; clay loam), and exposed to the target dsRNA by incorporating insect-protected maize biomass and purified (in vitro-transcribed) DvSnf7 RNA into soil. The GM and control (non-GM maize) materials were added to each soil and incubated at ca. 22 °C for 48 hours (h). Samples were collected at 12 time intervals during the incubation period, extracted, and analyzed using QuantiGene molecular analysis and insect bioassay methods. The DT50 (half-life) values for DvSnf7 RNA in IL, MO, and ND soils were 19, 28, and 15 h based on QuantiGene, and 18, 29, and 14 h based on insect bioassay, respectively. Furthermore, the DT90 (time to 90% degradation) values for DvSnf7 RNA in all three soils were <35 h. These results indicate that DvSnf7 RNA was degraded and biological activity was undetectable within approximately 2 days after application to soil, regardless of texture, pH, clay content and other soil differences. Furthermore, soil-incorporated DvSnf7 RNA was non-detectable in soil after 48 h, as measured by QuantiGene, at levels ranging more than two orders of magnitude (0.3, 1.5, 7.5 and 37.5 µg RNA/g soil). Results from this study indicate that the DvSnf7 dsRNA is unlikely to persist or accumulate in the environment. Furthermore, the rapid degradation of DvSnf7 dsRNA provides a basis to define relevant exposure scenarios for future RNA-based agricultural products.


Assuntos
Agricultura , Meio Ambiente , RNA de Cadeia Dupla/química , Solo/química , Animais , Biomassa , Hidrólise , Insetos , Cinética , RNA de Plantas
15.
Chem Biol ; 19(8): 963-71, 2012 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-22921063

RESUMO

1,25-Dihydroxyvitamin D(3) (1,25D), the hormonal form of vitamin D, and several analogs have failed as monotherapies for cancer because of poor efficacy or acquired resistance. However, 1,25D analogs are amenable to bifunctionalization. Preclinical studies have revealed combinatorial effects of 1,25D analogs and histone deacetylase inhibitors (HDACi). Secosteroidal hybrid molecules combining vitamin D receptor (VDR) agonism with HDACi displayed enhanced efficacy but are laborious to synthesize. Here, we have developed easily assembled, fully integrated, non-secosteroidal VDR agonist/HDACi hybrids. The most promising are full VDR agonists with ~10-fold lower potency than 1,25D. Structure/function studies revealed that antiproliferative activity against 1,25D-resistant squamous carcinoma cells required VDR agonism and HDACi. Remarkably, modeling and X-ray crystallography reveal non-secosteroidal hybrids bind in the VDR ligand binding domain in the opposite orientation of their secosteroidal counterparts.


Assuntos
Antineoplásicos/química , Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Receptores de Calcitriol/agonistas , Vitamina D/análogos & derivados , Animais , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Desenho de Drogas , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Histona Desacetilases/metabolismo , Estrutura Terciária de Proteína , Receptores de Calcitriol/metabolismo , Secoesteroides/química , Peixe-Zebra
16.
Org Lett ; 13(13): 3376-9, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21648396

RESUMO

A sequence of Sonogashira coupling, Pd(II)-catalyzed carbonylative annulation, and benzofuran reduction (Mg, MeOH, NH(4)Cl) provides a convergent and modular synthetic route to trans-2-aryl-2,3-dihydrobenzo[b]furan-3-carboxylates, which are a structural feature of numerous biologically active natural products. This versatile strategy was applied to the formal total synthesis of the anti-HIV natural product (+)-lithospermic acid.


Assuntos
Fármacos Anti-HIV/síntese química , Benzofuranos/síntese química , Depsídeos/síntese química , Produtos Biológicos/síntese química , Catálise , Estrutura Molecular , Oxirredução , Paládio/química
17.
Infect Immun ; 79(9): 3501-9, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21708995

RESUMO

After transmission by an infected tick, the Lyme disease spirochete, Borrelia burgdorferi sensu lato, colonizes the mammalian skin and may disseminate systemically. The three major species of Lyme disease spirochete--B. burgdorferi sensu stricto, B. garinii, and B. afzelii--are associated with different chronic disease manifestations. Colonization is likely promoted by the ability to bind to target tissues, and Lyme disease spirochetes utilize multiple adhesive molecules to interact with diverse mammalian components. The allelic variable surface lipoprotein decorin binding protein A (DbpA) promotes bacterial binding to the proteoglycan decorin and to the glycosaminoglycan (GAG) dermatan sulfate. To assess allelic variation of DbpA in GAG-, decorin-, and cell-binding activities, we expressed dbpA alleles derived from diverse Lyme disease spirochetes in B. burgdorferi strain B314, a noninfectious and nonadherent strain that lacks dbpA. Each DbpA allele conferred upon B. burgdorferi strain B314 the ability to bind to cultured kidney epithelial (but not glial or endothelial) cells, as well as to purified decorin and dermatan sulfate. Nevertheless, allelic variation of DbpA was associated with dramatic differences in substrate binding activity. In most cases, decorin and dermatan sulfate binding correlated well, but DbpA of B. afzelii strain VS461 promoted differential binding to decorin and dermatan sulfate, indicating that the two activities are separable. DbpA from a clone of B. burgdorferi strain N40 that can cause disseminated infection in mice displayed relatively low adhesive activity, indicating that robust DbpA-mediated adhesive activity is not required for spread in the mammalian host.


Assuntos
Adesinas Bacterianas/genética , Decorina/metabolismo , Dermatan Sulfato/metabolismo , Doença de Lyme/microbiologia , Adesinas Bacterianas/metabolismo , Alelos , Animais , Aderência Bacteriana/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Sequência de Bases , Grupo Borrelia Burgdorferi/genética , Grupo Borrelia Burgdorferi/imunologia , Grupo Borrelia Burgdorferi/patogenicidade , Adesão Celular/genética , Adesão Celular/fisiologia , Células Cultivadas , Variação Genética , Células HEK293 , Humanos , Ratos , Análise de Sequência de DNA , Pele/microbiologia
18.
Mutagenesis ; 24(4): 309-16, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19372135

RESUMO

The XPC protein (encoded by the xeroderma pigmentosum Xpc gene) is a key DNA damage recognition factor that is required for global genomic nucleotide excision repair (G-NER). In contrast to transcription-coupled nucleotide excision repair (TC-NER), XPC and G-NER have been reported to contribute only modestly to cell survival after DNA damage. Previous studies were conducted using fibroblasts of human or mouse origin. Since the advent of Xpc-/- mice, no study has focused on the bone marrow of these mice. We used carboplatin to induce DNA damage in Xpc-/- and strain-matched wild-type mice. Using several independent methods, Xpc-/- bone marrow was approximately 10-fold more sensitive to carboplatin than the wild type. Importantly, 12/20 Xpc-/- mice died while 0/20 wild-type mice died. We conclude that G-NER, and XPC specifically, can contribute substantially to cell survival. The data are important in the context of cancer chemotherapy, where Xpc gene status and G-NER may be determinants of response to DNA-damaging agents including carboplatin. Additionally, altered cell cycles and altered DNA damage signalling may contribute to the cell survival end point.


Assuntos
Células da Medula Óssea/citologia , Sobrevivência Celular , Proteínas de Ligação a DNA/genética , Animais , Antineoplásicos/farmacologia , Carboplatina/farmacologia , Ciclo Celular , Linhagem Celular Tumoral , Linhagem da Célula , Dano ao DNA , Marcadores Genéticos , Humanos , Camundongos , Camundongos Transgênicos , Modelos Genéticos
19.
J Bacteriol ; 190(24): 7885-91, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18849429

RESUMO

Borrelia burgdorferi is the causative agent of Lyme disease, the most common vector-borne illness in the Northern hemisphere. Low-passage-number infectious strains of B. burgdorferi exhibit extremely low transformation efficiencies-so low, in fact, as to hinder the genetic study of putative virulence factors. Two putative restriction-modification (R-M) systems, BBE02 contained on linear plasmid 25 (lp25) and BBQ67 contained on lp56, have been postulated to contribute to this poor transformability. Restriction barriers posed by other bacteria have been overcome by the in vitro methylation of DNA prior to transformation. To test whether a methylation-sensitive restriction system contributes to poor B. burgdorferi transformability, shuttle plasmids were treated with the CpG methylase M.SssI prior to the electroporation of a variety of strains harboring different putative R-M systems. We found that for B. burgdorferi strains that harbor lp56, in vitro methylation increased transformation by at least 1 order of magnitude. These results suggest that in vitro CpG methylation protects exogenous DNA from degradation by an lp56-contained R-M system, presumably BBQ67. The utility of in vitro methylation for the genetic manipulation of B. burgdorferi was exemplified by the ease of plasmid complementation of a B. burgdorferi B31 A3 BBK32 kanamycin-resistant (B31 A3 BBK32::Kan(r)) mutant, deficient in the expression of the fibronectin- and glycosaminoglycan (GAG)-binding adhesin BBK32. Consistent with the observation that several surface proteins may promote GAG binding, the B. burgdorferi B31 A3 BBK32::Kan(r) mutant demonstrated no defect in the ability to bind purified GAGs or GAGs expressed on the surfaces of cultured cells.


Assuntos
Borrelia burgdorferi/genética , Ilhas de CpG , Metilação de DNA , Plasmídeos/metabolismo , Transformação Bacteriana , Borrelia burgdorferi/metabolismo , DNA Bacteriano/metabolismo , DNA-Citosina Metilases/metabolismo , Eletroporação , Teste de Complementação Genética , Mutagênese Insercional , Mutação
20.
Mol Cancer Ther ; 6(1): 355-61, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17237294

RESUMO

Selenium in various chemical forms has been the subject of cancer chemoprevention trials, but, more recently, selenium has been used in combination with DNA-damaging chemotherapeutics. Specifically, selenium protected tissues from dose-limiting toxicity and, in fact, allowed delivery of higher chemotherapeutic doses. At the same time, selenium did not protect cancer cells. Therefore, we seek to define the genetic basis for the observed selectivity of selenium in combination chemotherapeutics. The tumor suppressor p53 is mutated in the vast majority of cancers, but is by definition wild-type in nontarget tissues such as bone marrow and gut epithelium, tissues that are often dose-limiting due to DNA damage. We used primary, low-passage mouse embryonic fibroblasts that are wild-type or null for p53 genes to test differential effects of selenium. Seleno-l-methionine, nontoxic by itself, was used to pretreat cell cultures before exposure to UV radiation or UV-mimetic cancer chemotherapy drugs. Seleno-l-methionine pretreatment caused a DNA repair response, which protected from subsequent challenge with DNA-damaging agents. The observed DNA repair response and subsequent DNA damage protection were p53 dependent as neither was observed in p53-null cells. The data suggest that (a) p53 may be an important genetic determinant that distinguishes normal cells from cancer cells, and (b) combinatorial chemotherapeutics that act by p53-dependent mechanisms may enhance chemotherapeutic efficacy by increasing the chemotherapeutic window distinguishing cancer cells from normal cells.


Assuntos
Reparo do DNA , Compostos Organoplatínicos/farmacologia , Selenometionina/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , DNA/biossíntese , DNA/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Genoma/efeitos dos fármacos , Genoma/efeitos da radiação , Humanos , Camundongos , Especificidade de Órgãos , Compostos Organosselênicos/metabolismo , Proteína Supressora de Tumor p53/deficiência , Raios Ultravioleta
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