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1.
Int J Mol Sci ; 21(6)2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32244989

RESUMO

The Center of Biomedical Research Excellence in Matrix Biology strives to improve our understanding of extracellular matrix at molecular, cellular, tissue, and organismal levels to generate new knowledge about pathophysiology, normal development, and regenerative medicine. The primary goals of the Center are to i) support junior investigators, ii) enhance the productivity of established scientists, iii) facilitate collaboration between both junior and established researchers, and iv) build biomedical research infrastructure that will support research relevant to cell-matrix interactions in disease progression, tissue repair and regeneration, and v) provide access to instrumentation and technical support. A Pilot Project program provides funding to investigators who propose applying their expertise to matrix biology questions. Support from the National Institute of General Medical Sciences at the National Institutes of Health that established the Center of Biomedical Research Excellence in Matrix Biology has significantly enhanced the infrastructure and the capabilities of researchers at Boise State University, leading to new approaches that address disease diagnosis, prevention, and treatment. New multidisciplinary collaborations have been formed with investigators who may not have previously considered how their biomedical research programs addressed fundamental and applied questions involving the extracellular matrix. Collaborations with the broader matrix biology community are encouraged.

2.
Regen Med ; 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31313975

RESUMO

Aim: Umbilical cord blood (UCB) sourced allografts are promising interventions for tissue regeneration. As applications of these allografts and regulations governing them continue to evolve, we were prompted to identify parameters determining their quality, safety and regenerative potential. Materials & methods: Flow-cytometry, mass-spectrometry, protein multiplexing, nanoparticle tracking analysis and standard biological techniques were employed. Results: Quality attributes of a uniquely processed UCB-allograft (UCBr) were enumerated based on identity (cell viability, immunophenotyping, proteomic profiling, and quantification of relevant cytokines); safety (bioburden and microbiological screening), purity (endotoxin levels) and potency (effect of UCBr on chondrocytes and mesenchymal stem cells derived exosomes). These attributes were stable up to 24 months in cryopreserved UCBr. Conclusion: We identified a comprehensive panel of tests to establish the clinical efficacy and quality control attributes of a UCB-sourced allograft.

3.
Sci Rep ; 8(1): 15515, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30341382

RESUMO

Notch signaling is a form of intercellular communication which plays pivotal roles at various stages in development and disease. Previous findings have hinted that integrins and extracellular matrix may regulate Notch signaling, although a mechanistic basis for this interaction had not been identified. Here, we reveal that the regulation of Notch by integrins and extracellular matrix is carried out by Src family kinases (SFKs) working downstream of integrins. We identify a physical interaction between the SFK member, c-Src, and the Notch intracellular domain (NICD) that is enhanced by ß3 integrin and the integrin binding ECM protein, MAGP2. Our results demonstrate that c-Src directly phosphorylates the NICD at specific tyrosine residues and that mutation of these phosphorylation sites increases Notch responsive transcriptional activity. Furthermore, we also find that phosphorylation of the NICD by SFKs attenuates Notch mediated transcription by decreasing recruitment of MAML to the Notch co-transcriptional complex. Finally, we also find that SFK activity decreases NICD half-life. Collectively, our results provide important mechanistic data that underlie the emerging role of Notch as a general sensor and responder to extracellular signals.


Assuntos
/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endotélio Vascular/fisiologia , Matriz Extracelular/metabolismo , Receptor Notch1/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular , Proteínas Contráteis/metabolismo , Endotélio Vascular/patologia , Meia-Vida , Humanos , Integrina beta3/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fosforilação , Ligação Proteica , Estabilidade Proteica , Transdução de Sinais
4.
Ann Biomed Eng ; 46(11): 1882-1895, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29873012

RESUMO

Ligament wound healing involves the proliferation of a dense and disorganized fibrous matrix that slowly remodels into scar tissue at the injury site. This remodeling process does not fully restore the highly aligned collagen network that exists in native tissue, and consequently repaired ligament has decreased strength and durability. In order to identify treatments that stimulate collagen alignment and strengthen ligament repair, there is a need to develop in vitro models to study fibroblast activation during ligament wound healing. The objective of this study was to measure gene expression and matrix protein accumulation in fibroblast-collagen gels that were subjected to different static stress conditions (stress-free, biaxial stress, and uniaxial stress) for three time points (1, 2 or 3 weeks). By comparing our in vitro results to prior in vivo studies, we found that stress-free gels had time-dependent changes in gene expression (col3a1, TnC) corresponding to early scar formation, and biaxial stress gels had protein levels (collagen type III, decorin) corresponding to early scar formation. This is the first study to conduct a targeted evaluation of ligament healing biomarkers in fibroblast-collagen gels, and the results suggest that biomimetic in-vitro models of early scar formation should be initially cultured under biaxial stress conditions.


Assuntos
Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Ligamentos , Modelos Biológicos , Cicatrização , Animais , Matriz Extracelular/patologia , Fibroblastos/patologia , Géis , Ligamentos/lesões , Ligamentos/metabolismo , Ligamentos/patologia , Camundongos , Células NIH 3T3
5.
Sci Rep ; 7(1): 2448, 2017 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-28550293

RESUMO

The ability of pore-forming proteins to interact with various analytes has found vast applicability in single molecule sensing and characterization. In spite of their abundance in organisms from all kingdoms of life, only a few pore-forming proteins have been successfully reconstituted in artificial membrane systems for sensing purposes. Lysenin, a pore-forming toxin extracted from the earthworm E. fetida, inserts large conductance nanopores in lipid membranes containing sphingomyelin. Here we show that single lysenin channels may function as stochastic nanosensors by allowing the short cationic peptide angiotensin II to be electrophoretically driven through the conducting pathway. Long-term translocation experiments performed using large populations of lysenin channels allowed unequivocal identification of the unmodified analyte by Liquid Chromatography-Mass Spectrometry. However, application of reverse voltages or irreversible blockage of the macroscopic conductance of lysenin channels by chitosan addition prevented analyte translocation. This investigation demonstrates that lysenin channels have the potential to function as nano-sensing devices capable of single peptide molecule identification and characterization, which may be further extended to other macromolecular analytes.


Assuntos
Angiotensina II/química , Bicamadas Lipídicas/química , Oligoquetos/química , Toxinas Biológicas/química , Angiotensina II/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Técnicas Biossensoriais/métodos , Quitosana/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Bicamadas Lipídicas/metabolismo , Esfingomielinas/química , Esfingomielinas/metabolismo , Toxinas Biológicas/metabolismo
6.
Artigo em Inglês | MEDLINE | ID: mdl-28533891

RESUMO

Although harboring the apolipoprotein E4 (APOE4) allele is a well known risk factor in Alzheimer's disease (AD), the mechanism by which it contributes to disease risk remains elusive. To investigate the role of proteolysis of apoE4 as a potential mechanism, we designed and characterized a site-directed cleavage antibody directed at position D151 of the mature form of apoE4 and E3. Characterization of this antibody indicated a high specificity for detecting synthesized recombinant proteins corresponding to the amino acid sequences 1-151 of apoE3 and E4 that would generate the 17 kDa (p17) fragment. In addition, this antibody also detected a ~17 kDa amino-terminal fragment of apoE4 following incubation with collagenase and matrix metalloproteinase-9 (MMP-9), but did not react with full-length apoE4. Application of this amino-terminal apoE cleavage-fragment (nApoECFp17) antibody, revealed nuclear labeling within glial cells and labeling of a subset of neurofibrillary tangles in the human AD brain. A quantitative analysis indicated that roughly 80% of labeled nuclei were microglia. To confirm these findings, cultured BV2 microglia cells were incubated with the amino-terminal fragment of apoE4 corresponding to the cleavage site at D151. The results indicated efficient uptake of this fragment and trafficking to the nucleus that also resulted in significant cell death. In contrast, a similarly designed apoE3 fragment showed no toxicity and primarily localized within the cytoplasm. These data suggest a novel cleavage event by which apoE4 is cleaved by the extracellular proteases, collagenase and MMP-9, generating an amino-terminal fragment that is then taken up by microglia, traffics to the nucleus and promotes cell death. Collectively, these findings provide important mechanistic insights into the mechanism by which harboring the APOE4 allele may elevate dementia risk observed in AD.

7.
Protein Expr Purif ; 134: 147-153, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28400296

RESUMO

The RNA-binding proteins that comprise the La-related protein (LARP) superfamily have been implicated in a wide range of cellular functions, from tRNA maturation to regulation of protein synthesis. To more expansively characterize the biological function of the LARP6 subfamily, we have recombinantly expressed the full-length LARP6 proteins from two teleost fish, platyfish (Xiphophorus maculatus) and zebrafish (Danio rerio). The yields of the recombinant proteins were enhanced to >2 mg/L using a tandem approach of an N-terminal His6-SUMO tag and an iterative solubility screening assay to identify structurally stabilizing buffer components. The domain topologies of the purified fish proteins were probed with limited proteolysis. The fish proteins contain an internal, protease-resistant 40 kDa domain, which is considerably more stable than the comparable domain from the human LARP6 protein. The fish proteins are therefore a lucrative model system in which to study both the evolutionary divergence of this family of La-related proteins and the structure and conformational dynamics of the domains that comprise the LARP6 protein.


Assuntos
Ciprinodontiformes/genética , Expressão Gênica , Proteínas de Ligação a RNA , Proteínas de Peixe-Zebra , Peixe-Zebra/genética , Animais , Ciprinodontiformes/metabolismo , Humanos , Domínios Proteicos , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/isolamento & purificação
8.
Toxicol Appl Pharmacol ; 311: 42-51, 2016 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-27693115

RESUMO

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant and high-affinity ligand for the aryl hydrocarbon receptor (AhR). Increasing evidence indicates that AhR signaling contributes to wound healing, which involves the coordinated deposition and remodeling of the extracellular matrix. In the liver, wound healing is attributed to the activation of hepatic stellate cells (HSCs), which mediate fibrogenesis through the production of soluble mediators and collagen type I. We recently reported that TCDD treatment increases the activation of human HSCs in vitro. The goal of this study was to determine how TCDD impacts HSC activation in vivo using a mouse model of experimental liver fibrosis. To elicit fibrosis, C57BL6/male mice were treated twice weekly for 8weeks with 0.5ml/kg carbon tetrachloride (CCl4). TCDD (20µg/kg) or peanut oil (vehicle) was administered once a week during the last 2weeks. Results indicate that TCDD increased liver-body-weight ratios, serum alanine aminotransferase activity, and hepatic necroinflammation in CCl4-treated mice. Likewise, TCDD treatment increased mRNA expression of HSC activation and fibrogenesis genes, namely α-smooth muscle actin, desmin, delta-like homolog-1, TGF-ß1, and collagen type I. However, TCDD treatment did not exacerbate fibrosis, nor did it increase the collagen content of the liver. Instead, TCDD increased hepatic collagenase activity and increased expression of matrix metalloproteinase (MMP)-13 and the matrix regulatory proteins, TIMP-1 and PAI-1. These results support the conclusion that TCDD increases CCl4-induced liver damage and exacerbates HSC activation, yet collagen deposition and the development of fibrosis may be limited by TCDD-mediated changes in extracellular matrix remodeling.


Assuntos
Células Estreladas do Fígado/efeitos dos fármacos , Inflamação/induzido quimicamente , Cirrose Hepática/induzido quimicamente , Dibenzodioxinas Policloradas/toxicidade , Animais , Tetracloreto de Carbono/toxicidade , Colágeno Tipo I/metabolismo , Colagenases/metabolismo , Células Estreladas do Fígado/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Cirrose Hepática/metabolismo , Masculino , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Índice de Gravidade de Doença
9.
Bioorg Med Chem ; 24(16): 3752-7, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27338657

RESUMO

Veratrum californicum, commonly referred to as corn lily or Californian false hellebore, grows in high mountain meadows and produces the steroidal alkaloid cyclopamine, a potent inhibitor of the Hedgehog (Hh) signaling pathway. The Hh pathway is a crucial regulator of many fundamental processes during vertebrate embryonic development. However, constitutive activation of the Hh pathway contributes to the progression of various cancers. In the present study, a direct correlation was made between the extraction efficiency for cyclopamine from root and rhizome by eight methods, and the associated biological activity in Shh-Light II cells using the Dual-Glo® Luciferase Assay System. Alkaloid recovery ranged from 0.39 to 8.03mg/g, with ethanol soak being determined to be the superior method for obtaining biologically active cyclopamine. Acidic ethanol and supercritical extractions yielded degraded or contaminated cyclopamine with lower antagonistic activity towards Hh signaling.


Assuntos
Alcaloides de Veratrum/farmacologia , Veratrum/química , Biomassa , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Transdução de Sinais/efeitos dos fármacos , Alcaloides de Veratrum/isolamento & purificação
10.
Toxicol Mech Methods ; 26(3): 196-201, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26982377

RESUMO

The Comet assay is a reproducible and sensitive assay for the detection of DNA damage in eukaryotic cells and tissues. Incorporation of lesion specific, oxidative DNA damage repair enzymes (for example, Fpg, OGG1 and EndoIII) in the standard alkaline Comet assay procedure allows for the detection and measurement of oxidative DNA damage. The Comet assay using white blood cells (WBC) has proven useful in monitoring DNA damage from environmental agents in humans. However, it is often impractical to performance Comet assay immediately after blood sampling. Thus, storage of blood sample is required. In this study, we developed and tested a simple storage method for very small amount of whole blood for standard and Fpg-modified modified Comet assay. Whole blood was stored in RPMI 1640 media containing 10% FBS, 10% DMSO and 1 mM deferoxamine at a sample to media ratio of 1:50. Samples were stored at -20 °C and -80 °C for 1, 7, 14 and 28 days. Isolated lymphocytes from the same subjects were also stored under the same conditions for comparison. Direct DNA strand breakage and oxidative DNA damage in WBC and lymphocytes were analyzed using standard and Fpg-modified alkaline Comet assay and compared with freshly analyzed samples. No significant changes in either direct DNA strand breakage or oxidative DNA damage was seen in WBC and lymphocytes stored at -20 °C for 1 and 7 days compared to fresh samples. However, significant increases in both direct and oxidative DNA damage were seen in samples stored at -20 °C for 14 and 28 days. No changes in direct and oxidative DNA damage were observed in WBC and lymphocytes stored at -80 °C for up to 28 days. These results identified the proper storage conditions for storing whole blood or isolated lymphocytes to evaluate direct and oxidative DNA damage using standard and Fpg-modified alkaline Comet assay.


Assuntos
Ensaio Cometa/métodos , Criopreservação/métodos , Dano ao DNA , DNA-Formamidopirimidina Glicosilase , Eritrócitos/patologia , Proteínas de Escherichia coli , Leucócitos/patologia , Adulto , DNA-Formamidopirimidina Glicosilase/isolamento & purificação , Proteínas de Escherichia coli/isolamento & purificação , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade
11.
Toxicology ; 344-346: 26-33, 2016 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-26860701

RESUMO

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a halogenated aromatic hydrocarbon that elicits toxicity through the aryl hydrocarbon receptor (AhR). In the liver, gross markers of TCDD toxicity are attributed to AhR activation in parenchymal hepatocytes. However, less is known regarding the consequences of TCDD treatment on non-parenchymal cells in the liver. Hepatic stellate cells (HSCs) are non-parenchymal cells that store vitamin A when quiescent. Upon liver injury, activated HSCs lose this storage ability and instead function in the development and maintenance of inflammation and fibrosis through the production of pro-inflammatory mediators and collagen type I. Reports that TCDD exposure disrupts hepatic retinoid homeostasis and dysregulates extracellular matrix remodeling in the liver led us to speculate that TCDD treatment may disrupt HSC activity. The human HSC line LX-2 was used to test the hypothesis that TCDD treatment directly activates HSCs. Results indicate that exposure to 10nM TCDD almost completely inhibited lipid droplet storage in LX-2 cells cultured with retinol and palmitic acid. TCDD treatment also increased LX-2 cell proliferation, expression of α-smooth muscle actin, and production of monocyte chemoattractant protein-1 (MCP-1), all of which are characteristics of activated HSCs. However, TCDD treatment had no effect on Col1a1 mRNA levels in LX-2 cells stimulated with the potent profibrogenic mediator, transforming growth factor-ß. The TCDD-mediated increase in LX-2 cell proliferation, but not MCP-1 production, was abolished when phosphoinositide 3-kinase was inhibited. These results indicate that HSCs are susceptible to direct modulation by TCDD and that TCDD likely increases HSC activation through a multi-faceted mechanism.


Assuntos
Proliferação de Células/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Dibenzodioxinas Policloradas/toxicidade , Linhagem Celular , Proliferação de Células/fisiologia , Humanos , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/metabolismo
12.
Environ Toxicol ; 31(12): 1808-1818, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26332274

RESUMO

The induction of oxidative stress and damage appears to be involved in acrylonitrile induction of brain astrocytomas in rat. The present study examined the effects of dietary antioxidant supplementation on acrylonitrile-induced oxidative stress and oxidative damage in rats in vivo. To assess the effects of antioxidants on biomarkers of acrylonitrile-induced oxidative stress, female F344 rats were provided with diets containing vitamin E (0.05%), green tea polyphenols (GTP, 0.4%), N-acetyl cysteine (NAC, 0.3%), sodium selenite (0.1mg/kg), and taurine (10g/kg) for 7 days, and then co-administered with 0 and 100 ppm acrylonitrile in drinking water for 28 days. Significant increase in oxidative DNA damage in brain, evidenced by elevated 8OHdG levels, was seen in acrylonitrile-exposed rats. Supplementation with vitamin E, GTP, and NAC reduced acrylonitrile-induced oxidative DNA damage in brain while no protective effects were seen with the selenium or taurine supplementation. Acrylonitrile increased oxidative DNA damage, measured by the fpg-modified alkaline Comet assay in rat WBCs, which was reduced by supplementation of Vitamin E, GTP, NAC, selenium, and taurine. In addition to stimulation of oxidative DNA damage, acrylonitrile triggered induction of pro-inflammatory cytokines Tnfα, Il-1ß, and Ccl2, and the growth stimulatory cyclin D1 and cyclin D2 genes, which were effectively down-regulated with antioxidant treatment. Antioxidant treatment also was able to stimulate the pro-apoptotic genes Bad, Bax, and FasL and DNA repair genes Xrcc6 and Gadd45α. The results of this study support the involvement of oxidative stress in the development of acrylonitrile-induced astrocytomas and suggest that antioxidants block acrylonitrile-mediated damage through mechanisms that may involve in the suppression of inflammatory responses, inhibition of cell proliferation and stimulation of apoptosis. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1808-1818, 2016.


Assuntos
Acrilonitrila/toxicidade , Antioxidantes/farmacologia , Encéfalo/efeitos dos fármacos , Carcinógenos/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Acetilcisteína/farmacologia , Animais , Biomarcadores/metabolismo , Encéfalo/metabolismo , Camellia sinensis/química , Dano ao DNA/efeitos dos fármacos , Suplementos Nutricionais , Feminino , Polifenóis/farmacologia , Ratos Endogâmicos F344 , Selênio/farmacologia , Taurina/farmacologia , Vitamina E/farmacologia
13.
West N Am Nat ; 75(1): 78-87, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-26582971

RESUMO

Sagebrush (Artemisia spp.) in North America is an abundant native plant species that is ecologically and evolutionarily adapted to have a diverse array of biologically active chemicals. Several of these chemicals, specifically polyphenols, have antioxidant activity that may act as biomarkers of biotic or abiotic stress. This study investigated the spatial variation of antioxidant capacity, as well as the relationship between a mammalian herbivore and antioxidant capacity in Wyoming big sagebrush (Artemisia tridentata wyomingensis). We quantified and compared total polyphenols and antioxidant capacity of leaf extracts from sagebrush plants from different spatial scales and at different levels of browsing by a specialist mammalian herbivore, the pygmy rabbit (Brachylagus idahoensis). We found that antioxidant capacity of sagebrush extracts was positively correlated with total polyphenol content. Antioxidant capacity varied spatially within and among plants. Antioxidant capacity in sagebrush was not related to either browsing intensity or duration of association with rabbits. We propose that the patterns of antioxidant capacity observed in sagebrush may be a result of spatial variation in abiotic stress experienced by sagebrush. Antioxidants could therefore provide a biomarker of environmental stress for sagebrush that could aid in management and conservation of this plant in the threatened sagebrush steppe.

14.
Methods Mol Biol ; 1340: 263-78, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26445845

RESUMO

Tissue engineering holds promise for the treatment of damaged and diseased tissues, especially for those tissues that do not undergo repair and regeneration readily in situ. Many techniques are available for cell and tissue culturing and differentiation of chondrocytes using a variety of cell types, differentiation methods, and scaffolds. In each case, it is critical to demonstrate the cellular phenotype and tissue composition, with particular attention to the extracellular matrix molecules that play a structural role and that contribute to the mechanical properties of the resulting tissue construct. Mass spectrometry provides an ideal analytical method with which to characterize the full spectrum of proteins produced by tissue-engineered cartilage. Using normal cartilage tissue as a standard, tissue-engineered cartilage can be optimized according to the entire proteome. Proteomic analysis is a complementary approach to biochemical, immunohistochemical, and mechanical testing of cartilage constructs. Proteomics is applicable as an analysis approach to most cartilage constructs generated from a variety of cellular sources including primary chondrocytes, mesenchymal stem cells from bone marrow, adipose tissue, induced pluripotent stem cells, and embryonic stem cells. Additionally, proteomics can be used to optimize novel scaffolds and bioreactor applications, yielding cartilage tissue with the proteomic profile of natural cartilage.


Assuntos
Cartilagem/metabolismo , Condrócitos/metabolismo , Condrogênese , Proteínas da Matriz Extracelular/metabolismo , Proteômica/métodos , Medicina Regenerativa/métodos , Engenharia Tecidual/métodos , Animais , Biomarcadores/metabolismo , Cartilagem/citologia , Bovinos , Técnicas de Cultura de Células , Células Cultivadas , Biologia Computacional , Bases de Dados de Proteínas , Eletroforese em Gel de Poliacrilamida , Nanotecnologia , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
15.
Curr Protoc Toxicol ; 65: 3.12.1-3.12.11, 2015 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-26250399

RESUMO

Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies.


Assuntos
Ensaio Cometa/métodos , Dano ao DNA , Linfócitos/efeitos dos fármacos , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Substâncias Perigosas , Humanos , Estresse Oxidativo , Sensibilidade e Especificidade
16.
Biochemistry ; 53(39): 6231-42, 2014 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-25215658

RESUMO

The acyl-homoserine lactone (AHL) autoinducer mediated quorum sensing regulates virulence in several pathogenic bacteria. The hallmark of an efficient quorum sensing system relies on the tight specificity in the signal generated by each bacterium. Since AHL signal specificity is derived from the acyl-chain of the acyl-ACP (ACP = acyl carrier protein) substrate, AHL synthase enzymes must recognize and react with the native acyl-ACP with high catalytic efficiency while keeping reaction rates with non-native acyl-ACPs low. The mechanism of acyl-ACP substrate recognition in these enzymes, however, remains elusive. In this study, we investigated differences in catalytic efficiencies for shorter and longer chain acyl-ACP substrates reacting with an octanoyl-homoserine lactone synthase Burkholderia mallei BmaI1. With the exception of two-carbon shorter hexanoyl-ACP, the catalytic efficiencies of butyryl-ACP, decanoyl-ACP, and octanoyl-CoA reacting with BmaI1 decreased by greater than 20-fold compared to the native octanoyl-ACP substrate. Furthermore, we also noticed kinetic cooperativity when BmaI1 reacted with non-native acyl-donor substrates. Our kinetic data suggest that non-native acyl-ACP substrates are unable to form a stable and productive BmaI1·acyl-ACP·SAM ternary complex and are thus effectively discriminated by the enzyme. These results offer insights into the molecular basis of substrate recognition for the BmaI1 enzyme.


Assuntos
Proteína de Transporte de Acila/metabolismo , Acil-Butirolactonas/metabolismo , Proteínas de Bactérias/metabolismo , Ligases/metabolismo , Proteínas de Bactérias/genética , Biocatálise , Burkholderia mallei/enzimologia , Burkholderia mallei/genética , Burkholderia mallei/metabolismo , Cromatografia Líquida de Alta Pressão , Cinética , Ligases/genética , Especificidade por Substrato
17.
PLoS One ; 9(3): e90052, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24651674

RESUMO

Several risk factors have been identified as potential contributors to pancreatic cancer development, including environmental and lifestyle factors, such as smoking, drinking and diet, and medical conditions such as diabetes and pancreatitis, all of which generate oxidative stress and DNA damage. Oxidative stress status can be modified by environmental factors and also by an individual's unique genetic makeup. Here we examined the contribution of environment and genetics to an individual's level of oxidative stress, DNA damage and susceptibility to pancreatic cancer in a pilot study using three groups of subjects: a newly diagnosed pancreatic cancer group, a healthy genetically-unrelated control group living with the case subject, and a healthy genetically-related control group which does not reside with the subject. Oxidative stress and DNA damage was evaluated by measuring total antioxidant capacity, direct and oxidative DNA damage by Comet assay, and malondialdehyde levels. Direct DNA damage was significantly elevated in pancreatic cancer patients (age and sex adjusted mean ± standard error: 1.00 ± 0.05) versus both healthy unrelated and related controls (0.70 ± 0.06, p<0.001 and 0.82 ± 0.07, p = 0.046, respectively). Analysis of 22 selected SNPs in oxidative stress and DNA damage genes revealed that CYP2A6 L160H was associated with pancreatic cancer. In addition, DNA damage was found to be associated with TNFA -308G>A and ERCC4 R415Q polymorphisms. These results suggest that measurement of DNA damage, as well as select SNPs, may provide an important screening tool to identify individuals at risk for development of pancreatic cancer.


Assuntos
Meio Ambiente , Predisposição Genética para Doença , Neoplasias Pancreáticas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Estudos de Casos e Controles , Dano ao DNA , Demografia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Neoplasias Pancreáticas/patologia , Polimorfismo de Nucleotídeo Único/genética , Adulto Jovem
18.
J Mammal ; 95(4): 834-842, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-26366011

RESUMO

For herbivores, nutrient intake is limited by the relatively low nutritional quality of plants and high concentrations of potentially toxic defensive compounds (plant secondary metabolites, PSMs) produced by many plants. In response to phytochemical challenges, some herbivores selectively forage on plants with higher nutrient and lower PSM concentrations relative to other plants. Pygmy rabbits (Brachylagus idahoensis) are dietary specialists that feed on sagebrush (Artemisia spp.) and forage on specific plants more than others within a foraging patch. We predicted that the plants with evidence of heavy foraging (browsed plants) would be of higher dietary quality than plants that were not browsed (unbrowsed). We used model selection to determine which phytochemical variables best explained the difference between browsed and unbrowsed plants. Higher crude protein increased the odds that plants would be browsed by pygmy rabbits and the opposite was the case for certain PSMs. Additionally, because pygmy rabbits can occupy foraging patches (burrows) for consecutive years, their browsing may influence the nutritional and PSM constituents of plants at the burrows. In a post hoc analysis, we did not find a significant relationship between phytochemical concentrations, browse status and burrow occupancy length. We concluded that pygmy rabbits use nutritional and chemical cues while making foraging decisions.

19.
J Chem Ecol ; 37(12): 1285-93, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22116690

RESUMO

The plant secondary metabolite papyriferic acid (PA) deters browsing by snowshoe hares (Lepus americanus) on the juvenile developmental stage of the Alaska paper birch (Betula neoalaskana). However, the physiological mechanism that reduces browsing remains unknown. We used pharmacological assays and molecular modeling to test the hypothesis that inhibition of succinate dehydrogenase (SDH) is a mode of action (MOA) of toxicity of PA in snowshoe hares. We tested this hypothesis by measuring the effect of PA on the activity of SDH in liver mitochondria isolated from wild hares. In addition, we used molecular modeling to determine the specific binding site of PA on SDH. We found that PA inhibits SDH from hares by an uncompetitive mechanism in a dose-dependent manner. Molecular modeling suggests that inhibition of SDH is a result of binding of PA at the ubiquinone binding sites in complex II. Our results provide a MOA for toxicity that may be responsible for the concentration-dependent anti-feedant effects of PA. We propose that snowshoe hares reduce the dose-dependent toxic consequences of PA by relying on efflux transporters and metabolizing enzymes that lower systemic exposure to dietary PA.


Assuntos
Betula/química , Lebres/metabolismo , Malonatos/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Succinato Desidrogenase/antagonistas & inibidores , Triterpenos/farmacologia , Alaska , Animais , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Herbivoria , Masculino , Malonatos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Modelos Moleculares , Oxirredução , Ratos , Ratos Wistar , Succinato Desidrogenase/metabolismo , Triterpenos/metabolismo
20.
Toxicol Appl Pharmacol ; 254(2): 86-99, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21296097

RESUMO

Reactive oxygen species (ROS) are induced through a variety of endogenous and exogenous sources. Overwhelming of antioxidant and DNA repair mechanisms in the cell by ROS may result in oxidative stress and oxidative damage to the cell. This resulting oxidative stress can damage critical cellular macromolecules and/or modulate gene expression pathways. Cancer induction by chemical and physical agents involves a multi-step process. This process includes multiple molecular and cellular events to transform a normal cell to a malignant neoplastic cell. Oxidative damage resulting from ROS generation can participate in all stages of the cancer process. An association of ROS generation and human cancer induction has been shown. It appears that oxidative stress may both cause as well as modify the cancer process. Recently association between polymorphisms in oxidative DNA repair genes and antioxidant genes (single nucleotide polymorphisms) and human cancer susceptibility has been shown.


Assuntos
Carcinógenos/toxicidade , Dano ao DNA/fisiologia , Neoplasias/metabolismo , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/fisiologia , Humanos , Neoplasias/induzido quimicamente , Neoplasias/etiologia , Oxirredução , Estresse Oxidativo/efeitos dos fármacos
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