Biomedical subjects

Alan R Buckpitt

Publications and source records attributed to Alan R Buckpitt.

13 recordsLinked to original sources

PubMed2006

Use of a fluorescent internal protein standard to achieve quantitative two-dimensional gel electrophoresis.

2-DE is a powerful separation method for complex protein mixtures. However, large intergel variations in spot intensity limit its use for quantitative proteomics studies. To address this issue, we developed a fluorescent internal protein standard for use in 2-DE analysis. Protein samples are spiked with an Alexa-labeled internal standard (ALIS) prior to separation with 2-DE. Due to the high extinction coefficient of the Alexa-fluor, incorporation of 0.1% of total protein is sufficient to allow visualization of the internal standard yet low enough to avoid interference in subsequent quantification and identification steps. Following 2-DE, total proteins are visualized with fluorescent postelectrophoretic stains spectrally separated from ALIS. Four protein stains, Deep Purple, Sulforhodamine G, ruthenium II-tris(bathophenanthroline disulfonate) (RuTBS), and SYPRO Ruby, including improved purification and staining protocols for RuTBS and ten-fold dilutions of SYPRO Ruby were evaluated. All staining protocols were compatible with the ALIS method and had similar LODs (1-4 ng) and dynamic ranges (10(3)). ALIS is a powerful normalization method for quantitative 2-DE which avoids potential problems associated with dual spot migration patterns observed in the DIGE method. Furthermore, ALIS provides significantly improved normality in the distribution of spot abundance-variance compared to normalization through division by the total spot volume.

Animals↗

PubMed2006

Identification of proteins adducted by reactive metabolites of naphthalene and 1-nitronaphthalene in dissected airways of rhesus macaques.

Naphthalene and 1-nitronaphthalene are ambient air pollutants, which undergo P450-dependent bioactivation in the lung. Reactive metabolites of naphthalene and 1-nitronaphthalene covalently bind to proteins, and the formation of covalent adducts correlates with airway epithelial cell injury in rodent models. These studies were designed to identify protein adducts generated from these reactive metabolites within distal respiratory airways. Distal bronchioles and parenchyma from rhesus monkeys were incubated with [(14)C]naphthalene or [(14)C]1-nitronaphthalene. Proteins were separated by 2-DE, blotted to PVDF membranes, and adducted proteins imaged by storage phosphor analysis. MS of in-gel tryptic digests identified numerous adducted proteins including: eight cytoskeletal proteins, two chaperone proteins, seven metabolic enzymes, one redox protein, two proteins involved in ion balance and cell signaling, and two extracellular proteins. While many proteins are adducted by both naphthalene and 1-nitronaphthalene, some are unique to the individual toxicant and airway subcompartment. Although the role which adduction of these proteins plays in cytotoxicity was not evaluated, these studies provide candidate proteins for future work designed to determine the importance of protein adducts in the mechanisms of toxicity and for developing biomarkers useful in determining the relevance of findings in animal models to exposed human populations.

Animals↗

PubMed2005

Frataxin deficiency alters heme pathway transcripts and decreases mitochondrial heme metabolites in mammalian cells.

Deficiency of the frataxin mRNA alters the transcriptome, triggering neuro- and cardiodegeneration in Friedreich's ataxia. We microarrayed murine frataxin-deficient heart tissue, liver tissue and cardiocytes and observed a transcript down-regulation to up-regulation ratio of nearly 2:1 with a mitochondrial localization of transcriptional changes. Combining all mouse and human microarray data for frataxin-deficient cells and tissues, the most consistently decreased transcripts were mitochondrial coproporphyrinogen oxidase (CPOX) of the heme pathway and mature T-cell proliferation 1, a homolog of yeast COX23, which is thought to function as a mitochondrial metallochaperone. Quantitative RT-PCR studies confirmed the significant down-regulation of Isu1, CPOX and ferrochelatase at 10 weeks in mouse hearts. We observed that mutant cells were resistant to aminolevulinate-dependent toxicity, as expected if the heme pathway was inhibited. Consistent with this, we observed increased cellular protoporphyrin IX levels, reduced mitochondrial heme a and heme c levels and reduced activity of cytochrome oxidase, suggesting a defect between protoporphyrin IX and heme a. Fe-chelatase activities were similar in mutants and controls, whereas Zn-chelatase activities were slightly elevated in mutants, supporting the idea of an altered metal-specificity of ferrochelatase. These results suggest that frataxin deficiency causes defects late in the heme pathway. As ataxic symptoms occur in other diseases of heme deficiency, the heme defect we observe in frataxin-deficient cells could be primary to the pathophysiological process.

Amino Acid Sequence↗

PubMed2005

In vivo effects of ozone exposure on protein adduct formation by 1-nitronaphthalene in rat lung.

The incidence of serious photochemical smog events is steadily growing in urban environments around the world. The electrophilic metabolites of 1-nitronaphthalene (1-NN), a common air pollutant in urban areas, have been shown to bind covalently to proteins. 1-NN specifically targets the airway epithelium, and the toxicity is synergized by prior long-term ozone exposure in rat. In this study we investigated the formation of 1-NN protein adducts in the rat airway epithelium in vivo and examined how prior long-term ozone exposure affects adduct formation. Eight adducted proteins, several involved in cellular antioxidant defense, were identified. The extent of adduction of each protein was calculated, and two proteins, peroxiredoxin 6 and biliverdin reductase, were adducted at high specific activities (0.36-0.70 and 1.0 nmol adduct/nmol protein). Furthermore, the N-terminal region of calreticulin, known as vasostatin, was adducted only in ozone-exposed animals. Although vasostatin was adducted at relatively low specific activity (0.01 nmol adduct/nmol protein), the adduction only in ozone-exposed animals makes it a candidate protein for elucidating the synergistic toxicity between ozone and 1-NN. These studies identified in vivo protein targets for reactive 1-NN metabolites that are potentially associated with the mechanism of 1-NN toxicity and the synergistic effects of ozone.

Air Pollutants↗

PubMed2005

Software-induced variance in two-dimensional gel electrophoresis image analysis.

Experimental variability in 2-DE is well documented, but little attention has been paid to variability arising from postexperimental quantitative analyses using various 2-DE software packages. The performance of two 2-DE analysis software programs, Phoretix 2D Expression v2004 (Expression) and PDQuest 7.2 (PDQuest), was evaluated in this study. All available background subtraction and smoothing algorithms were tested using both data generated from one single 2-DE gel image, thus excluding experimental variance, and with authentic sets of replicate gels (n = 5). A slight shift of the image boundaries (the "cropping area") caused both programs to induce variance in protein spot quantification of otherwise identical gel images. The resulting variance for PDQuest (CV(mean) = 8%) was approximately twice that for Expression (CV(mean) = 4%). In authentic sets of replicate 2-DE gels (n = 5), the experimental variance confounded the software-induced variance to some extent. However, Expression still outperformed PDQuest, which exhibited software-induced variance as high as 25% of the total observed variance. Surprisingly, the complete omission of background subtraction algorithms resulted in the least amount of software-based variance. These data indicate that 2-DE gel analysis software constitutes a significant source of the variance observed in quantitative proteomics, and that the use of background subtraction algorithms can further increase the variance.

Algorithms↗

PubMed2005

Characterization of a structurally intact in situ lung model and comparison of naphthalene protein adducts generated in this model vs lung microsomes.

Airway epithelial cells are a susceptible site for injury by ambient air toxicants such as naphthalene that undergo P450-dependent metabolic activation. The metabolism of naphthalene in Clara cells to reactive intermediates that bind covalently to proteins correlates with cell toxicity. Although several proteins adducted by reactive naphthalene metabolites were identified in microsomal incubations, new methods that maintain the structural integrity of the lung are needed to examine protein targets. Therefore, we developed a method that involves inflation of the lungs via the trachea with medium containing (14)C-naphthalene followed by incubation in situ. The viability of this preparation is supported by maintenance of glutathione levels, rates of naphthalene metabolism, and exclusion of ethidium homodimer-1 from airway epithelium. Following in situ incubation, the levels of adduct per milligram of protein were measured in proteins obtained from bronchoalveolar lavage, epithelial cells, and remaining lung. The levels of adducted proteins obtained in lavage and epithelial cells were similar and were 20-fold higher than those in residual lung tissue. (14)C-Labeled adducted proteins were identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) and quadrupole-TOF MS/MS. Major adducted proteins include cytoskeletal proteins, proteins involved in folding and translocation, ATP synthase, extracellular proteins, redox proteins, and selenium binding proteins. We conclude that in situ incubation maintains structural integrity of the lung while allowing examination of reactive intermediate activation and interaction with target cell proteins of the lung. The proteins adducted and identified from in situ incubations were not the same proteins identified from microsomal incubations.

Animals↗

PubMed2004

Bioactivation of the pulmonary toxicants naphthalene and 1-nitronaphthalene by rat CYP2F4.

Naphthalene, a ubiquitous environmental contaminant, produces cytotoxicity in nonciliated bronchiolar epithelial (Clara) cells in mice; rats are refractory to lung cytotoxicity from naphthalene. In contrast, 1-nitronaphthalene is a potent toxicant in both species. Naphthalene is metabolized by CYP2F to a 1,2-epoxide, the first and obligate step in events leading to cytotoxicity. 1-Nitronaphthalene is metabolized to both the 5,6- and the 7,8-epoxides with the 7,8-epoxide predominating in lung. Previous studies have demonstrated recombinant CYP2F2 (mouse) to efficiently metabolize both naphthalene and 1-nitronaphthalene. To better understand the mechanism for the unique toxicity profiles for both compounds, a CYP2F ortholog (CYP2F4) was isolated from rat lung and expressed using a baculovirus system. Recombinant CYP2F4 efficiently generates 1R,2S-naphthalene oxide (K(m) = 3 microM, V(max) = 107 min(-1)) and the 5,6- and 7,8-epoxides of 1-nitronaphthalene (K(m) = 18 microM, V(max) = 25 min(-1) based on total generated glutathione conjugates). Kinetics and regio/stereoselectivity of rat CYP2F4 were indistinguishable from mouse CYP2F2. These results, combined with our recent immunomapping studies demonstrating minimal pulmonary CYP2F expression in rats, indicate that CYP2F expression is the factor most clearly associated with susceptibility to naphthalene-induced pneumotoxicity. CYP2F4 failed to display an enhanced ability to bioactivate 1-nitronaphthalene, an ability that could have potentially compensated for the lower CYP2F pulmonary expression levels in the rat, yet equal species susceptibilities. These results suggest the importance of other P450 enzymes in the epoxidation/bioactivation of 1-nitronaphthalene. Expression of recombinant CYP2F1 (human) yielded an immunoreactive protein with no detectable CO-difference spectrum suggesting inadequate heme incorporation.

Animals↗

PubMed2004

Comparison of pulmonary/nasal CYP2F expression levels in rodents and rhesus macaque.

Naphthalene is a ubiquitous environmental contaminant that results in dose-dependent and tissue-, species-, and cell-selective necrosis of murine Clara cells upon exposure. Naphthalene is metabolized by CYP2F to a 1,2-epoxide, the first and obligate step in events leading to cytotoxicity. The studies reported here examine the relationship between levels of transcript (mRNA) and CYP2F protein in the respiratory tract of rodents with tissue susceptibility to injury. In both mice and rats, the lung contains more CYP2F transcript than liver; levels in kidney were undetectable. Mice expressed 4- and 8-fold greater CYP2F transcript in lung and liver tissue, respectively, than rats. Quantitative immunoblot blot analysis of CYP2F in airway subcompartments revealed mice to have 30- (minor daughters/terminal bronchioles), 20- (major daughter), 40- (trachea), and 6- (parenchyma) fold higher levels of CYP2F protein than rats. Within the lungs of both rodent species, the highest CYP2F expression was found in the distal airways. The kidney contained undetectable amounts of CYP2F; multiple immunoreactive bands in liver precluded quantification. The olfactory epithelium contains the greatest amount of cytochrome P450 protein of all tissues studied in the rat, consistent with the observed pattern of in vivo injury. Overall, these studies in rodents demonstrate a strong association between CYP2F expression levels and susceptibility to naphthalene-induced cytotoxicity. Of all primate tissues studied, only the nasal ethmoturbinates contain quantifiable amounts of CYP2F, roughly 10- and 20-fold less than the corresponding tissues in rats and mice, respectively. These results suggest that rhesus macaques may be refractory to naphthalene-induced pulmonary injury.

Animals↗

PubMed2003

Assessing gene expression in lung subcompartments utilizing in situ RNA preservation.

The mechanisms of toxicant-mediated lung injury and repair are influenced by the considerable spatial heterogeneity that exists within the conducting airways of the lungs. As a result of this heterogeneity, significant differences and similarities in gene expression are observed throughout lung subcompartments. RNA-based technologies such as real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and cDNA microarray analysis of gene expression provide valuable clues to understanding the mechanisms of toxicant-induced injury. Isolating RNA from lung subcompartments has previously involved considerable time and labor-intensive processes that limit the number of animals that could be processed in a day. The aim of this study was to determine if intact, high-quality RNA could be preserved in situ over a period of time to delay the need to immediately perform site-specific lung subcompartment microdissections and RNA isolations. Two hours after 1-nitronaphthalene treatment, rat lungs were inflated with and stored in RNA preservation solution and stored at 4 degrees C for 7 days. RNA was isolated from the lung subcompartments isolated by microdissection. After 7 days of storage, the RNA was intact, of high quality, and could be used for real-time RT-PCR to examine heterogeneous gene expression in the lung subcompartments. In summary, this simplified technique of in situ RNA preservation and site-specific lung subcompartment microdissection allows the isolation of intact, high-quality RNA that may be used with molecular RNA-based technologies that will significantly accelerate our understanding of pulmonary injury and repair mechanisms.

Animals↗

PubMed2003

Isolation of rodent airway epithelial cell proteins facilitates in vivo proteomics studies of lung toxicity.

Recent developments in genomics, proteomics, and metabolomics hold substantial promise for understanding cellular responses to toxicants. Gene expression profiling is now considered standard procedure, but numerous publications reporting a lack of correlation between mRNA and protein expression emphasize the importance of conducting parallel proteomics studies. The cellular complexity of the lung presents great challenges for in vivo proteomics, and improved isolation methods for proteins from specific lung cell phenotypes are required. To address this issue, we have developed a novel method for isolation of rodent airway epithelial cell proteins that facilitates in vivo proteomics studies of two target-cell pheno-types of the lung, Clara cells and ciliated cells. The airway epithelial cell proteins are reproducibly solubilized, leaving the underlying basement membrane and smooth muscle intact as shown by histopathological analyses. The method yields epithelial cell-specific proteins in fivefold higher concentrations and reduces the yield of nonepithelial cell proteins 13-fold compared with homogenates from microdissected airways. In addition, 36% more protein spots were detectable by two-dimensional gel electrophoresis.

Actins↗

PubMed2003

Gene expression analysis in response to lung toxicants: I. Sequencing and microarray development.

A key challenge in measuring gene expression changes in the lung in response to site-selective toxicants is differentiating between target and nontarget areas. The toxicity for the cytotoxicant 1-nitronaphthalene is highly localized in the airway epithelium. Target cells comprise but a fraction of the total lung cell mass; measurements from whole lung homogenates are not likely to reflect what occurs at the target site. Additionally, the use of generic microarrays to measure expression in airway epithelium may not provide a good representation of transcripts present at the site of toxic action. cDNA libraries from airway and alveolar subcompartments of rat lung were sequenced for the development of a custom microarray representative of these lung regions. We identified 7,460 nonredundant rat lung sequences. Nearly 30% of the sequences on this array are not present on the Affymetrix Rat GeneChip 230. A 20,000-element microarray was developed that delineates differences in gene expression between subcompartments. This is the first in a series of articles employing this microarray for detecting gene expression changes during acute injury produced by 1-nitronaphthalene and subsequent repair.

Animals↗

PubMed2002

Pharmacokinetics and metabolism of a novel antifibrotic drug pirfenidone, in mice following intravenous administration.

The present study describes the pharmacokinetics and metabolism of pirfenidone (PD), a compound which has been shown to have significant antifibrotic effects in rodent models of pulmonary and cardiac fibrosis. Despite the fact that this compound is currently in phase II clinical trials, little data are available on the metabolism and disposition of this agent in rodents or humans. Radioactive PD [benzene ring (14)C(U)] was administered i.v. to mice at 40 mg PD/kg body weight, and animals were killed at varying times for determination of parent compound and metabolites in various tissues. The disappearance of parent compound from the plasma followed apparent 2-compartment elimination kinetics with a terminal elimination half-life of 8.6 min. Cl (0.10 ml/min/g) and V(d(ss)) (0.67 ml/g) indicated that PD was rapidly distributed in body water. This is consistent with the finding that peak tissue radioactivity occurred within 5 min following the i.v. administration of [(14)C]-PD and that well-perfused tissues, kidney>liver>lung have much higher levels of parent compound and metabolites than did fat. Two peaks isolated from plasma samples by HPLC yielded mass spectra that were consistent with initial oxidation to the alcohol followed by further metabolism to the carboxylic acid. The radioactivity recovered in the 24 h urine samples averaged 97% of the administered dose and none of that was associated with the parent compound. The short plasma half-life of parent compound in mice supports the need for additional studies in humans where the compound has been shown to have clinical benefits.

Animals↗

PubMed2002

Induction of tolerance to naphthalene in Clara cells is dependent on a stable phenotypic adaptation favoring maintenance of the glutathione pool.

Repeated exposures to the Clara cell cytotoxicant naphthalene (NA) result in target cell populations that become refractory to further injury. To determine whether tolerance occurs from specific adaptations favoring glutathione (GSH) resynthesis without broad shifts in cellular phenotype, mice were administered NA for 21 days. We found that gamma-glutamylcysteine synthetase (gamma-GCS) was induced in tolerant Clara cells by repeated exposures to NA. Treating tolerant mice with buthionine sulfoximine, a gamma-GCS inhibitor, eliminates resistance acquired by repeated exposures to NA. Broad phenotypic shifts were not present. Marker proteins of differentiation declined over the first 3 days in the development of tolerance, but returned to control levels at 14 and 21 days. Epithelial organizational structure and internal organelle composition in Clara cells from tolerant mice were similar compared to corn oil-treated controls, while subtle shifts in organelle distribution were present. We conclude that induction of gamma-GCS expression is coordinated with the development of NA tolerance, but induction of NA tolerance does not markedly alter Clara cell differentiation, epithelial organization, or organelle composition in bronchiolar epithelium.

Adaptation, Physiological↗