Biomedical subjects

Brian D Thrall

Publications and source records attributed to Brian D Thrall.

16 recordsLinked to original sources

PubMed2006

Particokinetics in vitro: dosimetry considerations for in vitro nanoparticle toxicity assessments.

The rapid growth in the use of in vitro methods for nanoparticle toxicity assessment has proceeded with limited consideration of the unique kinetics of these materials in solution. Particles in general and nanoparticles specifically, diffuse, settle, and agglomerate in cell culture media as a function of systemic and particle properties: media density and viscosity and particle size, shape, charge and density, for example. Cellular dose then is also a function of these factors as they determine the rate of transport of nanoparticles to cells in culture. Here we develop and apply the principles of dosimetry in vitro and outline an approach for simulation of nanoparticle particokinetics in cell culture systems. We illustrate that where equal mass concentrations (mug/ml) imply equal doses for dissimilar materials, the corresponding particle number or surface area concentration doses differ by orders of magnitude. More importantly, when rates of diffusional and gravitational particle delivery are accounted for, trends and magnitude of the cellular dose as a function of particle size and density differ significantly from those implied by "concentration" doses. For example, 15-nm silver nanoparticles appear approximately 4000 times more potent than micron-sized cadmium oxide particles on a cm(2)/ml media basis, but are only approximately 50 times more potent when differences in delivery to adherent cells are considered. We conclude that simple surrogates of dose can cause significant misinterpretation of response and uptake data for nanoparticles in vitro. Incorporating particokinetics and principles of dosimetry would significantly improve the basis for nanoparticle toxicity assessment, increasing the predictive power and scalability of such assays.

Cells, Cultured↗

PubMed2006

Data merging for integrated microarray and proteomic analysis.

The functioning of even a simple biological system is much more complicated than the sum of its genes, proteins and metabolites. A premise of systems biology is that molecular profiling will facilitate the discovery and characterization of important disease pathways. However, as multiple levels of effector pathway regulation appear to be the norm rather than the exception, a significant challenge presented by high-throughput genomics and proteomics technologies is the extraction of the biological implications of complex data. Thus, integration of heterogeneous types of data generated from diverse global technology platforms represents the first challenge in developing the necessary foundational databases needed for predictive modelling of cell and tissue responses. Given the apparent difficulty in defining the correspondence between gene expression and protein abundance measured in several systems to date, how do we make sense of these data and design the next experiment? In this review, we highlight current approaches and challenges associated with integration and analysis of heterogeneous data sets, focusing on global analysis obtained from high-throughput technologies.

Animals↗

PubMed2006

Functional link between TNF biosynthesis and CaM-dependent activation of inducible nitric oxide synthase in RAW 264.7 macrophages.

Inflammatory responses stimulated by bacterial endotoxin LPS involve Ca2+-mediated signaling, yet the cellular sensors that determine cell fate in response to LPS remain poorly understood. We report that exposure of RAW 264.7 macrophage-like cells to LPS induces a rapid increase in CaM abundance, which is associated with the modulation of the inflammatory response. Increases in CaM abundance precede nuclear localization of key transcription factors (i.e., NF-kappaB p65 subunit, phospho-c-Jun, Sp1) and subsequent increases in the proinflammatory cytokine TNF-alpha and inducible nitric oxide synthase (iNOS). Cellular apoptosis after LPS challenge is blocked upon inhibition of iNOS activity using the pharmacological inhibitor 1400W. LPS-mediated iNOS expression and apoptosis also were inhibited by siRNA-mediated silencing of TNF induction, indicating TNF induction both precedes and is necessary for subsequent regulation of iNOS expression. Increasing the level of cellular CaM by stable transfection results in reductions in LPS-induced expression of TNF and iNOS, along with reduced activation of their transcriptional regulators and concomitant protection against apoptosis. Thus the level of CaM available for Ca2+-dependent signaling regulation plays a key role in determining the expression of the proinflammatory and proapoptotic cascade during cellular activation by LPS. These results indicate a previously unrecognized central role for CaM in maintaining cellular homeostasis in response to LPS such that, under resting conditions, cellular concentrations of CaM are sufficient to inhibit the biosynthesis of proinflammatory mediators associated with macrophage activation. Although CaM and iNOS protein levels are coordinately increased as part of the oxidative burst, limiting cellular concentrations of CaM due to association with iNOS (and other high-affinity binders) commit the cell to an unchecked inflammatory cascade leading to apoptosis.

Animals↗

PubMed2005

Improved proteome coverage by using high efficiency cysteinyl peptide enrichment: the human mammary epithelial cell proteome.

Automated multidimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly applied in various large scale proteome profiling efforts. However, comprehensive global proteome analysis remains technically challenging due to issues associated with sample complexity and dynamic range of protein abundances, which is particularly apparent in mammalian biological systems. We report here the application of a high efficiency cysteinyl peptide enrichment (CPE) approach to the global proteome analysis of human mammary epithelial cells (HMECs) which significantly improved both sequence coverage of protein identifications and the overall proteome coverage. The cysteinyl peptides were specifically enriched by using a thiol-specific covalent resin, fractionated by strong cation exchange chromatography, and subsequently analyzed by reversed-phase capillary LC-MS/MS. An HMEC tryptic digest without CPE was also fractionated and analyzed under the same conditions for comparison. The combined analyses of HMEC tryptic digests with and without CPE resulted in a total of 14 416 confidently identified peptides covering 4294 different proteins with an estimated 10% gene coverage of the human genome. By using the high efficiency CPE, an additional 1096 relatively low abundance proteins were identified, resulting in 34.3% increase in proteome coverage; 1390 proteins were observed with increased sequence coverage. Comparative protein distribution analyses revealed that the CPE method is not biased with regard to protein M(r) , pI, cellular location, or biological functions. These results demonstrate that the use of the CPE approach provides improved efficiency in comprehensive proteome-wide analyses of highly complex mammalian biological systems.

Amino Acid Sequence↗

PubMed2005

Low-LET microbeam investigation of the track-end dependence of electron-induced damage in normal human diploid fibroblasts.

Using a pulsed electron beam, we investigated the dependence of micronucleus formation on the incident electron energy in AG01522 human diploid fibroblasts after nontargeted irradiations at 25 and 80 keV. Examining the dose response, we found that 25 keV electrons are more effective than 80 keV electrons at producing biological damage for a given dose. Our results demonstrating the induction of micronuclei as a function of incident electron energy offer direct support for the hypothesis that the electron track end is responsible for the biological damage occurring in the cell.

Cells, Cultured↗

PubMed2004

High-throughput comparative proteome analysis using a quantitative cysteinyl-peptide enrichment technology.

A new quantitative cysteinyl-peptide enrichment technology (QCET) was developed to achieve higher efficiency, greater dynamic range, and higher throughput in quantitative proteomics that use stable-isotope labeling techniques combined with high-resolution liquid chromatography (LC)-mass spectrometry (MS). This approach involves (18)O labeling of tryptic peptides, high-efficiency enrichment of cysteine-containing peptides, and confident protein identification and quantification using the accurate mass and time tag strategy. Proteome profiling of naïve and in vitro-differentiated human mammary epithelial cells using QCET resulted in the identification and quantification of 603 proteins in a single LC-Fourier transform ion cyclotron resonance MS analysis. Advantages of this technology include the following: (1) a simple, highly efficient method for enriching cysteinyl-peptides; (2) a high-throughput strategy suitable for extensive proteome analysis; and (3) improved labeling efficiency for better quantitative measurements. This technology enhances both the functional analysis of biological systems and the detection of potential clinical biomarkers.

Cells, Cultured↗

PubMed2004

Calmodulin involvement in stress-activated nuclear localization of albumin in JB6 epithelial cells.

We report that albumin is translocated to the nucleus in response to oxidative stress. Prior measurements have demonstrated that in concert with known transcription factors albumin binds to an antioxidant response element, which controls the expression of glutathione S-transferase and other antioxidant enzymes that function to mediate adaptive cellular responses [Holderman, M. T., Miller, K. P., Dangott, L. J., and Ramos, K. S. (2002) Mol. Pharmacol. 61, 1174-1183]. To investigate the mechanisms underlying this adaptive cell response, we have identified linkages between calcium signaling and the nuclear translocation of albumin in JB6 epithelial cells. Under resting conditions, albumin and the calcium regulatory protein calmodulin (CaM) co-immunoprecipitate using antibodies against either protein, indicating a tight association. Calcium activation of CaM disrupts the association between CaM and albumin, suggesting that transient increases in cytosolic calcium levels function to mobilize intracellular albumin to facilitate its translocation into the nucleus. Likewise, nuclear translocation of albumin is induced by exposure of cells to hydrogen peroxide or a phorbol ester, indicating a functional linkage between reactive oxygen species, calcium, and PKC-signaling pathways. Inclusion of an antioxidant enzyme (i.e., superoxide dismutase) blocks nuclear translocation, suggesting that the oxidation of sensitive proteins functions to coordinate the adaptive cellular response. These results suggest that elevated calcium transients and associated increases in reactive oxygen species contribute to adaptive cellular responses through the mobilization and nuclear translocation of cellular albumin.

Albumins↗

PubMed2004

Induced autocrine signaling through the epidermal growth factor receptor contributes to the response of mammary epithelial cells to tumor necrosis factor alpha.

In contrast to the well known cytotoxic effects of tumor necrosis factor (TNF) alpha in many mammary cancer cells, we have found that TNF stimulates the proliferation and motility of human mammary epithelial cells (HMECs). Since the response of HMECs to TNF is similar to effects mediated by epidermal growth factor receptor (EGFR) activation, we explored the potential role of cross-talk through the EGFR signaling pathways in mediating cellular responses to TNF. Using a microarray enzyme-linked immunoassay, we found that exposure to TNF stimulated the dose-dependent shedding of the EGFR ligand transforming growth factor alpha (TGFalpha). Both proliferation and motility of HMECs induced by TNF was prevented either by inhibiting membrane protein shedding with a metalloprotease inhibitor, by blocking epidermal growth factor receptor (EGFR) kinase activity, or by limiting ligand-receptor interactions with an antagonistic anti-EGFR antibody. EGFR activity was also necessary for TNF-induced release of matrix metalloprotease-9, thought to be an essential regulator of mammary cell migration. The cellular response to TNF was associated with a biphasic temporal pattern of extracellular signal-regulated kinase (ERK) phosphorylation, which was EGFR-dependent and modulated by inhibition of metalloprotease-mediated shedding. Significantly, the late phase of ERK phosphorylation, detectable within 4 h after exposure, was blocked by the metalloprotease inhibitor batimastat, indicating that autocrine signaling through ligand shedding was responsible for this secondary wave of ERK activity. Our results indicate a novel and important role for metalloprotease activation and EGFR transmodulation in mediating the cellular response to TNF.

Autocrine Communication↗

PubMed2004

Simultaneous 1H PFG-NMR and confocal microscopy of monolayer cell cultures: effects of apoptosis and necrosis on water diffusion and compartmentalization.

We induced apoptosis and necrosis in monolayer cultures of Chinese hamster ovary cells using okadaic acid and hydrogen peroxide (H2O2), respectively, and examined the effect on water diffusion and compartmentalization using pulsed-field-gradient (PFG) 1H-NMR and simultaneous confocal microscopy. In PFG experiments characterized by a fixed diffusion time (<4.7 ms) and variable b-values (0-27000 s/mm2), 1H-NMR data collected with untreated cells exhibited multiexponential behavior. Analysis with a slow-exchange model revealed two distinct cellular water compartments with different apparent diffusion coefficients (ADCs; 0.56, 0.06 x 10(-3) mm2/s) and volume fractions (0.96 and 0.04). During the first 12 hr of necrosis or apoptosis, the amount of water in the smallest compartment increased twofold before significant changes in cell density or plasma membrane integrity occurred. Over the same period, water content in the largest compartment decreased by a factor of >2 in apoptotic cells, in accordance with observed cell shrinkage, and changed little in necrotic counterparts, where only slight swelling was evident. These results indicate that PFG 1H-NMR serves as a sensitive indicator of early cell death in monolayer cultures, and can be used to distinguish apoptosis from necrosis. Measurements of restricted diffusion and water exchange are presented to elucidate the compartment origins and justify the model assumptions.

Animals↗

PubMed2003

Detection of in situ labeled cell surface proteins by mass spectrometry: application to the membrane subproteome of human mammary epithelial cells.

Characterization of the surface exposed membrane subproteome of human mammary epithelial cells (strain 184 A1L5) implemented lysine specific in situ labeling of the proteins using sulfosuccinimidyl-6-(biotinamido)hexanoate, followed by enrichment of the biotinylated, tryptically digested peptides, and then liquid chromatography-tandem mass spectrometry analysis of the labeled peptides. Probing the membrane subproteome in this manner yielded unambiguous identification of proteins situated on the cell surface. The method reported can be adapted to include stable isotope labeling of proteins for quantitation of changes occurring on the cell surface in response to specific perturbations.

Amino Acid Sequence↗

PubMed2003

Probing conformational changes of gramicidin ion channels by single-molecule patch-clamp fluorescence microscopy.

Complex conformational changes influence and regulate the dynamics of ion channels. Such conformational changes are stochastic and often inhomogeneous, which makes it extremely difficult, if not impossible, to characterize them by ensemble-averaged experiments or by single-channel recordings of the electric current that report the open-closed events but do not specifically probe the associated conformational changes. Here, we report our studies on ion channel conformational changes using a new approach, patch-clamp fluorescence microscopy, which simultaneously combines single-molecule fluorescence spectroscopy and single-channel current recordings to probe the open-closed transitions and the conformational dynamics of individual ion channels. We demonstrate patch-clamp fluorescence microscopy by measuring gramicidin ion channel conformational changes in a lipid bilayer formed at a patch-clamp micropipette tip under a buffer solution. By measuring single-pair fluorescence resonance energy transfer and fluorescence self-quenching from dye-labeled gramicidin channels, we observed that the efficiency of single-pair fluorescence resonance energy transfer and self-quenching is widely distributed, which reflects a broad distribution of conformations. Our results strongly suggest a hitherto undetectable correlation between the multiple conformational states of the gramicidin channel and its closed and open states in a lipid bilayer.

Anti-Bacterial Agents↗

PubMed2003

Mode of action and pharmacokinetic studies of 2-butoxyethanol in the mouse with an emphasis on forestomach dosimetry.

Chronic inhalation studies with 2-butoxyethanol (BE) conducted by the National Toxicology Program identified the forestomach and liver of B6C3F1 mice as target organs for tumorigenicity (NTP, 2000). Previous studies have shown that the liver tumors likely resulted from chronic hemolysis-induced oxidative stress. For the forestomach lesions seen in mice, chronic contact irritation (cytotoxicity) and regenerative hyperplasia are hypothesized to result in forestomach tumor development. To test this hypothesis, several experiments were conducted to address the sensitivity of the mouse forestomach to BE administered by various routes. Oral administration of undiluted BE was shown to cause irritation and a compensatory proliferative response in the mouse forestomach, confirming that direct contact between the forestomach and BE, which can occur via grooming of BE condensed on the fur during inhalation exposures, can cause irritation. However, only small amounts of BE (<10 mg/kg) were detected on the fur of mice at the end of 6-h, whole-body or nose-only inhalation exposures to the highest concentration used in the NTP chronic inhalation studies (250 ppm). Furthermore, no significant differences were detected in the end-exposure blood concentrations of BE and butoxyacetic acid (BAA) between these types of exposures. In addition, parenteral administration of BE (ip and sc injection) also resulted in forestomach lesions, indicating that there may be sources other than grooming for BE- or BAA-induced forestomach irritation. In the pharmacokinetic study, BE and, to a lesser extent, BAA was eliminated more slowly from the forestomach tissue of mice than from blood or other tissues, following either oral gavage or ip injection. The forestomach was the only tissue with detectable levels of BE at 24 h. BE and BAA were both excreted in the saliva and were present in stomach contents for a prolonged period of time following these routes of exposure, which may further contribute to forestomach tissue dosimetry. Thus, there appear to be multiple mechanisms behind the increased levels of BE and BAA in the forestomach tissue of mice, which together can contribute to a prolonged contact irritation, compensatory hyperplasia, and tumorigenicity in mice. The relevance of these effects in humans, who lack a forestomach, is questionable.

Animals↗

PubMed2002

Establishment of a novel clonal murine bone marrow stromal cell line for assessment of p53 responses to genotoxic stress.

The p53 protein is widely regarded as an important sensor of genotoxic damage in cells, and mutations in p53 are the most frequent observed in human cancers. Rapid assays for evaluating the potential of a chemical or physical agent to alter the transcriptional regulatory role of p53 may therefore serve as useful tools in toxicological research. In this study, the use of enhanced green fluorescent protein (EGFP) as a live cell reporter to assess the transactivation response of p53 to chemical and physical agents was evaluated. A stable murine bone marrow stromal cell line (D2XRIIGFP24) expressing EGFP under control of p53 response elements was established. D2XRIIGFP24 cells displayed low constitutive background fluorescence which was significantly enhanced in response to exposure to agents that induced p53 protein levels. Increases in EGFP fluorescence in response to oxidative and nitrosative stress as well as UVC irradiation were dose-dependent, detectable within 3 h of exposure and correlated closely with the amount of p53 protein accumulated within the cell. The results demonstrate the potential for rapid and early detection of p53 transactivation using the EGFP reporter approach and indicate this approach is adaptable to a variety of fluorescent assay techniques and a useful cell model for molecular toxicology research.

Animals↗

PubMed2002

Contribution of dichloroacetate and trichloroacetate to liver tumor induction in mice by trichloroethylene.

Determining the key events in the induction of liver cancer in mice by trichloroethylene (TRI) is important in the determination of how risks from this chemical should be treated at low doses. At least two metabolites can contribute to liver cancer in mice, dichloroacetate (DCA) and trichloroacetate (TCA). TCA is produced from metabolism of TRI at systemic concentrations that can clearly contribute to this response. As a peroxisome proliferator and a species-specific carcinogen, TCA may not be important in the induction of liver cancer in humans at the low doses of TRI encountered in the environment. Because DCA is metabolized much more rapidly than TCA, it has not been possible to directly determine whether it is produced at carcinogenic levels. Unlike TCA, DCA is active as a carcinogen in both mice and rats. Its low-dose effects are not associated with peroxisome proliferation. The present study examines whether biomarkers for DCA and TCA can be used to determine if the liver tumor response to TRI seen in mice is completely attributable to TCA or if other metabolites, such as DCA, are involved. Previous work had shown that DCA produces tumors in mice that display a diffuse immunoreactivity to a c-Jun antibody (Santa Cruz Biotechnology, SC-45), whereas TCA-induced tumors do not stain with this antibody. In the present study, we compared the c-Jun phenotype of tumors induced by DCA or TCA alone to those induced when they are given together in various combinations and to those induced by TRI given in an aqueous vehicle. When given in various combinations, DCA and TCA produced a few tumors that were c-Jun+, many that were c-Jun-, but a number with a mixed phenotype that increased with the relative dose of DCA. Sixteen TRI-induced tumors were c-Jun+, 13 were c-Jun-, and 9 had a mixed phenotype. Mutations of the H-ras protooncogene were also examined in DCA-, TCA-, and TRI-induced tumors. The mutation frequency detected in tumors induced by TCA was significantly different from that observed in TRI-induced tumors (0.44 vs 0.21, p < 0.05), whereas that observed in DCA-induced tumors (0.33) was intermediate between values obtained with TCA and TRI, but not significantly different from TRI. No significant differences were found in the mutation spectra of tumors produced by the three compounds. The presence of mutations in H-ras codon 61 appeared to be a late event, but ras-dependent signaling pathways were activated in all tumors. These data are not consistent with the hypothesis that all liver tumors induced by TRI were produced by TCA.

Animals↗

PubMed2002

Dichloroacetate stimulates glycogen accumulation in primary hepatocytes through an insulin-independent mechanism.

Dichloroacetate (DCA), a by-product of water chlorination, causes liver cancer in B6C3F1 mice. A hallmark response observed in mice exposed to carcinogenic doses of DCA is an accumulation of hepatic glycogen content. To distinguish whether the in vivo glycogenic effect of DCA was dependent on insulin and insulin signaling proteins, experiments were conducted in isolated hepatocytes where insulin concentrations could be controlled. In hepatocytes isolated from male B6C3F1 mice, DCA increased glycogen levels in a dose-related manner, independently of insulin. The accumulation of hepatocellular glycogen induced by DCA was not the result of decreased glycogenolysis, since DCA had no effect on the rate of glucagon-stimulated glycogen breakdown. Glycogen accumulation caused by DCA treatment was not hindered by inhibitors of extracellular-regulated protein kinase kinase (Erk1/2 kinase or MEK) or p70 kDa S6 protein kinase (p70(S6K)), but was completely blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin. Similarly, insulin-stimulated glycogen deposition was not influenced by the Erk1/2 kinase inhibitor, PD098509, or the p70(S6K) inhibitor, rapamycin. Unlike DCA-stimulated glycogen deposition, PI3K-inhibition only partially blocked the glycogenic effect of insulin. DCA did not cause phosphorylation of the downstream PI3K target protein, protein kinase B (PKB/Akt). The phosphorylation of PKB/Akt did not correlate to insulin-stimulated glycogenesis either. Similar to insulin, DCA in the medium decreased IR expression in isolated hepatocytes. The results indicate DCA increases hepatocellular glycogen accumulation through a PI3K-dependent mechanism that does not involve PKB/Akt and is, at least in part, different from the classical insulin-stimulated glycogenesis pathway. Somewhat surprisingly, insulin-stimulated glycogenesis also appears not to involve PKB/Akt in isolated murine hepatocytes.

Androstadienes↗

PubMedDate not supplied

Multidimensional proteome analysis of human mammary epithelial cells.

Recent multidimensional liquid chromatography MS/MS studies have contributed to the identification of large numbers of expressed proteins for numerous species. The present study couples size exclusion chromatography of intact proteins with the separation of tryptically digested peptides using a combination of strong cation exchange and high resolution, reversed phase capillary chromatography to identify proteins extracted from human mammary epithelial cells (HMECs). In addition to conventional conservative criteria for protein identifications, the confidence levels were additionally increased through the use of peptide normalized elution times (NET) for the liquid chromatographic separation step. The combined approach resulted in a total of 5838 unique peptides identified covering 1574 different proteins with an estimated 4% gene coverage of the human genome, as annotated by the National Center for Biotechnology Information (NCBI). This database provides a baseline for comparison against variations in other genetically and environmentally perturbed systems. Proteins identified were categorized based upon intracellular location and biological process with the identification of numerous receptors, regulatory proteins, and extracellular proteins, demonstrating the usefulness of this application in the global analysis of human cells for future comparative studies.

Cell Line↗