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T J Kavanagh

Publications and source records attributed to T J Kavanagh.

At least 37 records · Page 2Linked to original sources
PubMed1999

Reduction in apolipoprotein-mediated removal of cellular lipids by immortalization of human fibroblasts and its reversion by cAMP: lack of effect with Tangier disease cells.

High density lipoprotein (HDL) phospholipids and apolipoproteins remove cellular lipids by two distinct mechanisms, but their relative contribution to reverse cholesterol transport is unknown. Whereas phospholipid-mediated cholesterol efflux from cultured cells reflects the activity of the HDL receptor SR-BI, apolipoprotein-mediated lipid removal is regulated in response to changes in cellular cholesterol content (positive) and cell proliferation rates (negative). Here we show that immortalization of human skin fibroblast lines with the papillomavirus E6/E7 oncogenes increased their proliferation rates and selectively reduced the activity of the apolipoprotein-mediated lipid removal pathway. This reduction was accompanied by a decrease in cellular cAMP levels and was reversed by treatment with a cAMP analog. The stimulatory effect of cAMP was independent of changes in cellular phenotype or activities of cholesteryl ester cycle enzymes. The severely impaired apolipoprotein-mediated lipid removal pathway in Tangier disease fibroblasts, which persisted after immortalization, was not improved by treatment with a cAMP analog, implying that the cellular defect in Tangier disease is upstream from this cAMP-dependent signaling pathway.These results indicate that papillomavirus-induced immortalization of fibroblasts selectively reduces the activity of the apolipoprotein-mediated lipid removal pathway by a cAMP-dependent process, perhaps to prevent loss of cellular lipids needed for continual membrane synthesis.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

The role of intracellular glutathione in methylmercury-induced toxicity in embryonic neuronal cells.

Previous studies indicate that the ability of cells to up-regulate levels of intracellular glutathione (GSH) synthesis may determine their sensitivity to MeHg exposure. The purpose of the current study is two-fold. First, we determined whether the vulnerability of the developing central nervous system (CNS) to MeHg lies in its intracellular GSH content. The intracellular GSH content and the activity of gamma-glutamyl cysteine synthetase (GCS) were determined with and without MeHg exposure in primary cultures of rat embryonic CNS cells. In addition, the effect of GSH modulation on MeHg-induced cytotoxicity was determined. Second, we characterized the mechanism of GCS regulation, initially by studying the GCS heavy chain subunit (GCS-HC). Primary embryonic limb bud cells were used as a reference cell type for comparing the response of CNS cells. The results indicate that constitutive intracellular GSH content, GCS activity, and GCS-HC mRNA and protein levels of CNS cells were approximately ten-, two-, five-, and ten-fold higher, respectively, than those in limb bud cells. A dose-dependent increase in GSH levels and GCS activity was observed in CNS and limb bud cells following 1 and 2 microM MeHg exposure for 20 hr. Further characterization of GCS up-regulation in CNS cells showed that the increase in GCS activity following MeHg exposure, unlike limb bud cells, was not accompanied by an elevation of GCS-HC mRNA and protein levels. Pretreatment with N-acetylcysteine led to a significant increase in intracellular GSH, while L-buthionine-(S,R)-sulfoximine (BSO) resulted in decreased GSH levels, however neither pretreatment had a significant impact on MeHg-induced cytotoxicity in either cell type. Our results suggest that although oxidative stress may mediate aspects of MeHg toxicity, disruption of GSH homeostasis alone is not responsible for the sensitivity of embryonic CNS cells to MeHg.

Acetylcysteine↗
PubMed1998

Human signaling protein 14-3-3zeta interacts with platelet glycoprotein Ib subunits Ibalpha and Ibbeta.

The initiation of primary hemostasis is mediated by interaction of the platelet glycoprotein Ib (GPIb) surface receptor and its arterial subendothelial von Willebrand factor (vWF) ligand. The intracellular signaling immediately following GPIb receptor occupancy connecting the adhesive event to platelet activation and aggregation has not been well characterized. The 14-3-3 proteins are a 27- to 30-kD ubiquitous protein family with diverse biologic roles, including functional modulation of several prominent signaling proteins. We used the yeast two-hybrid system and confocal microscopy to characterize the recently described interaction between GPIb and platelet 14-3-3zeta, and provide evidence for the potential signaling role of this protein. Two-hybrid interactions suggest that platelet 14-3-3zeta associates with the cytoplasmic domain of GPIb subunits Ibalpha and Ibbeta in transformed yeast cells. The 14-3-3 interaction with GPIbbeta may be partly mediated through the latter's phosphorylated serine 166 residue as its mutagenesis results in 20% to 40% reduced interaction. There was 51% to 59% reduced interaction between GPIb and three 14-3-3zeta deletion mutants compared with full-length 14-3-3zeta, suggesting that either the N-terminal dimerization or membrane-binding domains or more than one noncontiguous 14-3-3zeta element may be required for optimal GPIb interaction. Confocal studies of platelets and a megakaryocyte cell line provided additional evidence for interaction of 14-3-3zeta with GPIbalpha and GPIbbeta. We also found that, similar to the signaling mediators phosphatidylinositol 3-kinase and Src, platelet cytoskeletal 14-3-3zeta content is increased following vWF and ristocetin stimulation. We suggest that platelet 14-3-3zeta interacts with GPIbalpha and Ibbeta, that this interaction may be partly mediated through phosphoserine recognition, and that 14-3-3zeta cytoskeletal translocation may serve as a GPIb post-receptor occupancy signaling event.

14-3-3 Proteins↗
PubMed1998

Analysis of c-kit receptor dimerization by fluorescence resonance energy transfer.

Stem cell factor (SCF) binding to the c-kit receptor triggers homodimerization and intermolecular tyrosine phosphorylation of the c-kit receptor, thus initiating signal transduction. Receptor dimerization is a critical early step in this process. Prior biochemical studies of c-kit receptor dimerization have mainly used affinity cross-linking techniques, which are beset with problems including low efficiency of cross-linking and the usual requirement for radiolabeled SCF to detect the cross-linked complex. We used the fluorescence resonance energy transfer (FRET) technique to examine the effects of SCF and other hematopoietic cytokines on c-kit receptor dimerization. The nonneutralizing anti-c-kit receptor monoclonal antibody 104D2 was directly conjugated to fluorescein isothiocyanate (FITC) or to the carbocyanine dye Cy3 and used to label cytokine-responsive human hematopoietic cell lines. The ability of SCF to induce c-kit receptor dimerization was assessed by flow cytometric analysis of FRET between the donor fluorochrome FITC and the acceptor fluorochrome Cy3. SCF induced a dose-dependent increase in c-kit receptor dimerization that correlated well with the concentrations of SCF required to stimulate cell proliferation. Receptor dimerization was detectable within 3 minutes after the addition of SCF and was maximal 30 minutes after the addition of SCF. Confocal microscopy showed redistribution of the c-kit receptor (from a diffuse distribution on the cell surface to "caps" at one end of the cell) within 3 minutes after SCF addition, followed by receptor internalization. Reappearance of the c-kit receptor on the cell surface required new protein synthesis, suggesting that the c-kit receptor is not recycled to the cell surface after internalization. Finally, erythropoietin (Epo), but not the structurally and functionally related cytokine thrombopoietin (Tpo), stimulated c-kit receptor dimerization detectable by FRET, and tyrosine phosphorylation of the c-kit receptor. These results suggest that exposure to Epo can activate the c-kit receptor and provide further evidence for cross-talk between the Epo and c-kit receptors in human hematopoietic cell lines. Studies with progeny of burst-forming unit-erythroid (BFU-E) suggest that the FRET technique is sufficiently sensitive to detect c-kit receptor dimerization on normal human hematopoietic cells.

Carbocyanines↗
PubMed1998

Uncertainty analysis methods for comparing predictive models and biomarkers: A case study of dietary methyl mercury exposure.

Biologically based markers (biomarkers) are currently used to provide information on exposure, health effects, and individual susceptibility to chemical and radiological wastes. However, the development and validation of biomarkers are expensive and time consuming. To determine whether biomarker development and use offer potential improvements to risk models based on predictive relationships or assumed values, we explore the use of uncertainty analysis applied to exposure models for dietary methyl mercury intake. We compare exposure estimates based on self-reported fish intake and measured fish mercury concentrations with biomarker-based exposure estimates (i.e., hair or blood mercury concentrations) using a published data set covering 1 month of exposure. Such a comparison of exposure model predictions allowed estimation of bias and random error associated with each exposure model. From these analyses, both bias and random error were found to be important components of uncertainty regarding biomarker-based exposure estimates, while the diary-based exposure estimate was susceptible to bias. Application of the proposed methods to a simple case study demonstrates their utility in estimating the contribution of population variability and measurement error in specific applications of biomarkers to environmental exposure and risk assessment. Such analyses can guide risk analysts and managers in the appropriate validation, use, and interpretation of exposure biomarker information.

Animals↗
PubMed1998

Alterations in immune parameters associated with low level methylmercury exposure in mice.

Methylmercury (MeHg) is a known toxicant and continues to be a significant environmental contaminant. While the neurotoxicity and developmental toxicity of MeHg are well established, the immunotoxic effects of MeHg are just now being studied and described. This study evaluated strain and gender specific effects of low level, prolonged MeHg exposure in mice. Mice were exposed to MeHg in the drinking water (0, 3 or 10 ppm) for 4 weeks. Splenocytes and thymocytes were evaluated for alterations in immunophenotype, GSH levels, and intracellular Ca2+ flux after mitogen stimulation. MeHg exposure resulted in alterations in splenocyte and thymocyte subsets and a dose dependent decrease in GSH levels (as measured by monochlorobimane fluorescence and flow cytometry) of all splenocyte subsets. This decrease in GSH was further confirmed by biochemical assay in splenocytes. In addition, there was a dose response related decrease in mitogen stimulated Ca2+ flux and in the percentages of CD4+ splenocytes and CD8+ splenocytes from mice exposed to 10 ppm of MeHg. These results suggest that low-level chronic MeHg exposure may cause immune disfunction by disturbing thiol redox balance, transmembrane signaling and splenic cellularity.

Analysis of Variance↗
PubMed1997

Molecular cloning and sequencing of the cDNA encoding mouse glutamate-cysteine ligase regulatory subunit.

Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify and clone the regulatory subunit of mouse glutamate-cysteine ligase (Glclr) using primers adapted from the published rat Glclr cDNA sequence, and from mouse genomic DNA. Amplified cDNA was cloned into a plasmid vector, and additional RT-PCR reactions coupled with 3' RACE were used to amplify and sequence 3' regions covered by the rat primer. Comparison of the mouse Glclr cDNA sequence and predicted protein sequence with that of rat Glclr and human GLCLR revealed extensive homology in cDNA and amino acid sequences among these species.

Amino Acid Sequence↗
PubMed1997

Molecular cloning and sequencing of the cDNA encoding the catalytic subunit of mouse glutamate-cysteine ligase.

Reverse transcription-polymerase chain reaction (RT-PCR) was used for the enzymatic synthesis of cDNA sequences encompassing the open reading frame for the catalytic subunit of mouse kidney glutamate-cysteine ligase (Glclc). Comparison of the mouse Glclc cDNA sequence and predicted protein sequence with that of rat Glclc and human GLCLC revealed between 94.8% and 88.4% cDNA homology and 98.4% to 95% amino acid identity, respectively.

Amino Acid Sequence↗
PubMed1997

Role of oxidative stress in the action of vanadium phosphotyrosine phosphatase inhibitors. Redox independent activation of NF-kappaB.

The role of intracellular oxidative stress in the mechanism of action of phosphotyrosine phosphatase (PTP) inhibitors was studied using three vanadium-based compounds. Sodium orthovanadate (Na3VO4), sodium oxodiperoxo(1,10-phenanthroline)vanadate(V) (pV(phen), and bis(maltolato)-oxovanadium(IV) (BMOV) differentially induced oxidative stress in lymphocytes. Treatment with pV(phen), which caused intracellular oxidation, induced strong protein tyrosine phosphorylation compared with Na3VO4 and BMOV. Syk family kinases and the mitogen-activated protein kinase erk2 were rapidly activated by pV(phen) but not by BMOV or Na3VO4. In contrast, both BMOV and pV(phen) strongly activated NF-kappaB. The antioxidant pyrrolidine dithiocarbamate (PDTC) greatly diminished the intracellular oxidation and protein phosphotyrosine accumulation induced by pV(phen). Pretreatment of cells with PDTC reduced and delayed the activation of Syk kinases and erk2. However, NF-kappaB activation by pV(phen) was markedly enhanced in lymphocytes pretreated with PDTC, and another antioxidant, N-acetylcysteine, did not prevent the activation of NF-kappaB by BMOV. These results indicate a role for oxidative stress in the biological effects of some PTP inhibitors, whereas NF-kappaB activation by PTP inhibitors is mediated by mechanisms independent of intracellular redox status.

Antioxidants↗
PubMed1997

Induction of growth arrest and DNA damage-inducible genes Gadd45 and Gadd153 in primary rodent embryonic cells following exposure to methylmercury.

Methylmercury (MeHg) is recognized as a significant environmental hazard, particularly to the development of the nervous system. Studies on the mechanism of MeHg-induced toxicity reveal that inhibition of cell cycle progression may be one way by which MeHg interferes with normal development. In this study, we utilized primary rodent embryonic neuronal cell (CNS) and limb bud (LB) cultures to determine the mRNA expression level of two genes involved in cell cycle arrest, Gadd45 and Gadd153, both during cellular differentiation and in response to MeHg exposure. A differential expression pattern of Gadd45 and Gadd153 was observed during CNS and LB differentiation in culture. However, both CNS and LB cells responded to MeHg exposure with a concentration-dependent increase in Gadd45 and Gadd153 mRNA. Previous studies have shown that MeHg exposure (2 microm) of CNS cells for 24 hr causes a fourfold decrease in the number of cells passing through the cell cycle. The present study shows that at the same exposure concentration, a five- to eightfold increase in Gadd45 mRNA levels and a two- to fourfold increase of Gadd153 was observed. Induction of Gadd45 was also noted in adult female mice chronically exposed to 10 ppm MeHg, a dose that caused developmental toxicity in vivo. Based on the known involvement of the Gadd genes in cell cycle arrest, activation of these genes could be one mechanism by which MeHg interferes with the cell cycle in adult and developing organisms.

Animals↗
PubMed1996

Probenicid inhibition of fluorescence extrusion after MCB-staining of rat-1 fibroblasts.

The intracellular fluorescence level of cells stained continuously with monochlorobimane was monitored by flow cytometry in order to assess the initial rate of glutatione to monochlorobimane conjugation as a measure of glutathione S-transferase activity. In addition to a rapid initial increase and a plateau level, a decline in fluorescence intensity was found upon prolonged flow cytometric monitoring. Exposure to probenicid, an inhibitor of an ATP-dependent organic anion pump, prevented this decrease. Incubation with vanadate and verapamil was without effect. Thus, extrusion of fluorescentglutathione-conjugate perturbs the proportionality between initial glutathione level and monochlorobimane-dependent fluorescence intensity. Monitoring by flow cytometry the decrease in monochlorobimane-dependent fluorescence may be useful to detect multidrug resistant cells.

3T3 Cells↗
PubMed1996

Localization by in situ hybridization of gamma-glutamylcysteine synthetase mRNA expression in rat kidney following acute methylmercury treatment.

In previous studies we reported that prolonged treatment of rats with subtoxic levels of mercury as methylmercury hydroxide (MMH) elicited a two- to threefold increase in renal glutathione (GSH) content and a three- to fourfold increase in the mRNA encoding the catalytically active heavy subunit of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis. In the present studies, we demonstrate that enhanced expression of GCS mRNA and GSH synthesis rapidly occur following acute MMH treatment and, moreover, that increased expression of renal GCS mRNA is localized predominantly to regions of the kidney cortex consistent with the principal distribution of mercury in the kidney. Previous studies have demonstrated that resistance to mercury toxicity during prolonged MMH exposure may be associated with the ability to up-regulate GSH synthesis subsequent to intracellular dealkylation of MMH to Hg2+. The present finding that GCS mRNA and GSH levels are rapidly increased in kidney cells which are most susceptible to mercury toxicity supports the view that up-regulation of GSH synthesis occurs as an initial adaptive response to Hg2(+)-mediated cytotoxicity following acute as well as prolonged mercury exposure.

Animals↗
PubMed1996

Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry.

Characterizing constitutive activities and inducibility of various cytochrome P450 isozymes is important for elucidating species and individual differences in susceptibility to many toxicants. Although expression of certain P450s has been studied in homogenized tissues, the ability to assess functional enzyme activity without tissue disruption would further our understanding of interactive factors that modulate P450 activities. We used precision-cut, viable rat liver slices and confocal laser cytometry to determine the regional enzyme activities of P450 isozymes in situ. Livers from control and beta-naphthoflavone (beta NF)-treated rats were sectioned with a Krumdieck tissue slicer into 250-microns thick sections. A slice perfusion chamber that mounts on the cytometer stage was developed to allow for successive measurement of region-specific P450-dependent O-dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzyloxyresorufin (EROD, PROD, and BROD activity, respectively) in the same liver slice. Images of the accumulated fluorescent resorufin product within the tissue were acquired using a confocal laser cytometer in confocal mode. As expected, slices isolated from beta NF-treated rats showed high levels of centrilobular EROD activity compared to slices from control rats, whereas PROD and BROD activities remained at control levels. These techniques should allow for the accurate quantification of regional and cell-specific P450 enzyme activity and, with subsequent analysis of the same slice, the ability to correlate specific P450 mRNAs or other factors with enzymatic activity. Moreover, these techniques should be amenable to examination of similar phenomena in other tissues such as lung and kidney, where marked heterogeneity in cellular P450 expression patterns is also known to occur.

Analysis of Variance↗
PubMed1995

De novo synthesis of glutathione is required for both entry into and progression through the cell cycle.

To study the putative role of de novo synthesis of glutathione (GSH) in the regulation of the cell cycle, we exposed NIH-3T3 cells to buthionine sulfoximine (BSO) and analysed cell cycle kinetics with continuous bromodeoxyuridine (BrdU) labeling and bivariate Hoechst 33258/ethidium bromide flow cytometry. Treating quiescent cells, which themselves had a low GSH content, with BSO did not affect subsequent entry into and progression through the cell cycle. Adding BSO during serum stimulation, however, provoked a dose-dependent inhibition of cell growth and a delayed increase in GSH level. The cell kinetic mechanism underlying BSO-induced growth inhibition is a diminished entry into the cell cycle and a permanent arrest in the S and G2 phase of the cell cycle. Our results are consistent with the hypothesis that GSH de novo synthesis is required for cell activation and proper S and G2 phase transit.

3T3 Cells↗
PubMed1995

Ethanol inhibits muscarinic receptor-stimulated phosphoinositide metabolism and calcium mobilization in rat primary cortical cultures.

In recent years, it has been hypothesized that muscarinic receptor-stimulated phosphoinositide (PI) metabolism may represent a relevant target for the developmental neurotoxicity of ethanol. Age-, brain region-, and receptor-specific inhibitory effects of ethanol on this system have been found, both in vitro and after in vivo administration. As a direct consequence of this action, alterations of calcium homeostasis would be expected, through alterations of inositol trisphosphate formation, which mediates intracellular calcium mobilization. In the present study, the effects of ethanol (50-500 mM) on carbachol-stimulated PI metabolism and free intracellular calcium levels were investigated in rat primary cortical cultures, by measuring release of inositol phosphates and utilizing the two calcium probes fluo-3 and indo-1 on an ACAS (Adherent Cell Analysis and Sorting) Laser Cytometer. Ethanol exerted a concentration-dependent inhibition of carbachol-stimulated PI metabolism. In addition, ethanol's inhibitory effect paralleled the temporal development of the muscarinic receptor signal transduction system, with the strongest inhibition (25-50%) occurring when maximal stimulation by carbachol occurs (days 5-7). Ethanol also exerted a concentration-dependent decrease in free intracellular calcium levels following carbachol stimulation. Both initial calcium spike amplitude, seen in all responsive cells, as well as the total number of cells responding to carbachol, were decreased by ethanol. The inhibitory effects of ethanol seemed dependent upon preincubation time, in that a longer preincubation (30 min) with the lowest dose (50 mM), showed almost the same decrease in responding cell number and reduction in spike amplitude in responding cells, as a shorter incubation (10 min) with the highest ethanol dose (500 mM). The specificity of the response to carbachol was demonstrated by blocking the response with 10 microM atropine. Moreover, experiments with carbachol in calcium-free buffer with 1 mM EGTA indicated that the initial calcium spike was due to intracellular calcium mobilization from intracellular stores. Since calcium is believed to play important roles in cell proliferation and differentiation, these results support the hypothesis that this intracellular signal-transduction pathway may be a target for ethanol, contributing to its developmental neurotoxicity.

Action Potentials↗
PubMed1995

Expression of SPARC during development of the chicken chorioallantoic membrane: evidence for regulated proteolysis in vivo.

SPARC is a secreted glycoprotein that has been shown to disrupt focal adhesions and to regulate the proliferation of endothelial cells in vitro. Moreover, peptides resulting from the proteolysis of SPARC exhibit angiogenic activity. Here we describe the temporal synthesis, turnover, and angiogenic potential of SPARC in the chicken chorioallantoic membrane. Confocal immunofluorescence microscopy revealed specific expression of SPARC protein in endothelial cells, and significantly higher levels of SPARC were observed in smaller newly formed blood vessels in comparison to larger, developmentally older vessels. SPARC mRNA was detected at the earliest stages of chorioallantoic membrane morphogenesis and reached maximal levels at day 13 of embryonic development. Interestingly, steady-state levels of SPARC mRNA did not correlate directly with protein accumulation; moreover, the protein appeared to undergo limited degradation during days 10-15. Incubation of [125I]-SPARC with chorioallantoic membranes of different developmental ages confirmed that extracellular proteolysis occurred during days 9-15, but not at later stages (e.g., days 17-21). Comparison of peptides produced by incubation with chorioallantoic membranes with those generated by plasmin showed an identical pattern of proteolysis. Plasmin activity was present throughout development, and in situ zymography identified sites of plasminogen activator activity that corresponded to areas exhibiting high levels of SPARC expression. Synthetic peptides from a plasmin-sensitive region of SPARC, between amino acids 113-130, stimulated angiogenesis in the chorioallantoic membrane in a dose-dependent manner; in contrast, intact SPARC was inactive in similar assays. We have shown that SPARC is expressed in endothelial cells of newly formed blood vessels in a manner that is both temporally and spatially restricted. Between days 9 and 15 of chorioallantoic membrane development, the protein undergoes proteolytic cleavage that is mediated, in part, by plasmin. SPARC peptides released specifically by plasmin induce angiogenesis in vivo. We therefore propose that SPARC acts as an intrinsic regulator of angiogenesis in vivo.

Allantois↗
PubMed1995

Activation of murine T-cells via phospholipase-C gamma 1-associated protein tyrosine phosphorylation is reduced with aging.

Cross-linking of the T-cell receptor (CD3) induces activation of tyrosine kinases and the subsequent phosphorylation of intracellular protein substrates. We examined whether early events in signal transduction through CD3 or CD3 x CD4 receptor ligation were altered in aged murine T-lymphocytes. Both calcium mobilization and tyrosine phosphorylation of phospholipase C gamma 1 (PLC gamma 1) were decreased in T-lymphocytes from old mice. In addition, there was less tyrosine phosphorylation of a 35/36 kDa protein both in whole cell lysates and in PLC gamma 1 immunoprecipitates from old mice. This 35/36 kDa phosphoprotein binds specifically to the SH2 domains of PLC gamma 1. Using a fusion protein containing the SH2 domains of PLC gamma 1 and human IgG1 heavy chain, we identified three additional proteins that bind to the SH2 domains which were tyrosine phosphorylated following CD3 x CD4 ligation to a lesser degree with age. The tyrosine phosphorylation of two phosphoproteins binding to a fusion protein consisting of the SH2 domains of GAP (ras GTPase-activating protein) and human IgG1 heavy chain was also reduced with aging. The observed binding to SH2 domains was thiol redox sensitive. Thus, decreases in antioxidants with age may be responsible for inhibitory effects on PLC gamma 1-phosphatidylinositol signaling through redox regulation of tyrosine phosphoproteins.

Aging↗