John J Lemasters

Medical University of South Carolina, Charleston, South Carolina, United States

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Publications (424)2150.28 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Inclusion of liver grafts from cardiac death donors (CDD) would increase the availability of donor livers but is hampered by a higher risk of primary non-function. Here, we seek to determine mechanisms that contribute to primary non-function of liver grafts from CDD with the goal to develop strategies for improved function and outcome, focusing on c-Jun-N-terminal kinase (JNK) activation and mitochondrial depolarization, two known mediators of graft failure. Livers explanted from wild-type, iNOS(-/-), JNK1(-/-) or JNK2(-/-) mice after 45-min aorta clamping were implanted into wild-type recipients. Mitochondrial depolarization was detected by intravital confocal microscopy in living recipients. After transplantation of wild-type CDD livers, graft iNOS expression and 3-nitrotyrosine adducts increased, but hepatic endothelial NOS expression was unchanged. Graft injury and dysfunction were substantially higher in CDD grafts than in non-CDD grafts. iNOS-deficiency and inhibition attenuated injury and improved function and survival of CDD grafts. JNK1/2 and apoptosis signal-regulating kinase-1 activation increased markedly in wild-type CDD grafts, which was blunted by iNOS-deficiency. JNK inhibition and JNK2-deficiency, but not JNK1-deficiency, decreased injury and improved function and survival of CDD grafts. Mitochondrial depolarization and binding of phospho-JNK2 to Sab, a mitochondrial protein linked to the mitochondrial permeability transition, were higher in CDD than in non-CDD grafts. iNOS-deficiency, JNK inhibition and JNK2-deficiency all decreased mitochondrial depolarization and blunted ATP depletion in CDD grafts. JNK inhibition and deficiency did not decrease 3-nitrotyrosine adducts in CDD grafts. The iNOS-JNK2-Sab pathway promotes CDD graft failure via increased mitochondrial depolarization and is an attractive target to improve liver function and survival in CDD liver transplantation. Copyright © 2015. Published by Elsevier B.V.
    Journal of Hepatology 02/2015; DOI:10.1016/j.jhep.2015.02.017 · 10.40 Impact Factor
  • Journal of the American Society of Nephrology 11/2014; 25:389A. · 9.47 Impact Factor
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    ABSTRACT: Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as /`accidental cell death/' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. /`Regulated cell death/' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects i
    Cell death and differentiation 09/2014; DOI:10.1038/cdd.2014.137 · 8.39 Impact Factor
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    Eduardo N Maldonado, John J Lemasters
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    ABSTRACT: Non-proliferating cells generate the bulk of cellular ATP by fully oxidizing respiratory substrates in mitochondria. Respiratory substrates cross the mitochondrial outer membrane through only one channel, the voltage dependent anion channel (VDAC). Once in the matrix, respiratory substrates are oxidized in the tricarboxylic acid cycle to generate mostly NADH that is further oxidized in the respiratory chain to generate a proton motive force comprised mainly of membrane potential (ΔΨ) to synthesize ATP. Mitochondrial ΔΨ then drives release of ATP(-4) from the matrix in exchange for ADP(-3) in the cytosol via the adenine nucleotide translocator (ANT) located in the mitochondrial inner membrane. Thus, mitochondrial function in non-proliferating cells drives a high cytosolic ATP/ADP ratio, essential to inhibit glycolysis. By contrast, the bioenergetics of the Warburg phenotype of proliferating cells is characterized by enhanced aerobic glycolysis and suppression of mitochondrial metabolism. Suppressed mitochondrial function leads to lower production of mitochondrial ATP and hence lower cytosolic ATP/ADP ratios that favor enhanced glycolysis. Thus, cytosolic ATP/ADP ratio is a key feature that determines if cell metabolism is predominantly oxidative or glycolytic. Here, we describe two novel mechanisms to explain the suppression of mitochondrial metabolism in cancer cells: the relative closure of VDAC by free tubulin and inactivation of ANT. Both mechanisms contribute to low ATP/ADP ratios that activate glycolysis.
    Mitochondrion 09/2014; 19. DOI:10.1016/j.mito.2014.09.002 · 3.52 Impact Factor
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    ABSTRACT: First-line therapy for pancreatic cancer is gemcitabine. Although tumors may initially respond to the gemcitabine treatment, soon tumor resistance develops leading to treatment failure. Previously, we demonstrated in human MIA PaCa-2 pancreatic cancer cells that N-acetyl-l-cysteine (NAC), a glutathione (GSH) precursor, prevents NFκB activation via S-glutathionylation of p65-NFκB, thereby blunting expression of survival genes. In this study, we documented the molecular sites of S-glutathionylation of p65, and we investigated whether NAC can suppress NFκB signaling and augment a therapeutic response to gemcitabine in vivo. Mass spectrometric analysis of S-glutathionylated p65-NFκB protein in vitro showed post-translational modifications of cysteines 38, 105, 120, 160 and 216 following oxidative and nitrosative stress. Circular dichroism revealed that S-glutathionylation of p65-NFκB did not change secondary structure of the protein, but increased tryptophan fluorescence revealed altered tertiary structure. Gemcitabine and NAC individually were not effective in decreasing MIA PaCa-2 tumor growth in vivo. However, combination treatment with NAC and gemcitabine decreased tumor growth by approximately 50%. NAC treatment also markedly enhanced tumor apoptosis in gemcitabine-treated mice. Compared to untreated tumors, gemcitabine treatment alone increased p65-NFκB nuclear translocation (3.7-fold) and DNA binding (2.5-fold), and these effects were blunted by NAC. In addition, NAC plus gemcitabine treatment decreased anti-apoptotic XIAP protein expression compared to gemcitabine alone. None of the treatments, however, affected extent of tumor hypoxia, as assessed by EF5 staining. Together, these results indicate that adjunct therapy with NAC prevents NFκB activation and improves gemcitabine chemotherapeutic efficacy.
    Biomedecine [?] Pharmacotherapy 08/2014; DOI:10.1016/j.biopha.2014.08.007 · 2.11 Impact Factor
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    Biochimica et Biophysica Acta (BBA) - Bioenergetics 07/2014; 1837:e111. DOI:10.1016/j.bbabio.2014.05.267 · 4.83 Impact Factor
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    ABSTRACT: Ca(2+)-induced permeability transition pore (mPTP) opening in isolated rat brain mitochondria is promoted through targeting of connexin43. After a threshold Ca(2+) load, mitochondrial membrane potential drops and efflux of accumulated Ca(2+) from the mitochondrial matrix occurs, indicating the mPTP opening. Specific antibodies were used to assess the role of the translocator protein (18 kDa; TSPO) and connexin43 in swelling of isolated rat liver and brain mitochondria induced by carbenoxolone and the endogenous TSPO ligand protoporphyrin IX. Mitochondrial membrane potential, Ca(2+) transport and oxygen consumption were determined using selective electrodes. All the parameters were detected simultaneously in a chamber with the selective electrodes. The phosphorylation state of mitochondrial protein targets was assessed. We report that Ca(2+)-induced mitochondrial swelling was strengthened in the presence of both carbenoxolone and protoporphyrin IX. The carbenoxolone- and protoporphyrin IX-accelerated mPTP induction in brain mitochondria was completely prevented by antibodies specific for the mitochondrial translocator protein (TSPO). The anti-TSPO antibodies were more effective than anti-сonnexin43 antibodies. Moreover, carbenoxolone-stimulated phosphorylation of mitochondrial proteins was inhibited by anti-TSPO antibodies. Taken together, the data suggests that, in addition to acting via connexion43, carbenoxolone may exert its effect on mPTP via mitochondrial outer membrane TSPO.
    Archives of Biochemistry and Biophysics 07/2014; 558. DOI:10.1016/j.abb.2014.06.027 · 3.04 Impact Factor
  • Biochimica et Biophysica Acta (BBA) - Bioenergetics 07/2014; 1837:e112-e113. DOI:10.1016/j.bbabio.2014.05.271 · 4.83 Impact Factor
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    ABSTRACT: Despite recovery of hemodynamics by fluid resuscitation after hemorrhage, development of the systemic inflammatory response and multiple organ dysfunction syndromes can nonetheless lead to death. Minocycline and doxycycline are tetracycline derivatives that are protective in models of hypoxic, ischemic and oxidative stress. Our Aim was to determine whether minocycline and doxycycline protect liver and kidney and improve survival in a mouse model of hemorrhagic shock and resuscitation.
    Shock (Augusta, Ga.) 06/2014; DOI:10.1097/SHK.0000000000000213 · 2.73 Impact Factor
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    ABSTRACT: Polycystic kidney disease (PKD) is a common genetic disorder leading to cyst formation in the kidneys and other organs that ultimately results in kidney failure and death. Currently, there is no therapy for slowing down or stopping the progression of PKD. In this study we identified the disintegrin metalloenzyme 17 (ADAM17) as a key regulator of cell proliferation in kidney tissues of conditional knockout Ift88-/- mice and collecting duct epithelia cell from Ift88orpk mice, animal models of autosomal recessive polycystic kidney disease (ARPKD). Using Western blotting, an enzyme activity assay and a growth factor shedding assay in the presence or absence of the specific ADAM17 inhibitor TMI-005, we show that increased expression and activation of ADAM17 in cystic kidney and in collecting duct epithelial cells originating from the Ift88orpk mice (designated as PKD cells) leads to constitutive shedding of several growth factors including heparin-binding EGF-like growth factor (HB-EGF), amphiregulin and transforming growth factor-alpha (TGFα). Increased growth factor shedding induces activation of the EGFR/MAPK/ERK pathway and maintains higher cell proliferation rate in PKD cells compared to control cells. PKD cells also displayed increased lactate formation and extracellular acidification indicative of aerobic glycolysis (Warburg effect) that was blocked by ADAM17 inhibition. We propose that ADAM17 is a key promoter of cellular proliferation in PKD cells by activating the EGFR/ERK axis and a pro-proliferative glycolytic phenotype.
    American journal of physiology. Renal physiology 06/2014; 307(5). DOI:10.1152/ajprenal.00218.2014 · 3.30 Impact Factor
  • Gastroenterology 05/2014; 146(5):S928. DOI:10.1016/S0016-5085(14)63372-2 · 13.93 Impact Factor
  • Gastroenterology 05/2014; 146(5):944. DOI:10.1016/S0016-5085(14)63427-2 · 13.93 Impact Factor
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    ABSTRACT: An increase of ethanol metabolism and hepatic mitochondrial respiration occurs in vivo after a single binge of alcohol. Here, our aim was to determine how ethanol intake affects hepatic mitochondrial polarization status in vivo in relation to ethanol metabolism and steatosis. Hepatic mitochondrial polarization, permeability transition (MPT), and reduce pyridine nucleotides, and steatosis in mice were monitored by intravital confocal/multiphoton microscopy of the fluorescence of rhodamine 123 (Rh123), calcein, NAD(P)H, and BODIPY493/503, respectively, after gavage with ethanol (1-6 g/kg). Mitochondria depolarized in an all-or-nothing fashion in individual hepatocytes as early as 1 h after alcohol. Depolarization was dose- and time-dependent, peaked after 6 to 12 h and maximally affected 94% of hepatocytes. This mitochondrial depolarization was not due to onset of the MPT. After 24 h, mitochondria of most hepatocytes recovered normal polarization and were indistinguishable from untreated after 7 days. Cell death monitored by propidium iodide staining, histology and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was low throughout. After alcohol, mitochondrial NAD(P)H autofluorescence increased and decreased, respectively, in hepatocytes with polarized and depolarized mitochondria. Ethanol also caused steatosis mainly in hepatocytes with depolarized mitochondria. Depolarization was linked to ethanol metabolism, since deficiency of alcohol dehydrogenase and cytochrome-P450 2E1 (CYP2E1), the major ethanol-metabolizing enzymes, decreased mitochondrial depolarization by ∼70% and ∼20%, respectively. Activation of aldehyde dehydrogenase decreased depolarization, whereas inhibition of aldehyde dehydrogenase enhanced depolarization. Activation of aldehyde dehydrogenase also markedly decreased steatosis. Acute ethanol causes reversible hepatic mitochondrial depolarization in vivo that may contribute to steatosis and increased mitochondrial respiration. Onset of this mitochondrial depolarization is linked, at least in part, to metabolism of ethanol to acetaldehyde.
    PLoS ONE 03/2014; 9(3):e91308. DOI:10.1371/journal.pone.0091308 · 3.53 Impact Factor
  • AJP Heart and Circulatory Physiology 03/2014; 306(5):H778-9. DOI:10.1152/ajpheart.00021.2014 · 4.01 Impact Factor
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    John J. Lemasters
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    ABSTRACT: Mitophagy (mitochondrial autophagy), which removes damaged, effete and superfluous mitochondria, has several distinct variants. In Type 1 mitophagy occurring during nutrient deprivation, preautophagic structures (PAS) grow into cup-shaped phagophores that surround and sequester individual mitochondria into mitophagosomes, a process requiring phosphatidylinositol-3-kinase (PI3K) and often occurring in coordination with mitochondrial fission. After sequestration, the outer compartment of the mitophagosome acidifies, followed by mitochondrial depolarization and ultimately hydrolytic digestion in lysosomes. Mitochondrial damage stimulates Type 2 mitophagy. After photodamage to single mitochondria, depolarization occurs followed by decoration and then coalescence of autophagic LC3-containing structures on mitochondrial surfaces. Vesicular acidification then occurs. By contrast to Type 1 mitophagy, PI3K inhibition does not block Type 2 mitophagy. Further, Type 2 mitophagy is not associated with phagophore formation or mitochondrial fission. A third form of self-eating of mitochondria is formation of mitochondria-derived vesicles (MDVs) enriched in oxidized mitochondrial proteins that bud off and transit into multivesicular bodies. Topologically, the internalization of MDV by invagination of the surface of multivesicular bodies followed by vesicle scission into the lumen is a form of microautophagy, or micromitophagy (Type 3 mitophagy). Cell biological distinctions are the basis for these three types of mitophagy. Future studies are needed to better characterize the molecular and biochemical differences between Types 1, 2 and 3 mitophagy.
    01/2014; 2. DOI:10.1016/j.redox.2014.06.004
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    ABSTRACT: Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
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    ABSTRACT: Recent studies suggest that an increase in apoptosis within the myocardium may be a contributing factor for the progression of adverse left ventricular (LV) remodeling following myocardial infarction (MI). Given that apoptosis is often triggered by induction of the mitochondrial permeability transition (MPT) pore, the goal of this study was to evaluate the therapeutic efficacy of cyclosporin A (CsA), an MPT blocker, to prevent cells from undergoing apoptosis and consequently attenuate LV remodeling post-MI. MI was induced in C57BL/6 mice and then randomized to either vehicle or CsA groups. Beginning 48 h after surgery, mice were gavaged with CsA (2 mg/kg) or vehicle once daily. LV end diastolic volume and LV ejection fraction were assessed by echocardiography before MI induction and terminally at either 7 days (n=7) or 28 days (n=8) post-MI. LV end diastolic volume was increased and LV ejection fraction was decreased in all MI groups with no difference between the CsA-treated and untreated groups. After vehicle and CsA, areas of necrosis were present at 7 and 28 days post MI with no difference between MI groups. Caspase-3 activity and TUNEL-positive cells in myocardium from the non-necrotic areas were both increased after MI but were lower in CsA-treated mice compared to vehicle (p<0.05). In conclusion, CsA decreased apoptosis occurring after MI, confirming involvement of the MPT. However, CsA-mediated reduction in apoptosis in non-MI myocardium was not beneficial in reducing LV volume or improving LV pump function.
    AJP Heart and Circulatory Physiology 10/2013; 306(1). DOI:10.1152/ajpheart.00079.2013 · 4.01 Impact Factor
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    ABSTRACT: Minocycline, a tetracycline-derived compound, mitigates damage caused by ischemia/reperfusion (I/R) injury. Here, 19 tetracycline-derived compounds were screened in comparison to minocycline for their ability to protect hepatocytes against damage from chemical hypoxia and I/R injury. Cultured rat hepatocytes were incubated with 50μM of each tetracycline-derived compound 20min prior to exposure to 500μM iodoacetic acid plus 1mM KCN (chemical hypoxia). In other experiments, hepatocytes were incubated in anoxic Krebs-Ringer-Hepes buffer (KRH) at pH6.2 for 4h prior to reoxygenation at pH7.4 (simulated I/R). Tetracycline-derived compounds were added 20min prior to reperfusion. Ca(2+) uptake was measured in isolated rat liver mitochondria incubated with Fluo-5N. Cell killing after 120min of chemical hypoxia measured by propidium iodide (PI) fluorometery was 87%, which decreased to 28% and 42% with minocycline and doxycycline, respectively. After I/R, cell killing at 120min decreased from 79% with vehicle to 43% and 49% with minocycline and doxycycline. No other tested compound decreased killing. Minocycline and doxycycline also inhibited mitochondrial Ca(2+) uptake and suppressed the Ca(2+)-induced mitochondrial permeability transition (MPT), the penultimate cause of cell death in reperfusion injury. Ru360, a specific inhibitor of the mitochondrial calcium uniporter (MCU), also decreased cell killing after hypoxia and I/R and blocked mitochondrial Ca(2+) uptake and the MPT. Other proposed mechanisms, including mitochondrial depolarization and matrix metalloprotease inhibition could not account for cytoprotection. Taken together, these results indicate that minocycline and doxycycline are cytoprotective by way of inhibition of MCU.
    Toxicology and Applied Pharmacology 09/2013; 273(1). DOI:10.1016/j.taap.2013.08.027 · 3.63 Impact Factor
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    ABSTRACT: Sub-100 nm colloidal particles which are surface-functionalized with multiple environmentally-sensitive moieties have the potential to combine imaging, early detection, and the treatment of cancer with a single type of long-circulating “nanodevice”. Deep tissue imaging is achievable through the development of particles which are surface-modified with fluorophores that operate in the near-infrared (NIR) spectrum and where the fluorophore's signal can be maximized by “turning-on” the fluorescence only in the targeted tissue. We present a general approach for the synthesis of NIR emitting nanoparticles that exhibit a protein triggered activation/deactivation of the emission. Dispersing the particles into an aqueous solution, such as phosphate buffered saline (PBS), resulted in an aggregation of the hydrophobic fluorophores and a cessation of emission. The emission can be reinstated, or activated, by the conversion of the surface-attached fluorophores from an aggregate to a monomeric species with the addition of an albumin. This activated probe can be deactivated and returned to a quenched state by a simple tryptic digestion of the albumin. The methodology for emission switching offers a path to maximize the signal from the typically weak quantum yield inherent in NIR fluorophores.
    08/2013; 1(36). DOI:10.1039/C3TB20681E
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    ABSTRACT: Our previous studies showed that an extract from Camellia sinenesis (green tea), which contains several polyphenols, attenuates nephrotoxicity caused by cyclosporine A (CsA). Since polyphenols are stimulators of mitochondrial biogenesis (MB), this study investigated whether stimulation of MB plays a role in green tea polyphenol protection against CsA renal toxicity. Rats were fed a powdered diet containing green tea polyphenolic extract (0.1%) starting 3 days prior to CsA treatment (25 mg/kg, i.g. daily for 3 weeks). CsA alone decreased renal nuclear DNA-encoded oxidative phosphorylation (OXPHOS) protein ATP synthase-β (AS-β) by 42%, mitochondrial DNA (mtDNA)-encoded OXPHOS protein NADH dehydrogenase-3 (ND3) by 87% and their associated mRNAs. Mitochondrial DNA copy number was also decreased by 78% by CsA. Immunohistochemical analysis showed decreased cytochrome c oxidase subunit IV (COX-IV), an OXPHOS protein, in tubular cells. Peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, the master regulator of MB, and mitochondrial transcription factor-A (Tfam), the transcription factor that regulates mtDNA replication and transcription, were 42% and 90% lower, respectively, in the kidneys of CsA-treated than in untreated rats. These results indicate suppression of MB by chronic CsA treatment. Green tea polyphenols alone and following CsA increased AS-β, ND3, COX-IV, mtDNA copy number, PGC-1α mRNA and protein, decreased acetylated PGC-1α, and increased Tfam mRNA and protein. In association with suppressed MB, CsA increased serum creatinine, caused loss of brush border and dilatation of proximal tubules, tubular atrophy, vacuolization, apoptosis, calcification, and increased neutrophil gelatinase-associated lipocalin expression, leukocyte infiltration, and renal fibrosis. Green tea polyphenols markedly attenuated CsA-induced renal injury and improved renal function. Together, these results demonstrate that green tea polyphenols attenuate CsA-induced kidney injury, at least in part, through the stimulation of MB.
    PLoS ONE 06/2013; 8(6):e65029. DOI:10.1371/journal.pone.0065029 · 3.53 Impact Factor

Publication Stats

22k Citations
2,150.28 Total Impact Points

Institutions

  • 2006–2015
    • Medical University of South Carolina
      • • Department of Drug Discovery and Biomedical Sciences
      • • Center of Cell Death, Injury and Regeneration
      • • Department of Biochemistry and Molecular Biology (College of Medicine)
      Charleston, South Carolina, United States
  • 2012
    • University of Michigan
      • Life Sciences Institute
      Ann Arbor, MI, United States
  • 2010–2011
    • Kansas City VA Medical Center
      Kansas City, Missouri, United States
  • 1978–2008
    • University of North Carolina at Chapel Hill
      • • Department of Nutrition
      • • Department of Surgery
      • • Department of Pharmacology
      • • Department of Medicine
      North Carolina, United States
  • 2004–2006
    • The University of Arizona
      • College of Medicine
      Tucson, AZ, United States
    • Akita University
      Akita, Akita, Japan
  • 2002
    • University of Arkansas at Little Rock
      Little Rock, Arkansas, United States
  • 2000
    • University of Texas Health Science Center at San Antonio
      • Department of Cellular and Structural Biology
      San Antonio, TX, United States
  • 1997
    • Case Western Reserve University
      • Department of Anatomy
      Cleveland, OH, United States
  • 1996
    • University of Pavia
      Ticinum, Lombardy, Italy
  • 1990
    • Universität des Saarlandes
      Saarbrücken, Saarland, Germany
  • 1983
    • University of North Carolina at Charlotte
      Charlotte, North Carolina, United States