John J. Lemasters

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

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Publications (488)2549.32 Total impact

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    Daniel J Klionsky · Kotb Abdelmohsen · Akihisa Abe · Md Joynal Abedin · Hagai Abeliovich · Abraham Acevedo Arozena · Hiroaki Adachi · Christopher M Adams · Peter D Adams · Khosrow Adeli · [...] · Xiao-Feng Zhu · Yuhua Zhu · Shi-Mei Zhuang · Xiaohong Zhuang · Elio Ziparo · Christos E Zois · Teresa Zoladek · Wei-Xing Zong · Antonio Zorzano · Susu M Zughaier ·
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    ABSTRACT: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are 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 monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.
    Full-text · Article · Jan 2016 · Autophagy
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    ABSTRACT: Acetaminophen (APAP) overdose causes hepatotoxicity involving mitochondrial dysfunction and c-jun N-terminal kinase (JNK) activation. Because the safe limit of APAP dosing is controversial, our aim was to evaluate the role of the mitochondrial permeability transition (MPT) and JNK in mitochondrial dysfunction after APAP dosing considered non-toxic by criteria of serum alanine aminotransferase (ALT) release and histological necrosis in vivo. C57BL/6 mice were given APAP with and without the MPT inhibitor, NIM811, or the JNK inhibitor, SP600125. Fat droplet formation, cell viability and mitochondrial function in vivo were monitored by intravital multiphoton microscopy. Serum ALT, liver histology, total JNK and activated phospho(p)JNK were also assessed. High APAP (300 mg/kg) caused ALT release, necrosis, irreversible mitochondrial dysfunction and hepatocellular death. By contrast, lower APAP (150 mg/kg) caused reversible mitochondrial dysfunction and fat droplet formation in hepatocytes without ALT release or necrosis. Mitochondrial protein N-acetyl-p-benzoquinone imine adducts correlated with early JNK activation, but irreversible mitochondrial depolarization and necrosis at high dose were associated with sustained JNK activation and translocation to mitochondria. NIM811 prevented cell death and/or mitochondrial depolarization after both high and low dose APAP. After low dose, SP600125 decreased mitochondrial depolarization. In conclusion, low dose APAP produces reversible MPT-dependent mitochondrial dysfunction and steatosis in hepatocytes without causing ALT release or necrosis, whereas high dose leads to irreversible mitochondrial dysfunction and cell death associated with sustained JNK activation. Thus, non-toxic APAP has the potential to cause transient mitochondrial dysfunction that may synergize with other stresses to promote liver damage and steatosis.
    No preview · Article · Dec 2015 · Toxicological Sciences

  • No preview · Article · Dec 2015 · Journal of the American Society of Nephrology
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    ABSTRACT: Permeability of the mitochondrial outer membrane is determined by the activity of voltage-dependent anion channels (VDAC) which are regulated by many factors and proteins. One of the main partner-regulator of VDAC is the 18 kDa translocator protein (TSPO), whose role in the regulation of membrane permeability is not completely understood. We show that TSPO ligands, 1 μM PPIX and PK11195 at concentrations of 50 μM, accelerate opening of permeability transition pores (mPTP) in Ca(2+)-overloaded rat brain mitochondria (RBM). By contrast, PK11195 at 100 nM and anti-TSPO antibodies suppressed pore opening. Participation of VDAC in these processes was demonstrated by blocking VDAC with G3139, an 18-mer phosphorothioate oligonucleotides, which sensitized mitochondria to Ca(2+)-induced mPTP opening. Despite the inhibitory effect of 100 nM PK11195 and anti-TSPO antibodies alone, their combination with G3139 considerably stimulated the mPTP opening. Thus, 100 nM PK11195 and anti-TSPO antibody can modify permeability of the VDAC channel and mPTP. When VDAC channels are closed and TSPO is blocked, permeability of the VDAC for calcium seems to be the highest, which leads to accelerated pore opening.
    Full-text · Article · Oct 2015 · Archives of Biochemistry and Biophysics
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    ABSTRACT: The effect of surface potential modulators on palmitate/Ca(2+)-induced formation of lipid pores was studied in liposomal and inner mitochondrial membranes. Pore formation was monitored by sulforhodamine B release from liposomes and swelling of mitochondria. ζ-potential in liposomes was determined from electrophoretic mobility. Replacement of sucrose as the osmotic agent with KCl decreased negative ζ-potential in liposomes and increased resistance of both mitochondria and liposomes to the pore inducers, palmitic acid and Ca(2+). Micromolar Mg(2+) also inhibited palmitate/Ca(2+)-induced permeabilization of liposomes. The rate of palmitate/Ca(2+)-induced, cyclosporin A-insensitive swelling of mitochondria increased 22% upon increasing pH from 7.0 to 7.8. At below the critical micelle concentration, the cationic detergent cetyltrimethylammonium bromide (10 μM) and the anionic surfactant sodium dodecylsulfate (10-50 μM) made the ζ-potential less and more negative, respectively, and inhibited and stimulated opening of mitochondrial palmitate/Ca(2+)-induced lipid pores. Taken together, the findings indicate that surface potential regulates palmitate/Ca(2+)-induced lipid pore opening. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · May 2015 · Biochimica et Biophysica Acta

  • No preview · Article · May 2015 · Gastroenterology

  • No preview · Article · May 2015 · Gastroenterology
  • Yasodha Krishnasamy · Zengdun Shi · John J. Lemasters · Don C. Rockey · Zhi Zhong

    No preview · Article · Apr 2015 · Gastroenterology

  • No preview · Article · Apr 2015 · Gastroenterology
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    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.
    Full-text · Article · Feb 2015 · Journal of Hepatology
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    Eduardo N. Maldonado · Monika Gooz · David N. DeHart · John J. Lemasters

    Full-text · Article · Jan 2015 · Biophysical Journal
  • Li Li · John J. Lemasters

    No preview · Article · Jan 2015 · Biophysical Journal
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    ABSTRACT: Hepatocytes exposed to ischemia/reperfusion (I/R) succumb to cell death after reperfusion. Sphingosine and ceramide profiles revealed substantial accumulation of sphingosine after 4h of ischemia to rat hepatocytes, whereas other sphingoid bases did not change. A lysosomotropic inhibitor of acid ceramidase suppressed I/R-induced death, indicating a lysosomal origin of sphingosine. Addition of exogenous sphingosine to hepatocytes increased cell death, which was insensitive to the ceramide synthase inhibitor, fumonisin B1. This finding indicates that accumulation of sphingosine, not ceramide formed from sphingosine, promoted cell death. Exogenous sphingosine also inhibited complex IV (cytochrome oxidase), the terminal component of the respiratory chain, in isolated mitochondria. Accordingly, we hypothesized that downstream respiratory inhibition by sphingosine leads to increased formation of O2•- radicals after reperfusion, which by themselves have only a moderately harmful effect. However when Fe2+ redistributes from lysosomes into mitochondria during ischemia, Fenton chemistry occurs after reperfusion, leading to formation of highly reactive OH• radicals, potent inducers of the mitochondrial permeability transition pore and cell death. This hypothesis was directly tested using bafilomycin, which induces the release of Fe2+ from lysosomes with subsequent uptake into mitochondria. Indeed, bafilomycin potentiated sphingosine-induced cell death. The data highlight a novel mechanism mediating I/R injury, which involves sphingosine accumulation and uptake of lysosomal iron into mitochondria during ischemia, leading to respiratory chain inhibition, iron-dependent oxidative stress, mitochondrial permeability transition and cell death after reperfusion. DK073336, DK037034 and 14.Z50.31.0028 (JJL) and NS083544 (TIG).
    No preview · Article · Jan 2015 · Biophysical Journal
  • John J. Lemasters

    No preview · Article · Jan 2015 · Biophysical Journal

  • No preview · Article · Jan 2015 · Biophysical Journal

  • No preview · Article · Nov 2014 · Journal of the American Society of Nephrology
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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
    Full-text · Article · Sep 2014 · Cell death and differentiation
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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.
    Full-text · Article · Sep 2014 · Mitochondrion
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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.
    Full-text · Article · Aug 2014 · Biomedecine [?] Pharmacotherapy
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    Full-text · Article · Jul 2014 · Biochimica et Biophysica Acta (BBA) - Bioenergetics

Publication Stats

28k Citations
2,549.32 Total Impact Points


  • 2006-2015
    • Medical University of South Carolina
      • • Department of Drug Discovery and Biomedical Sciences
      • • Department of Biochemistry and Molecular Biology (College of Medicine)
      • • Center of Cell Death, Injury and Regeneration
      Charleston, South Carolina, United States
  • 2012
    • University of Michigan
      • Life Sciences Institute
      Ann Arbor, MI, United States
  • 2010
    • The Children's Hospital of Philadelphia
      Filadelfia, Pennsylvania, United States
  • 2008
    • Research Triangle Park Laboratories, Inc.
      Raleigh, North Carolina, United States
  • 1978-2008
    • University of North Carolina at Chapel Hill
      • • Center for Gastrointestinal Biology and Disease
      • • Department of Surgery
      • • Department of Pharmacology
      • • Department of Medicine
      North Carolina, United States
  • 1995
    • University of Louisiana at Monroe
      Монро, Louisiana, United States
  • 1993
    • Mayo Clinic - Rochester
      • Department of Gastroenterology and Hepatology
      Рочестер, Minnesota, United States
  • 1990
    • Universität des Saarlandes
      Saarbrücken, Saarland, Germany
  • 1983
    • University of North Carolina at Charlotte
      Charlotte, North Carolina, United States
  • 1973
    • Johns Hopkins University
      Baltimore, Maryland, United States
  • 1971
    • National Institute of Allergy and Infectious Diseases
      Maryland, United States